We explored differential polarization of macrophages during infection using a rat model of Pneumocystis pneumonia. We observed enhanced pulmonary M1 macrophage polarization in immunosuppressed (IS) hosts, but an M2 predominant response in immunocompetent (IC) hosts following Pneumocystis carinii challenge. Increased inflammation and inducible nitric oxide synthase (iNOS) levels characterized the M1 response. However, macrophage ability to produce nitric oxide was defective. In contrast, the lungs of IC animals revealed a prominent M2 gene signature, and these macrophages effectively elicited an oxidative burst associated with clearance of Pneumocystis. In addition, during P. carinii infection the expression of Dectin-1, a critical receptor for recognition and clearance of P. carinii, was upregulated in macrophages of IC animals but suppressed in IS animals. In the absence of an appropriate cytokine milieu for M2 differentiation, Pneumocystis induced an M1 response both in vitro and in vivo. The M1 response induced by P. carinii was plastic in nature and reversible with appropriate cytokine stimuli. Finally, we tested whether macrophage polarization can be modulated in vivo and used to help manage the pathogenesis of Pneumocystis pneumonia by adoptive transfer. Treatment with both M1 and M2 cells significantly improved survival of P. carinii-infected IS hosts. However, M2 treatment provided the best outcomes with efficient clearance of P. carinii and reduced inflammation.

Pneumocystis pneumonia (PCP) remains a major cause of morbidity and mortality within immunocompromised patients. In this study, we examined the potential role of Mincle (Macrophage inducible C-type lectin) for host defense against Pneumocystis. Binding assays implementing soluble Mincle Carbohydrate Recognition Domain (CRD) fusion proteins demonstrated binding to intact Pneumocystis carinii (Pc) as well as to organism homogenates, and purified major surface glycoprotein/glycoprotein A derived from the organism. Additional experiments showed that rats with Pneumocystis pneumonia (PCP) expressed increased Mincle mRNA levels. Mouse macrophages over-expressing Mincle displayed increased binding to Pc life forms and enhanced protein tyrosine phosphorylation. The binding of Pc to Mincle resulted in activation of Fc receptor γ (FcRγ) mediated cell signaling. RNA silencing of Mincle in mouse macrophages resulted in decreased activation of Syk kinase after Pc challenge, critical in downstream inflammatory signaling. Mincle deficient CD-4 depleted (Mincle−/−) mice showing a significant defect in organism clearance from the lungs with higher organism burdens and altered lung cytokine responses during Pneumocystis murina (Pm) pneumonia. Interestingly, Mincle−/− did not demonstrate worsened survival during PCP compared to wild type mice, despite the markedly increased organism burdens. This may be related to increased expression of anti-inflammatory factors such as IL-1Ra during infection in the Mincle−/− mice. Of note, the Pm infected Mincle−/− mice demonstrated increased expression of known C-type lectin receptors Dectin-1, Dectin-2, and MCL compared to infected wild type mice. Taken together, these data support a significant role for Mincle in Pneumocystis modulating host defense during infection.

Inflammation is a major cause of respiratory impairment during Pneumocystis pneumonia. Studies support a significant role for cell wall ␤-glucans in stimulating inflammatory responses. Fungal ␤-glucans are comprised of D-glucose homopolymers containing ␤-1,3-linked glucose backbones with ␤-1,6-linked glucose side chains. Prior studies in Pneumocystis carinii have characterized ␤-1,3 glucan components of the organism. However, recent investigations in other organisms support important roles for ␤-1,6 glucans, predominantly in mediating host cellular activation. Accordingly, we sought to characterize ␤-1,6 glucans in the cell wall of Pneumocystis and to establish their activity in lung cell inflammation. Immune staining revealed specific ␤-1,6 localization in P. carinii cyst walls. Homology-based cloning facilitated characterization of a functional P. carinii kre6 (Pckre6) ␤-1,6 glucan synthase in Pneumocystis that, when expressed in kre6-deficient Saccharomyces cerevisiae, restored cell wall stability. Recently synthesized ␤-1,6 glucan synthase inhibitors decreased the ability of isolated P. carinii preparations to generate ␤-1,6 carbohydrate. In addition, isolated ␤-1,6 glucan fractions from Pneumocystis elicited vigorous tumor necrosis factor alpha (TNF-␣) responses from macrophages. These inflammatory responses were significantly dampened by inhibition of host cell plasma membrane microdomain function. Together, these studies indicate that ␤-1,6 glucans are present in the P. carinii cell wall and contribute to lung cell inflammatory activation during infection. P neumocystis organisms are opportunistic fungi that produce significant morbidity and mortality in immunocompromised hosts, with infection-related fatalities ranging between 10% and 45% (1). Pneumocystis jirovecii is the species known to infect humans, while Pneumocystis carinii represents the parallel species studied widely in rodents (2). Pneumocystis pneumonia remains a significant cause of mortality during AIDS, despite highly active antiretroviral therapy (3-5). Severe Pneumocystis pneumonia is characterized by intense lung inflammation involving CD8 ϩ cells and neutrophils, impairing gas exchange (6-9).The P. carinii cell walls contain abundant ␤-glucan molecules (10). Fungal cell wall ␤-glucans are homopolymers of D-glucose consisting of ␤-1,3 core chains with variable numbers of ␤-1,6 glucose side chains. The variable inflammatory activities of different glucan preparations have been postulated to be related to the relative amounts and configurations of these two major structures (␤-1,3 versus ␤-1,6) (11). Almost all of the initial studies in fungi have largely focused on unfractionated glucans (10). In fact, all prior studies in P. carinii previously utilized only unfractionated ␤-1,3/␤-1,6 glucans. Interestingly, recent investigations in Saccharomyces cerevisiae indicate major roles for ␤-1,6 glucans in strongly mediating cellular activation and inflammation (11). Our investigations of unfractionated P. carinii ␤-glucans indicate that innate ...

Pneumocystis is an important fungal pathogen that causes life-threatening pneumonia in patients with AIDS and malignancy. Lung fungal pathogens are recognized by C-type lectin receptors (CLRs), which bind specific ligands and stimulate innate immune responses. The CLR Dectin-1 was previously shown to mediate immune responses to Pneumocystis spp. For this reason, we investigated a potential role for Dectin-2. Rats with Pneumocystis pneumonia (PCP) exhibited elevated Dectin-2 mRNA levels. Soluble Dectin-2 carbohydrate-recognition domain fusion protein showed binding to intact Pneumocystis carinii (Pc) and to native Pneumocystis major surface glycoprotein/glycoprotein A (Msg/gpA). RAW macrophage cells expressing V5-tagged Dectin-2 displayed enhanced binding to Pc and increased protein tyrosine phosphorylation. Furthermore, the binding of Pc to Dectin-2 resulted in Fc receptor-γ-mediated intracellular signaling. Alveolar macrophages from Dectin-2-deficient mice (Dectin-2) showed significant decreases in phospho-Syk activation after challenge with Pc cell wall components. Stimulation of Dectin-2 alveolar macrophages with Pc components showed significant decreases in the proinflammatory cytokines IL-6 and TNF-α. Finally, during infection with Pneumocystis murina, Dectin-2 mice displayed downregulated mRNA expression profiles of other CLRs implicated in fungal immunity. Although Dectin-2 alveolar macrophages had reduced proinflammatory cytokine release in vitro, Dectin-2 deficiency did not reduce the overall resistance of these mice in the PCP model, and organism burdens were statistically similar in the long-term immunocompromised and short-term immunocompetent PCP models. These results suggest that Dectin-2 participates in the initial innate immune signaling response to Pneumocystis, but its deficiency does not impair resistance to the organism.

N-acetylglucosamine (GlcNAc) serves as an essential structural sugar on the cell surface of organisms. For example, GlcNAc is a major component of bacterial peptidoglycan, it is an important building block of fungal cell walls, including a major constituent of chitin and mannoproteins, and it is also required for extracellular matrix generation by animal cells. Herein, we provide evidence for a uridine diphospho (UDP)-GlcNAc pathway in Pneumocystis species. Using an in silico search of the Pneumocystis jirovecii and P. murina (Pm) genomic databases, we determined the presence of at least four proteins implicated in the Saccharomyces cerevisiae UDP-GlcNAc biosynthetic pathway. These genes, termed GFA1, GNA1, AGM1, and UDP-GlcNAc pyrophosphorylase (UAP1), were either confirmed to be present in the Pneumocystis genomes by PCR, or, in the case of Pm uap1 (Pmuap1), functionally confirmed by direct enzymatic activity assay. Expression analysis using quantitative PCR of Pneumocystis pneumonia in mice demonstrated abundant expression of the Pm uap1 transcript. A GlcNAc-binding recombinant protein and a novel GlcNAc-binding immune detection method both verified the presence of GlcNAc in P. carinii (Pc) lysates. Studies of Pc cell wall fractions using highperformance gas chromatography/mass spectrometry documented the presence of GlcNAc glycosyl residues. Pc was shown to synthesize GlcNAc in vitro. The competitive UDP-GlcNAc substrate synthetic inhibitor, nikkomycin Z, suppressed incorporation of GlcNAc by Pc preparations. Finally, treatment of rats with Pneumocystis pneumonia using nikkomycin Z significantly reduced organism burdens. Taken together, these data support an important role for GlcNAc generation in the cell surface of Pneumocystis organisms.

AbstractAimsSuccessful management of IPF will likely require multi-drug therapy as its pathogenesis is thought to be both driven by both pro-inflammatory and pro-fibrotic pathways. We hypothesized that the available anti-fibrotic agents, pirfenidone and nintedanib, may exhibit synergy in suppressing lung fibroblast extracellular matrix protein generation when administered in combination with other orally active agents.Materials and MethodsA fibroblastic cell line (AKR-2B) was stimulated with TGF-β1 and used to screen a library of over 1500 FDA approved drugs. Extracellular matrix protein generation was assessed via fibronectin ELISA assay and maintenance of cell viability confirmed with XTT assay.ResultsThe screening revealed sixty-two drugs from the repurposed drug-screening library that were shown to significantly suppress fibronectin expression and not result in cell death. Specifically drugs within the category of NSAIDs, steroids, azole antifungal agents, and antipyrine were associated with significant suppression of fibronectin on ELISA analysis. Surprisingly, we observed anti-fibrotic activity across a number of the azole antifungal compounds. We next assessed whether combination of azoles would exhibit synergy when combined with current anti-fibrotic therapies in the stimulated fibroblasts. As proof of concept, we demonstrated in vitro synergy between oxiconazole and nintedanib in suppressing fibroblast generation of extracellular matrix fibronectin.ConclusionsThese results suggest an approach to identify potential combinations of therapy that may improve patient outcomes by reducing cost and potential toxicities during treatment.