Limper, Andrew H.

Pneumocystis sp. are fungal pathogens and members of the Ascomycota phylum. Immunocompetent individuals can readily eliminate the fungus, whereas immunocompromised individuals can develop Pneumocystis jirovecii pneumonia (PJP). Currently, over 500,000 cases occur worldwide, and the organism is listed on the recently released WHO fungal priority pathogens list. Overall, the number of PJP cases over the last few decades in developed countries with the use of highly effective antiretroviral therapy has decreased, but the cases of non-HIV individuals using immunosuppressive therapies have significantly increased. Even with relatively effective current anti-Pneumocystis therapies, the mortality rate remains 30–60% in non-HIV patients and 10–20% during initial episodes of PJP in HIV/AIDS patients. Although the role of alveolar macrophages is well studied and established, there is also well-established and emerging evidence regarding the role of epithelial cells in the immune response to fungi. This mini review provides a brief overview summarizing the innate immune response of the lung epithelium and various continuously cultured mammalian cell lines to Pneumocystis.

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 β-(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.

BACKGROUND: Pulmonary function test (PFT) impairments are common after allogeneic hematopoietic stem cell transplantation. The prognostic significance of these declines on outcomes is not well understood. The objectives were to determine the frequency of declines in pulmonary function (FVC, FEV 1 , and diffusing capacity for carbon monoxide [D LCO ]) in the early post-transplantation period; and to determine the prognostic significance of these declines on mortality or development of bronchiolitis obliterans syndrome. METHODS: This was a retrospective cohort study conducted at Mayo Clinic, Rochester, Minnesota. PFTs were obtained at baseline and at day +100. Competing risk survival models were developed, which accounted for pre-transplantation pulmonary function and relapse status. RESULTS: Between January 1, 2005, and December 31, 2020, 1,145 subjects underwent allogeneic hematopoietic stem cell transplantation and had a pre-transplantation PFT performed. Of these, 900 (78.6%) survived to day 100 and had post-transplantation PFTs performed (median [interquartile range] 97 [94–103] d). A decline of ≥10% in FEV 1, FVC, or D LCO was seen in 401 of 900 subjects (44.5%). Declines of ≥20% in FEV 1 (hazard ratio 1.65, 95% CI 1.07–2.56; P = .02), FVC (hazard ratio 1.72, 95% CI [1.11–2.67]; P = .02), and D LCO (hazard ratio 1.46, 95% CI 1.04–2.07; P = .028) were all associated with reduced survival when compared with those with < 10% decline in PFT measures. These findings were independent of pre-transplantation pulmonary function or relapse status. Bronchiolitis obliterans syndrome was diagnosed in 118 subjects (10.3%), and there was no relationship between early PFT decline and a subsequent diagnosis of bronchiolitis obliterans syndrome. The subjects who received myeloablative conditioning with cyclophosphamide plus total body irradiation or cyclophosphamide plus fludarabine plus total body irradiation were more likely to have lower spirometry values after hematopoietic stem cell transplantation. The subjects who received reduced intensity conditioning or nonmyeloablative conditioning with fludarabine plus total body irradiation were more likely to have higher post–hematopoietic stem cell transplantation FEV 1 , FVC, and D LCO . CONCLUSIONS: An absolute decline of ≥20% in FEV 1 , FVC, or D LCO were associated with reduced survival independent of pre-transplantation pulmonary function or relapse status. In contrast to previous work, early declines in PFT measures were not associated with future development of bronchiolitis obliterans syndrome.

Abstract Pneumocystis pneumonia is caused, at least partly, by inflammatory action of β-glucan, the major component of Pneumocystis cell wall. Glucan induces release of pro-inflammatory cytokines in alveolar macrophages and lung epithelial cells. While macrophages recognize glucan mainly by dectin receptors, epithelial cells utilize membrane glycosphingolipid (GSL) lactosyl ceramide (LacCer) as glucan-binding molecule. The use of GSL inhibitors in vitro causes reduced NF-κB activation and inhibited IL-8 release in epithelial cells. To assess the impact of LacCer in lung inflammation, we treated Pneumocystis-infected mice with GSL inhibitor PDMP. The animals were immunosuppressed by GK 1.5 antibody, infected with Pneumocystis by intratracheal injection and, starting at 8 weeks post infection, treated with either 150 mg PDMP/kg of body weight - dissolved in saline, or equal volume of saline alone (daily i.p. injections for 10 days). The analysis of sacrificed animals showed that PDMP-treated mice had fewer leukocytes in the bronchoalveolar lavage fluid (BALF) than saline-treated controls with the most marked difference in the number of neutrophils. Interestingly, BALF concentrations of MIP-2 and TNF-α (determined by ELISA) did not differ between these two groups. The analysis of Pneumocystis burden in the lung, as determined by Q-PCR, showed a trend of decreased Pneumocystis-specific DNA copies in PDMP-treated animals. These results suggest that PDMP might have an impact on inflammatory response to Pneumocystis. Other GSL inhibitors are yet to be tested in similar in vivo experiments. This might lead to new options in treatment of Pneumocystis pneumonia. Supported by NIH RO1 HL62150 and Mayo

Abstract Background Acute respiratory exacerbations in idiopathic pulmonary fibrosis (IPF), often triggered by infection, are life threatening events characterized by rapid deterioration of lung function and associated with poor prognosis. Since microbial antigens modulate immunity by activating different pattern recognition receptors (PRR) expressed by innate immune cells (including B-cells), we sought to investigate how the activation of human B-cells through PRR stimulation by different microbial (CpG and β-glucan) and non-microbial (Mitochondrial DAMPs; MTDs) antigens resulted in the release of an inflammatory/fibrotic milieu that contributed to the pathogenesis of fibrosis in patients with IPF. Results Our results show that an inflammatory milieu is induced by stimulating circulating B-cells with both noninfectious (MTDs) and infectious antigens (CpG and β-glucan) commonly found in bacteria and fungi. Moreover, we show that the inflammatory milieu produced was specific to the inducing antigen, and that the microbial antigens (CpG and β-glucan) triggered distinct signaling pathways in B-cells. Specifically, CpG induced mTOR-activation while β-glucan was mTOR-independent and activated Src and p38. Furthermore, our results also show that B-cell aggregates are present within fibrotic areas of the lung in IPF patients and activated B-cells induce fibroblast migration, which was modified by antifibrotics (nintedanib and pirfenidone). Conclusion Our results highlight the potential contribution of activated B-cells through PRRs to the proinflammatory and profibrotic milieu seen in patients with idiopathic pulmonary fibrosis.

Abstract Glycosphingolipid (GSL) Lactosyl Ceramide (LacCer) is thought to be involved in Pneumocystis interaction with the host. Our previous results show that treatment with glycosphingolipid synthesis inhibitor PDMP reduces the fungus lung burden as well as symptoms of inflammation in mice with Pneumocystis pneumonia (PCP). Experiments were set up to resolve whether anti-inflammatory effects were due to the specific mechanisms involved in innate immune responses to Pneumocystis, or to some general anti-inflammatory property of PDMP. Animals were infected with live organisms, or challenged with either beta-glucan (the pro-inflammatory component of Pneumocystis cell wall) or LPS (inducer of lung inflammation unrelated to Pneumocystis). At the experimental endpoint the following inflammatory parameters were assessed: leukocyte recruitment to the lungs, production of pro-inflammatory cytokines and chemokines, histopathology and lung damage. Results show that glucan-induced acute inflammation partially mimics the inflammation caused by the whole organism, and PDMP suppresses some of the inflammatory parameters affected in PCP. In contrast, although LPS induces comparable intensity of inflammation as live Pneumocystis or glucan, PDMP does not suppress any inflammatory parameters measured in this group. These results suggest that the mechanism of PDMP action is related to the features specific to Pneumocystis and host recognition of the microorganism. Further studies will attempt to determine the role of LacCer in these processes. Supported by NIH-RO1-HL62150 and Mayo Clinic

Abstract Alveolar macrophages (AMS) initiate inflammation during Pneumocystis carinii pneumonia by releasing cytokines including TNF-alpha. Recent studies suggest that macrophage responses to P. carinii are enhanced by serum opsonization, but the mechanisms of enhancement are not well defined. To determine whether macrophage release of TNF-alpha in response to P. carinii was augmented by immune opsonization, alveolar macrophages obtained from rabbits were cultured with P. carinii that had been opsonized with either nonimmune rabbit serum, immune serum generated against P. carinii, or an affinity-purified polyclonal Ab recognizing the major P. carinii surface Ag gp120. Each experiment also included organisms maintained in media alone (nonopsonized P. carinii). Opsonization of P. carinii with immune serum or gp120 Ab significantly enhanced macrophage TNF-alpha release. Interestingly, however, opsonization with nonimmune serum also increased TNF-alpha response to the organism. Because P. carinii is known to interact with the adhesive glycoproteins, vitronectin (VN) and fibronectin (FN), we hypothesized that they might also augment TNF-alpha release. Opsonization of P. carinii with VN or FN resulted in significant potentiation of macrophage TNF-alpha liberation. We further determined that VN and FN were present in increased quantities in the lower respiratory tract of patients with P. carinii pneumonia compared with normal volunteers. Additionally, VN and FN were demonstrated on the surface of freshly isolated P. carinii organisms by immunoblot analysis. Our study suggests that immune and nonimmune opsonins contribute to host defenses during P. carinii pneumonia by enhancing regional TNF-alpha release in response to the organism.