https://orcid.org/0009-0009-9568-8998
Figures
Abstract
Tuberculosis (TB) kills an estimated 1.25 million people annually and is the leading cause of death in people with HIV (PWH) (1). The CD4+ T helper (Th) populations play significant roles in protective immunity to Mycobacterium tuberculosis (Mtb) and are essential hosts for HIV pathogenesis. Emerging evidence in blood and gastrointestinal mucosa of PWH suggests that, among Th cells, Th17 and Th22 may be preferentially depleted during HIV infection. Targeting of Th17 and Th22 cells by HIV could pose important and poorly understood risks for Mtb containment in those with co-infection. Mtb-driven activation of Th17 and Th22 immunity may also contribute to HIV proliferation and persistence. We employed a humanized mouse model of co-infection to assess changes in Th17 and Th22 frequency and function due to infection with HIV, Mtb, or both. In infected mice, Th17 cells were the predominant host for HIV in spleen and shown to be a source of HIV replication in pulmonary TB granulomas. Th17 cells were increased in lung of mice with TB or TB-HIV. Conversely, Th22 cells were reduced in mice with HIV or TB-HIV. Mtb infection increased the viral load in lungs of co-infected mice while HIV suppressed the pulmonary Th17 family cytokine response to Mtb including IL-6, IL-22, IL-23, and IL-1β. Differential transcriptome assessment demonstrated that HIV co-infection disrupted Th17 pathways activated by Mtb in lung. Overall, these results suggest that HIV may compromise Th22 immunity and exploit Th17 cells to promote viral pathogenesis in the setting of Mtb and HIV co-infection.
Author summary
Tuberculosis (TB), caused by the bacteria Mycobacterium tuberculosis (Mtb), is the world’s leading cause of death by infectious disease, as well as the leading cause of death in people with HIV. The cytokines interleukin (IL)-17 and IL-22 are chemical messengers that are essential for a protective immune response to Mtb infections and tissue repair following bacterial clearance, respectively. The CD4+ T cell subsets that are important sources of these cytokines, Th17 and Th22 cells, are also primary targets for HIV infection at mucosal surfaces like the gut. Our work reveals Th17 as important HIV viral reservoirs in organs during Mtb and HIV co-infection, exhibiting defective immune signals compared to Mtb single infection in the humanized mouse model. Th22 responses were also decreased by HIV as early as nine days after infection in mice with either HIV or Mtb-HIV infection. These results have potential applications for future TB therapeutic vaccine design and may implicate possible targets to augment TB therapy.
Citation: Martinez-Martinez YB, Huante MB, Naqvi KF, Shah MN, Lisinicchia JG, Files MA, et al. (2026) HIV impairs and exploits pulmonary Th17 and Th22 cell-mediated immune responses to Mycobacterium tuberculosis. PLoS Pathog 22(1): e1013897. https://doi.org/10.1371/journal.ppat.1013897
Editor: Jason M. Brenchley, NIH, NIAID, UNITED STATES OF AMERICA
Received: August 25, 2025; Accepted: January 13, 2026; Published: January 30, 2026
Copyright: © 2026 Martinez-Martinez 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: The original RNA-seq data from this study was uploaded to the GEO database (https://www.ncbi.nlm.nih.gov/geo/) with the accession number GSE314870.
Funding: This work was supported by the National Institutes of Health (NIH), National Institute for Allergy and Infectious Diseases (NIAID) R01AI147948 to J.J.E, NIAID T35AI078878 to M.N.S., and NIAID R25GM134990 to J.P. Fellowship support was provided by the Conacyt-I2T2-Contex in Mexico to Y.B.M.M., by the James W. McLaughlin Fellowship Fund to Y.B.M.M. and K.F.N and by the Sealy Institute for Vaccine Sciences Fellowship to M.A.F. The funders had no role in 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
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), kills 1.25 million people throughout the world every year, including 161,000 people with HIV (PWH) [1]. One-third of HIV deaths are due to TB, and the risk of developing active TB increases markedly in PWH, especially if latent TB is present [2]. Addressing health problems resulting from Mtb and HIV co-infection is challenging due to the limitations of experimental models and the poorly understood microbial synergy that exacerbates both diseases [3]. CD4 + T cells (T helper cells, Th) are critical in protection against TB [4,5] but are also the main replication targets for HIV. Th cells in the lung, especially lung interstitium, may be lost early during Mtb-HIV co-infection, even before systemic immune impairment, and residual impairments in Th cells persist despite anti-retroviral therapy [6].
The effects of HIV or SIV infection on the numbers and function of Th1 and Th2 subpopulations have been characterized in accessible human specimens and tissues of NHP (non-human primate) models, including Mtb co-infections [7,8]. In contrast, less is known about effects on other Th subpopulations such as Th17 and Th22 cells, especially in organs of co-infection, such as lung and spleen. Th17 cells promote inflammation and mediate protective immune responses to various bacterial and fungal pathogens [9–11]. These beneficial responses of Th17 occur mainly at mucosal barriers [12,13] through secretion of cytokines such as IL-17A, IL-17F, IL-22, and IL-21, and by neutrophil recruitment [14,15] in gut and lung mucosa [13,16]. Th22 cells secrete IL-22 but not IL-17. IL-22 targets epithelial cells, fibroblasts, and macrophages specifically involved in tissue repair, wound healing, and mucosal immunity [17]. Th17 are important in vaccine-induced protection against Mtb and strongly correlate with bacterial control in mouse and humans, while Th22 cells and their cytokines contribute to immunity through incompletely described mechanisms [18–25]. During active TB, both Th17 and Th22 are increased in lung but decreased in the blood [26–29].
Understanding the multifaceted effects of HIV on diverse T-cell populations is a challenge for vaccination and other host directed interventions. Simian Immunodeficiency Virus (SIV)-mediated reactivation of latent TB is associated with CD4 + T cell loss, changes in T cell cytokine responses, and CD4 + T cell loss-independent mechanisms in different NHP species [7,30,31]. During HIV mono-infection, lack of Th17 is linked to impairment in the Th17/Treg ratio [32] and disease progression [33], where memory Th17 (CD45RA-CCR6+) are preferentially infected and depleted [34–36]. This preferential depletion of Th17 among Th cells is due to higher levels of CD4, CCR5, and CXCR4 (receptors and co-receptors for HIV) and a lack of anti-HIV-RNases [37,38]. IL-22 is protective against HIV-induced gut epithelial damage [39]. However, both Th17 and Th22 are depleted in blood, sigmoid colon, jejunum, and colorectal mucosa following infection with HIV or SIV [39–42]. Mtb specific Th17 and Th22 cells, as well as IL-17 and IL-22 cytokines, are decreased in blood of Mtb-HIV co-infected subjects [43–46].
Collectively, observations from PWH and NHP models of SIV suggest that HIV may impair protective roles of Th17 and Th22 against Mtb in co-infections. However, experimental validation and extension to critical tissue sites of infection are limited to date. The human immune system (HIS) mouse model has gained acceptance due to the development of functional human immune compartments capable of reproducing important human immune responses to pathogens with restricted host range. Benefits of HIS mice compared to NHP models are the smaller animal size, the ability to use unmodified HIV strains, reduced regulatory burden, maintenance cost, and scalable sample size for statistical analysis [47]. HIS mouse models have demonstrated reliable aspects of human infection and disease progression in HIV and Mtb-HIV co-infection [48–51] as well as translational utility for assessment of host directed therapies in these diseases [52,53].
Here, using a cord-blood HIS infection model, we demonstrate that Th17 cells in tissues are the predominant host Th cell for HIV. Viral load was increased in mice with pulmonary TB compared to mice with HIV only, and RNAscope analysis identified cells co-expressing IL-17 and HIV gag transcripts in TB granulomas. Th17 cells remain elevated in lungs of mice with Mtb, or Mtb and HIV co-infection, while activation of Th1 cells was suppressed in co-infection. Ingenuity Pathway Analysis of bulk RNA-seq data from lungs of Mtb-infected mice predicts disruption of IL-17 signaling pathways due to HIV co-infection. We further demonstrate that HIV markedly suppressed Th22 responses in both HIV mono- or co-infected mice. Overall, these findings indicate that HIV could exploit Th17 cells to promote viral pathogenesis in the setting of Mtb-HIV co-infection, leading to an increased viral reservoir at important sites of immunity in the lung and further suppression of immunity mediated by events downstream of IL-17 receptor signaling pathways. Th22 protection against TB could be lost early during co-infection due to the generalized suppression of Th22 cells and IL-22 cytokine following HIV infection. Preventive and therapeutic interventions for TB should thus consider these effects of HIV on Th17 and Th22 responses (Fig 1) in Mtb-HIV co-infected individuals.
Graphical representation of Th17 and Th22 responses in the lung (left section) and spleen (right section) in the four conditions of each panel: uninfected (upper left), HIV (upper right), TB (lower left) and TB-HIV (lower right). In the lungs there is increased HIV viral load in TB-HIV compared to HIV, increased Th17 and IL-17A when there is infection with Mtb (TB and TB-HIV), and decreased Th22/IL-22 when there is HIV infection (HIV and TB-HIV). In spleen, Th17 are preferentially infected by HIV during HIV mono-infection, IL-17 cytokine is increased during TB, and Th22/IL-22 are impaired when there is HIV infection (HIV and TB-HIV groups). Created in BioRender. Martinez-martinez, Y. (2025) https://BioRender.com/0djmnlx.
Materials and methods
Ethics statement
All animal experiments were approved under protocol 1501001B, complying with the University of Texas Medical Branch (UTMB) Institutional Animal Care and Use Committee.
HIS mouse infection models
The NSG-hu-CD34 + HIS mouse model was used for both animal experiments (Figs 2A and 3A). NOD-SCID/γcnull female mice (strain 005557) were purchased from The Jackson Laboratory (JAX) following injection of with human CD34+ stem cells via an i.v. (intravenous) route and used at 36 weeks of age. For both experimental timepoints, human T-cell reconstitution was validated in peripheral blood via flow cytometry using anti-CD45 human-AmCyan, anti-CD45 mouse-PE, and anti-CD3 human-BUV395.
(A) Experimental design to generate HIS mice and assess HIV distribution among Th subpopulations and representative flow cytometry analysis. Created in BioRender. Martinez-martinez, Y. (2025) https://BioRender.com/0djmnlx. Immune-deficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were reconstituted with human immune cells by i.v. administration of cord blood CD34 + stem cells. After confirmation of human immune system reconstitution, mice were infected i.v. with 105 HIV-1 ADA TCID, and spleens collected after 14 days of infection for flow cytometry and histology analysis. Bottom: Representative examples of flow cytometry gating to assess HIV-FITC-A signal in non-infected (N.I., left) and HIV-infected (right) HIS mice. (B) Representative images of hematoxylin and eosin (H&E, top) and RNAscope-based detection of HIV gag (bottom, red substrate) in comparison between non-infected (left) and HIV-infected (right) spleen. (C-H) Preferential infection of Th17 and IL-17 + leukocytes in comparison to other cytokine producing cell subsets present in the spleen at 14 days post HIV infection. (C, F) Increase in intracellular signal for HIVp24 in leukocytes and CD3 + CD4 + T cells of HIV-infected animal spleen compared to background fluorescent signal in non-infected animals. (C-E) Leukocytes and (F-H) T helper (CD3 + CD4+) cell infection with HIV. (D, G) Similar proportions of cytokines (IL-4, IL-10, IL-17, IL-22 and IFNγ) are produced by total leukocytes and Th cells from spleen of uninfected and infected animals. (E, H) HIV infection bias towards IL-17 + leukocytes and Th17 in comparison to other cytokine-producing cells at day 14 p.i. n = 4/group. Differences between two groups were determined using a Student’s T-test while an ANOVA with the Benjamini FDR post-hoc analysis was used for analysis of data from three or more groups. Significance was considered for *p < 0.05 **p < 0.01.
(A) Experimental design to generate acute Mtb-HIV co-infection in HIS. After confirmation of human immune system reconstitution, mice were infected with 100 CFU Mtb HN878 via an aerosol route for a total of 30 days. At day 21 p.i., non- and Mtb-infected groups were further infected i.v. with HIV-1 ADA (105 TCID) to establish 4 groups: non-infected, HIV, TB, and HIV-TB. The study included N = 38 mice with n = 8-10/ group. Created in BioRender. Martinez-martinez, Y. (2025) https://BioRender.com/0djmnlx. (B, C) Representative images of lung (B) and spleen (C) tissue specimens showing granulomatous inflammation from Mtb-HIV co-infected tissues as visualized with hematoxylin and eosin (H&E) (D-E). Representative lung (D), or spleen (E) from Mtb-HIV co-infected HIS mice infected with Mtb bacilli, as visualized with Ziehl-Neelsen-based detection of AFB. Arrows indicate Mtb bacilli clusters. (F-I). HIV viral load as determined by ELISA-based detection of HIV p24 in lung (F), and spleen (H). HIV viral copies per gram of lung (G) and spleen (I) in mice infected with HIV or HIV-Mtb were determined by using RT-PCR. Differences between the two groups were determined by a Student’s T-test with significance considered for *p < 0.05, **p < 0.01, and ns = non-significant.
All animal and bacterial experiments were performed in Biosafety level 3 (BSL3) laboratory and animal facilities (ABSL3) approved by the Centers for Disease Control and Prevention. Experiments were conducted under established guidelines and protocols from the UTMB Department of Biosafety. Mtb strain HN878 (Beijing lineage) was cultured to mid-log phase for delivery of aerosol infection with 100 CFU/mouse as previously described [29]. HIV-ADA (NIH AIDS Reagent Program) was propagated in human PBMCs and concentrated by Lenti-virus concentrator (Origene, Cat. TR30025) for animal injections. Virus stock was quantified by GHOST cell infections, measuring the tissue culture infectious dose (TCID) by GFP quantification via flow cytometry, as described previously [54]. Virus stock was diluted with PBS to 1x106 TCID/mL, and further HIV infections were performed via tail vein injection with 100 µL containing 105 TCID/ mouse.
For the HIV mono-infection experiment (Fig 2A), mice were infected with HIV for 14 days, or non-infected, and humanely euthanized to collect spleen for histology and flow cytometry (n = 4 per group). For the acute Mtb-HIV co-infection (Fig 3A) mice were infected by the aerosol route with Mtb, or non-infected, as previously described [29]. On day 21, non- and Mtb-infected groups were further i.v. infected with HIV-1 (strain ADA) as described [53]. All mice were humanely euthanized at experimental day 30, establishing four experimental groups: non-infected, HIV (9 days), Mtb (30 days), and Mtb-HIV (30 days Mtb infection with HIV co-infection during the last 9 days) with 8–10 mice/group (N = 38). Lung, spleen, blood, and liver were collected for further assessment.
Assessment of cell populations by flow cytometry
The left lung lobe and one-third of the spleen were collected for flow cytometric analysis. Tissue was disrupted, supernatants were collected for cytokine measurements, and the cells were activated with human anti-CD3, anti-CD28, and GolgiStop (BD) to retain intracellular cytokines, as previously described [29]. Extracellular staining was performed with live/dead Near IR and antibodies specific to human markers: CD3-BUV395, CD4-PacBlue, CD45-AmCyan, and CD14-BV786. After fixation with Cytofix/Cytoperm, intracellular staining was performed with antibodies to human cytokines IFNγ-BV650, IL-10-AF594, IL-17A-PE, IL-22-APC, CD68-PerCP-Cy5.5, and to HIV-FITC. Fixation was performed with 4% ultrapure formaldehyde (FA) for 48 hours to ensure inactivation, followed by a change to 1% FA and acquisition of total cells on an LSR II (Fortessa) flow cytometer. Subsequent analysis was performed using FCS Express 6 software (de Novo, Inc).
Determination of viral burden
Quantification of HIV p24 capsid protein was used to estimate viral burden in lung and spleen extracellular supernatants, using an ELISA kit (Zeptometrix, Cat No. 0801111). To evaluate HIV RNA levels, the right inferior and post-caval lobes from lung, one-third of spleen, or a section of the left superior lobe from liver were collected. RNA extraction was performed using the Qiagen RNeasy mini kit (Cat. 74104), and the RNase-free DNase set (Cat. 79254), according to the manufacturer’s instructions. This RNA was used for assessment of viral copies by measuring the HIV gag mRNA per gram of tissue, detected by RT-PCR as previously described [55].
Measurement of human cytokines
Extracellular cytokine measurements were done following the collection of intracardiac blood and centrifugation at 3,000 rpm for 10 min. Supernatants from lungs and spleen were also assessed following tissue disaggregation and centrifugation at 350 × g for 5 min before cellular activation. Lung, blood, and spleen samples were collected in cryovials and frozen at -80°C for subsequent analysis. Frozen samples were inactivated by γ-irradiation on dry ice for biosafety as described [56] following approved UTMB biosafety protocols. The human cytokines from lungs and spleen were concentrated and incubated overnight to overcome a dilution challenge inherent to the tissue separation process for cellular analysis and potential for reduced sensitivity of detection in samples from xenochimeric mouse tissues. The six most representative animals from each group were selected, and the supernatants concentrated using the Pierce Protein Concentrator PES, 3K MWCO (Cat 88512). Human cytokines from plasma or concentrated lung and spleen samples were quantified using a Bio-rad multiplex ELISA, the Bio-Plex Pro Human Th17 cytokine assays (Catalog #: 171AA001M), following the manufacturer’s instructions, except for incubation of samples with beads overnight. IL-21 and IL-31 results from the kit were excluded, as they were below the detection limit.
Histopathology and RNAscope
The right superior and middle lobes from lung were inflated with and collected in 10% Neutral Buffered Formaldehyde (NBF) containers (No. 032–059) along with one-third of the spleen and the left superior lobe of the liver. After 48h and a formalin change, tissues were removed from BSL3 and sent to the Anatomic Pathology Laboratory at UTMB. Paraffin-embedded tissue sections were serially sectioned (5 µm) and stained at the same core facility with H&E and the Ziehl-Neelsen method to detect acid-fast bacilli (AFB).
Additional tissue sections were stained using in situ hybridization to detect HIV infection in Th17 cells or other T helpers surrounding the TB-granuloma. RNAscope 2.5 HD chromogenic-RED assay for single-plex HIV RNA detection, or RNAscope Multiplex Fluorescent reagent kit v2 assay (Advanced Cell Diagnostics, ACD Cat. 323100) were used following the manufacturer’s instructions. Targets detected were HIV RNA-gag-pol (probe 317691) for single-plex analysis. HIV RNA (non-gag-pol probe 317711-C2), human IL-17 RNA (probe 310931-C3), and human CD4 RNA (probe 605601) were detected for multiplex analysis. Imaging was later performed by the UTMB Optical Microscopy Core by using a Zeiss LSM 880 confocal microscope with Zen software, followed by use of the Fiji program.
Differential transcriptome analysis
High throughput RNA sequencing was performed for 24 HIS mouse lung samples from six replicates of four conditions (non-infected, Mtb, HIV, or Mtb-HIV). The samples selected for RNA-seq were from the same tissues used to assess cytokine levels and viral burden. RNA quality was assessed with an Agilent Bioanalyzer, and RIN (RNA Integrity Number) values ranged between 9.8 and 10. PolyA + RNA was purified from ~100 ng of total RNA. NEBNext Ultra II RNA library kit (New England Biolabs) was used to prepare the sequencing libraries following the manufacturer’s protocol. Libraries (twenty-four) were quantified by qPCR, pooled, and sequenced using a single-end 75 base run on a Next-Seq 550 High-output flow cell at the UTMB Next Generation Sequencing Core. Canonical Pathway studies and Th cell and cytokine pathway disruption analyses were performed by Ingenuity Pathway Analysis Software (IPA, Qiagen). A cut-off range of -1.2 to 1.2 fold change and a p ≤ 0.09 was set to allow detection of differential responses in an experimental system (HIS mouse) that is chimeric and associated with variability. The original RNA-seq data from this study was uploaded to the GEO database (https://www.ncbi.nlm.nih.gov/geo/) with the accession number GSE314870.
Statistical analysis
Statistical analysis and graphical presentations were developed using GraphPad Prism 10. software. Data is presented as the mean ± SEM. For comparison between two experimental groups, an unpaired T-test was used. One-way ANOVA was used for experiments with more than two groups, with post-hoc correction for multiple comparisons. The false discovery rate was controlled by using the two-stage test set-up method for Benjamini, Krieger, and Yekutieli. Significance was considered with a p-value <0.05, and a trend in significance was considered where p < 0.1.
Results
HIV preferentially infects Th17 cells in HIS mouse tissue
In PWH, peripheral Th17 appears to be a preferred Th cell target for HIV infection and may act as a viral reservoir in blood and colorectal mucosa [40,57,58]. To validate and extend these observations in the HIS mouse and determine the effects of Mtb co-infection, we assessed cellular distribution of HIV in Th populations using the spleen as a source of peripheral leukocytes. HIV infection was confirmed in the spleen at 14 days post-infection based on flow cytometric detection of intracellular HIV p24 protein (Figs 2A and S1B) and RNAscope-based detection of viral RNA (Fig 2B). HIV infection was detected in human CD45 + cells (leukocytes) as shown in Fig 2C. The percentage of leukocytes producing specific cytokines (IFN-γ, IL-4, IL-17, IL-22, and IL-10) did not significantly differ among non-infected and HIV-infected tissues (Fig 2D), indicating a lack of subset-specific depletion at this acute infection stage. A definitive bias for HIV infection of the IL-17 + leukocyte subset, however, was observed (Fig 2E). Analysis of the human Th subpopulations (CD3 + CD4+) demonstrated a pattern similar to human CD45 + cells with regard to HIV infection, cytokine production, and HIV infection bias among the cytokine-producing cells that generally define Th subpopulations (Fig 2F, 2G and 2H). These results demonstrate preferential infection of IL-17-producing leukocytes and Th17 cells by HIV in the spleen.
Co-infection with Mtb promotes an increase in pulmonary HIV replication
To demonstrate the clinical relevance of the model for HIV in the setting of established TB, HIS mice were infected with Mtb and subsequently co-infected with HIV. Before infections, reconstituted mice were distributed among four experimental groups normalized for human leukocyte reconstitution (S2 Fig). The average percentages of reconstitution in mice were 57% human among total leukocytes (S2A Fig) and 67% of human T cells among the human leukocytes (S2B Fig). Limitations of the HIS mouse that should be noted include fewer leukocytes overall compared to mice with intact murine immune system, reduced lymphoid tissue population, poor reconstitution of some human immune cell populations, and incomplete development of memory responses by lymphocytes [51].
Mice were infected with 100 CFU of Mtb HN878/mouse via an aerosol route, for 30 days. On day 21, non- and Mtb-infected groups were further infected with 105 TCID of HIV-1 ADA via an i.v. route to establish four groups: non-infected, HIV, TB, and TB-HIV, as described in Fig 3A. Assessment of tissue histology demonstrated development of organized granulomatous inflammation in lung, spleen (Fig 3B and 3C), and liver (S3A Fig). Abundant acid-fast bacilli (AFB) were detected throughout lesions and central areas of inflammation in lung, liver, and spleen (Figs 3D, 3E and S3B). Due to tissue constraints, enumeration of bacterial burden was not performed. Analysis of HIV infection of tissues by using ELISA and RT-PCR revealed greater HIVp24 protein in lung supernatants (Fig 3F) and increased viral RNA copies in lung of mice with Mtb co-infection (Fig 3G). The splenic viral burden was markedly increased compared to the lung in general (Fig 3F–3I) consistent with optimum HIV replication in lymphoid tissues. However, co-infection with Mtb did not result in significant changes to viral burden in the spleen and liver, although a trend (p = 0.05) towards increased viral transcription in spleen was observed (Figs 3I and S3C).
HIV co-infection promotes distinct shifts in pulmonary T cell and macrophage responses to Mtb infection
To determine the effect of acute HIV, Mtb, or HIV-Mtb infections on important immune populations in the lung and spleen, we performed flow cytometric analyses of single-cell suspensions from disrupted tissue. The representative flow cytometry gating is shown in S1A Fig. In the lung, there were fewer total human leukocytes in HIV and TB groups, compared to non-infected, and interestingly, to TB-HIV (Fig 4A). Among human lung leukocytes, there were fewer T cells as a percentage in the TB and TB-HIV, compared to HIV and non-infected groups. These effects in the TB and TB-HIV groups were independent of CD4 + T cell loss (Fig 4A), despite the increased viral load measured in the lung (Fig 3F and 3G). In contrast, a moderate and non-significant decrease in Th cells in the HIV-infected group corresponded with decreased leukocytes (Fig 4A). The proportional decrease in T cells was compensated by increased macrophages in the TB group. Interestingly, the percentage of macrophages in lung of the TB-HIV group remained similar to non-infected and HIV groups. This indicates that changes in another cell population may contribute to the shifts in the percentage of T cells in this group (Fig 4A). In contrast to the lung, the spleen did not present changes in the leukocyte populations, as observed in Fig 4B although HIV groups were generally, but non-significantly, reduced.
Comparison of lung (A) and spleen (B) immune populations in non-infected (N.I.), HIV, TB, and TB-HIV groups. Total leukocytes among viable singlet cells were selected based on detection of human CD45 as shown in S1 Fig. Among human CD45 + cells, T cells and macrophages were identified by surface expression of CD3 (Total T cells), CD3 and CD4 (Th cells) and CD68 + CD14lo (macrophages) and shown as % of human CD45 cells. Differences were determined by using one way ANOVA with the Benjamini FDR post-hoc used to determine differences among different groups. Significance was considered for *p < 0.05, **p < 0.01., ***p < 0.001, ****p < 0.0001.
The Th17 response to pulmonary TB differs from Th1 and Th22 in the setting of HIV co-infection
Th1, Th17, and Th22 are expanded in the lung during active TB in human and murine models [27–29]. Different responses have been described in the spleen and Th17 cells were shown to decrease while Th IL-10 + cells increased during active TB in a murine model [29]. To determine how HIV co-infection affects the Th populations (Th1, Th17, Th22, and ThIL10+) in lungs during Mtb-HIV co-infection, we assessed changes in functional Th subsets and the relative quantity of cytokine produced by flow cytometry and Bioplex analysis (Fig 5A–5F). As expected, Th1 percentages and extracellular IFNγ were increased in the TB group (Fig 5A), consistent with changes observed in human lung. However, expansion of Th1 due to Mtb infection is suppressed by HIV infection in accordance with Geldmacher’s findings in human subjects [59]. A similar decrease was observed for soluble IL-10 in the absence of changes in ThIL-10 + cells (S4F Fig). Interestingly, human Th17 cell and IL-17 cytokine increased in lungs of TB and remained elevated in the TB-HIV group (Fig 5C). In contrast, IL-22 cytokine was increased in the TB group while Th22 cells were similar in the TB and non-infected groups (Fig 5E). Both Th22 and IL-22 were suppressed by HIV in the mono- and co-infected groups (Fig 5E).
T helper subset comparison in lung (A, C, E) and spleen (B, D, F) between the four experimental groups: non-infected (N.I.), HIV, TB, and TB-HIV. For each organ (lung or spleen) subsection, the left panel represents the cell population, and the right panel shows the respective cytokine in tissue supernatant. Flow cytometry was used to identify Th subpopulations based on intracellular cytokine staining for IL-17, IL-22, and IFNγ, or IL-10 in viable CD45 + CD3 + CD4 + T cells using gating strategy shown in S1 Fig. Th22 cells were defined as those with an IL-22+ and IL-17- phenotype. Soluble cytokines from lung and spleen were measured by Bioplex and are depicted in pg/mL of organ supernatant. Significance was considered for *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 following one-way ANOVA and a Benjamini FDR correction for multiple comparisons.
Analysis of the spleen as a peripheral lymphoid compartment revealed a more variable and subdued response overall compared to the lung. Th17 cells were markedly more abundant in the spleen as a percentage of total T cells irrespective of infection status. No significant difference in Th1 cell or soluble IFNγ production was observed (Fig 5B). A unique observation in the spleen was the HIV-mediated suppression of IL-17 cytokine production in response to Mtb that occurred in the absence of changes in Th17 cells (Fig 5D). The suppressive effect of HIV on Th22 and IL-22 was more pronounced in the spleen compared to the lung Fig 5E and 5F and HIV mono-infection was also associated with increased ThIL-10 + cells in the spleen (S4G Fig). These outcomes may reflect the increased viral burden in spleen compared to lungs as we observed in Fig 3.
Cytokine determinants of Th17 and Th22 subset bias are differentially activated in Mtb and Mtb-HIV infections
To determine immune drivers of the unique subset activation and suppression in Mtb and HIV infections, we assessed a panel of cytokines and other soluble mediators (Bio-Plex Pro Human Th17 cytokine assay) that are key to Th17 and Th22 differentiation [17,60] across the lung, spleen, and blood compartments (Figs 6, S4 and S5). IL-1β and IL-6 were increased in response to Mtb infection across all compartments while a consistent suppressive effect of HIV co-infection was observed (Fig 6A). Of note, some effects were less robust (p = 0.05-0.07) due to animal variation that is inherent in the HIS model and can include some inflammation. In the lung, a consistent pattern of cytokine activation due to Mtb infection was observed nonetheless and included several significant differences (Figs 5, 6 and S4). Activation of IL-23 due to Mtb infection was markedly suppressed due to HIV co-infection in lung but was otherwise similar among treatment groups in the spleen and plasma (Fig 6C). The IL-17F response was similar to IL-1β and IL-6 across compartments except that plasma responses were low and did not differ among groups (Fig 6D). Mtb infection also increased IL-4 and the alarmins IL-33 and IL-25 (IL-17E) in the lung and IL-4 in the spleen in agreement with previous reports of increased Th2 during TB [29] (S4 Fig). However, HIV co-infection demonstrated moderate suppressive effects of the Mtb activation response in these cytokines (S4 Fig) although variability among HIS mice limited the determination of significance.
Comparison of extracellular human cytokines (pg/mL) from lung (left, n = 6/group), spleen (center, n = 6/group), or plasma (right, n = 8-10/group) between the four experimental groups: N.I., HIV, TB, and TB-HIV. (A) IL-1β, (B) IL-6, (C) IL-23, and (D) IL-17F differences due to infection or sample source were determined by using one way ANOVA with the Benjamini FDR corrections for multiple comparisons. Significance was considered for *p < 0.05, **p < 0.01, ***p < 0.001.
These cumulative results demonstrate that HIV infection disrupts cytokine networks whereby Th subpopulation bias and functional activation is regulated. The effects are most pronounced at the site of Mtb infection (i.e., lung) but are measurable in the periphery, suggesting a more potent response occurring in the lung that likely reflects the greater bacterial burden. Suppression of IL-1β by HIV (Fig 6A), along with the moderate suppression of TNF that reached significance in plasma (S4 and S5B Figs), could be especially significant with regard to reduction of Th22 populations in TB-HIV (Fig 5) given the key roles of these two cytokines for Th22 phenotype differentiation [61–63].
Th17, but not Th22, are important host cells for HIV in TB lung granulomas
Th17 are increased and protective during TB infection and are targets for TB vaccination efforts [64,65]. However, Mtb-specific Th17 responses are skewed by HIV in blood from PWH [66]. To determine if Th17 are preferentially infected in lung or spleen during Mtb-HIV co-infection we assessed differences in HIV infection of target cells. We observed that Th17 cells were uniquely resistant to HIV-mediated depletion in Mtb-infected HIS mouse lung, compared to Th1 and Th22 cells (Fig 5). Detection of intracellular p24 viral capsid protein by flow cytometry revealed a similar distribution of HIV among Th1, Th17, and Th-IL-10 + cells in the lung of mice infected with HIV (Fig 7A). Co-infection with Mtb did not significantly alter this pattern (Fig 7B). The marked bias toward Th17 as the preferential host for HIV in the spleen that we initially observed (Fig 2) was reproduced in this set of mice reconstituted with separate donor stem cells (Fig 7C). Following co-infection with Mtb, however, a highly variable distribution of p24 + cells that was similar among Th1, Th17, and ThIL-10 subsets was observed (Fig 7D). An especially interesting observation was the notable lack of detectable p24 in Th22 cells from the lung or spleen following HIV infection (Fig 7A and 7C). Co-infection with Mtb did not alter the susceptibility of Th22 cells to HIV infection in the lung or the spleen (Fig 7B and 7D).
(A-D) Percentage HIV-infected cells expressing IL-17, IL-22, IFNγ, or IL-10 among T helper (viable CD45 + CD3 + CD4 + singlets) lymphocytes, in lung (A-B) or spleen (C-D), during acute (9 days) HIV infection (orange, A, C) or TB-HIV co-infection (purple, B, D). *p < 0.05, **p < 0.01, ***p < 0.001 (One-Way ANOVA with Benjamini FDR correction). (E-H) Representative tissue analysis including H&E staining (F-G) and immunofluorescent RNAscope-stained images (E, H), processed through in situ hybridization to detect RNA of HIV (non-gag-pol) with OPAL 690 (purple), human IL-17 RNA paired with OPAL 620 (red), and human CD4 RNA with OPAL 520 (green). (E) Representative RNAscope image depicting possible combinations found in the lung TB-HIV granuloma. (F-G) H&E sections depicting the granulomatous areas selected for images in H. (H) High power images of Th17 cells in lung TB lesions (top) and infected by HIV in TB lesions of the TB-HIV group (bottom), assessed by RNAscope.
Since we had observed preferential infection of splenic Th17 by HIV (Fig 2), we examined whether Th17 are an important cellular niche for HIV pathogenesis in TB granulomas. We observed HIV-infected cells localized to TB granulomas (S6 Fig) as previously shown [67], as well as peribronchial areas and blood vessels in the lung during Mtb-HIV co-infection. As shown by using RNAscope multiplex immunofluorescent imaging (Fig 7E and 7H) HIV infects Th17 as well as other cells at sites of Mtb-driven inflammation in lung. Even though human IL-17A is increased in both TB and TB-HIV groups, IL-17 transcripts were more evident in the TB group by RNAscope multiplex immunofluorescent imaging (Fig 7H).
Differential transcriptome analysis reveals HIV-mediated disruption of pathways that regulate the Th subpopulation response to Mtb infection in the lung
In order to identify the different pathways affected during Mtb-HIV co-infection that may alter Th population bias and effector cytokines (Fig 6), we performed high throughput bulk RNA-seq of lung tissue (UTMB Molecular Genomics Core Facility) using six animals per experimental group. Differential transcription of human genes demonstrated both individual animal and experimental infection effects (S7A Fig). Ingenuity Pathway Analysis (IPA) of differentially expressed genes (S7B Fig) further identified the principal canonical pathways affected in the pertinent comparisons. Cytokine signaling, including several IL-17 pathways, were highly represented among the pathways disrupted due to HIV and TB (S7C Fig). In support of the differences in Th populations and related cytokines that we observed (Figs 5, 6, S4 and S5) IPA identified the pathogen-induced cytokine storm signaling pathway as the immune network most impacted by HIV, especially the pathways activated by Mtb (S7C Fig). The IL-17 and IL-6 signaling networks were shown to be disrupted individually in a pattern similar to the overall cytokine storm pathway. Predicted disruptions of Th17 and Th2 pathways by HIV infection also reflects the potential for transcriptional regulation of cytokines and other molecules that determine Th subset differentiation and activation (S7C Fig). HIV infection, compared to mock, was associated with increased interferon signaling that was absent in TB-HIV. Another interesting finding is the oxidative phosphorylation pathway, predicted to be decreased in almost all comparisons, except TB-HIV versus HIV. The Sirtuin Signaling Pathway presented the opposite effects, and predicted upregulation in TB-HIV versus TB and downregulation in TB-HIV versus HIV. Interestingly, the T cell receptor signaling pathway was downregulated in mono-infections but upregulated during co-infection.
Due to the prominent disruptions predicted in the cytokine, T cell, and IL-17 signaling networks, by IPA, an expanded analysis of genes contributing to these outcomes in TB versus TB-HIV was performed (Fig 8). In this focused comparison, IL-17 signaling in psoriasis and other IL-17 pathways contributed to five of the 20 pathways where the response to TB was most affected by HIV co-infection including predicted activation and mixed activation and inhibition profiles (Fig 8A). Genes primarily affected in the IL-17 pathways during TB were IL-17A, IL17RA, CXCL8, CXCL5, IL1B, CXCL3, and CCL2 (S8 Fig). Interestingly, a comparison of TB-HIV versus HIV revealed differential expression, primarily activation, of IL-13, RGS16, CSF2, MMP9, HSP90AA1, SRSF1, and TNFSF15 that was not observed in TB or HIV alone. Analysis of Th differentiation pathways between TB-HIV versus TB showed a mixed pattern of activation and inhibition of important receptors (e.g., CD28, TCR) or cytokines (e.g., IL-12, TGF-β), or transcription factors (e.g., GATA 3, RORC, FOXP3) that generally predict activation of Th1 and Th2 and inhibition of TH17 and Treg (Fig 8B). The directionality of these outcomes differs from the observed cellular and cytokine responses but supports the overall disruption of the pathways. Differential expression of the transcription factor associated with Th22 bias, the Aryl hydrocarbon Receptor (AhR) was not observed. The differences between the RNA-seq and the cellular or cytokine data are likely to due to timing, post-transcriptional regulation, and the complexity of the lung tissue compared to secreted proteins and isolated cellular populations.
(A) Principal 20 canonical pathways differentially altered in comparison of TB-HIV versus TB, demonstrating enrichment of five pathways related to IL-17. Blue color depicts a negative activation z-score, predicting downregulation of the pathway. Orange color predicts upregulation by activation z-scores. (B) Th differentiation during TB-HIV versus TB comparison. Red and green show an increased or decreased experimental measurement, respectively. Different orange shades show confidence in predicted activation, while blue shades demonstrate confidence in predicted inhibition. Predictions were calculated through Ingenuity Pathway Analysis.
Discussion
Our findings in an HIS mouse model of acute TB and HIV co-infection provide an important advancement in our understanding of how HIV perturbs and exploits different Th subpopulations in the setting of pulmonary TB. To date, HIV targeting of Th subsets is poorly defined in tissue compartments beyond the gut. Similarly, the impact of HIV on depletion and functional immune responses in those with important co-morbidities such as TB remain incompletely defined.
Our results (Fig 1) suggest that Th17 may be 1) an important and early target for HIV infection in the peripheral immune system, 2) a depletion-resistant niche for HIV replication at sites of Mtb-driven inflammation in the lung, and 3) an important mediator of effector function in lung that is compromised by HIV infection. Th22 cells, on the other hand, appear to be reduced due to HIV infection as associated with disruption of cytokine networks that may impair differentiation.
HIV increased in the lung during early Mtb-HIV co-infection compared to the HIV group, while the spleen and liver presented similar viral loads to the HIV mono-infected group. Th cells depletion due to HIV has been observed in other organs such as gut-associated lymphoid tissue in earlier stages before depletion in blood [68], in accordance with our results. The increased viral lung load that was observed in the absence of significant Th loss during co-infection could be due to the timing in our experiment and increased effector T cell recruitment to the lung by 21 days post Mtb infection, as observed in standard mouse models [69]. An increased Mtb burden due to HIV co-infection may promote a further increase in lung viral burden consistent with previous observations [49,51]. However, no differences in Mtb burden due to HIV co-infection were also reported using an infection strategy similar to the one described herein [70]. Bronchoalveolar lavage (BAL) Th cells of TB patients have been reported to express increased HIV co-receptor CCR5, which could lead to increased viral reservoirs and replication in the lung. Accordingly, CCR5 is downregulated by Mtb antigens in human splenic cells [71], which could limit additional HIV replication during co-infection. Nevertheless, extended infection periods or different conditions could promote increased replication of HIV in other organs due to Mtb co-infection [50].
Greater macrophage numbers and classical activation signaling pathways were observed during TB but not during TB-HIV. Antigen presentation by macrophages is essential for the control of mycobacterial growth [72]. Macrophages also play vital roles during HIV infection establishment and dissemination [73] and are known as HIV viral reservoirs in the absence of T cells [74]. Decreased macrophage percentages could compromise immunity to Mtb as a basis for previous observations that pulmonary bacterial load increased during co-infection [50]. The cause of decreased macrophages is unclear and may reflect fewer pro-attractant chemokines or non-apoptotic cell death given that HIV impairs macrophage apoptosis [75,76]. Our immunofluorescent results identified CD4-negative IL-17 producers infected with HIV in granulomas of Mtb-HIV co-infected lung. Macrophages can also produce IL-17 [77], suggesting that alveolar macrophages producing IL-17 could serve as potential viral reservoirs in the lung during co-infection. The localization of HIV to Th17 and other cells in the granuloma may also suppress immune activation at a key site for Mtb containment. It is important to note that, to the best of our knowledge, early entrance or localization of HIV in the lungs has not been reported due to tissue restrictions. Here, we were able to image the early HIV infection in the lung, as localized to TB granulomas in agreement with Foreman et al [67] in an NHP model as well as peribronchial areas of inflammation during Mtb-HIV co-infection.
HIV is known to functionally impair Mtb-specific Th-responses, even before the development of detectable impairment of systemic immunity [78,79]. Th cells in the gut mucosa are depleted during primary HIV infection, 2–4 weeks post-exposure in human subjects [68,80] and as soon as 7 days post-SIV infection in an NHP model [81]. Reduction of lung interstitial Th cells has been reported at four weeks post-HIV infection in animal models [6]. Loss of Th cells in TB granulomas is observed as early as 14 days post-SIV infection in NHPs and prior to depletion in blood [67]. Th17 are known to be preferentially depleted by HIV in human blood and different gastrointestinal regions [34–38,40]. However, preferential infection by HIV of Th cells in organs of Mtb-HIV co-infection, such as lungs and spleen, is not defined due to understandable constraints in human sample acquisition. In a HIS model of post-drug TB relapse due to HIV, levels of pulmonary IL-17 were previously shown to be suppressed [50]. Results of the current study demonstrate that Th17 are preferentially infected by HIV in the spleen, compared to other Th subsets.
Interestingly, Th17 are infected with HIV at similar frequencies to Th1 and ThIL-10 + cells in the lung regardless of whether animals also have Mtb infection. This was a surprising outcome since mice with co-infection displayed increased viral burden and greater Th17 percentages in the lungs, and harbored HIV in lung TB granulomas. In accordance with our results, Th17 from HIV-infected patients are not preferentially depleted in BAL [36]. T cells in BAL do not reflect lung interstitium [6] and the preferential infection of Th1Th17 by SIV in NHP granulomas was not observed in BAL, supporting a role for granuloma/localization-specific HIV responses [67]. Differences in microenvironment immune responses or other factors may contribute to these tissue-specific effects in lung and spleen. Another possible confounder for the lack of preferential infection of Th17 in the lung is the early stage of co-infection, which may not yet reflect preferential infection or depletion of specific Th cell subsets. A decrease in polyfunctional Th1Th17 cells (or Th1*) that was identified in Mtb-SIV co-infected granulomas [67] suggests the importance of assessments of polyfunctional cell populations as well as specific lung regions and different stages of infection.
Suppression of Th22 cells and IL-22 by HIV is an important observation relevant to the emerging role for IL-22 pathways in TB outcomes. Th22 cells are shown to increase in lung and blood following Mtb infection and have an important immune role during latent TB infection (LTBI) [29,41,44]. However, Mtb-specific Th22 cells are markedly reduced in the blood during co-infection, even more than Th1 and Th17 [44] and are depleted by HIV in blood of LTBI individuals [41]. Additionally, Th22 are depleted from blood and colorectal mucosa of SIV-mono-infected NHPs [40]. We expand on Makatsa’s and Bunjun’s co-infection studies to show that Th22 are not only depleted from blood during Mtb-HIV co-infection [41,44] but also in lung and spleen. This reduction was significant compared to other T-cell subsets in HIV mono- and co-infection and was observed very early in the co-infection process. Makatsa [44] offers a postulate that HIV may deplete Th22 as these cells present enhanced HIV permissiveness and higher viral co-receptor (CXCR4 and CCR5) expression. Another potential mechanism supported by our results is the reduction of cytokines that promote Th22 (such as IL-6, IL-23, IL-1β and TNF) that could impede differentiation of Th22 and Th17 subsets. Th22 are responsible for epithelial integrity and wound healing [17]. Therefore, in the context of granuloma pathology, the loss of Th22 due to HIV could increase immunopathogenesis and TB progression. Non-T-cell populations such as ILCs could rescue IL-22 production and maintain gut epithelial integrity during HIV infection, as demonstrated in the sigmoid colon [39,82]. Conversely, IL-22 production could not be stably rescued by other cells following CD4 + cell depletion in Mtb mono-infection [5]. Furthermore, IL-22 rescue has not been demonstrated in the lung or during co-infection.
Despite lack of preferential pulmonary infection in our experiments, Th17/IL-17 signaling pathways were well represented among the differential transcriptome in comparisons of Mtb-HIV versus TB. IL-17 signaling pathway genes (e.g., IL17RA, CXCL8 and CXCL5) and cytokine genes that regulate Th17/IL-17 bias (e.g., IL-6, IL-23, and IL-1β) were reduced during co-infection compared to TB alone. Due to the protective roles of IL-17 in TB [19,20,22–24], the loss of Th17-mediated IL-17 signaling is a previously underestimated risk for TB progression during Mtb-HIV co-infection, which potentially could not be rescued by other IL-17 producers, such as ILC3 [82], or CD8 T cells [5]. However, as our differential transcriptome results reflects total lung, we cannot assess if the loss of IL-17 signaling originates from IL-17 producers other than Th17, such as macrophages, γδ T cells, or neutrophils [77].
Among several interesting observations of IPA analyses was the predicted activation of the sirtuin pathway by TB-HIV versus TB. Sirtuins, histone deacetylases, play important roles in immune modulation during infections, such as T cell modulation, DNA repair, inflammation, aging, and responses to stress [83,84]. Interestingly, Sirtuin 1 and 7 activation, or inhibition of Sirt 2 lead to deacetylation of STAT3, reducing Th17 differentiation, IL-17 and IL-22 [85–89] which may inform our results of Th subpopulation pathway disruption. Activating Sirt1 and blocking Sirt2 may have exciting potential as host-directed therapies to treat Mtb-HIV co-infected people [90]. Further analysis of differential gene transcription revealed that the predicted activation of the sirtuin pathway is due to due to downregulation of genes that regulate mitochondrial ATP production (e.g., NADH dehydrogenase complex I) which limits sirtuin activation.
These comparative assessments that were performed across different Th populations and using an important opportunistic pathogen of those with HIV, also inform HIV viral reservoirs. The lungs serve as a critical HIV viral reservoir, and despite ART, support persisting infectious and non-infectious diseases [91]. Tissue reservoirs are an important reason why HIV infection remains incurable even with ART. Human lung parenchyma harbors an average of 6x109 lymphocytes as potential HIV viral reservoirs [92,93]. Th17 have been described as a long-lived HIV-viral reservoir in the gut [57]. Our results show that pulmonary Th17 cells were increased along with greater viral burden in the Mtb-HIV co-infected group, compared to HIV mono-infection, and frequently found to be infected with HIV in TB granulomas. Importantly, Th17 were the preferred Th target for HIV in the spleen. Together, these results could suggest Th17 as previously underestimated viral reservoirs that are increased in number during co-infection.
The TB vaccination field currently aims to boost Th17 as a protective TB response as reviewed by [18,64,65,94,95], with some vaccines achieving it [24,96–98]. Like other Th cells, Th17 cells have specificity for Mtb antigens [99], and are rapidly depleted by HIV [100]. Specifically, Th17 cells represent a predominant population with specificity to Mtb-antigens including PPD, ESAT-6/CFP-10 and Rv2031c which can be altered in HIV infection [43,101]. Furthermore, HIV infection can shift the Mtb-specific Th17IL-10 + “protective” responses to Th17Th1 “pathogenic” responses during Mtb-HIV co-infection [66]. Antigen specificity of Th cells was not determined in the current study, although our findings that Th17 are preferentially targeted by HIV in spleen and infected with HIV in lung granulomas suggest similar potential to alter the Mtb-specific Th responses. After boosting Th17 through vaccination, HIV could exploit an expanded population of Th17 memory cells to promote viral pathogenesis, leading to increased viral reservoirs, suppressed cytokine responses, and ultimately vaccine failure. This poses interesting new questions about the Th17 role during co-infection that could lead us to a potential paradigm shift in vaccination efforts, primarily in high-burden Mtb-HIV co-infection countries [1].
In conclusion, we identified targeting and impairment of Th17 and Th22 and defects in related cytokines in the lung and spleen due to HIV that could alter the antitubercular responses during co-infection. Additional studies are needed to understand Th17 infection by HIV in the lungs as it relates to viral reservoirs, functionality, and immune dysfunction in TB granulomas. The HIV-mediated loss of Th22/IL-22 during Mtb-HIV co-infection requires additional studies to understand the impact on immunity and regulation of immune-mediated pathology. Future interventions for HIV infection, including those focused on reservoir elimination, may consider the effects of HIV on Th22/IL-22 loss and the effects of Mtb co-infection on localized Th17 reservoirs. TB vaccination efforts should also consider the Th subpopulation depletion, infection bias, or functional defects relevant to use in Mtb-HIV co-infected populations.
Supporting information
S1 Fig. Representative flow cytometry gating strategy used for experiments.
(A) Gating used for selection of lung and spleen populations in both experiments. Sequential gating combinations of FSC-A, FSC-H, FSC-W, SSC-H, and SSC-W selected singlets. Among singlets (gate 3), Leukocytes were selected by size (FSC-A) and granularity (SSC-A). Viable (vital, negative for Live/dead Near IR) human CD45 + cells (leukocytes) were further gated for human macrophages (CD68 + CD14-). Among leukocytes, lymphocytes were selected for T cells (CD3+), and T helper (CD3 + CD4+) markers, which were additionally assessed for intracellular cytokines. In the example graphic, Th17 and Th22 are assessed by IL-17 and IL-22 intracellular markers, respectively, shown in the right panel of Fig A within a representative uninfected and infected mouse. (B) Representative gating used in spleen for identification of HIV in the different cytokine producer subsets in Fig 2E and 2H among spleen controls (N.I., non-infected) (top) and HIV-infected spleen samples at 14 dpi (bottom).
https://doi.org/10.1371/journal.ppat.1013897.s001
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S2 Fig. Reconstitution of human leukocytes in blood of HIS mice prior to initiation of co-infection experiment.
HIS mice were equally separated into four groups of 9–10 mice/group. Retroorbital blood was taken to assess distribution of human responses by flow cytometry before aerosol Mtb infection (or day 0) as in Fig 3A. (A) Reconstitution percentage, evaluated as % of human CD45+ (leukocyte) cells, among total leukocytes, using mouse and human CD45 markers. (B) Percentage of human T cells (CD3+), among the total human CD45 + leukocyte population. (C) Human CD45 leukocyte cell count. (D) Total human CD3 + cell count before infection among the four experimental groups: Non-infected, HIV, TB, and TB-HIV. No statistical difference was found before the infection (One-Way ANOVA with Benjamini FDR correction).
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S3 Fig. Liver histology and HIV infection in HIS mice.
(A) Representative H&E image of Mtb-infected liver, showing a granulomatous lesion. (B) Same histological area (arrow), with bacilli observed by AFB (magenta) staining. (C) HIV viral copies per gram of liver as detected by using RT-PCR. Ns = non-significant result based on Student’s T-test.
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S4 Fig. Soluble cytokines and ThIL-10 + cells in lung and spleen by infection status.
Supernatants were collected after cellular disaggregation for flow cytometry, frozen, and irradiated. After protein concentration, cytokines were quantified in supernatants as pg/mL by multiplex ELISA. In A-E we observe cytokines in lung (left panels) and spleen (right panels) including (A) IL-4, (B) IL-33, (C) soluble CD40 ligand, (D) IL-25 and (E) TNF in the four experimental groups: non-infected, HIV, TB, and TB-HIV groups. (F-G) Percentage of Th IL-10+ (IL-10 + IFNγ-)(left), or IL-10 cytokine production (right) in lung (F) or spleen (G). *p < 0.05, **p < 0.01, ***p < 0.001, (One-Way ANOVA with Benjamini FDR correction).
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S5 Fig. Human plasma cytokines produced during acute Mtb-HIV co-infection in the HIS mouse model.
Plasma samples were obtained after terminal intracardiac puncture, frozen, irradiated and cytokines were measured by multiplex ELISA. The different experimental groups are depicted with different color dots. From left to right, non-infected, HIV, TB, and TB-HIV. Plasma cytokines quantified in pg/mL were (A) IFNγ, (B) TNF, (C) IL-4, (D) IL-17A, (E) IL-25, (F) soluble CD40 ligand, (G) IL-22, (H) IL-10 and (I) IL-33. *p < 0.05, **p < 0.01, (One-Way ANOVA with Benjamini FDR correction).
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S6 Fig. HIV+ cells co-localize to TB granulomas and peribronchial areas of the lung in the HIS mouse model of co-infection.
(A) Organ scan overviews of lung in the TB-HIV group by H&E or (B) the respective tissue section RNAscope analysis , depicting HIV in cells surrounding the periphery of granulomas and the peribronchial areas of the lung by 9 days of HIV co-infection. In B, the representative immunofluorescent RNAscope image was stained through in situ hybridization, detecting RNA of HIV (non-gag-pol) with OPAL 690 (purple).
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S7 Fig. Human lung responses assessed by RNA sequencing demonstrates dysregulation of Th17/IL-17 pathways during Mtb-HIV co-infection in the HIS mouse model.
(A) Principal component analysis clusters the human lung responses according to infection in the HIS mouse model. Each dot represents an infected animal with HIV, Mtb, Mtb-HIV, or uninfected. (B) Heat map to observe the top 200 variable genes across all the samples, depicting transcriptome changes in the lungs. (C) Principal 50 canonical pathways affected by comparison of the four experimental groups, with arrows indicating IL-17 related pathways. The predictions were calculated through Ingenuity Pathway Analysis. Blue color depicts a negative activation z-score, predicting downregulation of the pathway. Orange color predicts upregulation by activation z-scores. n = 6/group.
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S8 Fig. Differential transcriptome analysis identifies specific changes in the IL-17 signaling pathway due to Mtb infection.
(A) Signaling pathway comparison of TB versus non-infected. Ingenuity Pathway Analysis predicts activation (orange) for most of the IL-17 signaling pathway due to observations of experimentally upregulated genes (red) in RNA-seq analysis.
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Acknowledgments
We thank the UTMB Flow Cytometry and Cell Sorting Core Facility, the Aerobiology Core Facility, and the Animal Resource Center for assistance in performing flow cytometry, aerosol infections with Mtb, and animal husbandry, respectively. We also thank the UTMB Optical Microscopy Core, the Molecular Genomics Core facility, and the UTMB Anatomical Pathology Laboratory for assisting in multifluorescent imaging, RNA sequencing, and histopathological services, respectively.
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