Little is known about the role of NK cells or their interplay with other immune cells during opportunistic infections. Using our murine model of Pneumocystis pneumonia, we found that loss of NK cells during immunosuppression results in substantial Pneumocystis lung burden. During early infection of C57B/6 CD4+ T cell depleted mice, there were significantly less NK cells in the lung tissue compared to CD4+ T cell intact animals, and the NK cells present demonstrated decreased upregulation of the activation marker NKp46 and production of the effector cytokine, IFN-γ. Furthermore, co-incubation studies revealed a significant increase in fungal killing when NK cells were combined with CD4+ T cells compared to either cell alone which was coincident with a significant increase in perforin production by NK cells. Finally, however, we found through adoptive transfer that memory CD4+ T cells are required for significant NK cell upregulation of the activation marker NKG2D and production of IFN-γ, granzyme B and perforin during Pneumocystis infection. To the best of our knowledge, this study is the first to not only demonstrate a role for NK cells in immunity to Pneumocystis pneumonia, but also to establish a functional relationship between CD4+ T cells and NK cells in the host response to an opportunistic fungal pathogen.

BackgroundRecent trials demonstrate increased pneumonia risk in chronic obstructive pulmonary disease patients treated with the inhaled corticosteroid (ICS) fluticasone propionate (FP). There is limited work describing FP effects on host defenses against bacterial pneumonia.MethodsC57BL/6 mice received daily, nose-only exposure to nebulized FP or vehicle for 8 days, followed by pulmonary challenge with Klebsiella pneumoniae. Bacterial burden, phagocytosis, leukocyte recruitment, cytokine expression, nitric oxide release, and survival were measured.ResultsInhaled FP increased bacterial burden in lungs and blood 48 h after infection but affected neither in vivo phagocytosis of bacteria by alveolar macrophages (AM) nor alveolar neutrophil recruitment. AM from FP-treated mice showed impaired expression of infection induced TNF-alpha, IP-10 (CXCL-10), and interleukin 6 (IL-6), and AM also showed a trend towards impaired intracellular pathogen control following in vivo infection. In vitro FP treatment resulted in a dose-dependent impairment of cytokine expression by AM. Furthermore, infection-induced nitric oxide (but not hydrogen peroxide) production was impaired by FP in vivo and in vitro. FP decreased survival in this model.ConclusionsExposure to inhaled FP impairs pulmonary clearance of K. pneumoniae in mice, an effect associated with greater systemic bacteremia and death. Decreased AM cytokine and nitric oxide expression parallel the failure to control infection. These results support the study of ICS effects on human pulmonary host defenses.

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Alcohol binge-drinking (acute ethanol consumption) is immunosuppressive and alters both the innate and adaptive arms of the immune system. Antigen presentation by macrophages (and other antigen presenting cells) represents an important function of the innate immune system that, in part, determines the outcome of the host immune response. Ethanol has been shown to suppress antigen presentation in antigen presenting cells though mechanisms of this impairment are not well understood. The constitutive and immunoproteasomes are important components of the cellular proteolytic machinery responsible for the initial steps critical to the generation of MHC Class I peptides for antigen presentation. In this study, we used an in-vitro cell culture model of acute alcohol exposure to study the effect of ethanol on the proteasome function in RAW 264.7 cells. Additionally, primary murine peritoneal macrophages obtained by peritoneal lavage from C57BL/6 mice were used to confirm our cell culture findings. We demonstrate that ethanol impairs proteasome function in peritoneal macrophages through suppression of chymotrypsin-like (Cht-L) proteasome activity as well as composition of the immunoproteasome subunit LMP7. Using primary murine peritoneal macrophages, we have further demonstrated that, ethanol-induced impairment of the proteasome function suppresses processing of antigenic proteins and peptides by the macrophage and in turn suppresses the presentation of these antigens to cells of adaptive immunity. The results of this study provide an important mechanism to explain the immunosuppressive effects of acute ethanol exposure.

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