Pneumocystis murina promotes inflammasome formation and NETosis during Pneumocystis pneumonia.

Pneumocystis jirovecii pneumonia (PjP) poses a serious risk to individuals with compromised immune systems, such as individuals with HIV/AIDS or undergoing immunosuppressive therapies for cancer or solid organ transplants. Severe PjP triggers excessive lung inflammation, resulting in lung function decline and consequential alveolar damage, potentially culminating in acute respiratory distress syndrome. Non-HIV patients face a 30%-60% mortality rate, emphasizing the need for a deeper understanding of inflammatory responses in PjP. Prior research emphasized macrophages in Pneumocystis infections, neglecting neutrophils' role in tissue damage. Consequently, the overemphasis on macrophages led to an incomplete understanding of the role of neutrophils and inflammatory responses. In the current investigation, our RNAseq studies on a murine surrogate model of PjP revealed heightened activation of the NLRP3 inflammasome and NETosis cell death pathways in their lungs. Immunofluorescence staining confirmed neutrophil extracellular trap (NET) presence in the lungs of the P. murina-infected mice, validating our findings. Moreover, isolated neutrophils exhibited NETosis when directly stimulated with P. murina. Isolated NETs compromised P. murina viability in vitro, highlighting the potential role of neutrophils in controlling fungal growth and promoting inflammation during P. murina pneumonia through NLRP3 inflammasome assembly and NETosis. These pathways, essential for inflammation and pathogen elimination, bear the risk of uncontrolled activation leading to excessive tissue damage and persistent inflammation. This pioneering study is the first to identify the formation of NETs and inflammasomes during Pneumocystis infection, paving the way for comprehensive investigations into treatments aimed at mitigating lung damage and augmenting survival rates for individuals with PjP.IMPORTANCEPneumocystis jirovecii pneumonia (PjP) affects individuals with weakened immunity, such as HIV/AIDS, cancer, and organ transplant patients. Severe PjP triggers lung inflammation, impairing function and potentially causing acute respiratory distress syndrome. Non-HIV individuals face a 30%-60% mortality rate, underscoring the need for deeper insight into PjP's inflammatory responses. Past research focused on macrophages in managing Pneumocystis infection and its inflammation, while the role of neutrophils was generally overlooked. In contrast, our findings in P. murina-infected mouse lungs showed neutrophil involvement during inflammation and increased expression of NLRP3 inflammasome and NETosis pathways. Detection of neutrophil extracellular traps further indicated their involvement in the inflammatory process. Although beneficial in combating infection, unregulated neutrophil activation poses a potential threat to lung tissues. Understanding the behavior of neutrophils in Pneumocystis infections is crucial for controlling detrimental reactions and formulating treatments to reduce lung damage, ultimately improving the survival rates of individuals with PjP.

Description of a Murine Model of Pneumocystis Pneumonia.

Pneumocystis pneumonia is a serious lung infection caused by an original ubiquitous fungus with opportunistic behavior, referred to as Pneumocystis jirovecii. P. jirovecii is the second most common fungal agent among invasive fungal infections after Candida spp. Unfortunately, there is still an inability to culture P. jirovecii in vitro, and so a great impairment to improve knowledge on the pathogenesis of Pneumocystis pneumonia. In this context, animal models have a high value to address complex interplay between Pneumocystis and the components of the host immune system. Here, we propose a protocol for a murine model of Pneumocystis pneumonia. Animals become susceptible to Pneumocystis by acquiring an immunocompromised status induced by iterative administration of steroids within drinking water. Thereafter, the experimental infection is completed by an intranasal challenge with homogenates of mouse lungs containing Pneumocystis murina. The onset of clinical signs occurs within 5 weeks following the infectious challenge and immunosuppression can then be withdrawn. At termination, lungs and bronchoalveolar lavage (BAL) fluids from infected mice are analyzed for fungal load (qPCR) and immune response (flow cytometry and biochemical assays). The model is a useful tool in studies focusing on immune responses initiated after the establishment of Pneumocystis pneumonia.

Endogenous IFN-γ facilitates Pneumocystis infection and downregulates carbohydrate receptors in CD4+ T cell-depleted mice.

IFN-γ plays a critical role in host defense against intracellular pathogens. IFN-γ is produced in the bronchoalveolar lavage fluid of mice infected with Pneumocystis, but the role of IFN-γ in host defense against Pneumocystis remains controversial. It has been previously reported that although exogenous IFN-γ has beneficial effects on eradication of Pneumocystis, endogenous IFN-γ has a negative impact on innate immunity in immunocompromised hosts. Surprisingly, CD4+ T cell-depleted IFN-γ deficient (GKO) mice exhibit resistance to Pneumocystis. Alveolar macrophages (AM) from GKO mice exhibit higher expression of macrophage mannose receptor (MMR) and Dectin-1. Concomitantly, they exhibited greater ability to phagocytize Pneumocystis, and this activity was suppressed by inhibitors of these receptors. Incubation with IFN-γ resulted in a reduction in both the expression of these receptors on AM and their Pneumocystis-phagocytic activity. These results indicate that endogenous IFN-γ facilitates Pneumocystis to escape from host innate immunity by attenuating the phagocytic activity of AM via downregulation of MMR and Dectin-1.

Retracing the evolution of Pneumocystis species, with a focus on the human pathogen Pneumocystis jirovecii.

SUMMARYEvery human being is presumed to be infected by the fungus Pneumocystis jirovecii at least once in his or her lifetime. This fungus belongs to a large group of species that appear to exclusively infect mammals, with P. jirovecii being the only one known to cause disease in humans. The mystery of P. jirovecii origin and speciation is just beginning to unravel. Here, we provide a review of the major steps of P. jirovecii evolution. The Pneumocystis genus likely originated from soil or plant-associated organisms during the period of Cretaceous ~165 million years ago and successfully shifted to mammals. The transition coincided with a substantial loss of genes, many of which are related to the synthesis of nutrients that can be scavenged from hosts or cell wall components that could be targeted by the mammalian immune system. Following the transition, the Pneumocystis genus cospeciated with mammals. Each species specialized at infecting its own host. Host specialization is presumably built at least partially upon surface glycoproteins, whose protogene was acquired prior to the genus formation. P. jirovecii appeared at ~65 million years ago, overlapping with the emergence of the first primates. P. jirovecii and its sister species P. macacae, which infects macaques nowadays, may have had overlapping host ranges in the distant past. Clues from molecular clocks suggest that P. jirovecii did not cospeciate with humans. Molecular evidence suggests that Pneumocystis speciation involved chromosomal rearrangements and the mounting of genetic barriers that inhibit gene flow among species.

Leukotriene B4-induced neutrophil extracellular traps impede the clearance of Pneumocystis.

Pneumocystis pneumonia (PCP) is a fungal pulmonary disease with high mortality in immunocompromised patients. Neutrophils are essential in defending against fungal infections; however, their role in PCP is controversial. Here we aim to investigate the effects of neutrophil extracellular traps (NETs) on Pneumocystis clearance and lung injury using a mouse model of PCP. Intriguingly, although neutrophils play a fundamental role in defending against fungal infections, NETs failed to eliminate Pneumocystis, but instead impaired the killing of Pneumocystis. Mechanically, Pneumocystis triggered Leukotriene B4 (LTB4)-dependent neutrophil swarming, leading to agglutinative NET formation. Blocking Leukotriene B4 with its receptor antagonist Etalocib significantly reduced the accumulation and NET release of neutrophils in vitro and in vivo, enhanced the killing ability of neutrophils against Pneumocystis, and alleviated lung injury in PCP mice. This study identifies the deleterious role of agglutinative NETs in Pneumocystis infection and reveals a new way to prevent NET formation, which provides new insights into the pathogenesis of PCP.

Role of the Fungus Pneumocystis in IL1ß Pathway Activation and Airways Collagen Deposition in Elastase-Induced COPD Animals.

Inflammation and mucus production are prevalent characteristics of chronic respiratory conditions, such as asthma and chronic chronic obstructive pulmonary disease (COPD). Biological co-factors, including bacteria, viruses, and fungi, may exacerbate these diseases by activating various pathways associated with airway diseases. An example is the fungus Pneumocystis, which is linked to severe COPD in human patients. Recent evidence has demonstrated that Pneumocystis significantly enhanced inflammation and mucus hypersecretion in a rat model of elastase-induced COPD. The present study specifically aims to investigate two additional aspects associated with the pathology induced by Pneumocystis infection: inflammation and collagen deposition around airways. To this end, the focus was to investigate the role of the IL-1ß pro-inflammatory pathway during Pneumocystis infection in COPD rats. Several airway pathology-related features, such as inflammation, mucus hypersecretion, and fibrosis, were evaluated using histological and molecular techniques. COPD animals infected with Pneumocystis exhibited elevated inflammation levels, including a synergistic increase in IL-1ß and Cox-2. Furthermore, protein levels of the IL-1ß-dependent transcription factor cAMP response element-binding (CREB) showed a synergistic elevation of their phosphorylated version in the lungs of COPD animals infected with Pneumocystis, while mucus levels were notably higher in the airways of COPD-infected animals. Interestingly, a CREB responsive element (CRE) was identified in the Muc5b promoter. The presence of CREB in the Muc5b promoter was synergistically increased in COPD animals infected with Pneumocystis compared to other experimental groups. Finally, an increment of deposited collagen was identified surrounding the airways of COPD animals infected with Pneumocystis compared with the other experimental animal groups and correlated with the increase of Tgfß1 mRNA levels. These findings emphasize the role of Pneumocystis as a potential biological co-factor in chronic respiratory diseases like COPD or asthma, warranting new perspectives in the treatment of chronic respiratory diseases.

Slicing through the challenge of maintaining Pneumocystis in the laboratory.

Pneumocystis jirovecii is a major fungal pathogen of humans that causes life-threatening lung infections in immunocompromised individuals. Despite its huge global impact upon human health, our understanding of the pathobiology of this deadly fungus remains extremely limited, largely because it is not yet possible to cultivate Pneumocystis in vitro, independently of the host. However, a recent paper by Munyonho et al. offers a major step forward (F. T. Munyonho, R. D. Clark, D. Lin, M. S. Khatun, et al., 2023, mBio 15:e01464-23, https://doi.org/10.1128/mbio.01464-23). They show that it is possible to maintain both the trophozoite and cyst forms of the mouse pathogen, Pneumocystis murina, in precision-cut lung slices for several weeks. Furthermore, they demonstrate that this offers the exciting opportunity to examine potential virulence factors such as possible biofilm formation as well as antifungal drug responses in the lung.

Targeting host tyrosine kinase receptor EphA2 signaling via small-molecule ALW-II-41-27 inhibits macrophage pro-inflammatory signaling responses to Pneumocystis carinii ß-glucans.

Pneumocystis jirovecii, the fungus that causes Pneumocystis jirovecii pneumonia (PJP), is a leading cause of morbidity and mortality in immunocompromised individuals. We have previously shown that lung epithelial cells can bind Pneumocystis spp. ß-glucans via the EphA2 receptor, resulting in activation and release of proinflammatory cytokines. Herein, we show that in vivo Pneumocystis spp. ß-glucans activation of the inflammatory signaling cascade in macrophages can be pharmacodynamically inhibited with the EphA2 receptor small-molecule inhibitor ALW-II-41-27. In vitro, when ALW-II-41-27 is administrated via intraperitoneal to mice prior to the administration of highly proinflammatory Saccharomyces cerevisiae ß-glucans in the lung, a significant reduction in TNF-alpha release was noted in the ALW-II-41-27 pre-treated group. Taken together, our data suggest that targeting host lung macrophage activation via EphA2 receptor-fungal ß-glucans interactions with ALW-II-41-27 or other EphA2 receptor kinase targeting inhibitors might be an attractive and viable strategy to reduce detrimental lung inflammation associated with PJP.

Characterization of the Pneumocystis jirovecii and Pneumocystis murina phosphoglucomutases (Pgm2s): a potential target for Pneumocystis therapy.

Pneumocystis cyst life forms contain abundant ß-glucan carbohydrates, synthesized using ß-1,3 and ß-1,6 glucan synthase enzymes and the donor uridine diphosphate (UDP)-glucose. In yeast, phosphoglucomutase (PGM) plays a crucial role in carbohydrate metabolism by interconverting glucose 1-phosphate and glucose 6-phosphate, a vital step in UDP pools for ß-glucan cell wall formation. This pathway has not yet been defined in Pneumocystis. Herein, we surveyed the Pneumocystis jirovecii and Pneumocystis murina genomes, which predicted a homolog of the Saccharomyces cerevisiae major PGM enzyme. Furthermore, we show that PjPgm2p and PmPgm2p function similarly to the yeast counterpart. When both Pneumocystis pgm2 homologs are heterologously expressed in S. cerevisiae pgm2Δ cells, both genes can restore growth and sedimentation rates to wild-type levels. Additionally, we demonstrate that yeast pgm2Δ cell lysates expressing the two Pneumocystis pgm2 transcripts individually can restore PGM activities significantly altered in the yeast pgm2Δ strain. The addition of lithium, a competitive inhibitor of yeast PGM activity, significantly reduces PGM activity. Next, we tested the effects of lithium on P. murina viability ex vivo and found the compound displays significant anti-Pneumocystis activity. Finally, we demonstrate that a para-aryl derivative (ISFP10) with known inhibitory activity against the Aspergillus fumigatus PGM protein and exhibiting 50-fold selectivity over the human PGM enzyme homolog can also significantly reduce Pmpgm2 activity in vitro. Collectively, our data genetically and functionally validate phosphoglucomutases in both P. jirovecii and P. murina and suggest the potential of this protein as a selective therapeutic target for individuals with Pneumocystis pneumonia.

Extracellular vesicles from Pneumocystis carinii-infected rats impair fungal viability but are dispensable for macrophage functions.

Pneumocystis spp. are host obligate fungal pathogens that can cause severe pneumonia in mammals and rely heavily on their host for essential nutrients. The lack of a sustainable in vitro culture system poses challenges in understanding their metabolism, and the acquisition of essential nutrients from host lungs remains unexplored. Transmission electron micrographs show that extracellular vesicles (EVs) are found near Pneumocystis spp. within the lung. We hypothesized that EVs transport essential nutrients to the fungi during infection. To investigate this, EVs from P. carinii- and P. murina-infected rodents were biochemically and functionally characterized. These EVs contained host proteins involved in cellular, metabolic, and immune processes as well as proteins with homologs found in other fungal EV proteomes, indicating that Pneumocystis may release EVs. Notably, EV uptake by P. carinii indicated their potential involvement in nutrient acquisition and a possibility for using engineered EVs for efficient therapeutic delivery. However, EVs added to P. carinii in vitro did not show increased growth or viability, implying that additional nutrients or factors are necessary to support their metabolic requirements. Exposure of macrophages to EVs increased proinflammatory cytokine levels but did not affect macrophages' ability to kill or phagocytose P. carinii. These findings provide vital insights into P. carinii and host EV interactions, yet the mechanisms underlying P. carinii's survival in the lung remain uncertain. These studies are the first to isolate, characterize, and functionally assess EVs from Pneumocystis-infected rodents, promising to enhance our understanding of host-pathogen dynamics and therapeutic potential.IMPORTANCEPneumocystis spp. are fungal pathogens that can cause severe pneumonia in mammals, relying heavily on the host for essential nutrients. The absence of an in vitro culture system poses challenges in understanding their metabolism, and the acquisition of vital nutrients from host lungs remains unexplored. Extracellular vesicles (EVs) are found near Pneumocystis spp., and it is hypothesized that these vesicles transport nutrients to the pathogenic fungi. Pneumocystis proteins within the EVs showed homology to other fungal EV proteomes, suggesting that Pneumocystis spp. release EVs. While EVs did not significantly enhance P. carinii growth in vitro, P. carinii displayed active uptake of these vesicles. Moreover, EVs induced proinflammatory cytokine production in macrophages without compromising their ability to combat P. carinii. These findings provide valuable insights into EV dynamics during host-pathogen interactions in Pneumocystis pneumonia. However, the precise underlying mechanisms remain uncertain. This research also raises the potential for engineered EVs in therapeutic applications.

Precision-cut lung slices as an ex vivo model to study Pneumocystis murina survival and antimicrobial susceptibility.

IMPORTANCE: Our study reveals the potential of precision-cut lung slices as an ex vivo platform to study the growth/survival of Pneumocystis spp. that can facilitate the development of new anti-fungal drugs.

[Progress of researches on developmental processes and reproduction mode of Pneumocystis].

Pneumocystis, an important opportunistic fungal pathogen that parasitizes in multiple mammalian lungs, may cause life-threatening Pneumocystis pneumonia (PCP) and even death among immunocompromised individuals. With the rapid development of high-throughput sequencing and multi-omics technologies, systematic comparative analyses of genome, transcriptome, and whole-genome sequencing results demonstrate that Pneumocystis is a type of obligate biotrophic fungi, and requires obtaining nutrition from hosts. In addition, sexual reproduction is an essential process for Pneumocystis survival, production and transmission, and asexual reproduction facilitates Pneumocystis survival, which provides new insights into understanding of the whole developmental process of Pneumocystis in the host lung and inter-host transmission of Pneumocystis. This review summarizes the advances in the reproduction mode of Pneumocystis and underlying mechanisms, which provides insights into prevention and treatment of PCP, notably for the prophylaxis against nosocomial transmission of PCP.

Relationship between clinical features and droplet digital PCR copy number in non-HIV patients with pneumocystis pneumonia.

OBJECTIVE: Droplet digital PCR (ddPCR) is a novel assay to detect pneumocystis jjrovecii (Pj) which has been defined to be more sensitive than qPCR in recent studies. We aimed to explore whether clinical features of pneumocystis pneumonia (PCP) were associated with ddPCR copy numbers of Pj. METHODS: A total of 48 PCP patients were retrospectively included. Pj detection was implemented by ddPCR assay within 4 h. Bronchoalveolar fluid (BALF) samples were collected from 48 patients with molecular diagnosis as PCP via metagenomic next generation sequencing (mNGS) or quantitative PCR detection. Univariate and multivariate logistic regression were performed to screen out possible indicators for the severity of PCP. The patients were divided into two groups according to ddPCR copy numbers, and their clinical features were further analyzed. RESULTS: Pj loading was a pro rata increase with serum (1,3)-beta-D glucan, D-dimmer, neutrophil percentage, procalcitonin and BALF polymorphonuclear leucocyte percentage, while negative correlation with albumin, PaO2/FiO2, BALF cell count, and BALF lymphocyte percentage. D-dimmer and ddPCR copy number of Pj were independent indicators for moderate/severe PCP patients with PaO2/FiO2 lower than 300. We made a ROC analysis of ddPCR copy number of Pj for PaO2/FiO2 index and grouped the patients according to the cut-off value (2.75). The high copy numbers group was characterized by higher level of inflammatory markers. Compared to low copy number group, there was lower level of the total cell count while higher level of polymorphonuclear leucocyte percentage in BALF in the high copy numbers group. Different from patients with high copy numbers, those with high copy numbers had a tendency to develop more severe complications and required advanced respiratory support. CONCLUSION: The scenarios of patients infected with high ddPCR copy numbers of Pj showed more adverse clinical conditions. Pj loading could reflect the severity of PCP to some extent.

Indication for a Pneumocystis Prophylaxis Therapy in Patients with Vascular Anomalies Treated with PIK3/AKT/mTOR Pathway Inhibitors: Experts' Opinion and Systematic Review from the Literature.

BACKGROUND: Vascular anomalies (VAs) are increasingly being treated with PI3K/AKT/mTOR pathway inhibitors. These drugs have immunosuppressive properties and thus theoretically overexpose patients to opportunistic infections, especially Pneumocystis jirovecii pneumonia (PJP). PJP prophylaxis use lacks consensus. We aimed to investigate the prevalence of PJP in patients receiving mTOR/PI3K/AKT inhibitors for VAs and determine any indication for pneumocystis prophylaxis in this population. METHODS: The study was conducted in 2 parts: (1) we sent a survey to a panel of international experts of VAs asking about their use of pneumocystis prophylaxis drugs and (2) we performed a systematic review of the literature of all published cases of patients receiving these drugs for VA to estimate the prevalence of PJP in this population. RESULTS: Answers from 68 experts were analyzed: 21 (30.9%) answered they always add PJP prophylaxis when prescribing mTOR inhibitors, 20 (29.4%) case-by-case, and 27 (39.7%) never. For the systematic review, among 3,053 reports screened, 217 were included involving 1,189 patients (1,143 received sirolimus, 38 everolimus, 4 alpelisib, 4 miransertib). Among the 1,189 cases, 2 (0.2%) PJP were reported: one under sirolimus and one under everolimus. Thus, the prevalence of PJP was estimated at 0.88 cases/1,000 patients under sirolimus (95% CI: -0.84 to 2.59) and 26.31 cases/1,000 under everolimus (95% CI: -24.58 to 77.18). Patients with PJP never received prophylaxis drugs. We found no PJP cases under alpelisib and miransertib. PJP prophylaxis was given in 218 (18.3%) cases, more frequently for children (91.3 vs. 77.2% in the non-prophylaxis group, p = 0.012), mostly trimethoprim-sulfamethoxazole (186 patients, 85.3%). CONCLUSION: Our study shows that even if PJP is a rare event, it may occur in patients with VAs treated with an mTOR inhibitor. Although our results cannot allow for revising guidelines, prophylaxis with TMP-SMX might be appropriate for a subgroup of patients with risk factors for PJP.

IFN-γ Limits Immunopathogenesis but Delays Fungal Clearance during Pneumocystis Pneumonia.

High levels of IFN-γ are produced in the lung during an adaptive immune response to Pneumocystis, but the effects of this prototypical Th1 cytokine on fungal clearance and immunopathogenesis have not been fully defined. Therefore, Pneumocystis-infected immunodeficient mice were immune reconstituted and administered control or anti-IFN-γ neutralizing Ab to determine how IFN-γ regulates the balance between host defense and immune-mediated lung injury. Mice treated with anti-IFN-γ demonstrated an initial worsening of Pneumocystis pneumonia-related immunopathogenesis, with greater weight loss, heightened lung inflammation, and more severe pulmonary function deficits than control mice. However, IFN-γ neutralization also enhanced macrophage phagocytosis of Pneumocystis and accelerated fungal clearance. When anti-IFN-γ-treated mice were also given IL-4 and IL-13 to promote a Th2-biased lung environment, the accelerated fungal clearance was preserved, but the severity of immunopathogenesis was reduced, and a more rapid recovery was observed. A direct suppressive effect of IFN-γ on macrophages was required but was not solely responsible for delayed fungal clearance, suggesting that IFN-γ acts through multiple mechanisms that likely include modulation of both macrophage and Th polarization. Enhanced Pneumocystis clearance in anti-IFN-γ-treated and IFN-γR-deficient mice was associated with significantly elevated IL-17+ CD4+ T cells and IL-17 protein in the lungs. Furthermore, neutralization of IL-17, but not IL-4, signaling blocked the accelerated fungal clearance observed in anti-IFN-γ-treated mice. Together, these data demonstrate that although IFN-γ delays fungal clearance by suppressing the lung Th17 response, it also serves an important regulatory role that limits immunopathogenesis and preserves pulmonary function.

Integrated multi-omics analyses reveal the altered transcriptomic characteristics of pulmonary macrophages in immunocompromised hosts with Pneumocystis pneumonia.

Introduction: With the extensive use of immunosuppressants, immunosuppression-associated pneumonitis including Pneumocystis jirovecii pneumonia (PCP) has received increasing attention. Though aberrant adaptive immunity has been considered as a key reason for opportunistic infections, the characteristics of innate immunity in these immunocompromised hosts remain unclear. Methods: In this study, wild type C57BL/6 mice or dexamethasone-treated mice were injected with or without Pneumocystis. Bronchoalveolar lavage fluids (BALFs) were harvested for the multiplex cytokine and metabolomics analysis. The single-cell RNA sequencing (scRNA-seq) of indicated lung tissues or BALFs was performed to decipher the macrophages heterogeneity. Mice lung tissues were further analyzed via quantitative polymerase chain reaction (qPCR) or immunohistochemical staining. Results: We found that the secretion of both pro-inflammatory cytokines and metabolites in the Pneumocystis-infected mice are impaired by glucocorticoids. By scRNA-seq, we identified seven subpopulations of macrophages in mice lung tissues. Among them, a group of Mmp12+ macrophages is enriched in the immunocompetent mice with Pneumocystis infection. Pseudotime trajectory showed that these Mmp12+ macrophages are differentiated from Ly6c+ classical monocytes, and highly express pro-inflammatory cytokines elevated in BALFs of Pneumocystis-infected mice. In vitro, we confirmed that dexamethasone impairs the expression of Lif, Il1b, Il6 and Tnf, as well as the fungal killing capacity of alveolar macrophage (AM)-like cells. Moreover, in patients with PCP, we found a group of macrophages resembled the aforementioned Mmp12+ macrophages, and these macrophages are inhibited in the patient receiving glucocorticoid treatment. Additionally, dexamethasone simultaneously impaired the functional integrity of resident AMs and downregulated the level of lysophosphatidylcholine, leading to the suppressed antifungal capacities. Conclusion: We reported a group of Mmp12+ macrophages conferring protection during Pneumocystis infection, which can be dampened by glucocorticoids. This study provides multiple resources for understanding the heterogeneity and metabolic changes of innate immunity in immunocompromised hosts, and also suggests that the loss of Mmp12+ macrophages population contributes to the pathogenesis of immunosuppression-associated pneumonitis.

CLEC4A and CLEC12B C-type lectin receptors mediate interactions with Pneumocystis cell wall components.

Introduction. C-type lectin receptors (CLRs) are prominently expressed on myeloid cells where they perform multiple functions including serving as pattern recognition receptors (PRRs) to drive innate as well as adaptive immunity to pathogens. Depending on the presence of a tyrosine-based signalling motif, CLR-microbial pathogen engagement may result in either anti- or pro-inflammatory signalling.Impact statement. In this manuscript, we report our laboratory study of two novel CLRs that recognize Pneumocystis murina cell wall homogenates (CWH) and a purified Pneumocystis carinii cell wall fraction (CWF).Aim. To study the potential of newly generated hFc-CLR fusions on binding to Pneumocystis murina CWHs and P. carinii CWFs and subsequent downstream inflammatory signalling analysis.Methods. Newly generated hFc-CLR fusion CLEC4A and CLEC12B were screened against P. murina CWHs and P. carinii CWFs preparations via modified ELISA. Immunofluorescence assay (IFA) was utilized to visualize hFc-CLR fusion binding against intact fixed fungal life forms to verify results. Quantitative PCR (q-PCR) analysis of lung mRNA from the mouse immunosuppressed Pneumocystis pneumonia (PCP) model versus uninfected mice was employed to detect possible changes in the respective Clec4a and Clec12b transcripts. Lastly, siRNA technology of both CLRs was conducted to determine effects on downstream inflammatory events in mouse macrophages stimulated in the presence of P. carinii CWFs.Results. We determined that both CLEC4A and CLEC12B hFc-CLRs displayed significant binding with P. murina CWHs and P. carinii CWFs. Binding events showed significant binding to both curdlan and laminarin, both polysaccharides containing ß-(1,3) glucans as well as N-acetylglucosamine (GlcNAc) residues and modest yet non-significant binding to the negative control carbohydrate dextran. IFA with both CLR hFc-fusions against whole P. murina life forms corroborated these findings. Lastly, we surveyed the mRNA expression profiles of both CLRs tested above in the mouse immunosuppressed Pneumocystis pneumonia (PCP) model and determined that both CLRs were significantly up regulated during infection. Lastly, siRNA of both CLRs in the mouse RAW macrophage cell line was conducted and results demonstrated that silencing of Clec4a resulted in no significant changes in TNF-alpha generation in P. carinii CWF stimulated macrophages. On the contrary, silencing of Clec12b CLR resulted in significant decreases in TNF-alpha in RAW cells stimulated with the same CWF.Conclusion. The data presented here provide new members of the CLRs family recognizing Pneumocystis. Future studies using CLEC4A and/or CLEC12B deficient mice in the PCP mouse model should provide further insights into the host immunological response to Pneumocystis.

Evaluation of the PneumoGenius® PCR assay for the diagnosis of Pneumocystis pneumonia and the detection of Pneumocystis dihydropteroate synthase mutations in respiratory samples.

Pneumocystis pneumonia (PCP) is the most frequent fungal opportunistic infection defining AIDS in HIV-infected patients, and is of growing importance in HIV-negative patients. In this latter category of patients, the diagnosis mainly relies on real-time polymerase chain reaction (qPCR) detection of Pneumocystis jirovecii (Pj) on respiratory samples. The PneumoGenius® kit (PathoNostics) allows the simultaneous detection of Pj mitochondrial large subunit (mtLSU) and dihydropteroate synthase (DHPS) polymorphisms, which could be of interest to anticipate therapeutic failure. This study aimed at evaluating its clinical performance on 251 respiratory specimens (239 patients), (i) for P. jirovecii detection in clinical samples, and (ii) for DHPS polymorphisms detection in circulating strains. Patients were classified according to modified European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria, as having proven PCP (n = 62), probable PCP (n = 87), Pneumocystis colonization (n = 37), and no PCP (n = 53). Compared with in-house qPCR, the sensitivity of PneumoGenius® assay for P. jirovecii detection reached 91.9% (182/198), the specificity was excellent (100%, 53/53) and the global concordance was 93.6% (235/253). A total of four diagnoses of proven/probable PCP were missed by the PneumoGenius® assay, reaching a 97.5% sensitivity (157/161) in this sub-group. The 12 other 'false-negative' results were obtained in patients diagnosed as colonized using the in-house PCR. DHPS genotyping was successful for 147/182 samples with PneumoGenius® and revealed dhps mutation in 8 samples, which were all confirmed by sequencing. In conclusion, PneumoGenius® assay missed the detection of low-burden PCP. This lower sensitivity for PCP diagnosis can be balanced by a higher specificity (P. jirovecii colonization less frequently detected) and the efficient detection of DHPS hot spot mutations.

The diagnosis of Pneumocystis pneumonia (PCP) relies on DNA detection of P. jirovecii in respiratory samples. In this study, we show that the commercial assay PneumoGenius® has a lower sensitivity than our in-house qPCR for PCP diagnosis, but provides accurate results for DHPS genotyping.