Human infections with highly pathogenic avian influenza A (H5N1) virus are frequently fatal but the mechanisms of disease remain ill-defined. H5N1 infection is associated with intense production of proinflammatory cytokines, but whether this cytokine storm is the main cause of fatality or is a consequence of extensive virus replication that itself drives disease remains controversial. Conventional intratracheal inoculation of a liquid suspension of H5N1 influenza virus in nonhuman primates likely results in efficient clearance of virus within the upper respiratory tract and rarely produces severe disease. We reasoned that small particle aerosols of virus would penetrate the lower respiratory tract and blanket alveoli where target cells reside. We show that inhalation of aerosolized H5N1 influenza virus in cynomolgus macaques results in fulminant pneumonia that rapidly progresses to acute respiratory distress syndrome with a fatal outcome reminiscent of human disease. Molecular imaging revealed intense lung inflammation coincident with massive increases in proinflammatory proteins and interferon-α in distal airways. Aerosolized H5N1 exposure decimated alveolar macrophages, which were widely infected and caused marked influx of interstitial macrophages and neutrophils. Extensive infection of alveolar epithelial cells caused apoptosis and leakage of albumin into airways, reflecting loss of epithelial barrier function. These data establish inhalation of aerosolized virus as a critical source of exposure for fatal human infection and reveal that direct viral effects in alveoli mediate H5N1 disease. This new nonhuman primate model will advance vaccine and therapeutic approaches to prevent and treat human disease caused by highly pathogenic avian influenza viruses.

Increasing evidence suggests that NK cells act to promote effective T cell–based antitumor responses. Using the B16-OVA melanoma model and an optimized Gram-positive bacteria–dendritic cell (DC) vaccination strategy, we determined that in vivo depletion of NK cells at time of tumor challenge abolished the benefit of DC immunotherapy. The contribution of NK cells to DC immunotherapy was dependent on tumor Ag presentation by DC, suggesting that NK cells act as helper cells to prime or reactivate tumor-specific T cells. The absence of NK cells at tumor challenge resulted in greater attenuation of tumor immunity than observed with selective depletion of either CD4 or CD8 T cell subsets. Although successful DC immunotherapy required IFN-γ, perforin expression was dispensable. Closer examination of the role of NK cells as helper cells in enhancing antitumor responses will reveal new strategies for clinical interventions using DC-based immunotherapy.

Interferon-gamma (IFN-γ) is a critical cytokine for the initiation of immune responses against a variety of infectious agents and malignancies. We found that a range of Gram-positive and Gram-negative bacteria stimulated the rapid release (<24 h) of IFN-γ from murine leukocytes. Using fluorescence activated cell sorting and cd1d(-/-) and rag1(-/-) mice, we determined that dendritic cells (DCs) and natural killer (NK) cells were primarily responsible for IFN-γ release by Streptococcus salivarius, a Gram-positive commensal, previously noted to possess potent interleukin-12 (IL-12)-inducing potential. IFN-γ release from NK cells required DC:NK membrane contact and IL-12/IL-18 expression, but was independent of lymphocyte function-associated antigen-1-mediated interactions. IFN-γ release in response to bacteria was maintained in mice deficient for Toll-like receptor (TLR)-2 and TLR-4, suggesting that bacteria activate antigen-presenting cells via multiple, redundant pathways. Together, our results suggest that Gram-positive bacteria may be useful in driving NK cell activation and T helper 1 polarization and have the potential for development as effective adjuvants.

The contribution of macrophages in the gastrointestinal tract to disease control or progression in HIV infection remains unclear. To address this question, we analyzed CD163+ macrophages in ileum and mesenteric lymph node (LN) from SIV-infected rhesus macaques with dichotomous expression of controlling MHC class I alleles predicted to be SIV controllers or progressors. Infection induced accumulation of macrophages into gut mucosa in the acute phase that persisted in progressors but was resolved in controllers. In contrast, macrophage recruitment to mesenteric LNs occurred only transiently in acute infection irrespective of disease outcome. Persistent gut macrophage accumulation was associated with CD163 expression on α4β7+CD16+ blood monocytes and correlated with epithelial damage. Macrophages isolated from intestine of progressors had reduced phagocytic function relative to controllers and uninfected macaques, and the proportion of phagocytic macrophages negatively correlated with mucosal epithelial breach, lamina propria E. coli density and plasma virus burden. Macrophages in intestine produced low levels of cytokines regardless of disease course, while mesenteric LN macrophages from progressors became increasingly responsive as infection advanced. These data indicate that non-inflammatory CD163+ macrophages accumulate in gut mucosa in progressive SIV infection in response to intestinal damage but fail to adequately phagocytose debris, potentially perpetuating their recruitment.

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