Project description:Pneumocystis is a pathogen of immunocompromised hosts but can also infect healthy hosts, in whom infection is rapidly controlled and cleared. To better understand the immune mechanisms contributing to clearance of infection, microarray methods were used to examine differential gene expression in the lungs of C57BL/6 and CD40 ligand knock-out (CD40L-KO) mice over time following exposure to Pneumocystis. Immuncompetent C57BL/6 mice, which control and clear infection efficiently, showed a robust response to infection characterized by the upregulation of 349 primarily immune-response associated genes. Temporal changes in the expression of these genes suggested that there was an early (week 2) primarily innate response, that waned without controlling infection; this were followed by primarily adaptive immune responses that peaked at week 5 and successfully cleared the infection. In conjunction with the latter, there was an increased expression of B cell associated (immunoglobulin) genes at week 6 that persisted through 11 weeks. In contrast, CD40L-KO mice, which are highly susceptible to developing severe Pneumocystis pneumonia, showed essentially no upregulation of immune-response associated genes at days 35 to 75. Immunohistochemical staining supported these observations by demonstrating an increase in CD4+, CD68+, and CD19+ cells in C57BL/6 but not CD40L-KO mice. Thus, the healthy host demonstrates a robust biphasic response to infection by Pneumocystis; CD40 ligand is an essential upstream regulator of the adaptive immune responses that efficiently control infection and prevent development of progressive pneumonia. Keywords: Time course response Pneumocystis murina infection wild type versus CD40L-KO mice In experiment 1, C57BL/6 and CD40L-KO mice were co-housed in 2 cages; one cage was exposed to a P. murina-infected seeder, while the second was unexposed. Mice (3 per group) were sacrificed at day 32. Because the initial study demonstrated very little change in gene expression in CD40L-KO mice, only C57BL/6 mice were used for experiments 2 and 3. In Experiment 2, three cages (10 animals/cage) were set up: 2 cages were exposed to P. murina, and one was unexposed. Animals (5 per cage) were sacrificed at days 34 and 41. In Experiment 3, which focused on gene expression early after exposure to P. murina, five cages (9-10 animals/cage) were set up, of which 3 were exposed and 2 were unexposed. Animals (1-3 per cage) were sacrificed at days 7, 14 and 21 of exposure. Experiment 4 was designed to verify the initial results in CD40L-KO mice, and included 5 unexposed controls and 4 mice exposed for 35 days.

Project description:Pneumocystis pneumonia is the most common serious opportunistic infection in patients with HIV/AIDS. Furthermore, Pneumocystis pneumonia is a feared complication of the immunosuppressive drug regimens used to treat autoimmunity, malignancy, and post-transplantation rejection. With an increasing at-risk population, there is a strong need for novel approaches to discover diagnostic and vaccine targets. There are multiple challenges to finding these targets, however. First, Pneumocystis has a largely unannotated genome. To address this, we evaluated each protein encoded within the Pneumocystis genome to that of other fungi using NCBI Blast. Second, Pneumocystis relies on a multiphasic life cycle, as both the transmissible form, the ascus, and the replicative form, the troph, reside within the alveolar space of the host. Towards that end, we purified asci and trophs from Pneumocystis murina and utilized transcriptomics to identify differentially regulated genes. Two such genes, Arp9 and Sp, are differentially regulated in the ascus and the troph, respectively, and can be utilized to characterize the state of the Pneumocystis life cycle in vivo. Gsc1, a β-1,3-glucan synthase with a large extracellular domain previously identified using surface proteomics, was more highly expressed on the ascus form of Pneumocystis. GSC-1 ectodomain immunization generated a strong antibody response capable of recognizing the surface of the Pneumocystis asci. GSC-1 ectodomain immunization was also capable of reducing ascus burden following primary challenge with Pneumocystis murina. Finally, mice immunized with the GSC-1 ectodomain had limited burden following natural transmission of Pneumocystis using a co-housing model. Pneumocystis asci and trophs were separated via flow cytometry and the transcriptome was sequenced, allowing to further understand the differential expression of various RNA transcripts. These data can be mined for life-form specific diagnostics and therapeutic targets.

Project description:Background: Pneumocystis jirovecii pneumonia (PJP) causes high morbidity and mortality in immunocompromised patients. Current therapeutic regimens for PJP are limited and thus alternative treatment options are needed. Results: In this study, we demonstrate that Pneumocystis murina (P. murina, a model of P. jirovecii infection) can survive and persist within bone marrow-derived macrophages (BMDMs), alveolar macrophages (AMs) and J774A macrophages. Modeling malnutrition, a risk factor for PJP, we found that nutrient deficient media enhanced the capacity for intracellular survival of the fungus. M-CSF boosted intracellular survival capability in starvation media in BMDMs. In vivo infection with P. murina promoted M2, not M1, macrophage polarization which was evident beginning. At 6 weeks post-infection, this polarization returned to baseline in wild type mice that clear the infection but remained elevated in IL-21R deficient mice, that fail to clear the infection. The activated M2 macrophages driven by IL4 and IL13 consisted of AMs and interstitial macrophages (IMs) and were derived from monocytes. AMs isolated from P. murina-infected mice supported continued survival of P. muria ex vivo, suggesting that AMs harbor P. murina in vivo. Immunofluorescence staining analysis showed co-localization of the P. murina antigen and the CD209-recognized mannan-containing major surface glycoprotein on the surface of P. murina within infected BMDMs and cultured AMs isolated from bronchoalveolar lavage fluid of infected mice. Immunohistochemistry analysis showed that P. murina staining colocalized with IMs in infected mouse lungs. The lung macrophage population and the lung burden are both significantly higher in Rag2-/- mice than that in WT mice at week 4. To determine if macrophages serve as a reservoir for the fungus we administered clodronate liposomes and observed a significant reduction of P. murina in vivo through depletion of lung macrophages. Conclusion: Our findings reveal that P. murina organisms are facultative intracellular fungal pathogens of macrophages. P. murina infection promoted monocyte-derived M2 macrophage activation, which comprises both AMs and IMs. Both Lung macrophage populations and fungal burdens in infected Rag2-/- mice are higher than that in infected C57BL/6 mice, suggestiong that lung macrophages are a reservoir and might be a proliferative niche for the fungus. Clodronate administration supports that macrophages are important host reservoir for the fungus, Therefore augmenting macrophage fungicidal activity is an important strategy for host directed therapies to enhance resolution of this infection.

			Action	DRS
	SRR12313714_1.fastq.gz	Fastqsanger.gz
	SRR12313714_2.fastq.gz	Fastqsanger.gz

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