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

Abstract

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.

Myeloid-Derived Suppressor Cells Impair Alveolar Macrophages through PD-1 Receptor Ligation during Pneumocystis Pneumonia

“…To confirm this result, PD-1 expression on the surface of AMs from uninfected and infected mice was examined by flow cytometry using antibodies against PD-1 and CD11c (Fig. 6B); the latter is a marker of AMs (54). Western blotting with anti-PD-1 antibody also was performed to further confirm PD-1 overexpression in AMs (Fig.…”

Differential Macrophage Polarization from Pneumocystis in Immunocompetent and Immunosuppressed Hosts: Potential Adjunctive Therapy during Pneumonia

“…An earlier study of modulating macrophage polarization during PcP using sulfasalazine demonstrated that this drug diminishes pulmonary inflammation while preserving the host defense by alternatively activating M2 lung macrophages (39). An additional study had shown that alternatively activated M2 macrophages are potent effector cells against Pneumocystis murina and suggests that enhancing M2a polarization may be an adjunctive therapy for the treatment of Pneumocystis infections (33).…”

Neither Classical nor Alternative Macrophage Activation Is Required for Pneumocystis Clearance during Immune Reconstitution Inflammatory Syndrome

“…The mouse model of PcP-related IRIS was used, as described previously (18,27). RAG2 Ϫ/Ϫ , RAG2/IL-4R␣ Ϫ/Ϫ , and RAG2/IFN-␥R Ϫ/Ϫ mice were intratracheally inoculated with 5 ϫ 10 5 purified Pneumocystis organisms based on cyst counts.…”

“…Our laboratory previously demonstrated that treatment with the immunomodulatory agent sulfasalazine (SSZ) reduced PcP-related immunopathogenesis and accelerated organism clearance. The beneficial effects of SSZ were associated with an increase in the abundance of macrophages with the M2 phenotype and enhanced AM phagocytosis of Pneumocystis (18). Other groups have also found that Th2 and M2 macrophages are effector cells that mediate immunity to Pneumocystis infection (22).…”

“…Interferon gamma (IFN-␥), a soluble cytokine produced by CD4 ϩ T helper 1 (Th1) cells, converts resting macrophages into cells with proinflammatory properties. These cells are known as classically activated or M1 macrophages (17,18). CD4 ϩ T helper 2 (Th2) responses produce the cytokines interleukin 4 (IL-4) and interleukin 13 (IL-13), which share the IL-4 receptor alpha (IL-4R␣) subunit (19,20).…”