Permissive lung neutrophils facilitate tuberculosis immunopathogenesis in male phagocyte NADPH oxidase-deficient mice

Eunsol Choi; Hong-Hee Choi; Kee Woong Kwon; Hagyu Kim; Ji-Hwan Ryu; Jung Joo Hong; Sung Jae Shin

doi:10.1371/journal.ppat.1012500

Abstract

NADPH oxidase 2 (NOX2) is an enzyme responsible for generating reactive oxygen species, primarily found in phagocytes. Chronic Granulomatous Disease (CGD), along with bacterial infections such as Mycobacterium tuberculosis (Mtb), is a representative NOX2-deficient X-linked disease characterized by uncontrolled inflammation. However, the precise roles of host-derived factors that induce infection-mediated hyperinflammation in NOX2-deficient condition remain incompletely understood. To address this, we compared Mtb-induced pathogenesis in Nox2^-/- and wild type (WT) mice in a sex-dependent manner. Among age- and sex-matched mice subjected to Mtb infection, male Nox2^-/- mice exhibited a notable increase in bacterial burden and lung inflammation. This was characterized by significantly elevated pro-inflammatory cytokines such as G-CSF, TNF-α, IL-1α, IL-1β, and IL-6, excessive neutrophil infiltration, and reduced pulmonary lymphocyte levels as tuberculosis (TB) progressed. Notably, lungs of male Nox2^-/- mice were predominantly populated with CD11b^intLy6G^intCXCR2^loCD62L^lo immature neutrophils which featured mycobacterial permissiveness. By diminishing total lung neutrophils or reducing immature neutrophils, TB immunopathogenesis was notably abrogated in male Nox2^-/- mice. Ultimately, we identified G-CSF as the pivotal trigger that exacerbates the generation of immature permissive neutrophils, leading to TB immunopathogenesis in male Nox2^-/- mice. In contrast, neutralizing IL-1α and IL-1β, which are previously known factors responsible for TB pathogenesis in Nox2^-/- mice, aggravated TB immunopathogenesis. Our study revealed that G-CSF-driven immature and permissive pulmonary neutrophils are the primary cause of TB immunopathogenesis and lung hyperinflammation in male Nox2^-/- mice. This highlights the importance of quantitative and qualitative control of pulmonary neutrophils to alleviate TB progression in a phagocyte oxidase-deficient condition.

Author summary

While tuberculosis (TB) remains a global threat to public health, the immunologic factors contributing to TB susceptibility are not yet fully defined. To enhance our understanding of TB immunopathogenesis, we utilized TB-susceptible male Nox2^-/- mice to dissect the immunologic factors accelerating TB pathogenesis. In this study, we observed that male Nox2^-/- mice infected with Mycobacterium tuberculosis (Mtb) exhibited increased lung hyperinflammation and mycobacterial burden compared to female Nox2^-/- mice and WT mice. Exacerbated TB immunopathogenesis in male Nox2^-/- mice was strongly correlated with an extreme infiltration of pulmonary neutrophils and the loss of pulmonary lymphocytes. The pulmonary neutrophils in male Nox2^-/- mice displayed immature phenotypes and mycobacterial permissiveness. The depletion of total neutrophils or the AM80-induced maturation of neutrophils ameliorated TB pathogenesis in Nox2^-/- mice, reducing pulmonary neutrophil counts, lung hyperinflammation, and mycobacterial load. Furthermore, neutralization of G-CSF mitigated TB pathogenesis in male Nox2^-/- mice by decreasing immature neutrophil counts. Thus, we identified that G-CSF-mediated generation of permissive immature neutrophils contributes to TB susceptibility in male Nox2^-/- mice.

Citation: Choi E, Choi H-H, Kwon KW, Kim H, Ryu J-H, Hong JJ, et al. (2024) Permissive lung neutrophils facilitate tuberculosis immunopathogenesis in male phagocyte NADPH oxidase-deficient mice. PLoS Pathog 20(8): e1012500. https://doi.org/10.1371/journal.ppat.1012500

Editor: Christopher M. Sassetti, University of Massachusetts Medical School, UNITED STATES OF AMERICA

Received: November 14, 2023; Accepted: August 12, 2024; Published: August 23, 2024

Copyright: © 2024 Choi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are presented in the paper and supporting information.

Funding: This work was supported by the National Research Foundation of Korea (NRF) grant, funded by the Ministry of Science and Information and Communication Technology (MSIT) under grant number RS-2023-00208115 to SJS and the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program under grant number KGM4572431 to SJS. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Mycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), which remains a significant public health concern worldwide. Approximately, one-quarter of the world’s population is estimated to have latent Mtb infection, with 5–10% of these individuals at risk of developing active TB at some point during their lifetime [1]. The factors that influence susceptibility to Mtb are still not completely understood, which hampers the control of this harmful pathogen [2–4]. There is extensive documentation indicating that males are more susceptible to TB than females, and this sexual bias is observed in various animal models including mice and humans [5,6]. The increased TB susceptibility in males is likely due to differences in immune cell functions as well as in their compositions [7,8]. Such host factor-mediated alterations in immune function may directly influence immune responses to Mtb infection. Considering the importance of immunological balance in maximizing anti-TB immunity, both excessive immune suppression or activation might be harmful to the host during TB progression [9–11]. These clues have led to the hypothesis that several host factors, such as biological sex and genetic distributions, which modulate inflammatory responses, may influence TB immunopathogenesis.

The phagocyte NADPH oxidase (NOX2, gp91^phox) is located in the lumen of phagosomes. Its primary function is to produce reactive oxygen species (ROS), which play a crucial role in protecting the host against a wide range of pathogens [12,13]. NOX2 also plays a vital role in regulating autophagy and cytokine/chemokine signaling [14–16]. The NOX2 complex is a multimeric enzyme composed of a single catalytic transmembrane heterodimer (gp91^phox / p22^phox) and four cytosolic subunits (p40^phox / p47^phox / p67^phox / Rac). Among the six major components of NOX2 complex, gp91^phox acts as the catalytic core for ROS generation [17,18]. Individuals with impaired NOX2 function may develop chronic granulomatous disease (CGD), characterized by an increased susceptibility to fungal and bacterial infections and impaired inflammation control [19–21]. Since Nox2 is located at Xp21.1 of the X chromosome, CGD caused by Nox2 deficiency is inherited in an X-linked recessive manner, primarily affecting young males [22,23]. Furthermore, individuals with X-linked CGD have a heightened vulnerability to mycobacterial infections [24]. This poses significant challenges for TB therapy and prevention in X-linked CGD patients as BCG vaccination cannot be implemented due to the high risks of BCGosis, which is a disseminated mycobacterial infection that can occur following BCG vaccination [25–27].

Although phagocyte NADPH oxidase-originated ROS play a crucial role in clearing intracellular pathogens, studies have shown that phagocyte NADPH oxidase deficiencies did not significantly alter mycobacterial growth compared to wild type (WT) animals. According to Cooper et al., p47^phox-deficient mice featured increase in lung mycobacterial load from 15 to 30 days post-infection. However, the increase in mycobacterial load was not sustained after 30 days post-infection [28]. Additionally, Olive et al. reported that male and female gp91^phox-deficient mice did not show an increase in lung mycobacterial load until 60 days or 20 weeks post-infection [29,30]. These researches give clues that NOX2 deficiency may modulate immune responses against Mtb, independently of ROS-mediated bactericidal effects. Recent studies support this idea as Thomas et al. demonstrated that gp91^phox-deficient mice displayed enhanced inflammation and lung neutrophil infiltration after Mtb challenge, which were mediated by IL-1 [12,31].

Dysregulated inflammatory responses and aberrant granuloma formation are hallmark features of X-linked CGD. X-linked CGD patients and NOX2-deficient mice exhibit uncontrolled formation of granulomas, characterized by an excessive influx of phagocytic myeloid cells to the site of infection [32,33]. NOX2-deficient phagocytes, such as macrophages and neutrophils, are major producers of pro-inflammatory cytokines [34,35], which can cause tissue damage [36,37]. Given that NOX2 deficiency-induced X-linked CGD can cause TB susceptibility along with lung hyperinflammation, we hypothesized that Mtb infection would cause uncontrolled inflammation and disruption of immune tolerance in NOX2-deficient mice. As the maintenance of immune tolerance is critical for effective anti-TB immunity, we further hypothesized that controlling the specific immunologic factor which causes Mtb infection-driven hyperinflammation would be crucial for ameliorating TB pathogenesis.

In this study, we established four experimental groups to investigate how two independent host factors (NOX2 deficiency and male sex) can modify the immunological responses against Mtb infection. As both male sex and NOX2 deficiency are risk factors for TB susceptibility, we hypothesized that male Nox2^-/- mice would exhibit severer TB progression than female Nox2^-/- mice and male WT mice. Additionally, we anticipated that specific immune factors would contribute to TB pathogenesis and hyperinflammation in male Nox2^-/- mice, which show a clear correlation to disease progression. Finally, we aimed to discover novel interventions that can effectively regulate these disease-promoting factors in male Nox2^-/- mice.

We found that male Nox2^-/- mice exhibited higher levels of lung inflammation and mycobacterial burden compared to female Nox2^-/- mice and WT mice following Mtb challenge. Male Nox2^-/- mice also featured elevated production of pro-inflammatory cytokines in the lungs, along with excessive infiltration of immature neutrophils and reduction of lymphocytes at infection sites. We also dissected the properties of aberrant permissive immature neutrophils, which were distinct from mature neutrophils and constituted the majority of immune cell populations in male Nox2^-/- mice. By reducing the number of total lung neutrophils or immature neutrophils, we could successfully mitigate TB pathogenesis in male Nox2^-/- mice. Finally, we determined that G-CSF, rather than other cytokines, is the dominant controller of immature neutrophil generation through in vivo neutralization of each cytokine. Collectively, we identified G-CSF driven permissive neutrophils as the key immunologic factor that facilitates TB immunopathogenesis and lung hyperinflammation in male Nox2^-/- mice. Furthermore, we suggested novel therapeutic interventions targeting immature neutrophils, which might be potential for controlling TB in NOX2-deficient condition.

Results

Male Nox2^-/- mice feature increased mycobacterial burden and lung hyperinflammation following aerosol infection with the Mtb K strain

Female WT, male WT, female Nox2^-/-, and male Nox2^-/- mice were aerosol-infected with the Mtb K. At four weeks post-infection, the mice were autopsied to assess the severity of TB and immune function (Fig 1A). The mice were euthanized at the four weeks post-infection time point, when the increases in lung bacterial load (S1A Fig) and neutrophil infiltration (S1B Fig)—key pathological features of Mtb-infected Nox2^-/- mice [12,31]—reached their peak. These measures began to increase from two weeks post infection, peaked at four weeks post-infection, and subsequently decreased until twelve weeks post-infection in Nox2^-/- mice. Male Nox2^-/- mice showed the highest lung bacterial load, spleen bacterial load, and lung inflammation. Female Nox2^-/- mice featured the second-highest severity, while male and female WT mouse groups were ranked as 3rd and 4th, respectively (Fig 1B and 1C). Pro-inflammatory cytokines including IFN-γ, IL-1α, IL-1β, IL-17A, IL-6, TNF-α, G-CSF, and BAFF were significantly upregulated in the lungs of male Nox2^-/- mice (Fig 1D). Conversely, anti-inflammatory cytokines including IL-4, IL-5, and TGF-β were not upregulated in the lungs of male Nox2^-/- mice, although IL-10 was significantly upregulated (Fig 1E). This indicates that NOX2 deficiency plays a significant role in accelerating TB immunopathogenesis, as both male and female Nox2^-/- mice feature exacerbated TB progression. Furthermore, we also found that male sex additionally promotes TB pathogenesis, as indicated by severer TB progression in male Nox2^-/- mice.

Download:

Fig 1. Male Nox2^-/- mice featured severe lung hyperinflammation and increased bacterial load upon Mtb infection.

(A) Experimental design for in vivo analysis of TB susceptibility. Six to seven-week-old female WT, male WT, female Nox2^-/-, and male Nox2^-/- mice (n = 6 per group) were aerosol-infected with Mtb K strain. At four weeks post-infection, all mice were autopsied, and immunological analysis, bacterial counting, and histopathological analysis were conducted (indicated by red arrow). Initial CFU = 288. (B) Mycobacterial CFUs in the lungs and spleens of each group at four weeks post-infection were analyzed by calculating the number of colonies and presented in bar graphs. (C) H&E staining was performed on the superior lobes of the right lung at four weeks post-infection to visualize the gross lung pathology. The inflamed area of the H&E-stained samples was quantified in terms of percentage and square millimeters and presented in bar graphs. (D) IFN-γ, IL-1α, IL-1β, IL-17A, IL-6, TNF-α, G-CSF, and BAFF levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex. (E) IL-10, IL-4, IL-5, and TGF-β levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex. The cytokine levels are presented in bar graphs. The experiment was conducted twice. The data are presented as the mean ± SD of six mice in each group. The significance of differences was determined, using the One-way ANOVA test. n.s., not significant. *p < 0.05, **p <0.01.

https://doi.org/10.1371/journal.ppat.1012500.g001

Pulmonary infiltration of neutrophils and loss of B lymphocytes are correlated with TB pathogenesis of male Nox2^-/- mice

To determine which immune cell population is associated with dysregulated pro-inflammatory cytokine production, live lung cell compositions of Mtb infected mice were examined according to the attached flow cytometry gating strategy (S1C Fig). Male Nox2^-/- mice exhibited the highest numbers and percentages of CD11b⁺Ly6G⁺ lung neutrophils, while female WT mice featured the lowest among the four groups (Fig 2A). On the other hand, male Nox2^-/- mice showed a significant reduction in pulmonary CD90.2⁺ T cell percentages (Fig 2B), CD19⁺B220⁺ B cell counts (Fig 2C), and CD19⁺B220⁺GL7⁺FAS⁺ Germinal Center B cell counts (Fig 2D).

The disparity in TB progression peaked at four weeks post-infection, as male Nox2^-/- mice exhibited significantly increased lung bacterial loads and pulmonary neutrophil counts at this time, compared to male WT mice and female Nox2^-/- mice. The increase in lung CFU (Colony-forming unit) s and pulmonary neutrophils in male Nox2^-/- mice began between two and three weeks post-infection and reached its maximum at four weeks post-infection. Interestingly, the disparity in pulmonary neutrophil counts between male Nox2^-/- mice and other groups was more pronounced than the differences in lung CFUs at two and three weeks post-infection, as the latter did not show significant statistical differences, suggesting that the influx of pulmonary neutrophils occurs earlier than the increase in lung bacterial load (Fig 2E). T cell responses against mycobacterial antigen stimulations were also examined, as IFN-γ producing CD4⁺ T cells take crucial roles in anti-TB immunity [38,39]. Although male Nox2^-/- mice featured the most exacerbated TB progression among the four groups, T cell responses against Mtb antigens were strongly maintained (S2 Fig). The numbers of pulmonary CD11c⁺Siglec-F⁺ alveolar macrophages, CD11b⁺CD64⁺ macrophages, and CD11c⁺MHCII⁺ dendritic cells were also significantly reduced in male Nox2^-/- mice (S3 Fig). The immune cell dynamics in the lungs of mice infected with Mtb were visually represented using pie-charts and t-SNE plots. We observed that neutrophils, B cells, and T cells comprise the majority of pulmonary immune cells (Fig 2E). Furthermore, we found strong correlations between these immune cell populations and TB severity. The percentages of neutrophils displayed a strong positive correlation with TB severity, with high correlation coefficients and low p-values. Conversely, the percentages of T cells did not exhibit a significant correlation with TB severity, while the percentages of B cells demonstrated a reverse correlation with TB severity. The loss of pulmonary B cells also showed a significant negative correlation with neutrophil infiltration (S4 Fig). Thus, we hypothesized that TB susceptibility of male Nox2^-/- mice is highly associated with the increase of pulmonary neutrophils.

Download:

Fig 2. Neutrophils are increased in the lungs of Mtb infected male Nox2^-/- mice while B cells are diminished.

Pulmonary (A) CD11b⁺Ly6G⁺ neutrophil, (B) CD90.2⁺ T cell, (C) CD19⁺ B220⁺ B cell, and (D) CD19⁺ B220⁺ GL7⁺ FAS⁺ Germinal Center B cell populations of Mtb-infected mice at four weeks post-infection. The percentages of each immune cell among lung CD45⁺ cells and total cell counts are presented in bar graphs. (E) Pulmonary CFUs and neutrophil counts of female WT, male WT, female Nox2^-/-, and male Nox2^-/- mice were enumerated in a time-course dependent manner. Six-week old mice (n = 5 per group) were aerosol infected with Mtb K strain. The number of lung neutrophils were calculated at 0, 2, 3, and 4 weeks post-infection. Red bars and symbols represent statistical analysis between male Nox2^-/- mice and male WT mice. Black bars and symbols represent statistical analysis between male Nox2^-/- mice and female Nox2^-/- mice. Initial CFU = 300. The experiment was conducted once. The data are presented as the mean ± SD of five or six mice in each group. The significance of differences was determined, using the One-way ANOVA test. n.s., not significant. **p <0.01. (F) The compositions of the six major lung immune cell populations in Mtb-infected mice are presented in pie charts and t-SNE plots. The six major immune cells consist of neutrophils (red), macrophages (orange), alveolar macrophages (bright brown), dendritic cells (purple), B cells (blue), and T cells (green). The percentages of each immune cell among the sum of the six major immune cells are presented in pie charts.

https://doi.org/10.1371/journal.ppat.1012500.g002

Pulmonary neutrophils of Mtb infected male Nox2^-/- mice are predominantly composed of aberrant CD11b^intLy6G^intCXCR2^loCD62L^lo permissive immature neutrophils

It was not only the number of neutrophils that were altered in male Nox2^-/- mice. Pulmonary neutrophils of male Nox2^-/- mice featured CD11b^intLy6G^int phenotypes, while neutrophils of other mouse groups featured CD11b^hiLy6G^hi phenotypes. Atypical lung neutrophils of male Nox2^-/- mice showed relatively lower expression of CD11b, Ly6G, Ly6C, CXCR2, and CD62L compared to WT neutrophils, along with higher expression of CXCR4 (Fig 3A). We categorized CD11b⁺Ly6G⁺ pulmonary neutrophils into two distinct populations based on CXCR2 expression. According to previous studies, Ly6G^hi or CXCR2^hi lung neutrophils are categorized as mature neutrophils, whereas Ly6G^int or CXCR2^lo lung neutrophils are categorized as immature neutrophils [40–43]. We revealed that CD11b⁺Ly6G⁺ pulmonary neutrophils of Mtb infected mice are consisted of CXCR2^hi and CXCR2^lo populations. Moreover, we figured out that CXCR2^hi mature pulmonary neutrophils highly expressed CD62L, while CXCR2^lo immature neutrophils featured low expression of CD62L through our own gating strategy (Fig 3B). The majority of Nox2^-/- pulmonary neutrophils were CXCR2^loCD62L^lo immature neutrophils, whereas WT pulmonary neutrophils comprised both immature and mature neutrophils. This suggests that the excessive accumulation of pulmonary neutrophils in male Nox2^-/- mice is primarily mediated by immature neutrophil-specific lung infiltration (Fig 3C). We also analyzed neutrophil profiles from uninfected WT and Nox2^-/- mice. The number of pulmonary total and immature neutrophils (S5A and S5B Fig), as well as bone marrow (BM) total and immature neutrophils (S5C and S5D Fig), were not increased in uninfected male Nox2^-/- mice. Therefore, we concluded that the extreme pulmonary infiltration of CXCR2^loCD62L^lo immature neutrophils is initiated by the pathogenic stimulus triggered by Mtb infection. CXCR2^loCD62L^lo immature lung neutrophils displayed significantly lower expression levels of CD11b and Ly6G, which are major activation markers of neutrophils. They also featured lower FSC-A and SSC-A values compared to mature lung neutrophils, indicating that CXCR2^loCD62L^lo neutrophils are characterized by their smaller size and reduced granularity when compared to mature neutrophils (Fig 3D). To evaluate the permissiveness of pulmonary neutrophils, we isolated lung neutrophils from Mtb-infected mice and quantified intracellular bacterial counts. Nox2^-/- pulmonary neutrophils, predominantly consisted of CXCR2^loCD62L^lo immature neutrophils, exhibited higher mycobacterial permissiveness compared to WT pulmonary neutrophils (Fig 3E). To examine if CXCR2^loCD62L^lo immature neutrophils are more permissive than CXCR2^hiCD62L^hi mature neutrophils, we infected male WT and Nox2^-/- mice with YFP-expressing Mtb K strain. At four weeks post-infection, the mice were euthanized, and neutrophil permissiveness was evaluated using flow cytometry analysis (S6A Fig). We noted that neutrophils constituted the majority of YFP⁺ cells in male Nox2^-/- mice, and the number of Mtb-containing neutrophils were also increased. The mean fluorescence intensity (MFI) values of YFP in Nox2^-/- lung neutrophils were substantially higher than those in WT mice, indicating increased mycobacterial permissiveness of Nox2^-/- lung neutrophils (S6B Fig). Both immature and mature Mtb-containing neutrophil populations were enlarged in Nox2^-/- mice. Notably, immature neutrophils displayed considerably higher YFP MFI values than mature neutrophils, suggesting their increased permissiveness to Mtb (S6C Fig). Conversely, the proportion of YFP⁺ alveolar macrophages was lower in male Nox2^-/- mice, and there was no significant difference in the number of YFP⁺ alveolar macrophages between WT and Nox2^-/- mice, although these cells exhibited relatively higher YFP MFI values compared to mature neutrophils (S6D Fig). The number of YFP⁺ macrophages was elevated in Nox2^-/- mice, but their YFP MFI values were relatively lower than those of immature neutrophils and alveolar macrophages (S6E Fig). In conclusion, we hypothesized that immature neutrophils are the primary permissive phagocytes in male Nox2^-/- mice, considering both their dominance in population and their significant bacterial permissiveness as indicated by MFI values. These results suggest that permissive immature lung neutrophils may play a key role in driving TB pathogenesis and lung hyperinflammation in male Nox2^-/- mice.

Download:

Fig 3. CD11b^intLy6G^int CXCR2^loCD62L^lo immature neutrophils were dominant among the pulmonary neutrophils of Mtb infected male Nox2^-/- mice.

(A) The expression levels of CD11b, Ly6G, Ly6C, CXCR4, CXCR2, and CD62L on CD11b⁺Ly6G⁺ neutrophils in the lungs of Mtb-infected mice at four weeks post-infection are presented. The mean fluorescent intensity (MFI) values of each molecule are presented in bar graphs. (B) The gating strategy used to distinguish immature and mature pulmonary neutrophils. CD11b⁺Ly6G⁺ neutrophils were classified into CXCR2^hi mature neutrophils (red) and CXCR2^lo immature neutrophils (bright blue). (C) CD62L expression levels of CXCR2^hi neutrophils and CXCR2^lo neutrophils are presented into contour plots, demonstrating that mature neutrophils feature CXCR2^hiCD62L^hi phenotypes while immature neutrophils feature CXCR2^loCD62L^lophenotypes. The numbers and percentages of immature and mature neutrophils among total pulmonary neutrophils in each group are presented in bar graphs, along with flow cytometry plots. (D) The expression levels of CD11b and Ly6G on mature and immature pulmonary neutrophils are presented in flow cytometry plots and bar graphs, along with FSC-A and SSC-A values of mature and immature neutrophils. (E) The purity of mouse lung neutrophils after Ly6G specific magnetic sorting and their bacterial permissiveness are presented in flow cytometry plots and bar graphs. Equal numbers of WT and Nox2^-/- lung neutrophils were lysed and plated onto 7H10 agar plates to enumerate bacterial permissiveness. The data are presented as the mean ± SD of six mice in each group. The significance of differences was determined, using the One-way ANOVA test and Mann-Whitney U test. *p < 0.05, **p <0.01.

https://doi.org/10.1371/journal.ppat.1012500.g003

Neutrophil depletion alleviates TB pathogenesis and lung hyperinflammation in male Nox2^-/- mice

The exacerbated TB pathogenesis of male Nox2^-/- mice was characterized by uncontrolled pro-inflammatory cytokine production and influx of permissive immature pulmonary neutrophils. Considering that neutrophils contribute to lung inflammation in Nox2^-/- mice [20,44,45], and they are responsible for the increased mycobacterial burden in TB susceptible mouse models [46,47], we hypothesized that reducing Ly6G⁺ neutrophils would alleviate TB pathogenesis in male Nox2^-/- mice. Starting from two weeks post-infection, a Ly6G-specific monoclonal antibody (mAb) was intraperitoneally injected to WT and Nox2^-/- mice to deplete neutrophils. At four weeks post-infection, the mice were autopsied (Fig 4A). Neutrophil depletion alleviated TB pathogenesis in both WT and Nox2^-/- mice, by effectively reducing bacterial load in the lungs and spleens (Fig 4B) and lung inflammation (Fig 4C). IFN-γ, IL-1 α, IL-1 β, IL-6, TNF-α, and G-CSF levels were significantly downregulated in the lungs of male Nox2^-/- mouse after neutrophil depletion (Fig 4D), while IL-10, IL-4, IL-5, and TGF-β levels were not significantly altered (Fig 4E). Therefore, we clarified that Ly6G⁺ neutrophils are the actual cause of TB progression and lung hyperinflammation in male Nox2^-/- mouse.

Download:

Fig 4. Neutrophil depletion ameliorated TB pathogenesis in male Nox2^-/- mice.

(A) Experimental design for in vivo depletion of neutrophils in Mtb infected mice. six-week old male WT and Nox2^-/- mice (n = 4 per group) were aerosol infected with Mtb K strain. Starting from two weeks post-infection, 250 μg of anti-Ly6G mAb was intraperitoneally injected into each mouse three times a week (indicated by blue bars). At four weeks post-infection, all mice were autopsied, and immunological analysis, bacterial counting, and histopathological analysis were conducted (indicated by red arrow). Initial CFU = 569. (B) Mycobacterial CFUs in the lungs and spleens of each group at four weeks post-infection were analyzed by calculating the number of colonies and presented in bar graphs. (C) H&E staining was performed on the superior lobes of the right lung at four weeks post-infection to visualize the gross lung pathology. The inflamed area of the H&E-stained samples was quantified in terms of percentage and square millimeters and presented in bar graphs. (D) IFN-γ, IL-1α, IL-1β, IL-17A, IL-6, TNF-α, G-CSF, and BAFF levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex. (E) IL-10, IL-4, IL-5, and TGF-β levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex. The cytokine levels are presented in bar graphs. The experiment was conducted twice. The data are presented as the mean ± SD of four mice in each group. The significance of differences was determined, using the One-way ANOVA and Mann-Whitney U test. n.s., not significant. *p < 0.05.

https://doi.org/10.1371/journal.ppat.1012500.g004

Neutrophil depletion reduces immature lung neutrophils and restores pulmonary lymphocytes in Mtb infected male Nox2^-/- mice

Ly6G⁺ neutrophil depletion not only ameliorated TB pathogenesis, but also altered immune cell compositions in the lungs of male Nox2^-/- mice. Neutrophil depletion removed most lung neutrophils in WT mice, and significantly reduced pulmonary neutrophil numbers in male Nox2^-/- mice (Fig 5A). Surprisingly, it was highly effective at reducing CXCR2^loCD62L^lo pulmonary immature neutrophils in both WT and Nox2^-/- mice (Fig 5B). Along with the reduction of immature pulmonary neutrophils, the number of B cells were increased in the lungs of Mtb infected male Nox2^-/- mice (Fig 5C). However, neutrophil depletion did not significantly alter lung T cell population in male Nox2^-/- mice (Fig 5D). This phenomenon was evident from the pie-charts and t-SNE plots illustrating lung immune cell dynamics, which clearly showed the reduction of pulmonary neutrophils and the restoration of B cells (Fig 5E). Consequently, we discovered that the reduction of pulmonary neutrophil counts can restore the balance between lung lymphocytes and neutrophils in male Nox2^-/- mice.

Download:

Fig 5. Neutrophil depletion removed immature neutrophils and restored lymphocytes in the lungs of Mtb infected Nox2^-/- mice.

Pulmonary (A) CD11b⁺Ly6G⁺ neutrophil, (B) CD11b⁺Ly6G⁺CXCR2^loCD62L^lo immature neutrophil, CD11b⁺Ly6G⁺CXCR2^hiCD62L^hi mature neutrophil, (C) CD19⁺MHC-II⁺ B cell, and (D) CD90.2⁺ T cell populations of Mtb-infected mice at four weeks post-infection. The percentages of each immune cell among lung CD45⁺ cells or total neutrophils, and total cell counts are presented in bar graphs, along with flow cytometry plots. (E) The compositions of the six major lung immune cell populations in Mtb-infected mice are presented in pie charts and t-SNE plots. The six major immune cells consist of neutrophils (red), macrophages (orange), alveolar macrophages (bright brown), dendritic cells (purple), B cells (blue), and T cells (green). The percentages of each immune cell among the sum of the six major immune cells are presented in pie charts. The data are presented as the mean ± SD of four mice in each group. The significance of differences was determined, using the One-way ANOVA and Mann-Whitney U test. n.s., not significant. *p < 0.05.

https://doi.org/10.1371/journal.ppat.1012500.g005

AM80 administration mitigates TB pathogenesis in male Nox2^-/- mice and reduces pulmonary immature neutrophils

Among various strategies for the specific control of immature neutrophils, we considered RA treatment as a promising option. Previous studies have examined that retinoic acid (RA) or retinoic acid receptor (RAR) agonist administration can effectively promote the generation of fully differentiated mature neutrophils, which feature segmented nuclei and enhanced bactericidal capacity [48,49]. Moreover, Yamada et al. suggested the potential of RAs such as all-trans retinoic acid (ATRA) as a therapeutic agent against mycobacterial infection [50,51]. To investigate whether immature neutrophil-specific intervention can ameliorate TB pathogenesis in male Nox2^-/- mouse, we utilized tamibarotene (AM80), a synthetic retinoid and a RAR agonist demonstrating 10 times greater potency than ATRA. AM80 induces the expression of key molecules, such as C/EBP, CD11b, and CXCR2 through the activation of RARα and RARβ [52], triggering a cascade of cellular pathways that generate fully functional mature neutrophils [53,54]. Starting from one-week post-infection, we orally administered 40 μg of AM80 per mouse, with a total of nine injections given over three weeks. At four weeks post-infection, the mice were autopsied (Fig 6A). AM80 administration significantly reduced mycobacterial load in the lungs and spleens (Fig 6B) and alleviated lung inflammation (Fig 6C) in male Nox2^-/- mice. However, despite a reduction in pulmonary bacterial load, its effects were not prominent in reducing lung inflammation of male WT mice. AM80 administration downregulated pulmonary expression of IFN-γ, IL-1 β, IL-17A, and G-CSF in male Nox2^-/- mice. It did not substantially alter the expression of pro-inflammatory cytokines in male WT mice, although the expression of TNF-α was significantly reduced (Fig 6D). AM80 intervention did not alter the expression of IL-10, IL-4, IL-5, and TGF-β in both WT and Nox2^-/- mice (Fig 6E). Total lung neutrophil counts (Fig 6F) and CXCR2^loCD62L^lo immature lung neutrophil counts (Fig 6G) were significantly decreased, and the portion of mature neutrophils among total lung neutrophils were increased in AM80-treated WT and Nox2^-/- mice. While AM80 treatment effectively modified the accumulation of immature lung neutrophils, it did not exhibit significant effects in promoting Mtb-specific T cell responses in WT and Nox2^-/- mice (S7 Fig). This suggests that the therapeutic potential of AM80 intervention in male Nox2^-/- mice is largely contingent on the conversion of neutrophil maturity. To summarize, we specified immature lung neutrophils as the key subpopulation responsible for accelerating TB pathogenesis in male Nox2^-/- mice.

Download:

Fig 6. AM80 administration mitigated TB pathogenesis in male Nox2^-/- mice by reducing immature neutrophils.

(A) Experimental design for in vivo administration of AM80 in Mtb infected mice. Six-week old male WT and Nox2^-/- mice (n = 5 per group) were aerosol infected with Mtb K strain. Starting from one week post-infection, 20 μg of AM80 was orally administered to each mouse three times a week (indicated by blue bars). At four weeks post-infection, all mice were autopsied, and immunological analysis, bacterial counting, and histopathological analysis were conducted (indicated by red arrow). Initial CFU = 320. (B) Mycobacterial CFUs in the lungs and spleens of each group at four weeks post-infection were analyzed by calculating the number of colonies and presented in bar graphs. (C) H&E staining was performed on the superior lobes of the right lung at four weeks post-infection to visualize the gross lung pathology. The inflamed area of the H&E-stained samples was quantified in terms of percentage and square millimeters and presented in bar graphs. (D) IFN-γ, IL-1α, IL-1β, IL-17A, IL-6, TNF-α, G-CSF, and BAFF levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex. (E) IL-10, IL-4, IL-5, and TGF-β levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex. The cytokine levels are presented in bar graphs. Pulmonary (F) CD11b⁺Ly6G⁺ neutrophil, (G) CD11b⁺Ly6G⁺CXCR2^loCD62L^lo immature neutrophil, and CD11b⁺Ly6G⁺CXCR2^hiCD62L^hi mature neutrophil populations of Mtb-infected mice at four weeks post-infection. The percentages of each immune cell among lung CD45⁺ cells or total neutrophils, and total cell counts are presented in bar graphs, along with flow cytometry plots. The experiment was conducted twice. The data are presented as the mean ± SD of five mice in each group. The significance of differences was determined, using the One-way ANOVA and Mann-Whitney U test. n.s., not significant. *p < 0.05. **p < 0.01.

https://doi.org/10.1371/journal.ppat.1012500.g006

G-CSF predominantly regulates the generation of permissive immature pulmonary neutrophils in Mtb infected male Nox2^-/- mice

To conclude the study, we aimed to identify specific immunologic factors responsible for the generation of CXCR2^loCD62L^lo permissive immature pulmonary neutrophils. Considering that pro-inflammatory cytokines upregulated in the lungs of Mtb-infected male Nox2^-/- mice, such as IL-1, IL-6, and G-CSF accelerate the production of immature neutrophils through emergency granulopoiesis [55–57], we hypothesized that uncontrolled production of pro-inflammatory cytokines may trigger an explosive generation of immature neutrophils. To identify the causative cytokines, we administered mAbs to neutralize each cytokine in Nox2^-/- mice. We confirmed that IFN-γ and IL-17A, key cytokines in anti-TB immunity for both innate and adaptive immune responses, are not associated with the generation of immature neutrophils. In vivo neutralization of IFN-γ or IL-17A increased mycobacterial burden, lung inflammation, pro-inflammatory cytokine levels, and pulmonary influx of immature neutrophils in female Nox2^-/- mice (S8 Fig). Furthermore, in vivo neutralization of IL-6, IL-1α, and IL-1β also aggravated immature neutrophil-mediated TB pathogenesis and lung hyperinflammation in Nox2^-/- mice, although IL-1 is reported to be crucial for mediating neutrophilic inflammation in NOX2-deficient conditions [12,36,37,44] (S9 Fig). Blockade of IL-1 receptor (IL-1R), which inhibits the function of both IL-1α, and IL-1β, exacerbated TB progression in Nox2^-/- mice as well. We implemented two different in vivo blockade models: a three-week IL-1R blockade on female Nox2^-/- mice and a two-week IL-1R blockade on male Nox2^-/- mice, following the protocols from Olive et al. [12]. Both blockade models resulted in exacerbated TB progression, as evidenced by increased pulmonary bacterial load, lung inflammation, and neutrophil counts in both female and male Nox2^-/- mice (S10 Fig). In contrast, in vivo neutralization of G-CSF, the main controller of granulopoiesis, remarkably ameliorated immature neutrophil-mediated TB pathogenesis in male Nox2^-/- mice. Starting from one-week post-infection, G-CSF specific mAb was intraperitoneally administered, with a total of nine injections over a three-week period. At four weeks post-infection, the mice were autopsied (Fig 7A). While G-CSF neutralization did not alleviate TB progression in WT mice, it significantly reduced mycobacterial load in the lungs and spleens (Fig 7B) and mitigated lung inflammation (Fig 7C) of male Nox2^-/- mice. G-CSF neutralization also downregulated IFN-γ, IL-1α, IL-1β, IL-17A, IL-6, TNF-α, G-CSF, and BAFF levels specifically in the lungs of male Nox2^-/- mice (Fig 7D), while IL-10. IL-4, IL-5, and TGF-β (Fig 7E) levels did not change. G-CSF neutralization decreased total lung neutrophil counts (Fig 7F) and CXCR2^loCD62L^lo immature lung neutrophil counts (Fig 7G) in male WT and Nox2^-/- mice. The reduction of immature lung neutrophils was particularly pronounced and dramatically reflected in G-CSF neutralized male Nox2^-/- mice. This underscores that the excessive G-CSF-mediated accumulation of pathogenic immature neutrophils is a specific event observed in TB susceptible male Nox2^-/- mice Ultimately, we concluded that upregulated G-CSF is the immunologic trigger for the generation of permissive immature pulmonary neutrophils, which facilitates TB immunopathogenesis and lung hyperinflammation in male Nox2^-/- mice (Fig 8).

Download:

Fig 7. G-CSF neutralization ameliorated TB pathogenesis of male Nox2^-/- mice by suppressing generation of immature pulmonary neutrophils.

(A) Experimental design for in vivo neutralization of G-CSF in Mtb infected mice. Six-week old male WT and Nox2^-/- mice (n = 6 per group) were aerosol infected with Mtb K strain. Starting from one week post-infection, 30 μg of anti-G-CSF mAb was intraperitoneally administered to each mouse three times a week (indicated by blue bars). At four weeks post-infection, all mice were autopsied, and immunological analysis, bacterial counting, and histopathological analysis were conducted (indicated by red arrow). Initial CFU = 225. (B) Mycobacterial CFUs in the lungs and spleens of each group at four weeks post-infection were analyzed by calculating the number of colonies and presented in bar graphs. (C) H&E staining was performed on the superior lobes of the right lung at four weeks post-infection to visualize the gross lung pathology. The inflamed area of the H&E-stained samples was quantified in terms of percentage and square millimeters and presented in bar graphs. (D) IFN-γ, IL-1α, IL-1β, IL-17A, IL-6, TNF-α, G-CSF, and BAFF levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex. (E) IL-10, IL-4, IL-5, and TGF-β levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex. The cytokine levels are presented in bar graphs. Pulmonary (F) CD11b⁺Ly6G⁺ neutrophil, (G) CD11b⁺Ly6G⁺CXCR2^loCD62L^lo immature neutrophil, and CD11b⁺Ly6G⁺CXCR2^hiCD62L^hi mature neutrophil populations of Mtb-infected mice at four weeks post-infection. The percentages of each immune cell among lung CD45⁺ cells or total neutrophils, and total cell counts are presented in bar graphs, along with flow cytometry plots. The experiment was conducted three times. The data are presented as the mean ± SD of six mice in each group. The significance of differences was determined, using the One-way ANOVA test. n.s., not significant. *p < 0.05. **p < 0.01.

https://doi.org/10.1371/journal.ppat.1012500.g007

Download:

Fig 8. The results of the study are summarized in a visual abstract.

CD11b^intLy6G^intCXCR2^loCD62L^lo permissive immature pulmonary neutrophils are responsible for lung hyperinflammation and increased mycobacterial load in male Nox2^-/- mice. Among the upregulated pro-inflammatory cytokines, G-CSF, rather than IL-1, plays a dominant role in generating immature neutrophils. The control of immature neutrophils through neutrophil depletion, AM80 intervention, and G-CSF neutralization has shown promising results in ameliorating TB immunopathogenesis in male Nox2^-/- mice.

https://doi.org/10.1371/journal.ppat.1012500.g008

Discussion

Our study aimed to identify disease-promoting factors that are responsible for triggering Mtb-induced immunopathogenesis in the absence of phagocyte oxidase. We found that pulmonary infiltration of aberrant immature neutrophils is strongly associated with increased mycobacterial burden, lung hyperinflammation, and the loss of pulmonary lymphocytes in male Nox2^-/- mice. We were able to remarkably alleviate TB progression in male Nox2^-/- mice by reducing immature neutrophil counts. Overall, we identified G-CSF, rather than other host factors, as the initiator of immature pulmonary neutrophil-mediated TB immunopathogenesis in male Nox2^-/- mice.

Progressed from previous studies on the roles of phagocyte oxidases in TB pathogenesis, our study proposed novel advocative findings. First, we have demonstrated that both sex difference and NOX2 deficiency can affect TB susceptibility in mice. In previous studies, gp91^phox-deficient mice did not feature a significant increase in mycobacterial load when compared to WT mice [12,29–31]. Similarly, Duox1 or p47^phox-deficient mice did not show an increase in lung mycobacterial load around four weeks post-infection [28,58]. Moreover, Olive et al. indicated that there was no variation in TB susceptibility between male and female gp91^phox-deficient mice [12,31]. However, we displayed that male Nox2^-/- mice exhibited a significantly higher lung mycobacterial load compared to WT mice and female Nox2^-/- mice. This could be attributed to the use of the Mtb K strain, recognized for its heightened virulence in comparison to the H37Rv and H37Ra strains [59,60]. Additionally, as we focused in vivo analysis at the four weeks post-infection period, when TB severity in Nox2^-/- mice is optimized, we were able to evaluate the exacerbated TB susceptibility of male Nox2^-/- mice. This observation of time-point-dependent alterations in TB progression in Nox2^-/- mice is particularly significant, as the pulmonary influx of neutrophils occurs before the explosive increase in mycobacterial CFUs. Such findings highlight the crucial role of constant pulmonary neutrophil influx, serving as a source of uncontrolled inflammation and a superior niche for Mtb, consequently driving TB pathogenesis. Our results newly indicate that biological sex is one of the crucial factors that accelerate TB pathogenesis in mice lacking phagocyte NADPH oxidase, which is inherited in an X-linked manner. Additionally, to the best of our knowledge, our study is the first to investigate the populations and functions of pulmonary lymphocytes in Nox2^-/- mice during Mtb infection. We revealed that IFN-γ-producing CD4⁺ T cells were abundant, and antigen-specific Th1 responses were highly maintained in the lungs of male Nox2^-/- mice. Hence, we concluded that the TB susceptibility of male Nox2^-/- mice is not solely due to the inadequacy of Th1 responses, which contributes to TB susceptibility in various mouse models [61–63]. Lastly, we emphasized G-CSF as the primary inducer of immature neutrophil-mediated TB pathogenesis in male Nox2^-/- mice, instead of other cytokines which remain essential for proper defense against Mtb infection. Chao et al. reported that the blockade of IL-1β or IL-1R can alleviate mycobacterial infection in phagocyte oxidase-deficient mice [12,37]. However, our data indicated that both IL-1α and IL-1β are required for proper defense against Mtb. IL-1α and IL-1β act as the upstream controllers of excessive G-CSF production in Nox2^-/- mouse model [36,44], but they also play protective roles of in anti-TB immunity, such as activating myeloid cells in general, beyond just neutrophils [64–66]. We suggest that such protective roles of IL-1 signaling are especially crucial in the early phases of mycobacterial infection. By demonstrating that initiating IL-1R blockade at either one week or two weeks post-infection time point exacerbates TB progression in Nox2^-/- mice, we highlighted the dominance of G-CSF over IL-1 in neutrophil-mediated TB pathogenesis.

Although Nox2^-/- mice are prone to autoimmune diseases such as rheumatoid arthritis [67,68], Mtb-induced lung hyperinflammation in Nox2^-/- mice was not primarily associated with impaired Th1 responses or autoreactive T cells. We observed that pulmonary neutrophils are the actual cause of lung hyperminflammation, and the loss of pulmonary B cells is more closely associated with neutrophilic inflammation rather than alterations in T cell responses. Our research may offer deeper insights into TB susceptible animal models that are not associated with altered T cell function. We explicitly characterized immature lung neutrophils and identified them as the primary drivers of TB immunopathology in male Nox2^-/- mice. While various studies have emphasized the importance of neutrophils in promoting lung inflammation in Nox2^-/- mice undergoing bacterial, parasitic, or fungal challenge [36,45,69–71], their properties were not dissected in detail. We uncovered that Nox2^-/- immature pulmonary neutrophils feature CD11b^intLy6G^int and CXCR2^loCD62L^lo phenotypes, altered size and granularity, and increased mycobacterial permissiveness. Still, we need transcriptomic or metabolomic analysis of CD11b^intLy6G^intCXCR2^loCD62L^lo immature neutrophils to elucidate the precise mechanisms underlying neutrophilic inflammation and mycobacterial permissiveness. We proposed novel interventions with therapeutic potentials to enable proper control of immature neutrophils. The effectiveness of neutrophil depletion in alleviating TB pathogenesis and Mtb-induced lung hyperinflammation was formerly demonstrated in TB susceptible Nos2^-/- and TLR2^-/- mice [72,73], as well as in WT mice suffering from chronic TB [74]. We firstly provided the therapeutic potential of neutrophil depletion in alleviating TB pathogenesis specifically in Nox2^-/- mice. We also demonstrated the effectiveness of AM80 in alleviating TB pathogenesis. While Yamada et al. illustrated that ATRA intervention mitigated TB progression by increasing pulmonary T cell counts or reducing pulmonary myeloid-derived suppressor cells (MDSCs) [50,51,75–77], AM80 has not been tested on animal models as a therapeutic agent against TB. We highlighted the efficacy of AM80 in controlling TB pathogenesis in a susceptible mouse model, focusing on its role in reducing inflammatory neutrophils rather than altering T cell or macrophage responses. We have newly revealed that AM80 intervention does not alter Mtb-specific T cell responses in Nox2^-/- mice. Although AM80 may affect the phagocytic capacities of Nox2^-/- macrophages, we have excluded the effects on macrophages because AM80 did not alter the phagocytosis of Nox2^-/- BMDMs in our setup studies. Since AM80 shows enhanced potency compared to ATRA and is free from the side effects associated with RAR-γ activation [78,79], AM80 intervention may offer superior outcomes for establishing host-directed TB therapies in susceptible models compared to ATRA. Furthermore, our study newly discovered that G-CSF neutralization can alleviate TB pathogenesis in Nox2^-/- mice. These attempts may provide insights into controlling TB progression in susceptible animal models with excessive pulmonary granulocyte influx.

Still, our study requires discussions on the following limitations. AM80 intervention was effective at mitigating TB pathogenesis in WT mice, while G-CSF neutralization did not clearly alter TB progression in this mouse model. This is considered to be originated from two different reasons. Firstly, unlike male Nox2^-/- mouse model, WT mice do not show excessive and constant G-CSF production in response to Mtb infection. Therefore, Mtb infected WT mice may have an insufficient amount of circulating G-CSF which should be depleted through G-CSF neutralization. The second reason is that G-CSF and AM80 promotes myeloid cell generation via independent signaling pathways. AM80 induces neutrophil differentiation and maturation through the activation of RAR, while G-CSF is not involved in RAR signaling or C/EBPβ activation, rapidly generating immature neutrophils [52]. Therefore, we conclude that AM80 featured therapeutic capacity in both mouse strains, while G-CSF neutralization specifically showed potency in male Nox2^-/- mouse model. While anti-Ly6G mAb injection successfully removed pulmonary neutrophils in WT mice, it failed to fully eliminate the cells in Nox2^-/- mice. This outcome is thought to stem from the nature of the intraperitoneal injection of anti-Ly6G mAb (1A8). Boivin et al. noted that intraperitoneal injection of anti-GR1 or Ly6G monoclonal antibodies may not fully eliminate neutrophils, as newly synthesized young neutrophils can replenish the blood or organs even after depletion [80]. Furthermore, anti-Ly6G mAb mediates slow neutrophil depletion through Fc-dependent opsonization and phagocytosis of neutrophils by macrophages. Considering that NOX2 deficiency leads to both G-CSF mediated constant neutrophil production and macrophage malfunction, we conclude that these two factors contributed to the incomplete depletion of pulmonary neutrophils in male Nox2^-/- mice. A similar explanation can be given for the persistence pro-inflammatory cytokines in the lungs, even after mAb-mediated cytokine neutralization. This inefficiency is likely due to the nature of intraperitoneal injections, which effectively target circulating cells or cytokines but may not completely reach lung-residing targets. Additionally, since Nox2^-/- neutrophils are inflammatory, they are likely the source of the cytokines that were still detected after intraperitoneal depletion. To optimally deplete such targets, a combination of intraperitoneal and intratracheal injections is likely necessary to effectively target and deplete cytokines residing in the lung. The disparity in the effects of IL-1R blockade on Nox2^-/- mice also warrants discussion. Despite following protocols from Olive et al. [12], initiating IL-1R blockade from two weeks post-infection exacerbated TB in male Nox2^-/- mice. We conclude that differences in the infection model system contributed to this discrepancy. Specifically, our study differed from previous ones in terms of the mycobacterial strain virulence, initial infectious doses, and the ages of the mice used. IL-1R blockade may suppress neutrophil-mediated TB pathogenesis in a less severe infection model, but it was ineffective in our system, which involved a severe infection with a high dose of the highly virulent Mtb K strain.

Despite the abundance of T cell responses, male Nox2^-/- mice displayed severe TB progression. Accumulating evidences suggest that disrupted T cell responses during Mtb infection may diminish protection against the infection. For instance, the deficiency of the T cell inhibitory receptor PD-1 leads to a significant enhancement of T cell responses and an increase in the pulmonary mycobacterial burden [81,82]. Given that neutrophil depletion and G-CSF neutralization decreased the pulmonary levels of IFN-γ and IL-17A in male Nox2^-/- mice, maintaining the balance between immune activation and suppression might be more crucial for the proper control of TB than the amount of cytokines produced. To gain a deeper understanding of TB pathogenesis, it is crucial to identify immunologic factors that disrupt the proper activation of Th1 responses. The absence of pulmonary B cells in the presence of BAFF, TNF-α, and IL-6, which are B cell activating cytokines, remains a question to be solved. As pulmonary B cells play crucial roles in suppressing neutrophil-mediated lung inflammation through direct interaction [83,84], their absence may contribute to TB susceptibility in Nox2^-/- mice. Thus, elucidating the association between the increase in neutrophils and the decrease in B cells may contribute to a better understanding of anti-TB immunity. We investigated the impacts of AM80 intervention at four weeks post-infection. However, considering the broad roles of retinoic acids in modulating macrophage and T cell responses [50,51], understanding the significance of non-neutrophil factors affected by AM80, such as T cell responses, recruited macrophage functions, and alveolar macrophage functions at early stages of Mtb infection, can significantly enhance our understanding of the pathogenesis of TB in susceptible animal models.

Collectively, our study revealed that phagocyte NADPH oxidase deficiency contributes to exacerbated TB immunopathogenesis in a sex-dependent manner. This is attributed to the excessive pulmonary infiltration of immature neutrophils in the presence of sufficient Th1 responses. Therapeutic interventions targeting the generation of G-CSF driven immature pulmonary neutrophils successfully ameliorated TB immunopathogenesis in male Nox2^-/- mice. Our study provides a comprehensive understanding of TB pathogenesis and highlights potential therapeutic targets for proper TB control in the absence of phagocyte-specific NADPH oxidase.

Materials and methods

Ethics statements and study approval

All animal experiments followed the regulations set by the Korean Food and Drug Administration. The Ethics Committee and Institutional Animal Care and Use Committee (2019–0174; C57BL/6N, Nox2^-/-, 2022–0140; C57BL/6N, Nox2^-/-) at Yonsei University Health System (Seoul, Korea) granted approval for each experimental protocol.

Mice

Six- to seven-week-old female and male C57BL/6N mice were obtained from Japan SLC, Inc. (Shizuoka, Japan). Six- to seven-week-old female and male Nox2^-/- mice were provided by Dr. Yun Soo Bae (Ewha Womans University, Seoul, South Korea). The mice were housed in a specific pathogen-free (SPF) environment with barriered conditions at the Yonsei University Medical Research Center SPF facility.

Bacterial culture and infection protocols

Purchase of the Mtb K strain, mycobacterial culture, and aerosol infection processes were executed according to the referenced studies [85,86]. The mycobacteria were cultured in 7H9 broth supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC; Difco Laboratories, MD) and 2 μg/ml of mycobactin J (Allied Monitor, Fayette, MO) for 4 weeks at 37°C. Following cultivation, all bacteria were washed three times with 10 mM phosphate-buffered saline (PBS; pH 7.4). Bacterial cell pellets were collected after centrifugation, and small aliquots were stored at -80°C until use. The Mtb K strain used for in vivo challenges was PDIM positive. YFP vectors for the generation of YFP⁺ Mtb K strain were kindly gifted by Dr. Christopher Sassetti. In order to deliver bacteria to the lungs of each mouse, all mice were exposed to at least 200 viable mycobacteria in an inhalation chamber for 60 minutes.

In vivo treatments for neutrophil control

From two weeks post-infection, 250 μg/mouse of anti-Ly6G mAb (clone: 1A8, Bio X Cell, West Lebanon, NH, USA), 200 μg/mouse of anti-IL-1R mAb (clone: JAMA-147, Bio X Cell, West Lebanon, NH, USA), 200 μg/mouse of anti-IFN-γ mAb (clone: H22, Bio X Cell, West Lebanon, NH, USA), and 250 μg/mouse of anti-IL-17A mAb (clone: 17F3, Bio X Cell, West Lebanon, NH, USA) mAb were intraperitoneally administered. The antibodies were diluted in PBS and administered three times a week for a total duration of two weeks. From one-week post-infection, 30 μg/mouse of anti-G-CSF mAb (clone: #67604, R&D Systems, Minneapolis, MN, USA), 200 μg/mouse of anti-IL-1R mAb (clone: JAMA-147, Bio X Cell, West Lebanon, NH, USA), 400 μg/mouse of anti-IL-6 mAb (clone: MP5-20F3, Bio X Cell, West Lebanon, NH, USA), 200 μg/mouse of anti-IL-1α mAb (clone: ALF-161, Bio X Cell, West Lebanon, NH, USA), and 200 μg/mouse of anti-IL-1β mAb (clone: B122, Bio X Cell, West Lebanon, NH, USA) were intraperitoneally administered. The antibodies were diluted in PBS and administered three times a week for a total duration of three weeks. Rat IgG2a, rat IgG1, Armenian hamster IgG, and mouse IgG1 isotype control antibodies were purchased from Bio X Cell (West Lebanon, NH, USA). Isotype control antibodies were administered to control mice in correspondence with the original antibodies. From one-week post-infection, 20 μg/mouse of AM80 (Sigma-Aldrich, St. Louis, MO, USA) was orally administered. AM80 was diluted in PBS and administered three times a week for a total duration of three weeks.

Antibodies and flow cytometry

Flow cytometric analysis of immune cells was conducted using the subsequent antibodies. LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit, Green Dead Cell Stain Kit, and Aqua Dead Cell Stain Kit were purchased from Molecular Probes (Carlsbad, CA, USA). Brilliant violet (BV) 605-conjugated mAb against Thy1.2 and CD19; Allophycocyanin (APC)-conjugated mAb against CD45R (B220); BV 421-conjugated mAb against CD45; APC-R700-conjugated mAb against Siglec-F; Fluorescein isothiocyanate (FITC)-conjugated mAb against CD95 (FAS); BV 786-conjugated mAb against CXCR2; and V450- conjugated mAb against Ly6G were purchased from BD Bioscience (San Jose, CA, USA). Phycoerythrin (PE)-conjugated mAb against CXCR2, CD64, IFN-γ, and GL7; Peridinin chlorophyll (PerCP)-Cy5.5-conjugated mAb against CD11b; APC-Cy7-conjugated mAb against MHC-II; Alexa fluor 700-conjugated mAb against CD62L; and PE-Dazzle conjugated mAb against CD11c were purchased from Biolegend (San Diego, CA, USA). Surface and intracellular staining processes were executed according to the referenced study [87].

Preparation of single cell suspensions and immune cell analysis

Four weeks post-infection, the mice were euthanized via inhalation of carbon dioxide and then underwent an autopsy. The mouse lungs were perfused before undergoing flow cytometry analysis. Using the methods described in previous studies [61,88], single-cell suspensions were obtained from the entire lung tissue. These cells were then stained with the mAbs mentioned in the previous section and subjected to flow cytometry analysis.

Measurement of cytokines

For the analysis of lung cytokines, lung lysates from the autopsied mice were collected, homogenized, dissolved into PBS, and then stored in a -80°C freezer. For the analysis of T cell cytokines, lung single cell suspensions were stimulated with two different Mtb antigens, PPD and ESAT-6 according to the referenced studies [87,88]. To detect cytokines, sandwich enzyme-linked immunosorbent assay (ELISA) and LEGENDplex kits were utilized. Briefly, mouse G-CSF and IL-1α ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). Mouse IFN-γ, IL-1β, IL-17A, and IL-5 ELISA kits were purchased from Invitrogen (San Diego, CA, USA). Mouse IL-10 ELISA kit, LEGENDplex mouse B cell panel (for the detection of TNF-α, IL-4, IL-6, BAFF, and TGF-β), and LEGENDplex mouse inflammation panel (for the detection of IL-1α, IL-1β) were purchased from Biolegend (San Diego, CA, USA).

Quantification of lung inflammation and mycobacterial CFU

The lungs and spleens of Mtb infected mice were harvested four weeks post-infection. The right superior lobes of the lungs were kept in 10% formalin overnight for preservation and later embedded in paraffin. To perform histopathologic analysis, the lungs were sectioned at 4–5 μm and stained with hematoxylin and eosin (H&E). To quantify pulmonary inflammation in each mouse, we employed Adobe Photoshop (Adobe, San Jose, California) and ImageJ (National Institutes of Health, USA) programs, following a previously described reference [89]. The complete lung lesion image was isolated using Adobe Photoshop and saved as a separate file. Another image, identical in size to the original lung image but filled with black, was also saved as a separate file. These images were then opened in ImageJ. By using the ’Split Channels’ function, the image from the green channel was processed to calculate the size of the area showing green positivity, which was originally purple-blue due to H&E staining. The black-colored image was analyzed through the green channel in the same manner to calculate the size of the entire lung lesion. The ’inflamed area (%)’ value, which indicates the proportion of inflamed tissue relative to the entire lung lesion, was computed by dividing the green positivity values of the first image by the green positivity values of the black-colored image. Subsequently, the actual size of the inflamed lesion was calculated by determining the pixel values in Adobe Photoshop. The pixel value corresponding to a single millimeter was calculated based on a 2mm scale bar included in each images. The pixel value for the entire lung lesion was also enumerated, and the value was divided by the square of the pixel value representing one millimeter to quantify the size (mm²) of each lung lesion. By multiplying the inflamed area (%) values by the total lung sizes (mm²) for each sample, we were able to precisely enumerate the exact size of each inflamed area (mm²) (S11 Fig). To enumerate mycobacterial growth, lung and spleen tissues were plated onto Middlebrook 7H10 agar (Becton Dickinson, Franklin Lakes, NJ, USA) after homogenization, following preparation methods from previous studies [61,90]. After a four-week incubation period at 37°C, mycobacterial colonies were counted.

Neutrophil purification and assessment of mycobacterial permissiveness

MACS magnetic cell sorting kit including anti-Ly6G magnetic beads and LS MACS columns (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to enrich Ly6G⁺ cells from the lungs of Mtb-infected mice. The manufacturer’s protocols were followed. After separating Ly6G⁺ lung cells, the cells were treated with Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) and plated onto Middlebrook 7H10 agar (Becton Dickinson, Franklin Lakes, NJ, USA). After a three-week incubation period at 37°C, mycobacterial colonies were counted.

Statistical analyses

The results were presented as the mean ± standard deviation (SD). To analyze the significance of differences between two selected groups, One-way ANOVA and Mann-Whitney U test were conducted using GraphPad Prism version 8 for Windows (GraphPad Software, La Jolla, CA, USA, www.graphpad.com). The statistical significance was determined using the following definitions: n.s.: not significant, *p < 0.05, and **p < 0.01. Correlation analysis and unpaired t-test were conducted using GraphPad Prism, and the correlation coefficients and p-values were reported for each graph. Flowjo V10 (Flowjo, Ashland, OR, USA) was employed to perform t-distributed stochastic neighbor embedding (t-SNE) analysis.

Supporting information

S1 Fig. The gating strategy for immune cell populations and the kinetics time-course data of pulmonary CFUs and neutrophils are provided.

(A) Pulmonary CFUs of female WT and Nox2^-/- mice were enumerated in a time-course dependent manner. six-week old female WT and Nox2^-/- mice (n = 5 per group) were aerosol infected with Mtb K strain. Mycobacterial CFUs in the lungs of Mtb infected mice were calculated at 0, 2, 3, 4, 8, and 12 weeks post-infection. Initial CFU = 373. The experiment was conducted once. (B) Pulmonary neutrophil counts were calculated at 0, 2, 3, 4, 8, and 12 weeks post-infection. The experiment was conducted once. The significance of differences was determined, using the One-way ANOVA and Mann-Whitney U test. n.s., not significant. *p < 0.05. **p < 0.01. (C) The gating strategy for immune cell populations is presented as flow cytometry plots. Firstly, Live cells stained with the LIVE/DEAD Aqua Dead Cell Stain Kit were gated among single cells. CD45⁺ immune cells were then gated in order to investigate lung immune cell compositions. Among CD45⁺ immune cells, CD19⁺CD90.2⁺ SSC-A^lo lymphocytes and CD19^-CD90.2^-SSC-A^hi myeloid cells were discriminated. Lymphocytes consisted of CD90.2⁺ T cells, CD19⁺B220⁺ B cells, and CD19⁺B220⁺GL7⁺FAS⁺ germinal center B cells. Myeloid cells consisted of CD11b⁺Ly6G⁺ neutrophils, CD11c⁺Siglec-F⁺ alveolar macrophages, CD11b⁺CD64⁺ macrophages, and CD11c⁺MHC-II⁺ dendritic cells.

https://doi.org/10.1371/journal.ppat.1012500.s001

(TIF)

S2 Fig. Pulmonary T cells of male Nox2^-/- mice maintained strong responses against mycobacterial antigens.

Live lung cell suspensions were cultured with or without the mycobacterial antigens ESAT-6 and PPD for 8 hours. (A) Lung IFN-γ levels and (B) Lung IL-17A levels after mycobacterial antigen stimulus are presented in bar graphs. (C) IFN-γ positive CD90.2⁺ CD4⁺ CD44⁺ CD62L⁺ effecter T cell populations are presented in bar graphs and flow cytometry plots. The data are presented as the mean ± SD of six mice in each group. The significance of differences was determined, using the One-way ANOVA test. n.s., not significant. **p <0.01.

https://doi.org/10.1371/journal.ppat.1012500.s002

(TIF)

S3 Fig. Alveolar macrophages, macrophages, and dendritic cells were reduced in the lungs of Mtb infected male Nox2^-/- mice.

Pulmonary (A) CD11c⁺Siglec-F⁺ alveolar macrophages, (B) CD11b⁺CD64⁺ macrophages, and (C) CD11c⁺MHCII⁺ dendritic cell populations of Mtb-infected mice at four weeks post-infection. The percentages of each immune cell among lung CD45⁺ cells and total cell counts are presented in bar graphs. The data are presented as the mean ± SD of six mice in each group. The significance of differences was determined, using the One-way ANOVA test. **p <0.01.

https://doi.org/10.1371/journal.ppat.1012500.s003

(TIF)

S4 Fig. TB severity featured strong correlation to increase of lung neutrophils and decrease of lung B cells.

The percentages of (A) neutrophils, (B) T cells, and (C) B cells among lung CD45⁺ cells were individually correlated with inflamed lung percentages, inflamed lung area, and lung bacterial loads of each mouse and presented in correlation graphs. Additionally, (D) the ratio of pulmonary B cells to neutrophils (B:N ratio) was also individually correlated with inflamed lung percentages, inflamed lung area, and lung bacterial loads of each mouse and presented in correlation graphs. The significance of differences was determined by unpaired t-test and correlation analysis, featuring r and p values of each correlation.

https://doi.org/10.1371/journal.ppat.1012500.s004

(TIF)

S5 Fig. BM and lung neutrophil profiles of uninfected WT and Nox2^-/- mice are illustrated.

Neutrophil counts and percentages from the lungs and bone marrows of uninfected WT and Nox2^-/- mice are provided (n = 4 per group). (A) The percentages of neutrophils among lung CD45⁺ cells and total cell counts are presented in bar graphs, along with flow cytometry plots. (B) Total cell counts and the percentages of CXCR2^loCD62L^lo immature neutrophils and CXCR2^hiCD62L^hi mature neutrophils among total lung neutrophils are presented in bar graphs, along with flow cytometry plots. (C) The percentages of neutrophils among CD45⁺ bone marrow cells and total cell counts are presented in bar graphs, along with flow cytometry plots. (D) Total cell counts and the percentages of CXCR2^loCD62L^lo immature neutrophils and CXCR2^hiCD62L^hi mature neutrophils among total bone marrow neutrophils are presented in bar graphs, along with flow cytometry plots. The data are presented as the mean ± SD of four mice in each group. The significance of differences was determined using the One-way ANOVA test and Mann-Whitney-U test. n.s., not significant. *p < 0.05. **p < 0.01. significant.

https://doi.org/10.1371/journal.ppat.1012500.s005

(TIF)

S6 Fig. Immature neutrophils were the most permissive cells to mycobacterial infection among phagocytes infected with YFP⁺ Mtb.

(A) Experimental design for the in vivo enumeration of permissiveness in phagocytes. Male WT and Nox2^-/- mice (n = 5 per group) were aerosol infected with YFP-expressing Mtb K strain. At four weeks post-infection, all mice were autopsied, and permissiveness of neutrophils were analysed via flow cytometry (indicated by red arrow). Initial CFU = 742. (B) The percentage of neutrophils among YFP⁺ cells are presented in bar graphs, along with flow cytometry plots. The number of YFP⁺ neutrophils and MFI values of YFP in concatenated neutrophils (10³ cells) are presented in bar graphs. (C) Total cell counts, percentages among YFP⁺ neutrophils, and MFI values of YFP (per 10³ concatenated cells) in CXCR2^loCD62L^lo immature neutrophils and CXCR2^hiCD62L^hi mature neutrophils are presented in bar graphs, along with flow cytometry plots. Total cell counts, percentages among YFP⁺ cells, and MFI values of YFP (per 10³ concatenated cells) in (D) CD11c⁺Siglec-F⁺ alveolar macrophages and (E) CD11b⁺CD64⁺ recruited macrophages are presented in bar graphs, along with flow cytometry plots. The experiment was conducted once. The data are presented as the mean ± SD of five mice in each group. The significance of differences was determined using the One-way ANOVA test and Mann-Whitney-U test. n.s., not significant. *p < 0.05. **p < 0.01.

https://doi.org/10.1371/journal.ppat.1012500.s006

(TIF)

S7 Fig. AM80 treatment did not significantly alter T cells responses against mycobacterial antigens.

Live lung cell suspensions were cultured with or without mycobacterial antigens ESAT-6 and PPD for 8 hours. (A) Lung IFN-γ levels and (B) Lung IL-17A levels after mycobacterial antigen stimulus are presented in bar graphs. (C) IFN-γ positive CD90.2⁺ CD4⁺ CD44⁺ CD62L⁺ effecter T cell populations are presented in bar graphs and flow cytometry plots. The data are presented as the mean ± SD of five mice in each group. The significance of differences was determined, using the One-way ANOVA test and Mann-Whitney test. n.s., not significant. *p < 0.05. **p < 0.01.

https://doi.org/10.1371/journal.ppat.1012500.s007

(TIF)

S8 Fig. Neutralization of IFN-γ and IL-17A exacerbated TB pathogenesis of female Nox2^-/- mice.

(A) Experimental design for in vivo neutralization of IFN-γ and IL-17A in Mtb infected mice. six-week old female Nox2^-/- mice (n = 5 per group) were aerosol infected with Mtb K strain. Starting from two weeks post-infection, 200 μg of anti-IFN-γ mAb or 250 μg of anti-IL-17A mAb was intraperitoneally administered to each mouse three times a week (indicated by blue bars). At four weeks post-infection, all mice were autopsied, and immunological analysis, bacterial counting, and histopathological analysis were conducted (indicated by red arrow). Initial CFU = 225. (B) Mycobacterial CFUs in the lungs and spleens of each group at four weeks post-infection were analyzed by calculating the number of colonies and presented in bar graphs. (C) H&E staining was performed on the superior lobes of the right lung at four weeks post-infection to visualize the gross lung pathology. The inflamed area of the H&E-stained samples was quantified in terms of percentage and square millimeters and presented in bar graphs. (D) IFN-γ, IL-1α, IL-1β, IL-17A, IL-6, TNF-α, G-CSF, and BAFF levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex (E) IL-10, IL-4, IL-5, and TGF-β levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex The cytokine levels are presented in bar graphs. Pulmonary (F) CD11b⁺Ly6G⁺ neutrophil, (G) CD11b⁺Ly6G⁺CXCR2^loCD62L^lo immature neutrophil, and CD11b⁺Ly6G⁺CXCR2^hiCD62L^hi mature neutrophil populations of Mtb-infected mice at four weeks post-infection. The percentages of each immune cell among lung CD45⁺ cells and total cell counts are presented in bar graphs, along with flow cytometry plots. The experiment was conducted once. The data are presented as the mean ± SD of five mice in each group. The significance of differences was determined, using the One-way ANOVA test. n.s., not significant. *p < 0.05. **p < 0.01.

https://doi.org/10.1371/journal.ppat.1012500.s008

(TIF)

S9 Fig. Neutralization of IL-6, IL-1α, and IL-1β exacerbated TB pathogenesis of male Nox2^-/- mice.

(A) Experimental design for in vivo neutralization of IL-6, IL-1α, and IL-1β in Mtb infected mice. six-week old male Nox2^-/- mice (n = 5 per group) were aerosol infected with Mtb K strain. Starting from one week post-infection, 400 μg of anti-IL-6 mAb or 200 μg of anti-IL-1α mAb or 200 μg of anti-IL-1β mAb was intraperitoneally administered to each mouse three times a week (indicated by blue bars). At four weeks post-infection, all mice were autopsied, and immunological analysis, bacterial counting, and histopathological analysis were conducted (indicated by red arrow). Initial CFU = 330. (B) Mycobacterial CFUs in the lungs and spleens of each group at four weeks post-infection were analyzed by calculating the number of colonies and presented in bar graphs. (C) H&E staining was performed on the superior lobes of the right lung at four weeks post-infection to visualize the gross lung pathology. The inflamed area of the H&E-stained samples was quantified in terms of percentage and square millimeters and presented in bar graphs. (D) IFN-γ, IL-1α, IL-1β, IL-17A, IL-6, TNF-α, G-CSF, and BAFF levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex (E) IL-10, IL-4, IL-5, and TGF-β levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex The cytokine levels are presented in bar graphs. Pulmonary (F) CD11b⁺Ly6G⁺ neutrophil, (G) CD11b⁺Ly6G⁺CXCR2^loCD62L^lo immature neutrophil, and CD11b⁺Ly6G⁺CXCR2^hiCD62L^hi mature neutrophil populations of Mtb-infected mice at four weeks post-infection. The percentages of each immune cell among lung CD45⁺ cells and total cell counts are presented in bar graphs, along with flow cytometry plots. The experiment was conducted once. The data are presented as the mean ± SD of five mice in each group. The significance of differences was determined, using the One-way ANOVA test. n.s., not significant. *p < 0.05. **p < 0.01.

https://doi.org/10.1371/journal.ppat.1012500.s009

(TIF)

S10 Fig. Blockade of IL-1R exacerbated TB pathogenesis of Nox2^-/- mice.

(A) Experimental design for in vivo blockade of IL-1R in Mtb infected female Nox2^-/- mice. six-week old female Nox2^-/- mice (n = 5 per group) were aerosol infected with Mtb K strain. Starting from one week post-infection, 200 μg of anti-IL-1R mAb was intraperitoneally administered to each mouse three times a week (indicated by blue bars). At four weeks post-infection, all mice were autopsied, and bacterial counting and histopathological analysis were conducted (indicated by red arrow). Initial CFU = 300. (B) Mycobacterial CFUs in the lungs and spleens of each group at four weeks post-infection were analyzed by calculating the number of colonies and presented in bar graphs. (C) H&E staining was performed on the superior lobes of the right lung at four weeks post-infection to visualize the gross lung pathology. The inflamed area of the H&E-stained samples was quantified in terms of percentage and square millimeters and presented in bar graphs. (D) Pulmonary CD11b⁺Ly6G⁺ neutrophil populations of Mtb-infected mice at four weeks post-infection. The percentages of neutrophils among lung CD45⁺ cells and total cell counts are presented in bar graphs, along with flow cytometry plots. (E) Experimental design for in vivo blockade of IL-1R in Mtb infected male Nox2^-/- mice. six-week old male Nox2^-/- mice (n = 5 per group) were aerosol infected with Mtb K strain. Starting from two weeks post-infection, 200 μg of anti-IL-1R mAb was intraperitoneally administered to each mouse three times a week (indicated by blue bars). At four weeks post-infection, all mice were autopsied, and bacterial counting and histopathological analysis were conducted (indicated by red arrow). Initial CFU = 342. (F) Mycobacterial CFUs in the lungs and spleens of each group at four weeks post-infection were analyzed by calculating the number of colonies and presented in bar graphs. (G) H&E staining was performed on the superior lobes of the right lung at four weeks post-infection to visualize the gross lung pathology. The inflamed area of the H&E-stained samples was quantified in terms of percentage and square millimeters and presented in bar graphs. (H) Pulmonary CD11b⁺Ly6G⁺ neutrophil populations of Mtb-infected mice at four weeks post-infection. The percentages of neutrophils among lung CD45⁺ cells and total cell counts are presented in bar graphs, along with flow cytometry plots. The experiments were conducted once. The data are presented as the mean ± SD of five mice in each group. The significance of differences was determined, using the Mann-Whitney test. n.s., not significant. *p < 0.05. **p < 0.01.

https://doi.org/10.1371/journal.ppat.1012500.s010

(TIF)

S11 Fig. Quantification method of lung inflammation is briefly described.

(A) As outlined in the Materials and Methods section, the inflamed area of the H&E-stained lung samples were quantified in terms of percentage and square millimetres. The values were calculated using Adobe Photoshop and ImageJ programs. New image files were created to determine the percentage of the inflamed (purple-stained) lesions, and the actual size of both total lung lesions and inflamed lesions was quantified in mm².

https://doi.org/10.1371/journal.ppat.1012500.s011

(TIF)

Acknowledgments

We appreciate the Medical Illustration & Design(MID) team, a member of Medical Research Support Services of Yonsei University College of Medicine, for their excellent support with medical illustration.

References

1. WHO. Global Tuberculosis Report 2022: World Health Organization; 2022.
2. Das S, Marin ND, Esaulova E, Ahmed M, Swain A, Rosa BA, et al. Lung Epithelial Signaling Mediates Early Vaccine-Induced CD4⁺ T Cell Activation and Mycobacterium tuberculosis Control. mBio. 2021;12(4):e0146821. pmid:34253059
- View Article
- PubMed/NCBI
- Google Scholar
3. Fu H, Lewnard JA, Frost I, Laxminarayan R, Arinaminpathy N. Modelling the global burden of drug-resistant tuberculosis avertable by a post-exposure vaccine. Nat Commun. 2021;12(1):424. pmid:33462224
- View Article
- PubMed/NCBI
- Google Scholar
4. Smith CM, Proulx MK, Olive AJ, Laddy D, Mishra BB, Moss C, et al. Tuberculosis Susceptibility and Vaccine Protection Are Independently Controlled by Host Genotype. mBio. 2016;7(5). pmid:27651361
- View Article
- PubMed/NCBI
- Google Scholar
5. Neyrolles O, Quintana-Murci L. Sexual inequality in tuberculosis. PLoS Med. 2009;6(12):e1000199. pmid:20027210
- View Article
- PubMed/NCBI
- Google Scholar
6. Nhamoyebonde S, Leslie A. Biological differences between the sexes and susceptibility to tuberculosis. J Infect Dis. 2014;209 Suppl 3:S100–6. pmid:24966189
- View Article
- PubMed/NCBI
- Google Scholar
7. Dibbern J, Eggers L, Schneider BE. Sex differences in the C57BL/6 model of Mycobacterium tuberculosis infection. Sci Rep. 2017;7(1):10957. pmid:28887521
- View Article
- PubMed/NCBI
- Google Scholar
8. Zhao Y, Ying H, Demei J, Xie J. Tuberculosis and sexual inequality: the role of sex hormones in immunity. Crit Rev Eukaryot Gene Expr. 2012;22(3):233–41. pmid:23140164
- View Article
- PubMed/NCBI
- Google Scholar
9. Chandra P, Grigsby SJ, Philips JA. Immune evasion and provocation by Mycobacterium tuberculosis. Nat Rev Microbiol. 2022;20(12):750–66. pmid:35879556
- View Article
- PubMed/NCBI
- Google Scholar
10. Kotzé LA, Young C, Leukes VN, John V, Fang Z, Walzl G, et al. Mycobacterium tuberculosis and myeloid-derived suppressor cells: Insights into caveolin rich lipid rafts. EBioMedicine. 2020;53:102670. pmid:32113158
- View Article
- PubMed/NCBI
- Google Scholar
11. Xu W, Snell LM, Guo M, Boukhaled G, Macleod BL, Li M, et al. Early innate and adaptive immune perturbations determine long-term severity of chronic virus and Mycobacterium tuberculosis coinfection. Immunity. 2021;54(3):526–41.e7. pmid:33515487
- View Article
- PubMed/NCBI
- Google Scholar
12. Olive AJ, Smith CM, Kiritsy MC, Sassetti CM. The Phagocyte Oxidase Controls Tolerance to Mycobacterium tuberculosis Infection. J Immunol. 2018;201(6):1705–16. pmid:30061198
- View Article
- PubMed/NCBI
- Google Scholar
13. Miller JL, Velmurugan K, Cowan MJ, Briken V. The type I NADH dehydrogenase of Mycobacterium tuberculosis counters phagosomal NOX2 activity to inhibit TNF-alpha-mediated host cell apoptosis. PLoS Pathog. 2010;6(4):e1000864. pmid:20421951
- View Article
- PubMed/NCBI
- Google Scholar
14. Sies H, Jones DP. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat Rev Mol Cell Biol. 2020;21(7):363–83. pmid:32231263
- View Article
- PubMed/NCBI
- Google Scholar
15. Xiao X, Bu H, Li Z, Li Z, Bai Q, Wang Z, et al. NADPH-Oxidase 2 Promotes Autophagy in Spinal Neurons During the Development of Morphine Tolerance. Neurochem Res. 2021;46(8):2089–96. pmid:34008119
- View Article
- PubMed/NCBI
- Google Scholar
16. Munnamalai V, Weaver CJ, Weisheit CE, Venkatraman P, Agim ZS, Quinn MT, et al. Bidirectional interactions between NOX2-type NADPH oxidase and the F-actin cytoskeleton in neuronal growth cones. J Neurochem. 2014;130(4):526–40. pmid:24702317
- View Article
- PubMed/NCBI
- Google Scholar
17. Brandes RP, Weissmann N, Schröder K. Nox family NADPH oxidases: Molecular mechanisms of activation. Free Radic Biol Med. 2014;76:208–26. pmid:25157786
- View Article
- PubMed/NCBI
- Google Scholar
18. Panday A, Sahoo MK, Osorio D, Batra S. NADPH oxidases: an overview from structure to innate immunity-associated pathologies. Cell Mol Immunol. 2015;12(1):5–23. pmid:25263488
- View Article
- PubMed/NCBI
- Google Scholar
19. Anjani G, Vignesh P, Joshi V, Shandilya JK, Bhattarai D, Sharma J, et al. Recent advances in chronic granulomatous disease. Genes Dis. 2020;7(1):84–92. pmid:32181279
- View Article
- PubMed/NCBI
- Google Scholar
20. Xu Q, Wang Q, Hu LT, Lin J, Jiang N, Peng XD, et al. NADPH oxidase 2 plays a protective role in experimental Aspergillus fumigatus keratitis in mice through killing fungi and limiting the degree of inflammation. Int J Ophthalmol. 2022;15(7):1044–52. pmid:35919314
- View Article
- PubMed/NCBI
- Google Scholar
21. Taylor JP, Tse HM. The role of NADPH oxidases in infectious and inflammatory diseases. Redox Biol. 2021;48:102159. pmid:34627721
- View Article
- PubMed/NCBI
- Google Scholar
22. Rojas Márquez JD, Li T, McCluggage ARR, Tan JMJ, Muise A, Higgins DE, et al. Cutting Edge: NOX2 NADPH Oxidase Controls Infection by an Intracellular Bacterial Pathogen through Limiting the Type 1 IFN Response. J Immunol. 2021;206(2):323–8. pmid:33288542
- View Article
- PubMed/NCBI
- Google Scholar
23. Kohn DB, Booth C, Kang EM, Pai SY, Shaw KL, Santilli G, et al. Lentiviral gene therapy for X-linked chronic granulomatous disease. Nat Med. 2020;26(2):200–6. pmid:31988463
- View Article
- PubMed/NCBI
- Google Scholar
24. García B, León-Lara X, Espinosa S, Blancas-Galicia L. [Mycobacterial disease in patients with chronic granulomatous disease]. Rev Alerg Mex. 2021;68(2):117–27. pmid:34525783
- View Article
- PubMed/NCBI
- Google Scholar
25. Conti F, Lugo-Reyes SO, Blancas Galicia L, He J, Aksu G, Borges de Oliveira E, Jr., et al. Mycobacterial disease in patients with chronic granulomatous disease: A retrospective analysis of 71 cases. J Allergy Clin Immunol. 2016;138(1):241–8.e3. pmid:26936803
- View Article
- PubMed/NCBI
- Google Scholar
26. Deffert C, Schäppi MG, Pache JC, Cachat J, Vesin D, Bisig R, et al. Bacillus calmette-guerin infection in NADPH oxidase deficiency: defective mycobacterial sequestration and granuloma formation. PLoS Pathog. 2014;10(9):e1004325. pmid:25188296
- View Article
- PubMed/NCBI
- Google Scholar
27. Ying W, Sun J, Liu D, Hui X, Yu Y, Wang J, et al. Clinical characteristics and immunogenetics of BCGosis/BCGitis in Chinese children: a 6 year follow-up study. PLoS One. 2014;9(4):e94485. pmid:24722620
- View Article
- PubMed/NCBI
- Google Scholar
28. Cooper AM, Segal BH, Frank AA, Holland SM, Orme IM. Transient loss of resistance to pulmonary tuberculosis in p47(phox^-/-) mice. Infect Immun. 2000;68(3):1231–4. pmid:10678931
- View Article
- PubMed/NCBI
- Google Scholar
29. Jung YJ, LaCourse R, Ryan L, North RJ. Virulent but not avirulent Mycobacterium tuberculosis can evade the growth inhibitory action of a T helper 1-dependent, nitric oxide Synthase 2-independent defense in mice. J Exp Med. 2002;196(7):991–8. pmid:12370260
- View Article
- PubMed/NCBI
- Google Scholar
30. Ng VH, Cox JS, Sousa AO, MacMicking JD, McKinney JD. Role of KatG catalase-peroxidase in mycobacterial pathogenesis: countering the phagocyte oxidative burst. Mol Microbiol. 2004;52(5):1291–302. pmid:15165233
- View Article
- PubMed/NCBI
- Google Scholar
31. Thomas SM, Olive AJ. High Lethality of Mycobacterium tuberculosis Infection in Mice Lacking the Phagocyte Oxidase and Caspase1/11. Infect Immun. 2023:e0006023. pmid:37314361
- View Article
- PubMed/NCBI
- Google Scholar
32. Goebel WS, Mark LA, Billings SD, Meyers JL, Pech N, Travers JB, et al. Gene correction reduces cutaneous inflammation and granuloma formation in murine X-linked chronic granulomatous disease. J Invest Dermatol. 2005;125(4):705–10. pmid:16185269
- View Article
- PubMed/NCBI
- Google Scholar
33. Yu JE, Azar AE, Chong HJ, Jongco AM 3rd, Prince BT. Considerations in the Diagnosis of Chronic Granulomatous Disease. J Pediatric Infect Dis Soc. 2018;7(suppl_1):S6–s11. pmid:29746674
- View Article
- PubMed/NCBI
- Google Scholar
34. Liao YC, Wu SY, Huang YF, Lo PC, Chan TY, Chen CA, et al. NOX2-Deficient Neutrophils Facilitate Joint Inflammation Through Higher Pro-Inflammatory and Weakened Immune Checkpoint Activities. Front Immunol. 2021;12:743030. pmid:34557202
- View Article
- PubMed/NCBI
- Google Scholar
35. Wang J, Ma MW, Dhandapani KM, Brann DW. Regulatory role of NADPH oxidase 2 in the polarization dynamics and neurotoxicity of microglia/macrophages after traumatic brain injury. Free Radic Biol Med. 2017;113:119–31. pmid:28942245
- View Article
- PubMed/NCBI
- Google Scholar
36. Bagaitkar J, Pech NK, Ivanov S, Austin A, Zeng MY, Pallat S, et al. NADPH oxidase controls neutrophilic response to sterile inflammation in mice by regulating the IL-1α/G-CSF axis. Blood. 2015;126(25):2724–33. pmid:26443623
- View Article
- PubMed/NCBI
- Google Scholar
37. Chao WC, Yen CL, Hsieh CY, Huang YF, Tseng YL, Nigrovic PA, et al. Mycobacterial infection induces higher interleukin-1β and dysregulated lung inflammation in mice with defective leukocyte NADPH oxidase. PLoS One. 2017;12(12):e0189453. pmid:29228045
- View Article
- PubMed/NCBI
- Google Scholar
38. Choi HG, Kwon KW, Choi S, Back YW, Park HS, Kang SM, et al. Antigen-Specific IFN-γ/IL-17-Co-Producing CD4⁺ T-Cells Are the Determinants for Protective Efficacy of Tuberculosis Subunit Vaccine. Vaccines (Basel). 2020;8(2). pmid:32545304
- View Article
- PubMed/NCBI
- Google Scholar
39. Qin S, Chen R, Jiang Y, Zhu H, Chen L, Chen Y, et al. Multifunctional T cell response in active pulmonary tuberculosis patients. Int Immunopharmacol. 2021;99:107898. pmid:34333359
- View Article
- PubMed/NCBI
- Google Scholar
40. Pfirschke C, Engblom C, Gungabeesoon J, Lin Y, Rickelt S, Zilionis R, et al. Tumor-Promoting Ly-6G⁺ SiglecF^high Cells Are Mature and Long-Lived Neutrophils. Cell Rep. 2020;32(12):108164. pmid:32966785
- View Article
- PubMed/NCBI
- Google Scholar
41. Rice CM, Davies LC, Subleski JJ, Maio N, Gonzalez-Cotto M, Andrews C, et al. Tumour-elicited neutrophils engage mitochondrial metabolism to circumvent nutrient limitations and maintain immune suppression. Nat Commun. 2018;9(1):5099. pmid:30504842
- View Article
- PubMed/NCBI
- Google Scholar
42. Kim MH, Yang D, Kim M, Kim SY, Kim D, Kang SJ. A late-lineage murine neutrophil precursor population exhibits dynamic changes during demand-adapted granulopoiesis. Sci Rep. 2017;7:39804. pmid:28059162
- View Article
- PubMed/NCBI
- Google Scholar
43. Evrard M, Kwok IWH, Chong SZ, Teng KWW, Becht E, Chen J, et al. Developmental Analysis of Bone Marrow Neutrophils Reveals Populations Specialized in Expansion, Trafficking, and Effector Functions. Immunity. 2018;48(2):364–79.e8. pmid:29466759
- View Article
- PubMed/NCBI
- Google Scholar
44. Song Z, Bhattacharya S, Huang G, Greenberg ZJ, Yang W, Bagaitkar J, et al. NADPH oxidase 2 limits amplification of IL-1β-G-CSF axis and an immature neutrophil subset in murine lung inflammation. Blood Adv. 2023;7(7):1225–40. pmid:36103336
- View Article
- PubMed/NCBI
- Google Scholar
45. Endo D, Fujimoto K, Hirose R, Yamanaka H, Homme M, Ishibashi KI, et al. Genetic Phagocyte NADPH Oxidase Deficiency Enhances Nonviable Candida albicans-Induced Inflammation in Mouse Lungs. Inflammation. 2017;40(1):123–35. pmid:27785664
- View Article
- PubMed/NCBI
- Google Scholar
46. Moreira-Teixeira L, Stimpson PJ, Stavropoulos E, Hadebe S, Chakravarty P, Ioannou M, et al. Type I IFN exacerbates disease in tuberculosis-susceptible mice by inducing neutrophil-mediated lung inflammation and NETosis. Nat Commun. 2020;11(1):5566. pmid:33149141
- View Article
- PubMed/NCBI
- Google Scholar
47. Lovewell RR, Baer CE, Mishra BB, Smith CM, Sassetti CM. Granulocytes act as a niche for Mycobacterium tuberculosis growth. Mucosal Immunol. 2021;14(1):229–41. pmid:32483198
- View Article
- PubMed/NCBI
- Google Scholar
48. Ding W, Shimada H, Li L, Mittal R, Zhang X, Shudo K, et al. Retinoid agonist Am80-enhanced neutrophil bactericidal activity arising from granulopoiesis in vitro and in a neutropenic mouse model. Blood. 2013;121(6):996–1007. pmid:23243275
- View Article
- PubMed/NCBI
- Google Scholar
49. Naskar D, Teng F, Felix KM, Bradley CP, Wu HJ. Synthetic Retinoid AM80 Ameliorates Lung and Arthritic Autoimmune Responses by Inhibiting T Follicular Helper and Th17 Cell Responses. J Immunol. 2017;198(5):1855–64. pmid:28130500
- View Article
- PubMed/NCBI
- Google Scholar
50. Bahlool AZ, Grant C, Cryan SA, Keane J, O’Sullivan MP. All trans retinoic acid as a host-directed immunotherapy for tuberculosis. Curr Res Immunol. 2022;3:54–72. pmid:35496824
- View Article
- PubMed/NCBI
- Google Scholar
51. Yamada H, Mizuno S, Ross AC, Sugawara I. Retinoic acid therapy attenuates the severity of tuberculosis while altering lymphocyte and macrophage numbers and cytokine expression in rats infected with Mycobacterium tuberculosis. J Nutr. 2007;137(12):2696–700. pmid:18029486
- View Article
- PubMed/NCBI
- Google Scholar
52. Li L, Qi X, Sun W, Abdel-Azim H, Lou S, Zhu H, et al. Am80-GCSF synergizes myeloid expansion and differentiation to generate functional neutrophils that reduce neutropenia-associated infection and mortality. EMBO Mol Med. 2016;8(11):1340–59. pmid:27737899
- View Article
- PubMed/NCBI
- Google Scholar
53. Tomita A, Kiyoi H, Naoe T. Mechanisms of action and resistance to all-trans retinoic acid (ATRA) and arsenic trioxide (As₂O₃) in acute promyelocytic leukemia. Int J Hematol. 2013;97(6):717–25. pmid:23670176
- View Article
- PubMed/NCBI
- Google Scholar
54. Cai W, Wang J, Hu M, Chen X, Lu Z, Bellanti JA, et al. All trans-retinoic acid protects against acute ischemic stroke by modulating neutrophil functions through STAT1 signaling. J Neuroinflammation. 2019;16(1):175. pmid:31472680
- View Article
- PubMed/NCBI
- Google Scholar
55. Basu S, Dunn A, Ward A. G-CSF: function and modes of action (Review). Int J Mol Med. 2002;10(1):3–10 pmid:12060844
- View Article
- PubMed/NCBI
- Google Scholar
56. Manz MG, Boettcher S. Emergency granulopoiesis. Nat Rev Immunol. 2014;14(5):302–14. pmid:24751955
- View Article
- PubMed/NCBI
- Google Scholar
57. Basu S, Hodgson G, Zhang HH, Katz M, Quilici C, Dunn AR. "Emergency" granulopoiesis in G-CSF-deficient mice in response to Candida albicans infection. Blood. 2000;95(12):3725–33 pmid:10845903
- View Article
- PubMed/NCBI
- Google Scholar
58. Gupta T, Sarr D, Fantone K, Ashtiwi NM, Sakamoto K, Quinn FD, et al. Dual oxidase 1 is dispensable during Mycobacterium tuberculosis infection in mice. Front Immunol. 2023;14:1044703. pmid:36936954
- View Article
- PubMed/NCBI
- Google Scholar
59. Kim WS, Kim JS, Cha SB, Han SJ, Kim H, Kwon KW, et al. Virulence-Dependent Alterations in the Kinetics of Immune Cells during Pulmonary Infection by Mycobacterium tuberculosis. PLoS One. 2015;10(12):e0145234. pmid:26675186
- View Article
- PubMed/NCBI
- Google Scholar
60. Kim WS, Kim H, Kwon KW, Cho SN, Shin SJ. Immunogenicity and Vaccine Potential of InsB, an ESAT-6-Like Antigen Identified in the Highly Virulent Mycobacterium tuberculosis Beijing K Strain. Front Microbiol. 2019;10:220. pmid:30809214
- View Article
- PubMed/NCBI
- Google Scholar
61. Kang TG, Kwon KW, Kim K, Lee I, Kim MJ, Ha SJ, et al. Viral coinfection promotes tuberculosis immunopathogenesis by type I IFN signaling-dependent impediment of Th1 cell pulmonary influx. Nat Commun. 2022;13(1):3155. pmid:35672321
- View Article
- PubMed/NCBI
- Google Scholar
62. Barbosa Bomfim CC, Pinheiro Amaral E, Santiago-Carvalho I, Almeida Santos G, Machado Salles É, Hastreiter AA, et al. Harmful Effects of Granulocytic Myeloid-Derived Suppressor Cells on Tuberculosis Caused by Hypervirulent Mycobacteria. J Infect Dis. 2021;223(3):494–507. pmid:33206171
- View Article
- PubMed/NCBI
- Google Scholar
63. Scanga CA, Bafica A, Feng CG, Cheever AW, Hieny S, Sher A. MyD88-deficient mice display a profound loss in resistance to Mycobacterium tuberculosis associated with partially impaired Th1 cytokine and nitric oxide synthase 2 expression. Infect Immun. 2004;72(4):2400–4. pmid:15039368
- View Article
- PubMed/NCBI
- Google Scholar
64. Silvério D, Gonçalves R, Appelberg R, Saraiva M. Advances on the Role and Applications of Interleukin-1 in Tuberculosis. mBio. 2021;12(6):e0313421. pmid:34809460
- View Article
- PubMed/NCBI
- Google Scholar
65. Juffermans NP, Florquin S, Camoglio L, Verbon A, Kolk AH, Speelman P, et al. Interleukin-1 signaling is essential for host defense during murine pulmonary tuberculosis. J Infect Dis. 2000;182(3):902–8. pmid:10950787
- View Article
- PubMed/NCBI
- Google Scholar
66. Mayer-Barber KD, Andrade BB, Oland SD, Amaral EP, Barber DL, Gonzales J, et al. Host-directed therapy of tuberculosis based on interleukin-1 and type I interferon crosstalk. Nature. 2014;511(7507):99–103. pmid:24990750
- View Article
- PubMed/NCBI
- Google Scholar
67. Lee K, Won HY, Bae MA, Hong JH, Hwang ES. Spontaneous and aging-dependent development of arthritis in NADPH oxidase 2 deficiency through altered differentiation of CD11b⁺ and Th/Treg cells. Proc Natl Acad Sci U S A. 2011;108(23):9548–53. pmid:21593419
- View Article
- PubMed/NCBI
- Google Scholar
68. Zhong J, Olsson LM, Urbonaviciute V, Yang M, Bäckdahl L, Holmdahl R. Association of NOX2 subunits genetic variants with autoimmune diseases. Free Radic Biol Med. 2018;125:72–80. pmid:29526808
- View Article
- PubMed/NCBI
- Google Scholar
69. Gonzalez A, Hung CY, Cole GT. Absence of phagocyte NADPH oxidase 2 leads to severe inflammatory response in lungs of mice infected with Coccidioides. Microb Pathog. 2011;51(6):432–41. pmid:21896326
- View Article
- PubMed/NCBI
- Google Scholar
70. Felmy B, Songhet P, Slack EM, Müller AJ, Kremer M, Van Maele L, et al. NADPH oxidase deficient mice develop colitis and bacteremia upon infection with normally avirulent, TTSS-1- and TTSS-2-deficient Salmonella Typhimurium. PLoS One. 2013;8(10):e77204. pmid:24143212
- View Article
- PubMed/NCBI
- Google Scholar
71. Segal BH, Han W, Bushey JJ, Joo M, Bhatti Z, Feminella J, et al. NADPH oxidase limits innate immune responses in the lungs in mice. PLoS One. 2010;5(3):e9631. pmid:20300512
- View Article
- PubMed/NCBI
- Google Scholar
72. Gopalakrishnan A, Dietzold J, Verma S, Bhagavathula M, Salgame P. Toll-like Receptor 2 Prevents Neutrophil-Driven Immunopathology during Infection with Mycobacterium tuberculosis by Curtailing CXCL5 Production. Infect Immun. 2019;87(3). pmid:30559223
- View Article
- PubMed/NCBI
- Google Scholar
73. Mishra BB, Lovewell RR, Olive AJ, Zhang G, Wang W, Eugenin E, et al. Nitric oxide prevents a pathogen-permissive granulocytic inflammation during tuberculosis. Nat Microbiol. 2017;2:17072. pmid:28504669
- View Article
- PubMed/NCBI
- Google Scholar
74. Scott NR, Swanson RV, Al-Hammadi N, Domingo-Gonzalez R, Rangel-Moreno J, Kriel BA, et al. S100A8/A9 regulates CD11b expression and neutrophil recruitment during chronic tuberculosis. J Clin Invest. 2020;130(6):3098–112. pmid:32134742
- View Article
- PubMed/NCBI
- Google Scholar
75. O’Connor G, Krishnan N, Fagan-Murphy A, Cassidy J, O’Leary S, Robertson BD, et al. Inhalable poly(lactic-co-glycolic acid) (PLGA) microparticles encapsulating all-trans-Retinoic acid (ATRA) as a host-directed, adjunctive treatment for Mycobacterium tuberculosis infection. Eur J Pharm Biopharm. 2019;134:153–65. pmid:30385419
- View Article
- PubMed/NCBI
- Google Scholar
76. Mourik BC, Leenen PJ, de Knegt GJ, Huizinga R, van der Eerden BC, Wang J, et al. Immunotherapy Added to Antibiotic Treatment Reduces Relapse of Disease in a Mouse Model of Tuberculosis. Am J Respir Cell Mol Biol. 2017;56(2):233–41. pmid:27654457
- View Article
- PubMed/NCBI
- Google Scholar
77. Knaul JK, Jörg S, Oberbeck-Mueller D, Heinemann E, Scheuermann L, Brinkmann V, et al. Lung-residing myeloid-derived suppressors display dual functionality in murine pulmonary tuberculosis. Am J Respir Crit Care Med. 2014;190(9):1053–66. pmid:25275852
- View Article
- PubMed/NCBI
- Google Scholar
78. Tobita T, Takeshita A, Kitamura K, Ohnishi K, Yanagi M, Hiraoka A, et al. Treatment with a new synthetic retinoid, Am80, of acute promyelocytic leukemia relapsed from complete remission induced by all-trans retinoic acid. Blood. 1997;90(3):967–73 pmid:9242525
- View Article
- PubMed/NCBI
- Google Scholar
79. Watanabe H, Bi J, Murata R, Fujimura R, Nishida K, Imafuku T, et al. A synthetic retinoic acid receptor agonist Am80 ameliorates renal fibrosis via inducing the production of alpha-1-acid glycoprotein. Sci Rep. 2020;10(1):11424. pmid:32651445
- View Article
- PubMed/NCBI
- Google Scholar
80. Boivin G, Faget J, Ancey PB, Gkasti A, Mussard J, Engblom C, et al. Durable and controlled depletion of neutrophils in mice. Nat Commun. 2020;11(1):2762. pmid:32488020
- View Article
- PubMed/NCBI
- Google Scholar
81. Barber DL, Mayer-Barber KD, Feng CG, Sharpe AH, Sher A. CD4 T cells promote rather than control tuberculosis in the absence of PD-1-mediated inhibition. J Immunol. 2011;186(3):1598–607. pmid:21172867
- View Article
- PubMed/NCBI
- Google Scholar
82. Ahmed M, Tezera LB, Elkington PT, Leslie AJ. The paradox of immune checkpoint inhibition re-activating tuberculosis. Eur Respir J. 2022;60(5). pmid:35595321
- View Article
- PubMed/NCBI
- Google Scholar
83. Kim JH, Podstawka J, Lou Y, Li L, Lee EKS, Divangahi M, et al. Aged polymorphonuclear leukocytes cause fibrotic interstitial lung disease in the absence of regulation by B cells. Nat Immunol. 2018;19(2):192–201. pmid:29335647
- View Article
- PubMed/NCBI
- Google Scholar
84. Podstawka J, Sinha S, Hiroki CH, Sarden N, Granton E, Labit E, et al. Marginating transitional B cells modulate neutrophils in the lung during inflammation and pneumonia. J Exp Med. 2021;218(9). pmid:34313733
- View Article
- PubMed/NCBI
- Google Scholar
85. Shin SJ, Lee BS, Koh WJ, Manning EJ, Anklam K, Sreevatsan S, et al. Efficient differentiation of Mycobacterium avium complex species and subspecies by use of five-target multiplex PCR. J Clin Microbiol. 2010;48(11):4057–62. pmid:20810779
- View Article
- PubMed/NCBI
- Google Scholar
86. Kim WS, Kim JS, Cha SB, Kim H, Kwon KW, Kim SJ, et al. Mycobacterium tuberculosis Rv3628 drives Th1-type T cell immunity via TLR2-mediated activation of dendritic cells and displays vaccine potential against the hyper-virulent Beijing K strain. Oncotarget. 2016;7(18):24962–82. pmid:27097115
- View Article
- PubMed/NCBI
- Google Scholar
87. Kwon KW, Aceves-Sánchez MJ, Segura-Cerda CA, Choi E, Bielefeldt-Ohmann H, Shin SJ, et al. BCGΔBCG1419c increased memory CD8⁺ T cell-associated immunogenicity and mitigated pulmonary inflammation compared with BCG in a model of chronic tuberculosis. Sci Rep. 2022;12(1):15824. pmid:36138053
- View Article
- PubMed/NCBI
- Google Scholar
88. Lee JM, Park J, Reed SG, Coler RN, Hong JJ, Kim LH, et al. Vaccination inducing durable and robust antigen-specific Th1/Th17 immune responses contributes to prophylactic protection against Mycobacterium avium infection but is ineffective as an adjunct to antibiotic treatment in chronic disease. Virulence. 2022;13(1):808–32. pmid:35499090
- View Article
- PubMed/NCBI
- Google Scholar
89. Jeon BY, Kim SC, Eum SY, Cho SN. The immunity and protective effects of antigen 85A and heat-shock protein X against progressive tuberculosis. Microbes Infect. 2011;13(3):284–90. pmid:21093603
- View Article
- PubMed/NCBI
- Google Scholar
90. Kwon KW, Kim LH, Kang SM, Lee JM, Choi E, Park J, et al. Host-directed anti-mycobacterial activity of colchicine, an anti-gout drug, via strengthened host innate resistance reinforced by the IL-1β/PGE(2) axis. Br J Pharmacol. 2022;179(15):3951–69. pmid:35301712
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. WHO. Global Tuberculosis Report 2022: World Health Organization; 2022.

[ref2] 2. Das S, Marin ND, Esaulova E, Ahmed M, Swain A, Rosa BA, et al. Lung Epithelial Signaling Mediates Early Vaccine-Induced CD4⁺ T Cell Activation and Mycobacterium tuberculosis Control. mBio. 2021;12(4):e0146821. pmid:34253059
View Article
PubMed/NCBI
Google Scholar

[3] View Article

[4] PubMed/NCBI

[5] Google Scholar

[ref3] 3. Fu H, Lewnard JA, Frost I, Laxminarayan R, Arinaminpathy N. Modelling the global burden of drug-resistant tuberculosis avertable by a post-exposure vaccine. Nat Commun. 2021;12(1):424. pmid:33462224
View Article
PubMed/NCBI
Google Scholar

[7] View Article

[8] PubMed/NCBI

[9] Google Scholar

[ref4] 4. Smith CM, Proulx MK, Olive AJ, Laddy D, Mishra BB, Moss C, et al. Tuberculosis Susceptibility and Vaccine Protection Are Independently Controlled by Host Genotype. mBio. 2016;7(5). pmid:27651361
View Article
PubMed/NCBI
Google Scholar

[11] View Article

[12] PubMed/NCBI

[13] Google Scholar

[ref5] 5. Neyrolles O, Quintana-Murci L. Sexual inequality in tuberculosis. PLoS Med. 2009;6(12):e1000199. pmid:20027210
View Article
PubMed/NCBI
Google Scholar

[15] View Article

[16] PubMed/NCBI

[17] Google Scholar

[ref6] 6. Nhamoyebonde S, Leslie A. Biological differences between the sexes and susceptibility to tuberculosis. J Infect Dis. 2014;209 Suppl 3:S100–6. pmid:24966189
View Article
PubMed/NCBI
Google Scholar

[19] View Article

[20] PubMed/NCBI

[21] Google Scholar

[ref7] 7. Dibbern J, Eggers L, Schneider BE. Sex differences in the C57BL/6 model of Mycobacterium tuberculosis infection. Sci Rep. 2017;7(1):10957. pmid:28887521
View Article
PubMed/NCBI
Google Scholar

[23] View Article

[24] PubMed/NCBI

[25] Google Scholar

[ref8] 8. Zhao Y, Ying H, Demei J, Xie J. Tuberculosis and sexual inequality: the role of sex hormones in immunity. Crit Rev Eukaryot Gene Expr. 2012;22(3):233–41. pmid:23140164
View Article
PubMed/NCBI
Google Scholar

[27] View Article

[28] PubMed/NCBI

[29] Google Scholar

[ref9] 9. Chandra P, Grigsby SJ, Philips JA. Immune evasion and provocation by Mycobacterium tuberculosis. Nat Rev Microbiol. 2022;20(12):750–66. pmid:35879556
View Article
PubMed/NCBI
Google Scholar

[31] View Article

[32] PubMed/NCBI

[33] Google Scholar

[ref10] 10. Kotzé LA, Young C, Leukes VN, John V, Fang Z, Walzl G, et al. Mycobacterium tuberculosis and myeloid-derived suppressor cells: Insights into caveolin rich lipid rafts. EBioMedicine. 2020;53:102670. pmid:32113158
View Article
PubMed/NCBI
Google Scholar

[35] View Article

[36] PubMed/NCBI

[37] Google Scholar

[ref11] 11. Xu W, Snell LM, Guo M, Boukhaled G, Macleod BL, Li M, et al. Early innate and adaptive immune perturbations determine long-term severity of chronic virus and Mycobacterium tuberculosis coinfection. Immunity. 2021;54(3):526–41.e7. pmid:33515487
View Article
PubMed/NCBI
Google Scholar

[39] View Article

[40] PubMed/NCBI

[41] Google Scholar

[ref12] 12. Olive AJ, Smith CM, Kiritsy MC, Sassetti CM. The Phagocyte Oxidase Controls Tolerance to Mycobacterium tuberculosis Infection. J Immunol. 2018;201(6):1705–16. pmid:30061198
View Article
PubMed/NCBI
Google Scholar

[43] View Article

[44] PubMed/NCBI

[45] Google Scholar

[ref13] 13. Miller JL, Velmurugan K, Cowan MJ, Briken V. The type I NADH dehydrogenase of Mycobacterium tuberculosis counters phagosomal NOX2 activity to inhibit TNF-alpha-mediated host cell apoptosis. PLoS Pathog. 2010;6(4):e1000864. pmid:20421951
View Article
PubMed/NCBI
Google Scholar

[47] View Article

[48] PubMed/NCBI

[49] Google Scholar

[ref14] 14. Sies H, Jones DP. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat Rev Mol Cell Biol. 2020;21(7):363–83. pmid:32231263
View Article
PubMed/NCBI
Google Scholar

[51] View Article

[52] PubMed/NCBI

[53] Google Scholar

[ref15] 15. Xiao X, Bu H, Li Z, Li Z, Bai Q, Wang Z, et al. NADPH-Oxidase 2 Promotes Autophagy in Spinal Neurons During the Development of Morphine Tolerance. Neurochem Res. 2021;46(8):2089–96. pmid:34008119
View Article
PubMed/NCBI
Google Scholar

[55] View Article

[56] PubMed/NCBI

[57] Google Scholar

[ref16] 16. Munnamalai V, Weaver CJ, Weisheit CE, Venkatraman P, Agim ZS, Quinn MT, et al. Bidirectional interactions between NOX2-type NADPH oxidase and the F-actin cytoskeleton in neuronal growth cones. J Neurochem. 2014;130(4):526–40. pmid:24702317
View Article
PubMed/NCBI
Google Scholar

[59] View Article

[60] PubMed/NCBI

[61] Google Scholar

[ref17] 17. Brandes RP, Weissmann N, Schröder K. Nox family NADPH oxidases: Molecular mechanisms of activation. Free Radic Biol Med. 2014;76:208–26. pmid:25157786
View Article
PubMed/NCBI
Google Scholar

[63] View Article

[64] PubMed/NCBI

[65] Google Scholar

[ref18] 18. Panday A, Sahoo MK, Osorio D, Batra S. NADPH oxidases: an overview from structure to innate immunity-associated pathologies. Cell Mol Immunol. 2015;12(1):5–23. pmid:25263488
View Article
PubMed/NCBI
Google Scholar

[67] View Article

[68] PubMed/NCBI

[69] Google Scholar

[ref19] 19. Anjani G, Vignesh P, Joshi V, Shandilya JK, Bhattarai D, Sharma J, et al. Recent advances in chronic granulomatous disease. Genes Dis. 2020;7(1):84–92. pmid:32181279
View Article
PubMed/NCBI
Google Scholar

[71] View Article

[72] PubMed/NCBI

[73] Google Scholar

[ref20] 20. Xu Q, Wang Q, Hu LT, Lin J, Jiang N, Peng XD, et al. NADPH oxidase 2 plays a protective role in experimental Aspergillus fumigatus keratitis in mice through killing fungi and limiting the degree of inflammation. Int J Ophthalmol. 2022;15(7):1044–52. pmid:35919314
View Article
PubMed/NCBI
Google Scholar

[75] View Article

[76] PubMed/NCBI

[77] Google Scholar

[ref21] 21. Taylor JP, Tse HM. The role of NADPH oxidases in infectious and inflammatory diseases. Redox Biol. 2021;48:102159. pmid:34627721
View Article
PubMed/NCBI
Google Scholar

[79] View Article

[80] PubMed/NCBI

[81] Google Scholar

[ref22] 22. Rojas Márquez JD, Li T, McCluggage ARR, Tan JMJ, Muise A, Higgins DE, et al. Cutting Edge: NOX2 NADPH Oxidase Controls Infection by an Intracellular Bacterial Pathogen through Limiting the Type 1 IFN Response. J Immunol. 2021;206(2):323–8. pmid:33288542
View Article
PubMed/NCBI
Google Scholar

[83] View Article

[84] PubMed/NCBI

[85] Google Scholar

[ref23] 23. Kohn DB, Booth C, Kang EM, Pai SY, Shaw KL, Santilli G, et al. Lentiviral gene therapy for X-linked chronic granulomatous disease. Nat Med. 2020;26(2):200–6. pmid:31988463
View Article
PubMed/NCBI
Google Scholar

[87] View Article

[88] PubMed/NCBI

[89] Google Scholar

[ref24] 24. García B, León-Lara X, Espinosa S, Blancas-Galicia L. [Mycobacterial disease in patients with chronic granulomatous disease]. Rev Alerg Mex. 2021;68(2):117–27. pmid:34525783
View Article
PubMed/NCBI
Google Scholar

[91] View Article

[92] PubMed/NCBI

[93] Google Scholar

[ref25] 25. Conti F, Lugo-Reyes SO, Blancas Galicia L, He J, Aksu G, Borges de Oliveira E, Jr., et al. Mycobacterial disease in patients with chronic granulomatous disease: A retrospective analysis of 71 cases. J Allergy Clin Immunol. 2016;138(1):241–8.e3. pmid:26936803
View Article
PubMed/NCBI
Google Scholar

[95] View Article

[96] PubMed/NCBI

[97] Google Scholar

[ref26] 26. Deffert C, Schäppi MG, Pache JC, Cachat J, Vesin D, Bisig R, et al. Bacillus calmette-guerin infection in NADPH oxidase deficiency: defective mycobacterial sequestration and granuloma formation. PLoS Pathog. 2014;10(9):e1004325. pmid:25188296
View Article
PubMed/NCBI
Google Scholar

[99] View Article

[100] PubMed/NCBI

[101] Google Scholar

[ref27] 27. Ying W, Sun J, Liu D, Hui X, Yu Y, Wang J, et al. Clinical characteristics and immunogenetics of BCGosis/BCGitis in Chinese children: a 6 year follow-up study. PLoS One. 2014;9(4):e94485. pmid:24722620
View Article
PubMed/NCBI
Google Scholar

[103] View Article

[104] PubMed/NCBI

[105] Google Scholar

[ref28] 28. Cooper AM, Segal BH, Frank AA, Holland SM, Orme IM. Transient loss of resistance to pulmonary tuberculosis in p47(phox^-/-) mice. Infect Immun. 2000;68(3):1231–4. pmid:10678931
View Article
PubMed/NCBI
Google Scholar

[107] View Article

[108] PubMed/NCBI

[109] Google Scholar

[ref29] 29. Jung YJ, LaCourse R, Ryan L, North RJ. Virulent but not avirulent Mycobacterium tuberculosis can evade the growth inhibitory action of a T helper 1-dependent, nitric oxide Synthase 2-independent defense in mice. J Exp Med. 2002;196(7):991–8. pmid:12370260
View Article
PubMed/NCBI
Google Scholar

[111] View Article

[112] PubMed/NCBI

[113] Google Scholar

[ref30] 30. Ng VH, Cox JS, Sousa AO, MacMicking JD, McKinney JD. Role of KatG catalase-peroxidase in mycobacterial pathogenesis: countering the phagocyte oxidative burst. Mol Microbiol. 2004;52(5):1291–302. pmid:15165233
View Article
PubMed/NCBI
Google Scholar

[115] View Article

[116] PubMed/NCBI

[117] Google Scholar

[ref31] 31. Thomas SM, Olive AJ. High Lethality of Mycobacterium tuberculosis Infection in Mice Lacking the Phagocyte Oxidase and Caspase1/11. Infect Immun. 2023:e0006023. pmid:37314361
View Article
PubMed/NCBI
Google Scholar

[119] View Article

[120] PubMed/NCBI

[121] Google Scholar

[ref32] 32. Goebel WS, Mark LA, Billings SD, Meyers JL, Pech N, Travers JB, et al. Gene correction reduces cutaneous inflammation and granuloma formation in murine X-linked chronic granulomatous disease. J Invest Dermatol. 2005;125(4):705–10. pmid:16185269
View Article
PubMed/NCBI
Google Scholar

[123] View Article

[124] PubMed/NCBI

[125] Google Scholar

[ref33] 33. Yu JE, Azar AE, Chong HJ, Jongco AM 3rd, Prince BT. Considerations in the Diagnosis of Chronic Granulomatous Disease. J Pediatric Infect Dis Soc. 2018;7(suppl_1):S6–s11. pmid:29746674
View Article
PubMed/NCBI
Google Scholar

[127] View Article

[128] PubMed/NCBI

[129] Google Scholar

[ref34] 34. Liao YC, Wu SY, Huang YF, Lo PC, Chan TY, Chen CA, et al. NOX2-Deficient Neutrophils Facilitate Joint Inflammation Through Higher Pro-Inflammatory and Weakened Immune Checkpoint Activities. Front Immunol. 2021;12:743030. pmid:34557202
View Article
PubMed/NCBI
Google Scholar

[131] View Article

[132] PubMed/NCBI

[133] Google Scholar

[ref35] 35. Wang J, Ma MW, Dhandapani KM, Brann DW. Regulatory role of NADPH oxidase 2 in the polarization dynamics and neurotoxicity of microglia/macrophages after traumatic brain injury. Free Radic Biol Med. 2017;113:119–31. pmid:28942245
View Article
PubMed/NCBI
Google Scholar

[135] View Article

[136] PubMed/NCBI

[137] Google Scholar

[ref36] 36. Bagaitkar J, Pech NK, Ivanov S, Austin A, Zeng MY, Pallat S, et al. NADPH oxidase controls neutrophilic response to sterile inflammation in mice by regulating the IL-1α/G-CSF axis. Blood. 2015;126(25):2724–33. pmid:26443623
View Article
PubMed/NCBI
Google Scholar

[139] View Article

[140] PubMed/NCBI

[141] Google Scholar

[ref37] 37. Chao WC, Yen CL, Hsieh CY, Huang YF, Tseng YL, Nigrovic PA, et al. Mycobacterial infection induces higher interleukin-1β and dysregulated lung inflammation in mice with defective leukocyte NADPH oxidase. PLoS One. 2017;12(12):e0189453. pmid:29228045
View Article
PubMed/NCBI
Google Scholar

[143] View Article

[144] PubMed/NCBI

[145] Google Scholar

[ref38] 38. Choi HG, Kwon KW, Choi S, Back YW, Park HS, Kang SM, et al. Antigen-Specific IFN-γ/IL-17-Co-Producing CD4⁺ T-Cells Are the Determinants for Protective Efficacy of Tuberculosis Subunit Vaccine. Vaccines (Basel). 2020;8(2). pmid:32545304
View Article
PubMed/NCBI
Google Scholar

[147] View Article

[148] PubMed/NCBI

[149] Google Scholar

[ref39] 39. Qin S, Chen R, Jiang Y, Zhu H, Chen L, Chen Y, et al. Multifunctional T cell response in active pulmonary tuberculosis patients. Int Immunopharmacol. 2021;99:107898. pmid:34333359
View Article
PubMed/NCBI
Google Scholar

[151] View Article

[152] PubMed/NCBI

[153] Google Scholar

[ref40] 40. Pfirschke C, Engblom C, Gungabeesoon J, Lin Y, Rickelt S, Zilionis R, et al. Tumor-Promoting Ly-6G⁺ SiglecF^high Cells Are Mature and Long-Lived Neutrophils. Cell Rep. 2020;32(12):108164. pmid:32966785
View Article
PubMed/NCBI
Google Scholar

[155] View Article

[156] PubMed/NCBI

[157] Google Scholar

[ref41] 41. Rice CM, Davies LC, Subleski JJ, Maio N, Gonzalez-Cotto M, Andrews C, et al. Tumour-elicited neutrophils engage mitochondrial metabolism to circumvent nutrient limitations and maintain immune suppression. Nat Commun. 2018;9(1):5099. pmid:30504842
View Article
PubMed/NCBI
Google Scholar

[159] View Article

[160] PubMed/NCBI

[161] Google Scholar

[ref42] 42. Kim MH, Yang D, Kim M, Kim SY, Kim D, Kang SJ. A late-lineage murine neutrophil precursor population exhibits dynamic changes during demand-adapted granulopoiesis. Sci Rep. 2017;7:39804. pmid:28059162
View Article
PubMed/NCBI
Google Scholar

[163] View Article

[164] PubMed/NCBI

[165] Google Scholar

[ref43] 43. Evrard M, Kwok IWH, Chong SZ, Teng KWW, Becht E, Chen J, et al. Developmental Analysis of Bone Marrow Neutrophils Reveals Populations Specialized in Expansion, Trafficking, and Effector Functions. Immunity. 2018;48(2):364–79.e8. pmid:29466759
View Article
PubMed/NCBI
Google Scholar

[167] View Article

[168] PubMed/NCBI

[169] Google Scholar

[ref44] 44. Song Z, Bhattacharya S, Huang G, Greenberg ZJ, Yang W, Bagaitkar J, et al. NADPH oxidase 2 limits amplification of IL-1β-G-CSF axis and an immature neutrophil subset in murine lung inflammation. Blood Adv. 2023;7(7):1225–40. pmid:36103336
View Article
PubMed/NCBI
Google Scholar

[171] View Article

[172] PubMed/NCBI

[173] Google Scholar

[ref45] 45. Endo D, Fujimoto K, Hirose R, Yamanaka H, Homme M, Ishibashi KI, et al. Genetic Phagocyte NADPH Oxidase Deficiency Enhances Nonviable Candida albicans-Induced Inflammation in Mouse Lungs. Inflammation. 2017;40(1):123–35. pmid:27785664
View Article
PubMed/NCBI
Google Scholar

[175] View Article

[176] PubMed/NCBI

[177] Google Scholar

[ref46] 46. Moreira-Teixeira L, Stimpson PJ, Stavropoulos E, Hadebe S, Chakravarty P, Ioannou M, et al. Type I IFN exacerbates disease in tuberculosis-susceptible mice by inducing neutrophil-mediated lung inflammation and NETosis. Nat Commun. 2020;11(1):5566. pmid:33149141
View Article
PubMed/NCBI
Google Scholar

[179] View Article

[180] PubMed/NCBI

[181] Google Scholar

[ref47] 47. Lovewell RR, Baer CE, Mishra BB, Smith CM, Sassetti CM. Granulocytes act as a niche for Mycobacterium tuberculosis growth. Mucosal Immunol. 2021;14(1):229–41. pmid:32483198
View Article
PubMed/NCBI
Google Scholar

[183] View Article

[184] PubMed/NCBI

[185] Google Scholar

[ref48] 48. Ding W, Shimada H, Li L, Mittal R, Zhang X, Shudo K, et al. Retinoid agonist Am80-enhanced neutrophil bactericidal activity arising from granulopoiesis in vitro and in a neutropenic mouse model. Blood. 2013;121(6):996–1007. pmid:23243275
View Article
PubMed/NCBI
Google Scholar

[187] View Article

[188] PubMed/NCBI

[189] Google Scholar

[ref49] 49. Naskar D, Teng F, Felix KM, Bradley CP, Wu HJ. Synthetic Retinoid AM80 Ameliorates Lung and Arthritic Autoimmune Responses by Inhibiting T Follicular Helper and Th17 Cell Responses. J Immunol. 2017;198(5):1855–64. pmid:28130500
View Article
PubMed/NCBI
Google Scholar

[191] View Article

[192] PubMed/NCBI

[193] Google Scholar

[ref50] 50. Bahlool AZ, Grant C, Cryan SA, Keane J, O’Sullivan MP. All trans retinoic acid as a host-directed immunotherapy for tuberculosis. Curr Res Immunol. 2022;3:54–72. pmid:35496824
View Article
PubMed/NCBI
Google Scholar

[195] View Article

[196] PubMed/NCBI

[197] Google Scholar

[ref51] 51. Yamada H, Mizuno S, Ross AC, Sugawara I. Retinoic acid therapy attenuates the severity of tuberculosis while altering lymphocyte and macrophage numbers and cytokine expression in rats infected with Mycobacterium tuberculosis. J Nutr. 2007;137(12):2696–700. pmid:18029486
View Article
PubMed/NCBI
Google Scholar

[199] View Article

[200] PubMed/NCBI

[201] Google Scholar

[ref52] 52. Li L, Qi X, Sun W, Abdel-Azim H, Lou S, Zhu H, et al. Am80-GCSF synergizes myeloid expansion and differentiation to generate functional neutrophils that reduce neutropenia-associated infection and mortality. EMBO Mol Med. 2016;8(11):1340–59. pmid:27737899
View Article
PubMed/NCBI
Google Scholar

[203] View Article

[204] PubMed/NCBI

[205] Google Scholar

[ref53] 53. Tomita A, Kiyoi H, Naoe T. Mechanisms of action and resistance to all-trans retinoic acid (ATRA) and arsenic trioxide (As₂O₃) in acute promyelocytic leukemia. Int J Hematol. 2013;97(6):717–25. pmid:23670176
View Article
PubMed/NCBI
Google Scholar

[207] View Article

[208] PubMed/NCBI

[209] Google Scholar

[ref54] 54. Cai W, Wang J, Hu M, Chen X, Lu Z, Bellanti JA, et al. All trans-retinoic acid protects against acute ischemic stroke by modulating neutrophil functions through STAT1 signaling. J Neuroinflammation. 2019;16(1):175. pmid:31472680
View Article
PubMed/NCBI
Google Scholar

[211] View Article

[212] PubMed/NCBI

[213] Google Scholar

[ref55] 55. Basu S, Dunn A, Ward A. G-CSF: function and modes of action (Review). Int J Mol Med. 2002;10(1):3–10 pmid:12060844
View Article
PubMed/NCBI
Google Scholar

[215] View Article

[216] PubMed/NCBI

[217] Google Scholar

[ref56] 56. Manz MG, Boettcher S. Emergency granulopoiesis. Nat Rev Immunol. 2014;14(5):302–14. pmid:24751955
View Article
PubMed/NCBI
Google Scholar

[219] View Article

[220] PubMed/NCBI

[221] Google Scholar

[ref57] 57. Basu S, Hodgson G, Zhang HH, Katz M, Quilici C, Dunn AR. "Emergency" granulopoiesis in G-CSF-deficient mice in response to Candida albicans infection. Blood. 2000;95(12):3725–33 pmid:10845903
View Article
PubMed/NCBI
Google Scholar

[223] View Article

[224] PubMed/NCBI

[225] Google Scholar

[ref58] 58. Gupta T, Sarr D, Fantone K, Ashtiwi NM, Sakamoto K, Quinn FD, et al. Dual oxidase 1 is dispensable during Mycobacterium tuberculosis infection in mice. Front Immunol. 2023;14:1044703. pmid:36936954
View Article
PubMed/NCBI
Google Scholar

[227] View Article

[228] PubMed/NCBI

[229] Google Scholar

[ref59] 59. Kim WS, Kim JS, Cha SB, Han SJ, Kim H, Kwon KW, et al. Virulence-Dependent Alterations in the Kinetics of Immune Cells during Pulmonary Infection by Mycobacterium tuberculosis. PLoS One. 2015;10(12):e0145234. pmid:26675186
View Article
PubMed/NCBI
Google Scholar

[231] View Article

[232] PubMed/NCBI

[233] Google Scholar

[ref60] 60. Kim WS, Kim H, Kwon KW, Cho SN, Shin SJ. Immunogenicity and Vaccine Potential of InsB, an ESAT-6-Like Antigen Identified in the Highly Virulent Mycobacterium tuberculosis Beijing K Strain. Front Microbiol. 2019;10:220. pmid:30809214
View Article
PubMed/NCBI
Google Scholar

[235] View Article

[236] PubMed/NCBI

[237] Google Scholar

[ref61] 61. Kang TG, Kwon KW, Kim K, Lee I, Kim MJ, Ha SJ, et al. Viral coinfection promotes tuberculosis immunopathogenesis by type I IFN signaling-dependent impediment of Th1 cell pulmonary influx. Nat Commun. 2022;13(1):3155. pmid:35672321
View Article
PubMed/NCBI
Google Scholar

[239] View Article

[240] PubMed/NCBI

[241] Google Scholar

[ref62] 62. Barbosa Bomfim CC, Pinheiro Amaral E, Santiago-Carvalho I, Almeida Santos G, Machado Salles É, Hastreiter AA, et al. Harmful Effects of Granulocytic Myeloid-Derived Suppressor Cells on Tuberculosis Caused by Hypervirulent Mycobacteria. J Infect Dis. 2021;223(3):494–507. pmid:33206171
View Article
PubMed/NCBI
Google Scholar

[243] View Article

[244] PubMed/NCBI

[245] Google Scholar

[ref63] 63. Scanga CA, Bafica A, Feng CG, Cheever AW, Hieny S, Sher A. MyD88-deficient mice display a profound loss in resistance to Mycobacterium tuberculosis associated with partially impaired Th1 cytokine and nitric oxide synthase 2 expression. Infect Immun. 2004;72(4):2400–4. pmid:15039368
View Article
PubMed/NCBI
Google Scholar

[247] View Article

[248] PubMed/NCBI

[249] Google Scholar

[ref64] 64. Silvério D, Gonçalves R, Appelberg R, Saraiva M. Advances on the Role and Applications of Interleukin-1 in Tuberculosis. mBio. 2021;12(6):e0313421. pmid:34809460
View Article
PubMed/NCBI
Google Scholar

[251] View Article

[252] PubMed/NCBI

[253] Google Scholar

[ref65] 65. Juffermans NP, Florquin S, Camoglio L, Verbon A, Kolk AH, Speelman P, et al. Interleukin-1 signaling is essential for host defense during murine pulmonary tuberculosis. J Infect Dis. 2000;182(3):902–8. pmid:10950787
View Article
PubMed/NCBI
Google Scholar

[255] View Article

[256] PubMed/NCBI

[257] Google Scholar

[ref66] 66. Mayer-Barber KD, Andrade BB, Oland SD, Amaral EP, Barber DL, Gonzales J, et al. Host-directed therapy of tuberculosis based on interleukin-1 and type I interferon crosstalk. Nature. 2014;511(7507):99–103. pmid:24990750
View Article
PubMed/NCBI
Google Scholar

[259] View Article

[260] PubMed/NCBI

[261] Google Scholar

[ref67] 67. Lee K, Won HY, Bae MA, Hong JH, Hwang ES. Spontaneous and aging-dependent development of arthritis in NADPH oxidase 2 deficiency through altered differentiation of CD11b⁺ and Th/Treg cells. Proc Natl Acad Sci U S A. 2011;108(23):9548–53. pmid:21593419
View Article
PubMed/NCBI
Google Scholar

[263] View Article

[264] PubMed/NCBI

[265] Google Scholar

[ref68] 68. Zhong J, Olsson LM, Urbonaviciute V, Yang M, Bäckdahl L, Holmdahl R. Association of NOX2 subunits genetic variants with autoimmune diseases. Free Radic Biol Med. 2018;125:72–80. pmid:29526808
View Article
PubMed/NCBI
Google Scholar

[267] View Article

[268] PubMed/NCBI

[269] Google Scholar

[ref69] 69. Gonzalez A, Hung CY, Cole GT. Absence of phagocyte NADPH oxidase 2 leads to severe inflammatory response in lungs of mice infected with Coccidioides. Microb Pathog. 2011;51(6):432–41. pmid:21896326
View Article
PubMed/NCBI
Google Scholar

[271] View Article

[272] PubMed/NCBI

[273] Google Scholar

[ref70] 70. Felmy B, Songhet P, Slack EM, Müller AJ, Kremer M, Van Maele L, et al. NADPH oxidase deficient mice develop colitis and bacteremia upon infection with normally avirulent, TTSS-1- and TTSS-2-deficient Salmonella Typhimurium. PLoS One. 2013;8(10):e77204. pmid:24143212
View Article
PubMed/NCBI
Google Scholar

[275] View Article

[276] PubMed/NCBI

[277] Google Scholar

[ref71] 71. Segal BH, Han W, Bushey JJ, Joo M, Bhatti Z, Feminella J, et al. NADPH oxidase limits innate immune responses in the lungs in mice. PLoS One. 2010;5(3):e9631. pmid:20300512
View Article
PubMed/NCBI
Google Scholar

[279] View Article

[280] PubMed/NCBI

[281] Google Scholar

[ref72] 72. Gopalakrishnan A, Dietzold J, Verma S, Bhagavathula M, Salgame P. Toll-like Receptor 2 Prevents Neutrophil-Driven Immunopathology during Infection with Mycobacterium tuberculosis by Curtailing CXCL5 Production. Infect Immun. 2019;87(3). pmid:30559223
View Article
PubMed/NCBI
Google Scholar

[283] View Article

[284] PubMed/NCBI

[285] Google Scholar

[ref73] 73. Mishra BB, Lovewell RR, Olive AJ, Zhang G, Wang W, Eugenin E, et al. Nitric oxide prevents a pathogen-permissive granulocytic inflammation during tuberculosis. Nat Microbiol. 2017;2:17072. pmid:28504669
View Article
PubMed/NCBI
Google Scholar

[287] View Article

[288] PubMed/NCBI

[289] Google Scholar

[ref74] 74. Scott NR, Swanson RV, Al-Hammadi N, Domingo-Gonzalez R, Rangel-Moreno J, Kriel BA, et al. S100A8/A9 regulates CD11b expression and neutrophil recruitment during chronic tuberculosis. J Clin Invest. 2020;130(6):3098–112. pmid:32134742
View Article
PubMed/NCBI
Google Scholar

[291] View Article

[292] PubMed/NCBI

[293] Google Scholar

[ref75] 75. O’Connor G, Krishnan N, Fagan-Murphy A, Cassidy J, O’Leary S, Robertson BD, et al. Inhalable poly(lactic-co-glycolic acid) (PLGA) microparticles encapsulating all-trans-Retinoic acid (ATRA) as a host-directed, adjunctive treatment for Mycobacterium tuberculosis infection. Eur J Pharm Biopharm. 2019;134:153–65. pmid:30385419
View Article
PubMed/NCBI
Google Scholar

[295] View Article

[296] PubMed/NCBI

[297] Google Scholar

[ref76] 76. Mourik BC, Leenen PJ, de Knegt GJ, Huizinga R, van der Eerden BC, Wang J, et al. Immunotherapy Added to Antibiotic Treatment Reduces Relapse of Disease in a Mouse Model of Tuberculosis. Am J Respir Cell Mol Biol. 2017;56(2):233–41. pmid:27654457
View Article
PubMed/NCBI
Google Scholar

[299] View Article

[300] PubMed/NCBI

[301] Google Scholar

[ref77] 77. Knaul JK, Jörg S, Oberbeck-Mueller D, Heinemann E, Scheuermann L, Brinkmann V, et al. Lung-residing myeloid-derived suppressors display dual functionality in murine pulmonary tuberculosis. Am J Respir Crit Care Med. 2014;190(9):1053–66. pmid:25275852
View Article
PubMed/NCBI
Google Scholar

[303] View Article

[304] PubMed/NCBI

[305] Google Scholar

[ref78] 78. Tobita T, Takeshita A, Kitamura K, Ohnishi K, Yanagi M, Hiraoka A, et al. Treatment with a new synthetic retinoid, Am80, of acute promyelocytic leukemia relapsed from complete remission induced by all-trans retinoic acid. Blood. 1997;90(3):967–73 pmid:9242525
View Article
PubMed/NCBI
Google Scholar

[307] View Article

[308] PubMed/NCBI

[309] Google Scholar

[ref79] 79. Watanabe H, Bi J, Murata R, Fujimura R, Nishida K, Imafuku T, et al. A synthetic retinoic acid receptor agonist Am80 ameliorates renal fibrosis via inducing the production of alpha-1-acid glycoprotein. Sci Rep. 2020;10(1):11424. pmid:32651445
View Article
PubMed/NCBI
Google Scholar

[311] View Article

[312] PubMed/NCBI

[313] Google Scholar

[ref80] 80. Boivin G, Faget J, Ancey PB, Gkasti A, Mussard J, Engblom C, et al. Durable and controlled depletion of neutrophils in mice. Nat Commun. 2020;11(1):2762. pmid:32488020
View Article
PubMed/NCBI
Google Scholar

[315] View Article

[316] PubMed/NCBI

[317] Google Scholar

[ref81] 81. Barber DL, Mayer-Barber KD, Feng CG, Sharpe AH, Sher A. CD4 T cells promote rather than control tuberculosis in the absence of PD-1-mediated inhibition. J Immunol. 2011;186(3):1598–607. pmid:21172867
View Article
PubMed/NCBI
Google Scholar

[319] View Article

[320] PubMed/NCBI

[321] Google Scholar

[ref82] 82. Ahmed M, Tezera LB, Elkington PT, Leslie AJ. The paradox of immune checkpoint inhibition re-activating tuberculosis. Eur Respir J. 2022;60(5). pmid:35595321
View Article
PubMed/NCBI
Google Scholar

[323] View Article

[324] PubMed/NCBI

[325] Google Scholar

[ref83] 83. Kim JH, Podstawka J, Lou Y, Li L, Lee EKS, Divangahi M, et al. Aged polymorphonuclear leukocytes cause fibrotic interstitial lung disease in the absence of regulation by B cells. Nat Immunol. 2018;19(2):192–201. pmid:29335647
View Article
PubMed/NCBI
Google Scholar

[327] View Article

[328] PubMed/NCBI

[329] Google Scholar

[ref84] 84. Podstawka J, Sinha S, Hiroki CH, Sarden N, Granton E, Labit E, et al. Marginating transitional B cells modulate neutrophils in the lung during inflammation and pneumonia. J Exp Med. 2021;218(9). pmid:34313733
View Article
PubMed/NCBI
Google Scholar

[331] View Article

[332] PubMed/NCBI

[333] Google Scholar

[ref85] 85. Shin SJ, Lee BS, Koh WJ, Manning EJ, Anklam K, Sreevatsan S, et al. Efficient differentiation of Mycobacterium avium complex species and subspecies by use of five-target multiplex PCR. J Clin Microbiol. 2010;48(11):4057–62. pmid:20810779
View Article
PubMed/NCBI
Google Scholar

[335] View Article

[336] PubMed/NCBI

[337] Google Scholar

[ref86] 86. Kim WS, Kim JS, Cha SB, Kim H, Kwon KW, Kim SJ, et al. Mycobacterium tuberculosis Rv3628 drives Th1-type T cell immunity via TLR2-mediated activation of dendritic cells and displays vaccine potential against the hyper-virulent Beijing K strain. Oncotarget. 2016;7(18):24962–82. pmid:27097115
View Article
PubMed/NCBI
Google Scholar

[339] View Article

[340] PubMed/NCBI

[341] Google Scholar

[ref87] 87. Kwon KW, Aceves-Sánchez MJ, Segura-Cerda CA, Choi E, Bielefeldt-Ohmann H, Shin SJ, et al. BCGΔBCG1419c increased memory CD8⁺ T cell-associated immunogenicity and mitigated pulmonary inflammation compared with BCG in a model of chronic tuberculosis. Sci Rep. 2022;12(1):15824. pmid:36138053
View Article
PubMed/NCBI
Google Scholar

[343] View Article

[344] PubMed/NCBI

[345] Google Scholar

[ref88] 88. Lee JM, Park J, Reed SG, Coler RN, Hong JJ, Kim LH, et al. Vaccination inducing durable and robust antigen-specific Th1/Th17 immune responses contributes to prophylactic protection against Mycobacterium avium infection but is ineffective as an adjunct to antibiotic treatment in chronic disease. Virulence. 2022;13(1):808–32. pmid:35499090
View Article
PubMed/NCBI
Google Scholar

[347] View Article

[348] PubMed/NCBI

[349] Google Scholar

[ref89] 89. Jeon BY, Kim SC, Eum SY, Cho SN. The immunity and protective effects of antigen 85A and heat-shock protein X against progressive tuberculosis. Microbes Infect. 2011;13(3):284–90. pmid:21093603
View Article
PubMed/NCBI
Google Scholar

[351] View Article

[352] PubMed/NCBI

[353] Google Scholar

[ref90] 90. Kwon KW, Kim LH, Kang SM, Lee JM, Choi E, Park J, et al. Host-directed anti-mycobacterial activity of colchicine, an anti-gout drug, via strengthened host innate resistance reinforced by the IL-1β/PGE(2) axis. Br J Pharmacol. 2022;179(15):3951–69. pmid:35301712
View Article
PubMed/NCBI
Google Scholar

[355] View Article

[356] PubMed/NCBI

[357] Google Scholar

Figures

Abstract

Author summary

Introduction

Results

Male Nox2-/- mice feature increased mycobacterial burden and lung hyperinflammation following aerosol infection with the Mtb K strain

Pulmonary infiltration of neutrophils and loss of B lymphocytes are correlated with TB pathogenesis of male Nox2-/- mice

Pulmonary neutrophils of Mtb infected male Nox2-/- mice are predominantly composed of aberrant CD11bintLy6GintCXCR2loCD62Llo permissive immature neutrophils

Neutrophil depletion alleviates TB pathogenesis and lung hyperinflammation in male Nox2-/- mice

Neutrophil depletion reduces immature lung neutrophils and restores pulmonary lymphocytes in Mtb infected male Nox2-/- mice

AM80 administration mitigates TB pathogenesis in male Nox2-/- mice and reduces pulmonary immature neutrophils

G-CSF predominantly regulates the generation of permissive immature pulmonary neutrophils in Mtb infected male Nox2-/- mice

Discussion

Materials and methods

Ethics statements and study approval

Mice

Bacterial culture and infection protocols

In vivo treatments for neutrophil control

Antibodies and flow cytometry

Preparation of single cell suspensions and immune cell analysis

Measurement of cytokines

Quantification of lung inflammation and mycobacterial CFU

Neutrophil purification and assessment of mycobacterial permissiveness

Statistical analyses

Supporting information

S1 Fig. The gating strategy for immune cell populations and the kinetics time-course data of pulmonary CFUs and neutrophils are provided.

S2 Fig. Pulmonary T cells of male Nox2-/- mice maintained strong responses against mycobacterial antigens.

S3 Fig. Alveolar macrophages, macrophages, and dendritic cells were reduced in the lungs of Mtb infected male Nox2-/- mice.

S4 Fig. TB severity featured strong correlation to increase of lung neutrophils and decrease of lung B cells.

S5 Fig. BM and lung neutrophil profiles of uninfected WT and Nox2-/- mice are illustrated.

S6 Fig. Immature neutrophils were the most permissive cells to mycobacterial infection among phagocytes infected with YFP+ Mtb.

S7 Fig. AM80 treatment did not significantly alter T cells responses against mycobacterial antigens.

S8 Fig. Neutralization of IFN-γ and IL-17A exacerbated TB pathogenesis of female Nox2-/- mice.

S9 Fig. Neutralization of IL-6, IL-1α, and IL-1β exacerbated TB pathogenesis of male Nox2-/- mice.

S10 Fig. Blockade of IL-1R exacerbated TB pathogenesis of Nox2-/- mice.

S11 Fig. Quantification method of lung inflammation is briefly described.

Acknowledgments

References

Male Nox2^-/- mice feature increased mycobacterial burden and lung hyperinflammation following aerosol infection with the Mtb K strain

Pulmonary infiltration of neutrophils and loss of B lymphocytes are correlated with TB pathogenesis of male Nox2^-/- mice

Pulmonary neutrophils of Mtb infected male Nox2^-/- mice are predominantly composed of aberrant CD11b^intLy6G^intCXCR2^loCD62L^lo permissive immature neutrophils

Neutrophil depletion alleviates TB pathogenesis and lung hyperinflammation in male Nox2^-/- mice

Neutrophil depletion reduces immature lung neutrophils and restores pulmonary lymphocytes in Mtb infected male Nox2^-/- mice

AM80 administration mitigates TB pathogenesis in male Nox2^-/- mice and reduces pulmonary immature neutrophils

G-CSF predominantly regulates the generation of permissive immature pulmonary neutrophils in Mtb infected male Nox2^-/- mice

S2 Fig. Pulmonary T cells of male Nox2^-/- mice maintained strong responses against mycobacterial antigens.

S3 Fig. Alveolar macrophages, macrophages, and dendritic cells were reduced in the lungs of Mtb infected male Nox2^-/- mice.

S5 Fig. BM and lung neutrophil profiles of uninfected WT and Nox2^-/- mice are illustrated.

S6 Fig. Immature neutrophils were the most permissive cells to mycobacterial infection among phagocytes infected with YFP⁺ Mtb.

S8 Fig. Neutralization of IFN-γ and IL-17A exacerbated TB pathogenesis of female Nox2^-/- mice.

S9 Fig. Neutralization of IL-6, IL-1α, and IL-1β exacerbated TB pathogenesis of male Nox2^-/- mice.

S10 Fig. Blockade of IL-1R exacerbated TB pathogenesis of Nox2^-/- mice.