Alveolar macrophages (AM) are one of the key cell types for initiating inflammatory and immune responses to influenza virus in the lung. However, the genome-wide changes in response to influenza infection in AM have not been defined. We performed gene profiling of human AM in response to H1N1 influenza A virus PR/8 using Affymetrix HG-U133 Plus 2.0 chips and verified the changes at both mRNA and protein levels by real-time RT-PCR and ELISA. We confirmed the response with a contemporary H3N2 influenza virus A/New York/238/2005 (NY/238). To understand the local cellular response, we also evaluated the impact of paracrine factors on virus-induced chemokine and cytokine secretion. In addition, we investigated the changes in the expression of macrophage receptors and uptake of pathogens after PR/8 infection. Although macrophages fail to release a large amount of infectious virus, we observed a robust induction of type I and type III interferons and several cytokines and chemokines following influenza infection. CXCL9, 10, and 11 were the most highly induced chemokines by influenza infection. UV-inactivation abolished virus-induced cytokine and chemokine response, with the exception of CXCL10. The contemporary influenza virus NY/238 infection of AM induced a similar response as PR/8. Inhibition of TNF and/or IL-1β activity significantly decreased the secretion of the proinflammatory chemokines CCL5 and CXCL8 by over 50%. PR/8 infection also significantly decreased mRNA levels of macrophage receptors including C-type lectin domain family 7 member A (CLEC7A), macrophage scavenger receptor 1 (MSR1), and CD36, and reduced uptake of zymosan. In conclusion, influenza infection induced an extensive proinflammatory response in human AM. Targeting local components of innate immune response might provide a strategy for controlling influenza A infection-induced proinflammatory response in vivo.

Alveolar Type II (ATII) cells are important targets for seasonal and pandemic influenza. To investigate the influenza-induced innate immune response in those cells, we measured the global gene expression profile of highly differentiated ATII cells infected with the influenza A virus at a multiplicity of infection of 0.5 at 4 hours and 24 hours after inoculation. Infection with influenza stimulated a significant increase in the mRNA concentrations of many host defense-related genes, including pattern/pathogen recognition receptors, IFN, and IFN-induced genes, chemokines, and suppressors of cytokine signaling. We verified these changes by quantitative real-time RT-PCR. At the protein level, we detected a robust virus-induced secretion of the three glutamic acid-leucine-arginine (ELR)-negative chemokines CXCL9, CXCL10, and CXCL11, according to ELISA. The ultraviolet inactivation of virus abolished the chemokine and cytokine response. Viral infection did not appear to alter the differentiation of ATII cells, as measured by cellular mRNA and concentrations of surfactant proteins. However, viral infection significantly reduced the secretion of surfactant protein (SP)-A and SP-D. In addition, influenza A virus triggered a time-dependent activation of phosphatidylinositol 3-kinase signaling in ATII cells. The inhibition of this pathway significantly decreased the release of infectious virus and the chemokine response, but did not alter virus-induced cell death. This study provides insights into influenza-induced innate immunity in differentiated human ATII cells, and demonstrates that the alveolar epithelium is a critical part of the initial innate immune response to influenza.

Fatal influenza (flu) virus infection often activates excessive inflammatory signals, leading to multi-organ failure and death, also referred to as cytokine storm. PPARγ (Peroxisome proliferator-activated receptor gamma) agonists are well-known candidates for cytokine storm modulation. The present study identified that influenza infection reduced PPARγ expression and decreased PPARγ transcription activity in human alveolar macrophages (AMs) from different donors. Treatment with PPARγ agonist Troglitazone ameliorated virus-induced proinflammatory cytokine secretion but did not interfere with the IFN-induced antiviral pathway in human AMs. In contrast, PPARγ antagonist and knockdown of PPARγ in human AMs further enhanced virus-stimulated proinflammatory response. In a mouse model of influenza infection, flu virus dose-dependently reduced PPARγ transcriptional activity and decreased expression of PPARγ. Moreover, PPARγ agonist troglitazone significantly reduced high doses of influenza infection-induced lung pathology. In addition, flu infection reduced PPARγ expression in all mouse macrophages, including AMs, interstitial macrophages, and bone-marrow-derived macrophages but not in alveolar epithelial cells. Our results indicate that the influenza virus specifically targets the PPARγ pathway in macrophages to cause acute injury to the lung.

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