https://orcid.org/0000-0001-5925-0168
Figures
Abstract
Cryptococcal meningitis is a fatal complication. Macrophages have been proposed to function as candidate “Trojan horse” cells, transferring Cryptococcus neoformans (C. neoformans) into the brain. The mechanisms of Trojan horses in cryptococcal meningitis are largely elusive. In this study, we performed scRNA-Seq on immune cells infiltrating the brain in a murine model of cryptococcal meningitis. Bioinformatics analysis revealed that phosphodiesterase 4B (PDE4B) is a candidate regulator associated with C. neoformans infected-macrophage. C. neoformans increases the total level of PDE4B in macrophages. However, virulent strains with increased production of melanin paradoxically decreased PDE4B expression in macrophages, implying that PDE4B in macrophages may be negatively associated with C. neoformans invasion. PDE4B inhibition increased Arg1, CXCR4 and CCR7 expression in macrophages, a process regulated by the cAMP/PKA signaling pathway. As expected, PDE4B inhibitors promote the ability of C. neoformans infected-macrophages to cross the blood–brain barrier (BBB) in vitro. Similarly, PDE4B inhibitors or PDE4B knockout increase the fungal burden in the brain, which is, at least partially, rescued by macrophage depletion, and adoptive transfer experiments further support macrophage-mediated fungal delivery to the brain. In contrast, PDE4B activation reduces fungal burden in the brain, including when administered after infection onset. Overall, this study revealed that PDE4B functions as an important regulator of macrophage functional programming during infection and supports a macrophage-mediated dissemination mechanism contributing to brain invasion, and is a potential therapeutic target for cryptococcal meningitis.
Author summary
Cryptococcus neoformans (C. neoformans), a facultative intracellular fungus, is the pathogen of destructive cryptococcal meningoencephalitis. Macrophages have been proposed to function as candidate “Trojan horse” cells, transferring C. neoformans across the blood‒brain barrier (BBB), although definitive in vivo evidence remains limited. Using unbiased scRNA-seq profiling of brain immune cells from C. neoformans-infected mice, we revealed that PDE4B is a candidate regulator associated with macrophage differentiation and migratory responses upon C. neoformans brain infection. In vitro, PDE4B inhibitors or PDE4B knockout promote the crossing of the BBB by C. neoformans-infected macrophages. In vivo, PDE4B inhibitors or PDE4B knockout aggravate C. neoformans brain infection, which is at least partially reversed by macrophage depletion. And adoptive transfer experiments further support macrophage-mediated fungal delivery to the brain. In contrast, PDE4B activation reduces fungal burden in the brain, including when administered after infection onset. PDE4B therefore emerges as a potential host-directed therapeutic target for the prevention and treatment of cryptococcal meningoencephalitis. Considering that PDE4B inhibitors have been widely used in clinical practice, our findings highlight the need for caution in their use in patients at risk for cryptococcal infection.
Citation: Gong Y, Wang T, Chen X, Li Y, Ye S, Liu S, et al. (2026) PDE4B deficiency aids macrophage differentiation and contributes to Cryptococcus neoformans brain infection. PLoS Pathog 22(3): e1014040. https://doi.org/10.1371/journal.ppat.1014040
Editor: Haoping Liu, University of California Irvine, UNITED STATES OF AMERICA
Received: November 8, 2025; Accepted: February 26, 2026; Published: March 10, 2026
Copyright: © 2026 Gong 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: scRNA-Seq data is accessible to GEO accession GSE287392 with full open access. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE287392 All other data is provided in the manuscript and Supporting information files.
Funding: This research was supported by the National Natural Science Foundation of China (82171738 to MZ), the Project of Nanjing Medical University (TZKY20230201 to YY) and Taizhou Science and Technology Support Plan (Social Development, No. TSL202503 to YY). 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
In 2022, the World Health Organization (WHO) published its first-ever list of pathogenic fungi, ranking Cryptococcus neoformans (C. neoformans) as the top priority among critical pathogenic fungi. C. neoformans is responsible for approximately 110,000 deaths annually, accounting for 19% of AIDS-related mortality cases [1]. Once inhaled, C. neoformans may cause pulmonary infections in immunocompromised hosts. The pathogen then crosses the alveolar‒capillary barrier to enter the bloodstream, causing fungemia [2–4]. C. neoformans subsequently traverses the blood‒brain barrier (BBB) to infect the central nervous system (CNS) and proliferate in the brain parenchyma, eventually leading to fatal meningoencephalitis, with a mortality rate reaching 61% [5,6]. The potential mechanisms by which C. neoformans crosses the BBB are complicated [7–9]. Direct interactions between fungi and brain endothelial cells may lead to endocytosis and subsequent transcellular migration of free fungi [10–12]. In a few cases, the disruption of endothelial cell junctions within the BBB permits the paracellular migration of free fungi [13,14], which may be accompanied by damage to the brain vasculature and hemorrhage [15]. In the “Trojan horse” model, fungi are transported across the BBB within infected macrophages [16,17].
The relative importance of each pathway for C. neoformans infiltration into the brain parenchyma is still unknown. However, evidence has indicated that the primary route for C. neoformans entry into the CNS relies on phagocytic cells, which occur mainly in postcapillary venules. Clearance of circulating monocytes has been shown to reduce the fungal burden in the brain [17,18]. Therefore, current research predominantly focuses on the role of the “Trojan horse” in facilitating C. neoformans CNS infection, while acknowledging that multiple dissemination mechanisms may coexist in vivo. Although arginase 1 [19] and myocilin [20] may regulate macrophages in brain dissemination, it remains unclear whether these “Trojan horse cells” exhibit specific molecular or functional characteristics upon acquiring this transport function. Identifying these potential specific markers could provide a basis for developing targeted interventions, allowing for precise labeling and disruption of these infected monocyte-derived macrophages, thereby reducing or preventing the dissemination of C. neoformans to the CNS.
Previous studies have shown that various pathogens, i.e., Bordetella pertussis, Brucella, and Vibrio cholerae, interfere with macrophage immune functions by elevating host cell cAMP levels, thereby increasing their capacity for survival and dissemination within the host [21–23]. M2 macrophages are abundant in conditions such as helminth infections and allergic airway diseases such as asthma [24] and play crucial roles in tissue repair in response to certain infectious agents [25]. cAMP can promote M2 polarization of macrophages by activating the PKA signaling pathway, which increases the expression of Arg1 [26]. Phosphodiesterase 4 (PDE4) is a key cAMP-metabolizing enzyme found predominantly in inflammatory and immune cells [27] and plays a critical role in signal transduction by regulating intracellular cyclic nucleotide concentrations. In human and rodent brain tissue monocytes and macrophages, PDE4 exists in three forms, PDE4a, PDE4b, and PDE4d, with PDE4b playing a major role in the bidirectional regulation of inflammatory responses [28,29]. In macrophage models stimulated with prostaglandin or bacterial lipopolysaccharide (LPS), the inhibition of PDE4 promotes macrophage migration through cAMP signaling [30,31]. Similarly, in LPS-induced lung injury models, the inhibition of PDE4 significantly increases the number of reparative alveolar macrophages [32].
A phase II clinical trial with BI 1015550 (an oral PDE4B inhibitor) suggested that selective inhibition of PDE4B exerts combined antifibrotic and anti-inflammatory effects, potentially preventing lung function decline in patients with idiopathic pulmonary fibrosis [33]. In contrast, research by Sarah and colleagues indicated that increasing PDE4B might provide a cardioprotective benefit by limiting the cardiotoxic effects of chronic sympathetic nerve stimulation and heart failure progression in mice, potentially counteracting the adverse effects of sympathetic overactivation in heart failure [34]. The diverse roles of PDE4B across different diseases suggest that PDE4B may exhibit bidirectional regulatory properties depending on the pathological context. Although PDE4B has been extensively studied and shown to play a key role in immune modulation, fibrosis prevention, and cardiovascular disease, its specific role in macrophage-mediated dissemination of C. neoformans across the BBB remains unexplored.
In this study, we identified a significant association between PDE4B expression and macrophage differentiation states in the C. neoformans brain infection through single-cell RNA sequencing (scRNA-Seq) analysis. We observed that, with increased production of melanin, an essential virulence factor for brain invasion, C. neoformans paradoxically reduces PDE4B expression in macrophages. PDE4B inhibition promotes macrophage M2 polarization by modulating the cAMP/PKA signaling pathway, with an increase in the expression of the chemokine receptors CXCR4 and CCR7 [35–38]. This regulation enhances macrophage migration and invasion, thereby supporting a potential macrophage-mediated dissemination mechanism across the BBB. Furthermore, PDE4B is critical for C. neoformans CNS infection, as mice treated with PDE4B inhibitors or lacking PDE4B exhibited significantly exacerbated brain infections with C. neoformans. Given that C. neoformans CNS infection can be reversed by MR-L2 (a PDE4B agonist) [39,40], PDE4B represents a potential therapeutic target for treating cryptococcal meningoencephalitis and may contribute to the pathogenesis of C. neoformans brain infection through modulation of macrophage function.
Results
scRNA-Seq analysis of C. neoformans infected-macrophages
To elucidate the characteristics of candidate culprit cells during C. neoformans brain infection, we isolated CD45+ cells from the brain tissue of H99Tdtomato-infected mice and conducted single-cell RNA sequencing (scRNA-Seq) analysis (Fig 1A). Clustering analysis of the transcriptomic data, which was based on known marker genes and literature, followed by UMAP dimensionality reduction revealed that the infiltrating leukocytes in the brain tissue postinfection were predominantly composed of myeloid cells (monocytes/macrophages and neutrophils) and lymphoid cells (T/NK cells, B cells, etc.) (Fig 1B). To further identify the cell types associated with cryptococcal infection, we analyzed the distribution of fungal RNA across different cell types. The results indicated that fungal RNA was enriched primarily in cell populations expressing high levels of monocyte/macrophage-specific genes, including marker genes such as CD68, Adgre1, and Lyz2 (Fig 1C and 1D). Further confirmation through immunofluorescence staining demonstrated that CD45+F4/80+ cells in the brain tissue of infected mice could phagocytose fluorescently labeled fungi, whereas CD45+F4/80- cells did not exhibit this phenomenon. These findings suggest a close association between monocytes/macrophages and the early stages of fungal infection (Fig 1E), implying that monocytes/macrophages may be involved with C. neoformans brain infection. To distinguish whether the macrophages phagocytosing fungi were peripheral infiltrating macrophages or resident macrophages, we conducted flow cytometry analysis and found that among the F4/80+Tdtomato+ macrophages containing Cryptococcus neoformans, the peripheral infiltrating macrophages (CD45highCCR2+Ly6C+) constituted the majority, with fungi predominantly located in the infiltrating macrophages (Fig 1F).
(A) Schematic representation of the single-cell sequencing protocol. C. neoformans was administered via tail vein infection, followed by the preparation of a single-cell suspension from brain tissue. CD45+ cells were sorted by flow cytometry for scRNA-Seq analysis. Created in BioRender. Gong, Y. (2026) https://BioRender.com/w68y60i (B) Dimensionality reduction plot of infiltrating leukocytes in brain tissue. (C) Analysis of fungal RNA distribution across different cell types. (D) Dimensionality reduction analysis showing specific marker genes of cell populations enriched with fungal RNA. (E) Observation of fungi within macrophages in brain tissue cryosections. Macrophages: CD45+F4/80+ (magenta and green); Cryptococcus neoformans: Tdtomato (dark red); DAPI (blue). Original magnification: 63X, scale bar: 10 μm. (F) The statistical graph illustrates the percentage of infiltrating macrophages (CD45highCCR2+Ly6C+) and resident macrophages (CD45lowCCR2-Ly6C-) within F4/80+Tdtomato+ macrophages that have phagocytosed fungi. Data are presented as mean ± standard error of the mean (SEM). ****P < 0.0001; independent sample t-test.
To further explore the candidate genes potentially involved in macrophage responses during infection, we applied weighted gene coexpression network analysis (WGCNA) to classify genes based on their weighted correlation coefficients. This method grouped 14,680 sequenced genes into 23 modules based on expression patterns and analyzed the correlation between these modules and fungal RNA (CNAG index) (Fig 2A and 2B). The results indicated that the green module presented the highest correlation with the fungus. Core hub genes in the green module included Pde4b, Sema4d, Clec4e, and Gbp3 (Fig 2C). Among these core genes, PDE4B was selected as the preferred target for subsequent studies because of its ability to regulate macrophage function and immune responses [28–32]. We assessed the expression of Pde4b along the trajectory and examined its association with both CD44 expression, a putative critical molecule in C. neoformans brain infection [41] and CNAG scores. Cells exhibiting high CD44 levels—indicative of enhanced infiltration—tended to show elevated Pde4b expression, particularly in those with increased CNAG scores (Fig 2D).
(A) Weighted gene coexpression network analysis (WGCNA) plot. The genes obtained from sequencing were grouped into 23 modules based on their expression patterns. (B) Correlation analysis between fungal RNA and the modules identified in (A), showing the green module with the highest correlation to the fungus. (C) Gene display of the green module. (D) The expression of Pde4b was evaluated along the pseudotime trajectory, and its relationship with CD44 expression levels and CNAG scores was analyzed. (E) Flow cytometry analysis of PDE4B expression levels in macrophages from the brains of Cryptococcus-infected mice, comparing macrophages that phagocytosed fungi with those that did not. Bottom left: macrophages without fungal phagocytosis; top right: macrophages with fungal phagocytosis. Right: Bar graph showing the percentage distribution of different PDE4B expression levels between phagocytosing and non-phagocytosing macrophages. n = 3 per group. (F) Western blot analysis of CD44 expression in RAW264.7 cells treated with nerandomilast.
To validate the crucial role of PDE4B in cryptococcal infection, we analyzed the expression levels of PDE4B in macrophages within the brain tissue of mice infected with C. neoformans. Compared with macrophages without phagocytosis (F4/80+Tdtomato-), those with C. neoformans phagocytosis (F4/80+Tdtomato+) presented biphasic expression of PDE4B, while the major population was negative for PDE4B (Fig 2E). Treatment with Nerandomilast resulted in a significant increase in CD44 expression in macrophages (Fig 2F). Overall, PDE4B may be involved with fungal traversal across the blood‒brain barrier by mediating macrophage differentiation and migration.
C. neoformans regulates PDE4B expression in macrophages
Considering the potential synergistic effects of various subtypes of the PDE4 family in immune responses, we further investigated the changes in the expression of PDE4B following C. neoformans infection via qPCR to measure the expression levels of PDE4A, PDE4B, and PDE4D. The results indicated that the expression of both PDE4A and PDE4D decreased, whereas PDE4B expression was significantly upregulated (Fig 3A–3C). Western blot analysis corroborated these findings at the protein level and provided further validation in primary mouse bone marrow-derived macrophages (BMDMs) (Fig 3D and 3E), supporting the critical role of PDE4B in macrophage responses to fungal infections. Meanwhile, we added inert beads to the culture system and observed that PDE4B expression was largely unaffected, indicating that its expression level is not influenced by macrophage phagocytosis (S1E Fig). Additionally, flow cytometry analysis revealed that, compared to non-phagocytosing macrophages, the PDE4B expression level in phagocytosing BMDMs was significantly elevated (Fig 3F). Interestingly, when macrophages were stimulated with a noncapsulated strain (Cap59) or a heat-killed strain (HKC), the expression of PDE4B was similarly increased (Fig 3G and 3H). In contrast, stimulating BMDMs with a strain of Cryptococcus expressing high levels of melanin (Fig 3I), an essential virulence factor for brain infection [42], decreased PDE4B expression in macrophages (Fig 3J).
(A-C) qPCR validation of PDE4a, PDE4b, and PDE4d expression levels in BMDMs 6 hours post infection with C. neoformans (n = 4 per group). (D) Western blot analysis of PDE4B expression in RAW264.7 cells treated with H99 (5 × 106) (n = 4 per group). (E) Western blot analysis of PDE4B expression in BMDMs treated with H99 (5 × 106) (n = 4 per group). (F) The representative data and statistical plots of PDE4B expression levels in phagocytosing and non-phagocytosing BMDM cells detected by flow cytometry (n = 4 per group). (G) Western blot analysis of PDE4B expression in BMDMs treated with CAP59 (5 × 106) and H99 (5 × 106) (n = 3 per group). (H) Western blot analysis of PDE4B expression in BMDMs treated with heat-killed Cryptococcus (HKC; 5 × 106) or H99 (5 × 106) (n = 3 per group). (I) Diagram of colonies of H99 and the melanin-rich Cryptococcus strain (M-H99). (J) Western blot analysis of PDE4B expression in BMDMs treated with M-H99 (5 × 106) or H99 (5 × 106) (n = 4 per group). The data are presented as the mean ± SEM. **P < 0.01, ****P < 0.0001; unpaired Student’s t test. ns, not significant.
PDE4B regulates macrophage differentiation
Building on previous research findings, we further investigated the specific effects of PDE4B inhibition on macrophage differentiation and function under conditions of C. neoformans H99 infection. We hypothesized that PDE4B may exert its effects by influencing the polarization state of macrophages. To validate this hypothesis, we observed that following treatment with a PDE4 inhibitor (Rolipram) or a selective PDE4B inhibitor (Nerandomilast), the expression of Arg1 significantly increased, whereas the expression of NOS2 decreased (Fig 4A–4C). Further, we assessed the expression of macrophage-related chemokines following Cryptococcus infection. PDE4B inhibitors significantly upregulated the expression of CCR7 and CXCR4 in macrophages, accompanied by a decrease in CCR2 expression levels (Fig 4G, 4I, 4J, 4L and 4M), which was further confirmed via flow cytometry analysis (Fig 4F, 4H and 4K).
(A-C) qPCR validation of Arg1 and NOS2 expression levels in BMDMs treated with Rolipram or Nerandomilast for 24 hours (n = 3 ~ 6 per group). Rolipram: PDE4 inhibitor; Nerandomilast: PDE4B-specific inhibitor. (D) Representative flow cytometry gating strategy for Arg1+ macrophages in BMDMs treated with Rolipram (n = 3 per group). (E) Bar chart showing the percentage of Arg1+ cells within the macrophage population. (F) Representative flow cytometry gating strategy and corresponding quantification of F4/80+CCR2+ macrophages in BMDMs following Rolipram treatment (n = 6 per group). (G) qPCR validation of CCR2 expression in BMDMs treated with Nerandomilast for 24 hours (n = 6 per group). (H) Representative flow cytometry gating strategy and quantitative analysis of CCR7+ macrophages in BMDMs treated with Rolipram (n = 4 per group). (I.J) qPCR validation of CCR7 expression in BMDMs treated with Rolipram and Nerandomilast, respectively, for 24 hours (n = 6 per group). (K) Representative flow cytometry gating strategy and quantitative analysis of CXCR4+ macrophages in BMDMs following Rolipram treatment (n = 8 per group). (L.M) qPCR validation of CXCR4 expression in BMDMs treated with Rolipram or Nerandomilast, respectively, for 24 hours (n = 6 per group). (N‒P) qPCR analysis of Arg1, CCR7, and CXCR4 expression levels in BMDMs treated with Nerandomilast for 24 hours, followed by PKA inhibition (n = 3 per group). The data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired Student’s t test. ns, not significant.
Given that PDE4B is associated primarily with the cAMP‒PKA signaling pathway, we explored the mechanism by which PDE4B regulates macrophage signaling pathways [43,44]. We added a PKA inhibitor for treatment with the PDE4B inhibitor Nerandomilast and subsequently infected the macrophages with C. neoformans. The results indicated that under conditions of PKA inhibition following Nerandomilast treatment, the expression levels of Arg1, CCR7, and CXCR4 significantly decreased. Additionally, in the vehicle + PKA inhibitor group, there was a marked decrease in the expression of Arg1, CCR7, and CXCR4 (Fig 4N–4P). These data suggest that PDE4B inhibitors can modulate macrophage polarization and chemokine expression through the cAMP‒PKA pathway and that the PKA pathway also contributes to maintaining macrophage homeostasis under basal conditions.
PDE4B inhibitors increase C. neoformans invasion in vitro
To further investigate the impacts of PDE4B inhibition on macrophage function during C. neoformans infection, we initially treated the fungus with varying concentrations of the PDE4 inhibitor Rolipram to assess its antifungal activity directly (Fig 5A). These results indicated that Rolipram itself did not kill C. neoformans directly. Consistently, Nerandomilast treatment also failed to inhibit fungal growth in the absence of host cells (S2A Fig), indicating that PDE4 inhibition does not exert direct antifungal effects. Next, we evaluated the influence of PDE4 inhibitors on the phagocytic and killing abilities of macrophages. We found that while the fungicidal activity of macrophages did not significantly change, their ability to phagocytose C. neoformans was increased (Fig 5B, 5C). Additionally, we designed a Transwell experiment to simulate the in vitro blood‒brain barrier (BBB) (Fig 5D) [7]. Specifically, we extracted and cultured mouse bone marrow-derived macrophages (BMDMs) in vitro and treated them with Rolipram after maturation, followed by the addition of H99 to promote phagocytosis. After washing away the free fungi, we collected the macrophages that had phagocytosed the fungal cells and placed them in the upper chamber of the Transwell system. After 24 hours of incubation, we captured images of the fluorescent fungi that had traversed to the lower chamber and conducted statistical analyses, along with counting the fungal cells in the lower chamber. Confocal microscopy imaging revealed that the macrophages had phagocytosed C. neoformans (Fig 5E). Compared with the control group (vehicle), the Rolipram and Nerandomilast groups presented significantly greater numbers of fluorescently labeled C. neoformans crossing the BBB model to reach the lower chamber, with a corresponding notable increase in the fungal count in the lower chamber (Fig 5F–5K). Furthermore, we introduced a CXCR4 inhibitor to evaluate its effect on migration. The results indicated that in macrophages treated with Nerandomilast, the addition of the CXCR4 inhibitor led to a significant reduction in the number of fluorescent fungi that crossed the barrier (Fig 5L‒5M).
(A) Colony-forming unit (CFU) counts of fungi to evaluate fungicidal capacity after stimulating BMDMs with different concentrations of Rolipram (n = 6 per group). (B) CFU counts for assessing fungicidal activity after treating RAW264.7 cells with Rolipram (40 μM) for 3 hours, followed by agitation to mix the culture medium (n = 6 per group). (C) CFU counts for evaluating phagocytic capacity after RAW264.7 cells were treated with Rolipram (40 μM) for 3 hours, followed by washing to remove free fungi (n = 8 per group). (D) Schematic diagram of the in vitro BBB model. Created in BioRender. Gong, Y. (2026) https://BioRender.com/94t24xp (E) Representative confocal image of F4/80+ macrophages phagocytosing the fluorescent fungus H99Tdtomato. Original magnification: 200 × ; scale bar: 20 μm. (F-G) Representative confocal microscopy image showing free fungi that had invaded the lower chamber in the Transwell in vitro BBB model (n = 3 per group). Original magnification: 200 × ; scale bar: 20 μm. (H, K, N) Representative confocal microscopy images showing macrophages, subjected to different treatments, carrying H99Tdtomato into the lower chamber of the Transwell in vitro BBB model (n = 3-4 per group). Nerandomilast (100 μM), the CXCR4 inhibitor Plerixafor (AMD3100) 8HCl (10 μM) Original magnification: 200 × ; scale bar: 20 μm. (G, I, L, O) Quantitative analysis of H99Tdtomato fungi that invaded the lower chamber of the Transwell model based on confocal microscopy images (n = 4 per group; each data point represents 3–4 microscopic fields). (J, M) After treating BMDMs with Rolipram or Nerandomilast, the number of fungi that invaded the lower chamber of the Transwell system was counted and statistically analyzed. The data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001; ns, not significant. Group comparisons were made using 2-tailed Student’s t test, while comparisons among more than two groups were analyzed using one-way ANOVA..
PDE4B deficiency promotes C. neoformans dissemination into the brain
To verify whether the effects of PDE4B inhibition on fungal infections in the brains of mice align with our in vitro findings, we preinjected mice with Rolipram or Nerandomilast, followed by infection with C. neoformans H99 via the tail vein. After 48 hours of infection, we collected brain tissue homogenates for colony-forming unit (CFU) counting. The results revealed a significant increase in cryptococcal infection in the brain tissue of the inhibitor-treated groups (Fig 6A, 6B). We further introduced PDE4B-/- mice for comparison and found that, compared with that of WT BMDMs, the fungicidal activity of PDE4B-/- BMDMs remained unchanged, whereas their phagocytic ability was significantly enhanced, which is consistent with previous in vitro BMDM results (Fig 6C, 6D). In parallel, to assess intracellular fungal retention and survival in WT and PDE4B-KO BMDMs, we performed an additional time-course intracellular fungal load assay. WT and PDE4B-KO BMDMs were exposed to fluorescently labeled C. neoformans, followed by extensive washing to remove extracellular fungi. Intracellular fungal burden was quantified by fluorescence microscopy at 3 h and 24 h post-infection, calculated as the ratio of total internalized fungi to total cells per high-power field. PDE4B-KO macrophages displayed a significantly higher intracellular fungal burden than WT macrophages at both time points, consistent with their enhanced phagocytic capacity (S2B and S2C Fig). In an in vitro BBB model, we also observed that the migration and invasion ability of macrophages from PDE4B KO mice was significantly increased, further confirming that PDE4B gene deletion enhances the migratory and invasive capabilities of macrophages (Fig 6E, 6F). To explore the regulatory role of PDE4B in cryptococcal infection, we conducted in vivo experiments with PDE4B+/- and PDE4B-/- mice. Following infection with Cryptococcus, we observed a marked increase in CFUs in brain tissue, indicating that PDE4B plays a crucial role in controlling cryptococcal infections (Fig 6G, 6H). Additionally, we found that after infection with C. neoformans, the proportion of Arg1+ cells in macrophages phagocytosed fungi was significantly increased in PDE4B-/- macrophages, whereas the proportion of iNOS+ cells remained unchanged (Fig 6I, 6J). In addition to the cryptococcal fungemia model, we also established a natural infection mouse model via intranasal inoculation. After intranasal administration of 5 × 10⁴ C. neoformans, we observed that mice treated with the PDE4B inhibitor showed prolonged median survival time (S1A Fig). However, both the control and PDE4B-inhibited groups exhibited minimal fungal burden in the brain and spleen, and no significant difference in pulmonary fungal burden was detected (S1B–S1D Fig). This may primarily result from the local immune barriers in the lungs effectively restricting the dissemination of C. neoformans, whereas the fungemia model more effectively amplifies the regulatory effects of PDE4B. Next, we designed an in vivo macrophage depletion experiment. We first injected macrophage-depleting agents (CLs) into the tail veins of WT and PDE4B-/- mice. The results indicated that after macrophage depletion, cryptococcal infection in the brain tissue was significantly reduced, with nearly no fungal infection in the spleen (Fig 6K, 6L). This finding underscores the important role of macrophages in the dissemination of cryptococcal infections and the process of brain infection. To further confirm the significance of PDE4B in regulating macrophage behavior, we conducted a macrophage adoptive transfer experiment. After the depletion of macrophages from all the experimental mice, the control WT mice received a mixture of WT BMDMs that phagocytosed H99GFP and WT BMDMs that phagocytosed H99Tdtomato, whereas another group of WT mice was injected with a mixture of WT BMDMs that phagocytosed H99GFP and PDE4B-/- BMDMs that phagocytosed H99Tdtomato. After 48 hours, we analyzed the proportion of infiltrating cryptococci in the brain tissue of the mice (Fig 6M). The results revealed a significant increase in the proportion of H99Tdtomato in the brain tissue of the group injected with PDE4B-/- BMDMs, suggesting that macrophages deficient in PDE4B more readily crossed the blood‒brain barrier and transmitted cryptococci into the brain parenchyma (Fig 6N). Additionally, fungal burden in the spleen was evaluated by CFU counting after adoptive transfer, and no significant difference was observed in the peripheral circulation among the different macrophage transfer groups (Fig 6O). In conjunction with the earlier in vitro results, the inhibition of PDE4B enhanced the migratory capacity of macrophages, particularly against the backdrop of C. neoformans infection. These findings support the hypothesis of the “Trojan horse effect” of infected macrophages in the intracranial dissemination of C. neoformans, whereby PDE4B-/- macrophages may carry and release cryptococci by enhancing their ability to traverse the blood‒brain barrier, thereby exacerbating infection in brain tissue.
(A-B) Brain fungal burdens in mice intraperitoneally pretreated with the PDE4 inhibitors Rolipram or Nerandomilast, compared with vehicle-treated controls, following infection with Cryptococcus neoformans. Both PDE4 inhibitor–treated groups exhibited increased fungal loads in brain tissue (n = 6–8 per group). (C) Assessment of the fungicidal capacity of BMDMs from PDE4b-/- mice 3 hours poststimulation with H99, as measured by colony-forming unit (CFU) counting (n = 6 per group). (D) Phagocytic capacity of PDE4b-/- BMDMs after 3 hours of H99 stimulation, following the removal of free fungi (n = 6 per group). (E) Quantification of macrophages with internalized H99Tdtomato in the lower chamber of a Transwell system modeling trans-endothelial migration, as determined by confocal microscopy (n = 4 per group). Each data point represents the mean of 3–4 fields of view. (F) Representative confocal images of macrophages in the lower chamber of the Transwell-based in vitro blood–brain barrier (BBB) model (green: F4/80+ macrophages; red: H99Tdtomato; DAPI: nuclei). Original magnification: 200 × ; scale bar: 20 μm. (G, H) After tail vein infection with Cryptococcus neoformans in PDE4b+/- and PDE4b-/- mice, the fungal burden in brain tissue was increased (n = 5/group). (I-J) After infection with Cryptococcus neoformans, the proportions of Arg1+ and iNOS+ cells within macrophages phagocytosed fungi were detected by flow cytometry (n = 5/group). (K) Flow cytometry gating strategy for F4/80+CD11b+ macrophages. After intraperitoneal injection of clodronate liposomes to deplete macrophages, the mice were infected with H99 (5 × 105) via the tail vein 48 hours later. Single-cell suspensions from brain tissue effectively deplete circulating macrophages. (K-L) Brain and spleen fungal burdens in PDE4b ⁻ / ⁻ mice after macrophage depletion, showing significantly reduced fungal loads (n = 4 per group). (M) Schematic of the macrophage adoptive transfer procedure. Created in BioRender. Zhang, M. (2026) https://BioRender.com/kg1txyb (N) Quantification of differentially labeled H99 infiltrating brain tissue 48 hours after adoptive transfer, expressed as percentage distributions, according to the procedure shown in (N). Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparison test (n = 4 per group). (O) Peripheral fungal burden assessment in mice 48 hours after adoptive transfer, with spleens collected and fungal load quantified by CFU counting, according to the procedure described in (M). The data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired Student’s t test. ns, not significant.
PDE4B is a target for controlling C. neoformans dissemination into the brain
Deficiency or inhibition of PDE4B enhances the ability of macrophages to transport C. neoformans across the blood‒brain barrier, thereby exacerbating infection in brain tissue. To further investigate the potential of PDE4B as a therapeutic target for treating cryptococcal meningitis, we used the PDE4B agonist MR-L2 for validation [40]. We administered an intraperitoneal injection of MR-L2 to the mice, followed by a tail vein injection of C. neoformans for infection. After 48 hours, we collected brain tissue homogenates for CFU counting (Fig 7A). The results indicated that preactivation of PDE4B significantly reduced the fungal burden in the brain tissue of the mice, with markedly decreased CFU counts (Fig 7B). Moreover, activation of PDE4B effectively prolonged the survival time of the mice (Fig 7C). To further evaluate the translational relevance of PDE4B activation, we next examined whether MR-L2 remains effective when administered after infection has been established. Mice were first infected intravenously with C. neoformans, and MR-L2 treatment was initiated 24 h post-infection. Notably, delayed administration of MR-L2 still significantly reduced fungal burden in brain tissue compared with vehicle-treated controls (Fig 7D), indicating that PDE4B activation confers therapeutic benefit even after fungal dissemination has begun.
(A) A flow chart of the experimental design is shown in Fig B. Created in BioRender. Zhang, M. (2026) https://BioRender.com/xh7vk3d (B) Fungal burden in brain tissue was significantly reduced after in vivo activation of PDE4B with the agonist MR-L2 (n = 5/group). Comparisons between the two groups were performed using an independent samples t-test. The data are presented as the mean ± SEM. ***P < 0.001. (C) Survival statistics of the PDE4B agonist MR-L2 (n = 10 per group). Survival analysis was performed using Kaplan–Meier survival curves, and survival differences between groups were compared using the log-rank (Mantel–Cox) test. (D) MR-L2 treatment initiated 24 h after C. neoformans infection significantly reduced fungal burden in brain tissue (n = 5 per group). (E, F) qPCR validation of Arg1 and Nos2 expression in BMDMs treated with MR-L2 for 24 hours (n = 3 per group). (G) After BMDMs were treated with MR-L2 (40 μM) for 3 hours, free fungi were removed by washing, and colony-forming unit (CFU) counts were performed to assess phagocytic capacity. (n = 6 per group). (H) After BMDMs were treated with MR-L2 (40 μM) for 3 hours, the culture medium was gently resuspended by pipetting, and CFU counts were conducted to evaluate fungicidal activity (n = 6 per group). (I) Quantitative analysis of H99Tdtomato fungi invading to the underside of the Transwell membrane (lower chamber side) based on confocal microscopy images (n = 4 per group; each data point represents three microscopic fields).
To explore the cellular mechanisms underlying this protective effect, we performed in vitro experiments using BMDMs treated with MR-L2. qPCR analysis showed that MR-L2 selectively enhanced the expression of Nos2, whereas Arg1 expression was not significantly altered (Fig 7E and 7F), suggesting that PDE4B activation preferentially promotes an iNOS-associated antimicrobial program rather than alternative macrophage activation. We then assessed whether MR-L2 modulates macrophage phagocytic uptake or fungal clearance. CFU-based phagocytosis assays revealed that MR-L2 treatment did not significantly affect the initial uptake of C. neoformans by BMDMs (Fig 7G). In contrast, MR-L2–treated macrophages exhibited markedly enhanced fungicidal activity, as evidenced by significantly reduced CFU counts in fungal killing assays (Fig 7H). These results indicate that PDE4B activation primarily strengthens intracellular antifungal killing rather than altering fungal internalization. In parallel, Transwell migration assays demonstrated that MR-L2 significantly impaired the ability of macrophages to migrate across the membrane, resulting in fewer fungi invading the underside of the Transwell system (Fig 7I). This finding suggests that PDE4B activation limits macrophage migratory capacity, thereby restricting fungal transport across barrier-like structures. These findings indicate that activating PDE4B may serve as a novel strategy for treating cryptococcal meningitis.
Discussion
Cryptococcal meningitis is a leading cause of death among AIDS patients [45], with one of the critical steps in disease progression being fungal dissemination and traversal of the BBB to invade the brain [9]. Previous studies have demonstrated that the primary route for C. neoformans entry into the CNS depends on macrophages [18]. Macrophages can phagocytose or kill C. neoformans, yet as facultative intracellular pathogens, C. neoformans can survive and proliferate within macrophages. These infected macrophages may potentially contribute to fungal dissemination to the central nervous system and could represent candidate “Trojan cells”. Our study identified specific marker molecules associated with candidate culprit cells in the C. neoformans brain infection. Through immunofluorescence and flow cytometry analyses, we observed that infiltrating monocyte-derived macrophages in brain tissue were the predominant population harboring fungal cells, further supporting their potential role as candidate culprit horse cells during early CNS infection. In this study, we found that the inhibition or knockout of PDE4B significantly increased Cryptococcus infection in the CNS. Following Cryptococcus infection in macrophages, the total level of PDE4B expression is upregulated; in contrast, a highly virulent Cryptococcus strain with melanin expression has reduced PDE4B expression. PDE4B inhibition regulates Arg1 via the cAMP/PKA signaling pathway, inducing macrophage polarization toward the M2 phenotype and significantly increasing the expression of chemokines such as CXCR4 and CCR7, thereby increasing the migratory capacity of infected macrophages and promoting CNS infection. Consistent with these findings, PDE4B inhibition also enhanced the ability of macrophages to traverse an in vitro BBB model, resulting in increased fungal transport across barrier-like structures, which may contribute to dissemination into the brain. Further studies revealed that treatment with MR-L2 in mice significantly reduced Cryptococcus infection in brain tissue, suggesting that PDE4B has potential as a therapeutic target for cryptococcal meningitis.
Through single-cell RNA sequencing, we found that fungal RNA is enriched primarily in monocyte-derived macrophages. Further analysis revealed a close association between genes such as PDE4B and macrophages. Previous studies have shown that PDE4B is involved in multiple inflammation-related signaling pathways, including but not limited to the cAMP/PKA, NF-κB, and MAPK/ERK pathways, and plays crucial roles in regulating macrophage function and the immune response [43,46]. Thus, PDE4B has emerged as a primary target for subsequent research. Feature plots along the macrophage differentiation trajectory further demonstrated overlapping enrichment of Cd44, Pde4b, and fungal RNA signals, suggesting that PDE4B activity may be linked to infiltrating macrophage subsets with high fungal burden. Compared with those in the control group, macrophages that phagocytosed Cryptococcus exhibited significant changes in PDE4B expression levels, with a marked reduction in the cell population with low PDE4B expression, which shifted predominantly toward negative or high PDE4B expression. This biphasic pattern may reflect heterogeneity among infected macrophages, indicating that distinct subsets may undergo different functional transitions upon fungal uptake. This phenomenon may indicate that a subset of macrophages, by upregulating PDE4B, shifts toward an anti-inflammatory state, i.e., M2 polarization, suppressing inflammatory responses to help the host manage tissue damage. However, this shift may also weaken their antimicrobial capabilities, allowing fungal survival within cells. Another subset of macrophages appears to lose PDE4B expression entirely (negative), likely owing to fungal manipulation, thereby losing the ability to regulate cAMP signaling and maintain a normal proinflammatory response. As a result, these phagocytes are no longer able to effectively clear pathogens, permitting fungal survival within the host. C. neoformans possesses a unique immune evasion mechanism, enabling it to survive within host macrophages and suppress their function. Changes in PDE4B expression may be part of this evasion strategy.
Previous studies have reported that PDE4B expression is significantly upregulated in macrophages following LPS stimulation, promoting an inflammatory response to perform immune defense functions [47]. This finding aligns with our finding that Cryptococcus infection also leads to a marked increase in PDE4B expression in macrophages. Additionally, the primary virulence factors of C. neoformans, i.e., the polysaccharide capsule, melanin, and secreted extracellular enzymes, not only support its pathogenic mechanisms but also play a role in modulating the host immune response [48]. Notably, Cryptococcus strains with high melanin expression stimulate macrophages, resulting in the downregulation of PDE4B, a phenomenon that is consistent with the immunosuppressive role of melanin in pathogens. Importantly, stimulation with noncapsulated or heat-killed strains also increased PDE4B expression, indicating that PDE4B induction may represent a broader innate response to cryptococcal components rather than depending strictly on fungal viability or capsule status. This downregulation may weaken macrophage activity or proinflammatory responses by inhibiting the cAMP signaling pathway, thus promoting fungal survival and immune evasion [49–51] and ultimately exacerbating infection.
Herein, we hypothesize that PDE4B may play a critical role in Cryptococcus CNS infection. Inhibition of PDE4B significantly upregulates Arg1 expression in macrophages, inducing their differentiation toward the M2 phenotype [52,53]. M2-type macrophages typically exhibit increased phagocytic activity and migratory capacity [54] and are essential in tissue repair and anti-inflammatory processes, helping maintain immune homeostasis and protecting neural tissue from damage due to excessive immune responses. In vitro assays further showed that PDE4B inhibition increased macrophage phagocytic uptake of C. neoformans, although fungicidal activity remained largely unchanged, suggesting that infected macrophages may serve as reservoirs facilitating dissemination. Additionally, PDE4B inhibition is associated with downregulation of CCR2 expression, indicating a reduction in proinflammatory responses and a shift of macrophages toward an anti-inflammatory phenotype [55]Notably, in this study, the expression of macrophage-associated chemokines such as CXCR4 and CCR7 increased significantly. Previous studies have indicated that CCR7 is associated primarily with M1 macrophages in proinflammatory environments, promoting immune cell migration to lymph nodes or sites of inflammation [56]. However, research by Zengxu Wang and colleagues suggested that CCR7 can also enhance M2 macrophage polarization, thereby increasing M2 macrophage migration and invasion capabilities [38]. The role of CXCR4 in regulating macrophage migration to hypoxic or damaged tissues has been widely studied, especially in CNS-related diseases and inflammatory responses; the CXCR4‒CXCL12 axis is crucial for cell migration across barriers [57–59]. Future studies will further explore the role of CXCR4 in PDE4B-regulated fungal CNS infection. Additionally, migration assays demonstrated a significant increase in the migratory and invasive abilities of macrophages following PDE4B inhibition. Therefore, we hypothesize that inhibiting PDE4B indirectly promotes macrophage migration and invasion, a process regulated by the cAMP/PKA signaling pathway.
In this study, compared with that in the control group, Cryptococcus infection in brain tissue was significantly increased in PDE4B KO mice as well as in mice treated with a PDE4 inhibitor or a selective PDE4B inhibitor, demonstrating consistent experimental stability. In further exploratory experiments, we observed that predepletion of peripheral monocyte-derived macrophages with liposomes significantly reduced CNS infection levels in PDE4B knockout mice, confirming the critical role of macrophages in the infection process. Furthermore, adoptive transfer experiments revealed that PDE4B-deficient macrophages more readily crossed the BBB and transmitted cryptococci into the brain parenchyma, supporting the Trojan horse model of macrophage-mediated dissemination. Additionally, in the macrophage adoptive transfer experiments, the proportion of H99Tdtomato phagocytosed in the brain was significantly greater in the mice that received PDE4B-/- BMDMs than in the control mice. These findings suggest that PDE4B-/- macrophages exhibit enhanced migratory and invasive capabilities. Furthermore, we found that treatment with a PDE4B agonist could reverse the level of Cryptococcus infection in the brain. In conclusion, this study demonstrated the central role of macrophages in PDE4B-regulated Cryptococcus infection. Although only a minority of brain-infiltrating F4/80 ⁺ macrophages were found to contain tdTomato⁺ C. neoformans at the analyzed time point, this observation does not preclude a meaningful contribution to CNS dissemination. The Trojan horse model does not require that the majority of fungal cells remain intracellular at steady state. Rather, transient carriage by a relatively small subset of infected monocyte/macrophage-lineage cells may be sufficient to facilitate fungal transit across the blood–brain barrier. Importantly, our interpretation is not based solely on co-localization analyses. Functional depletion of monocyte–macrophage lineage cells markedly reduced brain fungal burden, and adoptive transfer experiments demonstrated PDE4B ⁻ / ⁻ infected-macrophages preferentially delivered cryptococci into the brain parenchyma. Together, these findings support a contributory role for macrophage-mediated transport, while not excluding the possibility that extracellular dissemination or parallel invasion mechanisms also operate in vivo. Future intravital imaging approaches will be required to directly visualize the dynamics of fungal trafficking across the BBB.
This study has several limitations. First, while we observed that the Cryptococcus virulence factor melanin reduces PDE4B expression in macrophages, we did not investigate the specific mechanisms by which melanin regulates PDE4B. Melanin may hinder the capsule growth in the C. neoformans [60]. The individual and combined roles of different virulence factors of C. neoformans in the regulation of PDE4B warrant further research. Additionally, while our data support macrophage-mediated BBB traversal in vitro and in vivo, the precise identity of Trojan horse subpopulations during CNS infection remains to be fully defined. In the macrophage adoptive transfer experiments, we inferred the migratory behavior of different macrophage types on the basis of the proportion of differently fluorescently labeled Cryptococcus in brain tissue. However, owing to the lack of separate labeling for wild-type and PDE4B-/- macrophages, coupled with the complexity of Cryptococcus transmission, it was not possible to definitively determine the specific origin of macrophages. Furthermore, we did not validate the specific role of PDE4B-deficient macrophages in Cryptococcus CNS infection in macrophage-specific PDE4B knockout mice, nor did we identify an appropriate PDE4B agonist capable of significantly reducing or clearing brain tissue infection. The role of PDE4B in regulating macrophages requires further investigation. PDE4B deficiency may regulate macrophage differentiation and contributes to the brain infection. However, the role of infected macrophages as candidate “Trojan horse” cells in C. neoformans brain infection remains to be further defined.
Overall, this study demonstrated that PDE4B plays a critical role in C. neoformans CNS infection, likely through its involvement in macrophage differentiation and the upregulation of associated chemokines. Our findings provide valuable insights into the mechanisms of fungal‒host interactions and may offer new directions for therapeutic strategies targeting cryptococcal meningitis, while also identifying candidate genes potentially involved in macrophage responses during infection.
Materials and methods
Ethics statement
Wild-type (WT) C57BL/6J mice were obtained from the Experimental Animal Center of Nanjing Medical University (Nanjing, China). PDE4b-/- knockout mice on a C57BL/6 background were purchased from Cyagen Biosciences (Suzhou, China). All animal experiments in this study were conducted following the guidelines of the Institutional Animal Care and Use Committee of Nanjing Medical University (Nanjing, China, Approval Number: IACUC-2007017). The mice used in this study were all 6–8 weeks of age.
C. neoformans strains and growth media
The C. neoformans strain H99 used in this study was obtained from ATCC (208821, ATCC). For use, the strain was retrieved from liquid nitrogen, plated on Sabouraud dextrose agar (SDA, 210950; Becton Dickinson), and incubated overnight at 30°C to yield single colonies. A single colony was then transferred to Sabouraud dextrose broth (SDB, 238230; Becton Dickinson) and incubated in a shaker (200 rpm) at 30°C for 16–22 hours until it reached the stationary phase, after which it was harvested or transferred to experimental-specific media for further analysis. For both in vivo and in vitro experiments, yeast cells were washed twice with sterile phosphate-buffered saline (PBS) and diluted to the required concentration. Caffeic acid agar (CAA, M4205C, Tuopu Biotechnology) supplemented with L-DOPA (HY-N0304, MCE) and ferric citrate (F332743, FELIX) was used to culture Cryptococcus strains with high melanin expression (M-H99) [61]. The culture conditions for the acapsular strain CAP59 were identical to those for the H99 strain, and heat-killed HKC cells were prepared by inactivating the H99 strain at 50°C overnight [10].
Cryptococcal infection model and fungal burden assessment
Six- to eight-week-old female WT or Pde4b KO mice were administered an intraperitoneal injection of the PDE4 inhibitor Rolipram (5 mg/kg, HY-16900, MCE), the PDE4b inhibitor Nerandomilast (5 mg/kg, HY-153192, MCE), or the agonist MR-L2 (2.5 mg/kg, HY-128358, MCE). After 24 hours, 5 × 105 C. neoformans cells were resuspended in 100 μL of PBS and injected via the tail vein. Following infection for 48 hours, the mice were euthanized and placed in the supine position, with the thoracic cavity exposed. Approximately 20 mL of sterile saline solution (0.9% NaCl) was perfused through the heart until the effluent was clear to remove blood. The brain or spleen tissue was then harvested and homogenized in 1 mL of sterile PBS (60 Hz, 120 s). The tissue homogenates were serially diluted (1:10, 1:100, 1:1000), and 10 μL of each dilution was spread onto SDA agar plates, with six replicates per sample. The plates were incubated at 30°C for 36–48 hours until distinct colonies formed, after which colony-forming units (CFUs) were counted on plates with appropriate dilutions.
Cell preparation for single-cell RNA sequencing
Six- to eight-week-old WT mice were injected via the tail vein with C. neoformans (H99Tdtomato, expressing the fluorescent protein Tdtomato) to simulate fungal bloodstream infection. Six hours postinfection, the mice were euthanized, and cardiac perfusion with saline was performed to thoroughly remove free cells from the vasculature. The brain tissue was then harvested to prepare a single-cell suspension. Flow cytometry was used to sort CD45+ leukocytes for subsequent single-cell sequencing.
Single-cell RNA sequencing
The cell suspension was injected into the SCOPE-chip microfluidic chip. On the basis of the principle of “Poisson distribution”, single cells randomly settle into prefabricated microwells within the chip under the influence of gravity, ensuring that each microwell captures only one cell. Millions of barcoding beads carrying unique cell barcodes and molecular tags (UMIs) are added to the microwells. Through optimized design, it is ensured that each microwell contains only one bead coexisting with a single cell. Cell lysis is performed within the chip, releasing mRNA that binds to oligo(dT) sequences on the beads through its poly(A) tail, achieving mRNA capture. Simultaneously, each cell and its mRNA molecules are labeled with unique cell barcodes and UMIs. The beads containing the captured mRNA were collected, and the mRNA was reverse transcribed into cDNA, followed by amplification of the cDNA. The amplified cDNA is then fragmented, and adapter sequences are added to construct a sequencing library compatible with the Illumina sequencing platform. The constructed sequencing library was loaded onto the Illumina sequencing platform for high-throughput sequencing, ultimately obtaining gene expression data at the single-cell level.
Bio-informatics analysis
To investigate the molecular mechanisms underlying macrophage phagocytosis of fungal pathogens, we applied Monocle2’s plot_cell_trajectory function to single-cell RNA-seq data. We quantified fungal uptake by macrophages using a CNAG gene score, A composite score representing the abundance of fungal-derived transcripts within host cells.
We assessed the expression of Pde4b along the trajectory and examined its association with both CD44 expression and CNAG scores. Cells exhibiting high CD44 levels—indicative of enhanced infiltration—tended to show elevated Pde4b expression, particularly in those with increased CNAG scores. This suggests a potential link between Pde4b activity, macrophage infiltration, and fungal phagocytosis.
Cell culture and stimulation
RAW264.7 cells (CBP60533, ATCC), a murine macrophage leukemia cell line, were cultured in complete DMEM (HyClone, USA) supplemented with 10% fetal bovine serum (FBS, Lonsera, USA) and 1% penicillin/streptomycin (HyClone, USA).
THP-1 cells (BNCC358410, BeNa Culture Collection), a human acute monocytic leukemia cell line, were cultured in complete RPMI 1640 medium supplemented with β-mercaptoethanol (BNCC354257, BeNa Culture Collection). Prior to use, these cells were treated with 100 nM phorbol 12-myristate 13-acetate (PMA) for 48 hours to induce differentiation.
bEnd.3 cells (CBP61233, ATCC), a murine brain microvascular endothelial cell line, were cultured in complete DMEM (HyClone, USA) supplemented with 10% fetal bovine serum (FBS, Lonsera, USA) and 1% penicillin/streptomycin (HyClone, USA).
All the cell lines were cultured in a humidified incubator at 37°C with 5% CO₂. After overnight seeding in 24-well plates, the following treatments were added to the culture medium in different experiments: H99 (1 × 106), MH99 (1 × 106), CAP59 (1 × 106), HKC (1 × 106), Rolipram (40 μM, HY-16900, MCE), Nerandomilast (100 μM, HY-153192, MCE), the PKA inhibitor H 89 2HCl (30 μM, S1582, Selleck), the CXCR4 inhibitor Plerixafor (AMD3100) 8HCl (10 μM, S3013, Selleck), and GP120 (HY-P70907, MCE). The cells were collected for further analysis after treatment for 6--48 hours.
BMDM culture
After the mice were euthanized, the femurs and tibias were isolated. Under sterile conditions, both ends of the bones were trimmed, and the bone marrow cavity was flushed with sterile PBS to collect bone marrow cells in a centrifuge tube. The cell suspension was filtered through a 70 μm cell strainer (Falcon) and centrifuged, and the supernatant was discarded. The cells were then treated with red blood cell lysis buffer (00–4333–57, Invitrogen) to remove red blood cells. The extracted cells were resuspended in complete DMEM supplemented with 20 ng/mL macrophage colony-stimulating factor (M-CSF, 20 ng/mL, RP01216, ABclonal). During culture, a half-medium change was performed on day 3, a full-medium change was performed on day 6, and the cells were harvested on day 7 for subsequent experiments.
Flow cytometry
The mice were injected via the tail vein with C. neoformans (5 × 105 cells). After 48 hours, the mouse brain tissue was digested into a single-cell suspension via collagenase IV (C5138, Sigma) and DNase I (DN25, Sigma) at 37°C and 90 rpm for 20 minutes. The digestion was halted with PBS, and the suspension was filtered through a 70 μm cell strainer, centrifuged at 400 × g for 5 minutes, and resuspended in PBS for counting. Each sample included 1 × 106 cells in 100 μL of PBS and was stained with live/dead dye (423106, Biolegend; L34966, Invitrogen) at room temperature for 30 minutes. Next, 1 mL of 1% BSA (prepared in PBS) was added, and the cells were washed twice by centrifugation at 400 × g for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in 100 μL of 1% BSA, followed by the addition of an Fc blocker (101302, Biolegend) and blocking at room temperature for 15 minutes. Depending on the experimental objective, surface marker antibodies, such as CD45 (103114, 103137, Biolegend), F4/80 (53-4801-82, 17-4801-82, eBioscience), CD11b (11-0112-81, eBioscience), CCR2 (150605, Biolegend), CCR7 (120107, Biolegend), and CXCR4 (153805, Biolegend), were added and incubated at 4°C for 1 hour. The cells were then washed twice with 1 mL of 1% BSA by centrifugation at 400 × g for 5 minutes. For samples without intracellular labeling, 100 μL of flow cytometry fixation buffer was added, and the cells were fixed at 4°C for 1 hour before flow cytometry analysis. For intracellular staining, the supernatant was discarded, and the cells were resuspended in 400 μL of permeabilization buffer and incubated at 4°C for 1 hour, followed by two washes with 1 mL of permeabilization wash buffer and centrifugation at 400 × g for 5 minutes. The cells were then resuspended and stained with the intracellular antibodies PDE4B (ab170939, Abcam) or Arg1 (17-3697-82, eBioscience) at 4°C for 1 hour. After washing, the cells were resuspended in 300 μL of 1% BSA for acquisition on a flow cytometer (FACSVerse/FACSymphony A5 SORP, BD). For indirect antibody labeling, the cells were washed, resuspended, stained with secondary antibody at room temperature for 1 hour, centrifuged and prepared for analysis.
Quantitative real-time PCR
Total RNA was extracted from cells via an extraction kit (Vazyme), and the RNA concentration and purity were confirmed with a NanoDrop instrument (Thermo Fisher Scientific), ensuring an A260/A280 ratio between 1.8 and 2.0. A total of 1 µg of RNA was reverse transcribed into cDNA via a reverse transcription kit (11141ES60, Yeasen) following the manufacturer’s protocol. Reactions were prepared in a 96-well plate with a total volume of 10 µL, containing 1 µL cDNA template, 5 µL 2x SYBR Green qPCR Master Mix (11184ES08, Yeasen), 0.4 µL each of forward and reverse primers (final concentration 200 nM, Table 1), and nuclease-free water to complete the volume. The primer sequences were designed on the basis of published literature and verified for specificity via Primer-BLAST. The qPCRs were conducted on an Applied Biosystems 7500 Real-Time PCR System (ABI, USA) with the following protocol: 95°C for 3 minutes for initial denaturation, followed by 40 cycles of 95°C for 15 seconds (denaturation) and 60°C for 30 seconds (annealing/extension), with technical replicates for each sample. The Ct values obtained were analyzed via the ΔΔCt method for relative quantification. β-actin was used as the internal control gene, and gene expression levels were normalized to those of the control group; the results are presented as the standard error of the mean (SEM). The specificity of the amplified products was confirmed via melt curve analysis.
Western blot
The cells were collected for sample preparation, and total protein was extracted via RIPA lysis buffer (ST506, Beyotime Biotech) containing protease inhibitors (P0013B, Beyotime Biotech). The samples were sonicated for 5 seconds and centrifuged at 12,000 rpm for 10 minutes at 4°C to collect the supernatant. Equal amounts of protein were mixed with 5X protein loading buffer (20315ES, Yeasen) and heated at 100°C for 10 minutes to denature the proteins. The protein samples were then loaded onto an SDS‒PAGE gel for electrophoretic separation. Following electrophoresis, the proteins were transferred onto a 0.45 μm PVDF membrane (Merck Millipore) via a transfer apparatus. The membrane was blocked at room temperature for 1 hour with 5% nonfat milk to prevent nonspecific binding. The samples were then incubated overnight at 4°C with primary antibodies, including anti-PDE4B (ab170939; Abcam). The membrane was washed three times with TBST (5 minutes each) and incubated for 1 hour at room temperature with an HRP-conjugated anti-rabbit IgG secondary antibody (7074, Cell Signaling Technology). After additional washes with TBST, an enhanced chemiluminescence (ECL) detection reagent (36208-A, Yeasen) was applied, and the protein bands were visualized via a chemiluminescence imaging system (Tanon, 5200). Finally, grayscale analysis of the target protein bands was conducted via ImageJ software, and the results were normalized to those of the internal control protein (β-actin).
Immunofluorescence
After 48 hours of C. neoformans infection, the mice were sacrificed via cervical dislocation, and the brain tissue was harvested and fixed in 4% paraformaldehyde (G1101, Servicebio) before being embedded in paraffin. The brain sections were then deparaffinized, and antigen retrieval was performed, followed by immunofluorescence staining. For the Transwell samples, fixation was directly carried out at room temperature with 4% paraformaldehyde for 30 minutes, followed by three washes with PBS. The samples were then permeabilized with 0.5% Triton X-100 (diluted in PBS) for 15 minutes at room temperature and blocked with 5% fetal bovine serum (FBS, diluted in PBS) for 1 hour at room temperature. After the blocking solution was discarded, the primary antibodies anti-F4/80 (1:1000, GB113373, Servicebio) and anti-CD45 (GB113886, Servicebio) were applied, and the samples were incubated overnight at 4°C. The following day, the samples were washed three times with PBS for 5 minutes each, followed by incubation with appropriate secondary antibodies for 1 hour at room temperature. Finally, DAPI (36308ES20, Yeasen) was added for nuclear staining and coverslipping. Imaging was performed via a laser confocal microscope (LSM900, Leica) or a fluorescence microscope (IX73, Olympus).
Phagocytosis and killing assay
In the in vitro killing assay, BMDMs that had been pretreated with inhibitors were infected with C. neoformans strain H99. After a 24-h incubation, the culture medium was gently pipetted to resuspend the cells, and serial dilutions were then performed for fungal colony counting. For the phagocytosis assay, C. neoformans strain H99 was added to the cells, which were then incubated for 2 hours. After this incubation, any free extracellular fungi were washed away, and 1 mL of deionized water was added to lyse the cells. Fungal colony counting was then conducted.
Transwell assay
First, 70 µL of 8-fold diluted Matrigel (40187, Yeasen) was evenly spread onto the filter membrane of the upper chamber in an 8 µm pore-sized Transwell insert (Corning, USA) and incubated at 37°C for 3 hours to allow solidification. A total of 6 × 104 bEnd.3 cells were prepared, resuspended in 200 µL of complete medium, and seeded into the upper chamber of the Transwell insert. The cells were incubated for 3 days to form a confluent monolayer. And, we used 40% normal human serum as an opsonin to promote the phagocytosis of Cryptococcus neoformans. The fungi were first pretreated with 40% serum, washed thoroughly, and then co-incubated with phagocytic cells for subsequent analysis. Moreover, the macrophages were pretreated with inhibitors for 24 hours, followed by coincubation with H99Tdtomato at a 1:3 ratio to allow phagocytosis. After 24 hours, the free fungi were washed away. Next, 5 × 104 macrophages were counted, resuspended in 200 µL of medium containing 5% FBS, and added to the upper chamber of the insert. The lower chamber was filled with 600 µL of medium containing 20% FBS as a chemoattractant. After 24 hours, the number of fluorescent fungi in the lower chamber was counted, and the Transwell membrane was carefully removed for immunofluorescence staining. The number of fluorescent fungi on the underside of the membrane was visualized and quantified via a confocal microscope (LSM900, Leica) for statistical analysis.
Adoptive transfer of macrophages
Primary bone marrow-derived macrophages (BMDMs) from WT and PDE4b-/- mice were extracted and cultured in advance. On day 7, after macrophage maturation, 200 µL of macrophage depletion reagent (40337ES10, Yeasen) was injected into the tail vein of each mouse. A 4-fold greater quantity of H99GFP or H99Tdtomato was subsequently added to WT and PDE4b-/- BMDMs, respectively, to promote the phagocytosis of fluorescent fungi. After 24 hours, free fungi were removed, and primary macrophages containing different fluorescent fungi were collected, resuspended in PBS, and counted. A group of WT mice was injected with 200 µL of a cell mixture containing 1 × 106 WT BMDMs that had phagocytosed H99GFP and 1 × 106 WT BMDMs that had phagocytosed H99Tdtomato (1 × 106 WT BMDM-H99GFP + 1 × 106 WT BMDM-H99Tdtomato). Another group of WT mice was injected with a mixture containing 1 × 106 WT BMDM-H99GFP and 1 × 106 PDE4b-/- BMDM-H99Tdtomato. After 48 hours, the mice were euthanized, and cardiac perfusion was performed. The brain tissue was harvested, digested into a single-cell suspension, and analyzed via flow cytometry to assess the proportions of different fluorescent fungi in the brain tissue.
Statistical analysis
The data were summarized via basic descriptive statistics and are presented as the means ± SEMs. For normally distributed data, comparisons between two groups were conducted via an unpaired two-tailed Student’s t test, and comparisons involving more than two groups were analyzed via one-way ANOVA. For adoptive transfer flow cytometry data, two-way ANOVA with Sidak’s post hoc test was used for multiple comparisons. Statistical significance was set as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; and ns indicates “not significant”.
Supporting information
S1 Fig. Inhibition of PDE4B does not alter fungal burden in the brain in a murine intranasal infection model.
(A) Kaplan–Meier survival curves of mice intranasally infected with Cryptococcus neoformans and treated with the PDE4B inhibitor Nerandomilast or vehicle control. Survival differences between groups were analyzed using the log-rank (Mantel–Cox) test. (B–D) Fungal burdens in the lungs (B), spleen (C), and brain (D) of mice 7 days after intranasal inoculation with 5 × 10⁴ C. neoformans. Fungal burden was determined as colony-forming units (CFU) per organ. (E) Western blot analysis of PDE4B expression in BMDMs treated with inert beads (5 × 106) or H99 (5 × 106) (n = 3 per group). The data are presented as the mean ± SEM. unpaired Student’s t test. ns, not significant.
https://doi.org/10.1371/journal.ppat.1014040.s001
(TIF)
S2 Fig. PDE4B deficiency promotes macrophage phagocytosis of Cryptococcus neoformans.
(A) Colony-forming unit (CFU) assays were performed after C. neoformans was treated with different concentrations of Nerandomilast to exclude any direct fungicidal activity of the compound. Statistical analysis was performed using one-way ANOVA, ns indicates no statistically significant difference. (B-C) WT and PDE4B-KO BMDMs were exposed to fluorescently labeled Cryptococcus neoformans. After extensive washing at 3 h and 24 h to remove extracellular fungi, the intracellular fungal burden was quantitatively analyzed by fluorescence microscopy. The ratio of total internalized fungi to the total number of cells within each high-power field was subsequently calculated. Comparisons among groups were performed using two-way analysis of variance (two-way ANOVA) followed by Sidak’s post hoc multiple comparisons test. *P < 0.05 and **P < 0.001.
https://doi.org/10.1371/journal.ppat.1014040.s002
(TIF)
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