Samir P. Bhagwat

Sensitized CD8+ T Cells Fail To Control Organism Burden but Accelerate the Onset of Lung Injury during Pneumocystis carinii Pneumonia

ABSTRACT While CD8+ cells have been shown to contribute to lung injury during Pneumocystis carinii pneumonia (PCP), there are conflicting reports concerning the ability of CD8+ cells to kill P. carinii. To address these two issues, we studied the effect of the presence of CD8+ cells in two mouse models of PCP. In the reconstituted SCID mouse model, depletion of CD8+ cells in addition to CD4+ cells after reconstitution did not result in increased numbers of P. carinii cysts compared to the numbers of cysts in mice with only CD4+ cells depleted. This result was observed regardless of whether the mice were reconstituted with naïve or P. carinii-sensitized lymphocytes. In contrast, reconstitution with sensitized lymphocytes resulted in more rapid onset of lung injury that was dependent on the presence of CD8+ cells. The course of organism replication over a 6-week period was also examined in the CD4+-T-cell-depleted and CD4+- and CD8+-T-cell-depleted mouse model of PCP. Again, the organism burdens were identical at all times regardless of whether CD8+ cells were present. Thus, in the absence of CD4+ T cells, CD8+ T cells are a key contributor to the inflammatory lung injury associated with PCP. However, we were unable to demonstrate an in vivo effect of these cells on the course of P. carinii infection.

Immune Modulation with Sulfasalazine Attenuates Immunopathogenesis but Enhances Macrophage-Mediated Fungal Clearance during Pneumocystis Pneumonia

Although T cells are critical for host defense against respiratory fungal infections, they also contribute to the immunopathogenesis of Pneumocystis pneumonia (PcP). However, the precise downstream effector mechanisms by which T cells mediate these diverse processes are undefined. In the current study the effects of immune modulation with sulfasalazine were evaluated in a mouse model of PcP-related Immune Reconstitution Inflammatory Syndrome (PcP-IRIS). Recovery of T cell-mediated immunity in Pneumocystis-infected immunodeficient mice restored host defense, but also initiated the marked pulmonary inflammation and severe pulmonary function deficits characteristic of IRIS. Sulfasalazine produced a profound attenuation of IRIS, with the unexpected consequence of accelerated fungal clearance. To determine whether macrophage phagocytosis is an effector mechanism of T cell-mediated Pneumocystis clearance and whether sulfasalazine enhances clearance by altering alveolar macrophage phagocytic activity, a novel multispectral imaging flow cytometer-based method was developed to quantify the phagocytosis of Pneumocystis in vivo. Following immune reconstitution, alveolar macrophages from PcP-IRIS mice exhibited a dramatic increase in their ability to actively phagocytose Pneumocystis. Increased phagocytosis correlated temporally with fungal clearance, and required the presence of CD4+ T cells. Sulfasalazine accelerated the onset of the CD4+ T cell-dependent alveolar macrophage phagocytic response in PcP-IRIS mice, resulting in enhanced fungal clearance. Furthermore, sulfasalazine promoted a TH2-polarized cytokine environment in the lung, and sulfasalazine-enhanced phagocytosis of Pneumocystis was associated with an alternatively activated alveolar macrophage phenotype. These results provide evidence that macrophage phagocytosis is an important in vivo effector mechanism for T cell-mediated Pneumocystis clearance, and that macrophage phenotype can be altered to enhance phagocytosis without exacerbating inflammation. Immune modulation can diminish pulmonary inflammation while preserving host defense, and has therapeutic potential for the treatment of PcP-related immunopathogenesis.

Parenchymal Cell TNF Receptors Contribute to Inflammatory Cell Recruitment and Respiratory Failure in Pneumocystis carinii-Induced Pneumonia

The opportunistic organism Pneumocystis carinii (Pc) produces a life-threatening pneumonia (PcP) in patients with low CD4+ T cell counts. Animal models of HIV-AIDS-related PcP indicate that development of severe disease is dependent on the presence of CD8+ T cells and the TNF receptors (TNFR) TNFRsf1a and TNFRsf1b. To distinguish roles of parenchymal and hematopoietic cell TNF signaling in PcP-related lung injury, murine bone marrow transplant chimeras of wild-type, C57BL6/J, and TNFRsf1a/1b double-null origin were generated, CD4+ T cell depleted, and inoculated with Pc. As expected, C57 → C57 chimeras (donor marrow → recipient) developed significant disease as assessed by weight loss, impaired pulmonary function (lung resistance and dynamic lung compliance), and inflammatory cell infiltration. In contrast, TNFRsf1a/1b−/− → TNFRsf1a/1b−/− mice were relatively mildly affected despite carrying the greatest organism burden. Mice solely lacking parenchymal TNFRs (C57 → TNFRsf1a/1b−/−) had milder disease than did C57 → C57 mice. Both groups of mice with TNFR-deficient parenchymal cells had low bronchoalveolar lavage fluid total cell counts and fewer lavageable CD8+ T cells than did C57 → C57 mice, suggesting that parenchymal TNFR signaling contributes to PcP-related immunopathology through the recruitment of damaging immune cells. Interestingly, mice with wild-type parenchymal cells but TNFRsf1a/1b−/− hematopoietic cells (TNFRsf1a/1b−/− → C57) displayed exacerbated disease characterized by increased MCP-1 and KC production in the lung and increased macrophage and lymphocyte numbers in the lavage, indicating a dysregulated immune response. This study supports a key role of parenchymal cell TNFRs in lung injury induced by Pc and a potential protective effect of receptors on radiosensitive, bone marrow-derived cells.

Anti-CD3 Antibody Decreases Inflammation and Improves Outcome in a Murine Model of Pneumocystis Pneumonia

The T cell–mediated immune response elicited by Pneumocystis plays a key role in pulmonary damage and dysfunction during Pneumocystis carinii pneumonia (PcP). Mice depleted of CD4+ and CD8+ T cells prior to infection are markedly protected from PcP-related respiratory deficit and death, despite progressive lung infection. However, the therapeutic effectiveness of Ab-mediated disruption of T cell function in mice already displaying clinical symptoms of disease has not been determined. Therefore, a murine model of PcP-related immune reconstitution inflammatory syndrome was used to assess whether Ab to the pan-T cell molecule CD3 is effective for reducing the severity of PcP when administered after the onset of disease. Mice that received anti-CD3 Ab exhibited a rapid and dramatic halt in the PcP-associated pulmonary function decline within 1 week after treatment, and a striking enhancement of survival rate compared with mice receiving the control Ab. Physiologic improvement in anti-CD3 treated mice was associated with a significant reduction in the number of CD4+ and CD8+ T cells recovered in lung lavage fluid. This effectiveness of anti-CD3 was noted whether the mice also received antibiotic therapy with trimethoprim-sulfamethoxazole. These data suggest that monoclonal Ab-mediated disruption of T cell function may represent a specific and effective adjunctive therapy to rapidly reverse the ongoing pathologic immune response occurring during active PcP. Thus, the anti-human CD3 monoclonal Ab OKT3, which is already in clinical use, has the potential to be developed as an adjunctive therapy for PcP.

CD73 regulates anti-inflammatory signaling between apoptotic cells and endotoxin-conditioned tissue macrophages

The phagocytosis of apoptotic cells (efferocytosis) shifts macrophages to an anti-inflammatory state through a set of still poorly understood soluble and cell-bound signals. Apoptosis is a common feature of inflamed tissues, and efferocytosis by tissue macrophages is thought to promote the resolution of inflammation. However, it is not clear how the exposure of tissue macrophages to inflammatory cues (e.g., PAMPs, DAMPs) in the early stages of inflammation affects immune outcomes of macrophage-apoptotic cell interactions occurring at later stages of inflammation. To address this, we used low-dose endotoxin conditioning (LEC, 1 ng/ml LPS 18 h) of mouse resident peritoneal macrophages (RPMФ) to model the effects of suboptimal (i.e., non-tolerizing), antecedent TLR activation on macrophage inflammatory responses to apoptotic cells. Compared with unconditioned macrophages (MФ), LEC-MФ showed a significant enhancement of apoptotic cell-driven suppression of many inflammatory cytokines (e.g., TNF, MIP-1β, MCP-1). We then found that enzymatic depletion of adenosine or inhibition of the adenosine receptor A2a on LEC-MФ abrogated apoptotic cell suppression of TNF, and this suppression was entirely dependent on the ecto-enzyme CD73 (AMPadenosine) but not CD39 (ATPAMP), both of which are highly expressed on RPMФ. In addition to a requirement for CD73, we also show that Adora2a levels in macrophages are a critical determinant of TNF suppression by apoptotic cells. LEC treatment of RPMФ led to a ~3-fold increase in Adora2a and a ~28-fold increase in adenosine sensitivity. Moreover, in RAW264.7 cells, ectopic expression of both A2a and CD73 was required for TNF suppression by apoptotic cells. In mice, mild, TLR4-dependent inflammation in the lungs and peritoneum caused a rapid increase in macrophage Adora2a and Adora2b levels, and CD73 was required to limit neutrophil influx in this peritonitis model. Thus immune signaling via the CD73–A2a axis in macrophages links early inflammatory events to subsequent immune responses to apoptotic cells.

Neither classical nor alternative macrophage activation is required for Pneumocystis clearance during immune reconstitution inflammatory syndrome.

ABSTRACT Pneumocystis is a respiratory fungal pathogen that causes pneumonia (Pneumocystis pneumonia [PcP]) in immunocompromised patients. Alveolar macrophages are critical effectors for CD4+ T cell-dependent clearance of Pneumocystis, and previous studies found that alternative macrophage activation accelerates fungal clearance during PcP-related immune reconstitution inflammatory syndrome (IRIS). However, the requirement for either classically or alternatively activated macrophages for Pneumocystis clearance has not been determined. Therefore, RAG2−/− mice lacking either the interferon gamma (IFN-γ) receptor (IFN-γR) or interleukin 4 receptor alpha (IL-4Rα) were infected with Pneumocystis. These mice were then immune reconstituted with wild-type lymphocytes to preserve the normal T helper response while preventing downstream effects of Th1 or Th2 effector cytokines on macrophage polarization. As expected, RAG2−/− mice developed severe disease but effectively cleared Pneumocystis and resolved IRIS. Neither RAG/IFN-γR−/− nor RAG/IL-4Rα−/− mice displayed impaired Pneumocystis clearance. However, RAG/IFN-γR−/− mice developed a dysregulated immune response, with exacerbated IRIS and greater pulmonary function deficits than those in RAG2 and RAG/IL-4Rα−/− mice. RAG/IFN-γR−/− mice had elevated numbers of lung CD4+ T cells, neutrophils, eosinophils, and NK cells but severely depressed numbers of lung CD8+ T suppressor cells. Impaired lung CD8+ T cell responses in RAG/IFN-γR−/− mice were associated with elevated lung IFN-γ levels, and neutralization of IFN-γ restored the CD8 response. These data demonstrate that restricting the ability of macrophages to polarize in response to Th1 or Th2 cytokines does not impair Pneumocystis clearance. However, a cell type-specific IFN-γ/IFN-γR-dependent mechanism regulates CD8+ T suppressor cell recruitment, limits immunopathogenesis, preserves lung function, and enhances the resolution of PcP-related IRIS.

Intrinsic Programming of Alveolar Macrophages for Protective Antifungal Innate Immunity Against Pneumocystis Infection

Invasive fungal infections, including Pneumocystis Pneumonia (PcP), remain frequent life-threatening conditions of patients with adaptive immune defects. While innate immunity helps control pathogen growth early during infection, it is typically not sufficient for complete protection against Pneumocystis and other human fungal pathogens. Alveolar macrophages (AM) possess pattern recognition molecules capable of recognizing antigenic and structural determinants of Pneumocystis. However, this pathogen effectively evades innate immunity to infect both immunocompetent and immunosuppressed hosts, albeit with differing outcomes. During our studies of mouse models of PcP, the FVB/N strain was identified as unique because of its ability to mount a protective innate immune response against Pneumocystis infection. In contrast to other immunocompetent strains, which become transiently infected prior to the onset of adaptive immunity, FVB/N mice rapidly eradicated Pneumocystis before an adaptive immune response was triggered. Furthermore, FVB/N mice remained highly resistant to infection even in the absence of functional T cells. The effector mechanism of innate protection required the action of functional alveolar macrophages, and the adoptive transfer of resistant FVB/N AMs, but not susceptible CB.17 AMs, conferred protection to immunodeficient mice. Macrophage IFNγ receptor signaling was not required for innate resistance, and FVB/N macrophages were found to display markers of alternative activation. IFNγ reprogrammed resistant FVB/N macrophages to a permissive M1 biased phenotype through a mechanism that required direct activation of the macrophage IFNγR. These results demonstrate that appropriately programmed macrophages provide protective innate immunity against this opportunistic fungal pathogen, and suggest that modulating macrophage function may represent a feasible therapeutic strategy to enhance antifungal host defense. The identification of resistant and susceptible macrophages provides a novel platform to study not only the mechanisms of macrophage-mediated antifungal defense, but also the mechanisms by which Pneumocystis evades innate immunity.