Pneumocystis Murina Infection Research Articles

CD4, but not Cxcr6, is necessary for control of Pneumocystis murina infection

CD4+ T cells are critical to control of Pneumocystis infection, and Cxcr6 has been shown to be upregulated in these cells during infection, but the roles of CD4 and Cxcr6 in this setting are undefined. To explore this, mice deficient in CD4 or Cxcr6 expression were utilized in a co-housing mouse model that mimics the natural route of Pneumocystis infection. Organism load and anti-Pneumocystis antibodies were assayed over time, and immunohistochemistry, flow cytometry, and quantitative PCR were used to characterize host immune responses during infection. CD4 was found to be necessary for clearance of Pneumocystis murina, though partial control was seen in it's absence; based on ThPOK expression, double negative T cells with T helper cell characteristics may be contributing to this control. Using a Cxcr6 deficient mouse expressing gfp, control of infection in the absence of Cxcr6 was similar to that in heterozygous control mice. It is noteworthy that gfp + cells were seen in the lungs with similar frequencies between the 2 strains. Interferon-ɣ and chemokine/ligands Cxcr3, Cxcl9, and Cxcl10 increased during P. murina infection in all models. Thus, CD4, but not Cxcr6, is needed for clearance of P. murina infection.

AIDμS double knockout mice but not AID single knockout mice are highly susceptible to PneumocystisInfection

Abstract Pneumocystispneumonia can be a life-threatening disease to individuals who are immunocompromised due to infection, such as HIV, receiving immunotherapy drugs including corticosteroids or due to genetic defect such as hyper IgM syndrome. B cells are important to the defense and clearance of Pneumocystisinfection; however, the exact role has yet to be elucidated. Prior studies have demonstrated that B cell antigen presentation and priming of CD4 +T cells plays a major role in Pneumocystisclearance, while passive transfer of immune serum can prevent Pneumocystisinfection in mice. To better understand the B cell role in control and clearance of Pneumocystisinfection, using a natural exposure model, we compared the kinetics of Pneumocystis murinainfection in activation-induced cytidine deaminase (AID −/−), AIDμS −/−double knockout, and wild-type C57BL/6 mice. By 5 – 6 weeks post initial exposure all three murine strains were infected with Pneumocystis murina, detected by qPCR of the single copy gene dihydrofolate reductase (DHFR). However, after 10 weeks of infection only the AIDμS −/−double knockout mice continued to have high levels of Pneumocystisinfection. These data suggests that deficiency of B cells to class switch, such as in the AID −/−strain, is not in itself enough to confer increased susceptibility to Pneumocystis. However, in combination with μS deficiency, which inhibits the secretion of antibodies, it is enough to debilitate the immune response resulting in uncontrolled Pneumocystisinfection. Intramural Research Program of the NIH Clinical Center

The Effects of Sex and Strain on Pneumocystis murina Fungal Burdens in Mice.

Many preclinical studies of infectious diseases have neglected experimental designs that evaluate potential differences related to sex with a concomitant over-reliance on male model systems. Hence, the NIH implemented a monitoring system for sex inclusion in preclinical studies. Methods: Per this mandate, we examined the lung burdens of Pneumocystis murina infection in three mouse strains in both male and female animals at early, mid, and late time points. Results: Females in each strain had higher infection burdens compared to males at the later time points. Conclusion: Females should be included in experimental models studying Pneumocystis spp.

Pulmonary Interleukin-17-Positive Lymphocytes Increase during Pneumocystis murina Infection but Are Not Required for Clearance of Pneumocystis.

Pneumocystis remains an important pathogen of immunosuppressed patients, causing a potentially life-threatening pneumonia. Despite its medical importance, the immune responses required to control infection, including the role of interleukin-17 (IL-17), which is important in controlling other fungal infections, have not been clearly defined. Using flow cytometry and intracellular cytokine staining after stimulation with phorbol myristate acetate and ionomycin, we examined gamma interferon (IFN-γ), IL-4, IL-5, and IL-17 production by lung lymphocytes in immunocompetent C57BL/6 mice over time following infection with Pneumocystismurina We also examined the clearance of Pneumocystis infection in IL-17A-deficient mice. The production of both IFN-γ and IL-17 by pulmonary lymphocytes increased during infection, with maximum production at approximately days 35 to 40, coinciding with peak Pneumocystis levels in the lungs, while minimal changes were seen in IL-4- and IL-5-positive cells. The proportion of cells producing IFN-γ was consistently higher than for cells producing IL-17, with peak levels of ∼25 to 30% of CD3+ T cells for the former compared to ∼15% for the latter. Both CD4+ T cells and γδ T cells produced IL-17. Administration of anti-IFN-γ antibody led to a decrease in IFN-γ-positive cells, and an increase in IL-5-positive cells, but did not impact clearance of Pneumocystis infection. Despite the increases in IL-17 production during infection, IL-17A-deficient mice cleared Pneumocystis infection with kinetics similar to C57BL/6 mice. Thus, while IL-17 production in the lungs is increased during Pneumocystis infection in immunocompetent mice, IL-17A is not required for control of Pneumocystis infection.

Characterization of chemokine and chemokine receptor expression during Pneumocystis infection in healthy and immunodeficient mice

We examined gene expression levels of multiple chemokines and chemokine receptors during Pneumocystis murina infection in wild-type and immunosuppressed mice, using microarrays and qPCR. In wild-type mice, expression of chemokines that are ligands for Ccr2, Cxcr3, Cxcr6, and Cxcr2 increased at days 32–41 post-infection, with a return to baseline by day 75–150. Concomitant increases were seen in Ccr2, Cxcr3, and Cxcr6, but not in Cxcr2 expression. Induction of these same factors also occurred in CD40-ligand and CD40 knockout mice but only at a much later time-point, during uncontrolled Pneumocystis pneumonia (PCP). Expression of CD4 Th1 markers was increased in wild-type mice during clearance of infection. Ccr2 and Cx3cr1 knockout mice cleared Pneumocystis infection with kinetics similar to wild-type mice, and all animals developed anti-Pneumocystis antibodies. Upregulation of Ccr2, Cxcr3, and Cxcr6 and their ligands supports an important role for T helper cells and mononuclear phagocytes in the clearance of Pneumocystis infection. However, based on the current and prior studies, no single chemokine receptor appears to be critical to the clearance of Pneumocystis.

Alterations of dendritic cells, inflammatory monocytes and macrophages in mice during Pneumocystis murina infection

Objective To investigate the alterations and phenotypes of dendritic cells, inflammatory monocytes and macrophages in immunocompetent mice during Pneumocystis murina (P.murina) infection for further analysis of the function of these cells during P. murina infection. Methods Wild type male C57BL/6 mice at age 6-8 weeks were randomly divided into two groups including the group with P. murina infection and the group receiving sham surgery. The mice without any intervention were used to set up the blank control group. The loads of P. murina strains in lung tissues of each mouse were quantified by TaqMan real-time fluorescence polymerase chain reaction after the infection. Histopathological examination was performed to evaluate the degree of inflammation in lung tissues. The numbers of dendritic cells, inflammatory monocytes and macrophages in lung tissues, peripheral blood and bone marrow samples, and the changes of inflammatory monocytes in spleen tissues were measured by flow cytometry analysis. The expression of major histocompatability complex Ⅱ (MHCⅡ), CX3C chemokine receptor 1 (CX3CR1) and CC chemokine receptor 2 (CCR2) by dendritic cells, inflammatory monocytes and macrophages in lung tissues during P. murina infection were analyzed by flow cytometry analysis. All of the data were collected one, two, three and four weeks after the corresponding treatments. Results The loads of P. murina strains in P. murina infected mice were elevated after two and three weeks infection, but decline at week 4 (P>0.05). Significant pathological changes including the alveolar destruction, inflammatory cell infiltration and thickened alveolar septum in mice with P. murina infection were observed under a microscope at week 3 and week 4. Compared to the sham surgery treatment group, the number of CD11c+ CD11b+ dendritic cells were increased in lung tissues, but decreased in blood samples during P. murina infection (P 0.05). The CD11c+ CD11b+ dendritic cells in lung tissues of mice with P. murina infection expressed high levels of MHCⅡ and CX3CR1, and low levels of CCR2. The inflammatory monocytes in lung tissues of mice expressed high levels of CCR2, moderate levels of MHCⅡ and low levels of CX3CR1 during P. murina infection. High levels of CX3CR1 and low levels of MHCⅡ and CCR2 were observed in macrophages from lung tissues of mice with P. murina infection. Conclusion Highly expressed CD11c+ CD11b+ dendritic cells and MHCⅡ were detected in lung tissues of mice during P. murina infection, indicating that CD11c+ CD11b+ dendritic cells were involved in the host defense against P. murina infection. Key words: Pneumocystis murina infection; Dendritic cells; Inflammatory monocytes; Macrophages

Clearance of Pneumocystis murina infection is not dependent on MyD88

To determine if myeloid differentiation factor 88 (MyD88), which is necessary for signaling by most TLRs and IL-1Rs, is necessary for control of Pneumocystis infection, MyD88-deficient and wild-type mice were infected with Pneumocystis by exposure to infected seeder mice and were followed for up to 106 days. MyD88-deficient mice showed clearance of Pneumocystis and development of anti-Pneumocystis antibody responses with kinetics similar to wild-type mice. Based on expression levels of select genes, MyD88-deficient mice developed immune responses similar to wild-type mice. Thus, MyD88 and the upstream pathways that rely on MyD88 signaling are not required for control of Pneumocystis infection.

B Cell Production of Tumor Necrosis Factor in Response to Pneumocystis murina Infection in Mice

Pneumocystis species are opportunistic fungal pathogens that induce tumor necrosis factor (TNF) production by alveolar macrophages. Here we report that B cells from the draining lymph nodes as well as lung CD4(+) T cells are important producers of TNF upon Pneumocystis murina infection. To determine the importance of B cell-derived TNF in the primary response to P. murina, we generated bone marrow chimeras whose B cells were unable to produce TNF. The lung P. murina burden at 10 days postinfection in TNF knockout (TNFKO) chimeras was significantly higher than that in wild-type (WT) chimeras, which corresponded to reduced numbers of activated CD4(+) T cells in the lungs at this early time point. Furthermore, CD4(+) T cells isolated from P. murina-infected TNFKO chimeras were unable to stimulate clearance of P. murina upon adoptive transfer to recombinase-deficient (RAG1KO) hosts. Together, these data indicate that B cell-derived TNF plays an important function in promoting CD4(+) T cell expansion and production of TNF and facilitating protection against P. murina infection.

Alveolar Macrophages in Neonatal Mice Are Inherently Unresponsive to Pneumocystis murina Infection

Pneumocystis pneumonia was first diagnosed in malnourished children and has more recently been found in children with upper respiratory symptoms. We previously reported that there is a significant delay in the immune response in newborn mice infected with Pneumocystis compared to adults (Garvy BA, Harmsen AG, Infect. Immun. 64:3987-3992, 1996, and Garvy BA, Qureshi M, J. Immunol. 165:6480-6486, 2000). This delay is characterized by the failure of neonatal lungs to upregulate proinflammatory cytokines and attract T cells into the alveoli. Here, we report that regardless of the age at which we infected the mice, they failed to mount an inflammatory response in the alveolar spaces until they were 21 days of age or older. Anti-inflammatory cytokines had some role in dampening inflammation, since interleukin-10 (IL-10)-deficient pups cleared Pneumocystis faster than wild-type pups and the neutralization of transforming growth factor beta (TGF-β) with specific antibody enhanced T cell migration into the lungs at later time points. However, the clearance kinetics were similar to those of control pups, suggesting that there is an intrinsic deficiency in the ability of innate immunity to control Pneumocystis. We found, using an adoptive transfer strategy, that the lung environment contributes to association of Pneumocystis organisms with alveolar macrophages, implying no intrinsic deficiency in the binding of Pneumocystis by neonatal macrophages. Using both in vivo and in vitro assays, we found that Pneumocystis organisms were less able to stimulate translocation of NF-κB to the nucleus of alveolar macrophages from neonatal mice. These data indicate that there is an early unresponsiveness of neonatal alveolar macrophages to Pneumocystis infection that is both intrinsic and related to the immunosuppressive environment found in neonatal lungs.

Thymopoietic and Bone Marrow Response to Murine Pneumocystis Pneumonia

CD4(+) T cells play a key role in host defense against Pneumocystis infection. To define the role of naïve CD4(+) T cell production through the thymopoietic response in host defense against Pneumocystis infection, Pneumocystis murina infection in the lung was induced in adult male C57BL/6 mice with and without prior thymectomy. Pneumocystis infection caused a significant increase in the number of CCR9(+) multipotent progenitor (MPP) cells in the bone marrow and peripheral circulation, an increase in populations of earliest thymic progenitors (ETPs) and double negative (DN) thymocytes in the thymus, and recruitment of naïve and total CD4(+) T cells into the alveolar space. The level of murine signal joint T cell receptor excision circles (msjTRECs) in spleen CD4(+) cells was increased at 5 weeks post-Pneumocystis infection. In thymectomized mice, the numbers of naïve, central memory, and total CD4(+) T cells in all tissues examined were markedly reduced following Pneumocystis infection. This deficiency of naïve and central memory CD4(+) T cells was associated with delayed pulmonary clearance of Pneumocystis. Extracts of Pneumocystis resulted in an increase in the number of CCR9(+) MPPs in the cultured bone marrow cells. Stimulation of cultured bone marrow cells with ligands to Toll-like receptor 2 ([TLR-2] zymosan) and TLR-9 (ODN M362) each caused a similar increase in CCR9(+) MPP cells via activation of the Jun N-terminal protein kinase (JNK) pathway. These results demonstrate that enhanced production of naïve CD4(+) T lymphocytes through the thymopoietic response and enhanced delivery of lymphopoietic precursors from the bone marrow play an important role in host defense against Pneumocystis infection.

Galleria mellonella are Resistant to Pneumocystis murina Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.

Efficient resolution of Pneumocystis murina infection in surfactant protein A-deficient mice following withdrawal of corticosteroid-induced immunosuppression

Following withdrawal of immunosuppression, surfactant protein A (SP-A)-deficient and wild-type mice cleared Pneumocystis murina infection in a similar manner, but exhibited significant differences in lymphocyte populations, interleukin (IL)-6 levels and chemokine expression levels. A higher percentage of lymphocytes were detected in lung lavage fluid from SP-A-deficient mice, but more CD4+ T cells were isolated from lung tissue of wild-type mice. Higher concentrations of IL-6 were detected in lavage fluid and enhanced expression of lymphotactin and RANTES were detected in the lungs of wild-type mice. Equal levels of surfactant protein D were detected in SP-A-deficient and wild-type mice and no differences were detected in markers of lung injury between the two strains of mice. Thus, SP-A does not enhance organism clearance, but does modulate the host immune response during resolution of P. murina infection.

Resolution of Pneumocystis murina infection following withdrawal of corticosteroid induced immunosuppression

The host response during resolution of Pneumocystis murina infection following withdrawal of Dexamethasone (Dex) induced immunosuppression was analyzed. Mice were inoculated with P. murina and treated with Dex for 4 weeks. Treatment was stopped and mice were sacrificed at d1, d7, and d14. Control mice were treated in the same manner, but were inoculated with nonviable P. murina. P. murina was actively cleared from the lungs following withdrawal of Dex treatment. No P. murina was detected in control mice. Significantly more neutrophils, lymphocytes, macrophages, and eosinophils were recovered from the lungs of mice that had been infected with P. murina than from control mice at d7, but only neutrophils remained significantly elevated at d14. Significantly more CD4 + and CD8 +T cells were purified from the lungs of mice that had been infected with P. murina mice at d7 and d14. Cytokine levels were measured in lung lavage fluid by ELISA. TNF-α, IFN-γ, IL-1, and IL-6 levels were higher in mice that had been inoculated with P. murina at all three time points. TNF-α and IL-1 levels did not change significantly following withdrawal of Dex treatment. Low levels of IL 6 were detected at d1, but increased significantly by d7 and d14. IFN-γ levels peaked at d14. Chemokine message levels were measured in lung tissue by ribonuclease protection assay. MIP-1β and IP-10 message increased between d1 and d7 and then decreased by d14. RANTES message levels increased from d1 to d7 and remained elevated at d14. Withdrawal of Dex induced immunosuppression from P. murina infected mice resulted in activation of many arms of the host response that lead to resolution of the infection.

Surfactant protein A limits Pneumocystis murina infection in immunosuppressed C3H/HeN mice and modulates host response during infection

The development of Pneumocystis murina pneumonia and host response were characterized over time and at different levels of infection in corticosteroid immunosuppressed surfactant protein A (SP-A) knockout and wild-type (WT) mice. Infection increased over time in both strains of mice; however, significantly more cyst forms were detected in the knockout mice at intermediate and late stages of infection. In mice with heavy infections, TNF-α and IFN-γ protein concentrations were significantly higher in pulmonary lavage fluid from knockout mice. There was a significant positive correlation between TNF-α and IFN-γ concentrations and the level of infection in knockout mice, but not in WT mice. No significant differences were detected in IL-1 levels between the two strains of mice at any of the time points or at any level of infection. At heavier infection levels, significantly more MIP-2 protein was detected in the lungs of knockout mice, but a significant positive correlation between MIP-2 concentrations and the infection level was detected in both groups of mice. At the intermediate stage of infection, a significantly higher percentage of neutrophils was detected in the lungs of knockout mice than in WT mice. There was no difference in SP-D levels between WT and KO mice with identical levels of infection. These data support a protective role for SP-A in host defense against Pneumocystis and suggest that the effects of SP-A on the host response vary based on the intensity of the infection.