Limper, Andrew H

Abstract Background Alveolar macrophages (AMs) are central to host defense against Pneumocystis, mediating organism clearance while also contributing to lung inflammation. EphA2, a transmembrane receptor that binds fungal β-glucans, has emerged as a regulator of host–pathogen interactions, but its role in AM responses during Pneumocystis pneumonia (PCP) is unknown. Methods We evaluated AM inflammatory responses to Pneumocystis β-glucans in the presence and absence of EphA2. In vivo studies used a CD4-depleted mouse model of PCP comparing wild-type and EphA2-deficient (EphA2−/−) mice. Lung cytokine responses and organism burden were assessed. Additionally, EphA2 signaling was pharmacologically inhibited using ALW-II-41-27 during trimethoprim-sulfamethoxazole treatment. Results EphA2 deficiency significantly reduced AM proinflammatory cytokine responses to Pneumocystis β-glucans. In CD4-depleted PCP mice, EphA2−/− animals demonstrated decreased lung inflammatory cytokines, but increased organism burden compared to controls. Pharmacologic inhibition of EphA2 during antimicrobial therapy significantly suppressed lung inflammation. Conclusions EphA2 signaling plays a critical role in AM-mediated host defense against Pneumocystis by promoting inflammatory responses necessary for organism control. However, this pathway also contributes to lung inflammation. Targeting EphA2 may represent a novel strategy to modulate inflammation during severe PCP while preserving antimicrobial defense.

Abstract Candida auris is an emerging fungal pathogen with high rates of multidrug resistance, posing a significant global health threat. Immunosuppressed patients are particularly vulnerable to multidrug-resistant (MDR) C. auris infections. This systematic review and meta-analysis (SR-MA) aimed to assess the global prevalence of MDR-C. auris isolates in humans and their resistance to antifungal drugs, primarily fluconazole (FLZ), voriconazole (VOR), and amphotericin B (AmB). A comprehensive search was conducted in PubMed, Scopus, and ScienceDirect databases on published studies regarding clinical C. auris isolates and the use of antifungal susceptibility testing assay from 17 March 2015 to 1 February 2024. Meta-analysis was conducted using random-effects models to determine the estimated pooled prevalence of MDR-C. auris isolates and their antifungal resistance profiles from immunosuppressed humans. A total of 81 research studies were included in the final analysis, with the overall pooled prevalence of the three most important antifungal drugs; FLZ (92.5% | 95% CI: 89.5–94.7), VOR (49.0% | 95% CI: 37.6–60.4), and AmB (51.0% | 95% CI: 42.3–59.7). According to the subgroup analyses, FLZ-resistant C. auris was the most prevalent isolate observed in the five continents reporting compared to the VOR- and AmB-resistant C. auris having lower pooled prevalence. Further, higher pooled prevalence of MDR-C. auris isolates were observed from males (68.2%, n = 578) compared to females (37.1%, n = 283). Finally, the global pooled prevalence for mortality in immunosuppressed patients with MDR-C. auris from 13 research studies was 41.5% (95% CI: 29.9–54.2). This SR-MA study establishes the variation of MDR-C. auris resistance patterns observed from different continents and the importance to conduct further research studies using potent antifungal drugs. Accordingly, it is imperative to establish stringent surveillance on the increasing prevalence of antifungal resistance particularly in low-income regions with limited research resources to prevent increasing prevalence of MDR-C. auris.

ABSTRACT Pneumocystis jirovecii pneumonia (PJP) remains a significant cause of morbidity and mortality during AIDS. In AIDS, the absence of CD4 immunity results in exuberant and often fatal PJP. In addition, organism clearance requires a balanced macrophage response since excessive inflammation promotes lung injury and respiratory failure. Corticosteroids given in addition to antibiotics significantly improve outcomes during PJP. However, concerns exist that corticosteroids further suppress immunity and increase co-infections. New strategies to promote killing and clearance of Pneumocystis while balancing lung inflammation are required. Prior studies have shown that innate immunity to Pneumocystis is mediated by C-type lectin receptors (CLRs) on macrophages and involves downstream CARD9 activation. CARD9 can be targeted by a novel specific small molecule inhibitor (BRD5529) that significantly reduces inflammatory signaling by macrophages. CARD9 serves as the central intracellular molecule through which Dectin-1, Dectin-2, Mincle, and other CLRs signal. Dectin-1 CLR is activated through its own intracytoplasmic domain, whereas other innate CLRs (e.g., Dectin-2 and Mincle) require interactions with a common Fc-gamma receptor (FcγR) accessory chain to mediate responses. We now observe that mice double deficient in both Dectin-1 and Fcer1g (which lack the FcγR gamma chain) exhibit markedly reduced organism clearance compared with Card9 −/− infected animals. These mice also possess deficiencies in immunoglobulin (Ig) Fc receptors directly mediating antibody responses, further implicating altered humoral responses in Pneumocystis killing. We further demonstrate in the Pneumocystis pneumonia (PCP) mouse model that BRD5529 administration successfully suppresses inflammatory cytokines. Our data support that innate immune responses through the CLR-CARD9 axis and humoral response act together to mediate effective responses resulting in optimal organism killing and generation of host inflammatory responses. Furthermore, host lung inflammation during PCP may be successfully reduced with a novel CARD9 small molecule inhibitor. IMPORTANCE Pneumocystis pneumonia (PCP) causes severe respiratory impairment in hosts with suppressed immunity, particularly those with CD4 deficiencies, such as HIV. In addition to lymphocytic immunity, both innate and humoral immunities also participate in host defense against Pneumocystis . In the current studies, we defined the relative roles of CLR receptor-mediated inflammation, as well as FcgR-related inflammation and clearance of Pneumocystis organisms. Our studies reveal important roles for CLR activities for inducing lung inflammation, which can be ameliorated with a novel small molecule inhibitor of the CARD9 adaptor protein that is necessary for CLR signaling. In contrast, FcgR has a dominant role in organism clearance, underscoring an integral role of humoral responses for the elimination of this infection.

ABSTRACT Pneumocystis cyst life forms contain abundant β-glucan carbohydrates, synthesized using β-1,3 and β-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 β-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.

ABSTRACT 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. β-glucans via the EphA2 receptor, resulting in activation and release of proinflammatory cytokines. Herein, we show that in vivo Pneumocystis spp. β-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 β-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 β-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.

Introduction. Pathogen-associated molecular patterns’ (PAMPs) are microbial signatures that are recognized by host myeloid C-type lectin receptors (CLRs). These CLRs interact with micro-organisms via their carbohydrate recognition domains (CRDs) and engage signalling pathways within the cell resulting in pro-inflammatory and microbicidal responses. Gap statement. In this article, we extend our laboratory study of additional CLRs that recognize fungal ligands against Pneumocystis murina and Pneumocystis carinii and their purified major surface glycoproteins (Msgs). Aim. To study the potential of newly synthesized hFc-CLR fusions on binding to Pneumocystis and its Msg. Methods. A library of new synthesized hFc-CLR fusions was screened against Pneumocystis murina and Pneumocystis carinii organisms and their purified major surface glycoproteins (Msgs) found on the respective fungi via modified ELISA. Immunofluorescence assay (IFA) was implemented and quantified to verify results. mRNA expression analysis by quantitative PCR (q-PCR) was employed to detect respective CLRs found to bind fungal organisms in the ELISA and determine their expression levels in the mouse immunosuppressed Pneumocystis pneumonia (PCP) model. Results. We detected a number of the CLR hFc-fusions displayed significant binding with P. murina and P. carinii organisms, and similarly to their respective Msgs. Significant organism and Msg binding was observed for CLR members C-type lectin domain family 12 member A (CLEC12A), Langerin, macrophage galactose-type lectin-1 (MGL-1), and specific intracellular adhesion molecule-3 grabbing non-integrin homologue-related 3 (SIGNR3). Immunofluorescence assay (IFA) with the respective CLR hFc-fusions against whole P. murina life forms corroborated these findings. Lastly, we surveyed the mRNA expression profiles of the respective CLRs tested above in the mouse immunosuppressed Pneumocystis pneumonia (PCP) model and determined that macrophage galactose type C-type lectin (Mgl-1), implicated in recognizing terminal N-acetylgalactosamine (GalNAc) found in the glycoproteins of microbial pathogens was significantly up-regulated during infection. Conclusion. The data herein add to the growing list of CLRs recognizing Pneumocystis and provide insights for further study of organism/host immune cell interactions.

AbstractBackgroundPneumocystis jirovecii pneumonia (PJP) is a life‐threatening infection occurring in patients receiving bendamustine. The poorly defined incidence, particularly when utilizing polymerase chain reaction (PCR)‐based diagnostic techniques, precipitates unclear prophylaxis recommendations. Our objective was to determine the cumulative incidence of PJP diagnosed by single copy target, non‐nested PCR in patients receiving bendamustine.MethodsPatients were evaluated for PJP from initiation of bendamustine through 9 months after the last administration. The cumulative incidence of PJP was estimated using the Aalen–Johansen method. Cox proportional hazard models were used to demonstrate the strength of association between the independent variables and PJP risk.ResultsThis single‐center, retrospective cohort included 486 adult patients receiving bendamustine from 1 January 2006 through 1 August 2019. Most patients received bendamustine‐based combination therapy (n = 461, 94.9%), and 225 (46.3%) patients completed six cycles. Rituximab was the most common concurrent agent (n = 431, 88.7%). The cumulative incidence of PJP was 1.7% (95% CI 0.8%–3.3%, at maximum follow‐up of 2.5 years), after the start of bendamustine (n = 8 PJP events overall). Prior stem cell transplant, prior chemotherapy within 1 year of bendamustine, and lack of concurrent chemotherapy were associated with the development of PJP in univariate analyses. Anti‐Pneumocystis prophylaxis was not significantly associated with a reduction in PJP compared to no prophylaxis (HR 0.37, 95% CI (0.05, 3.04), p = 0.36).ConclusionsOur incidence of PJP below 3.5%, the conventional threshold for prophylaxis implementation, indicates routine anti‐Pneumocystis prophylaxis may not be necessary in this population. Factors indicating a high‐risk population for targeted prophylaxis require further investigation.

Pneumocystis jirovecii , the opportunistic fungus that causes Pneumocystis pneumonia (PCP) in humans, is a significant contributor to morbidity and mortality in immunocompromised patients. Given the profound deleterious inflammatory effects of the major β-glucan cell wall carbohydrate constituents of Pneumocystis through Dectin-1 engagement and downstream caspase recruitment domain-containing protein 9 (CARD9) immune activation, we sought to determine whether the pharmacodynamic activity of the known CARD9 inhibitor BRD5529 might have a therapeutic effect on macrophage innate immune signaling and subsequent downstream anti-inflammatory activity.