Alveolar macrophages (AM) are critical for defense against bacterial and fungal infections. However, a definitive role of AM in viral infections remains unclear. We here report that AM play a key role in survival to influenza and vaccinia virus infection by maintaining lung function and thereby protecting from asphyxiation. Absence of AM in GM-CSF-deficient (Csf2 −/−) mice or selective AM depletion in wild-type mice resulted in impaired gas exchange and fatal hypoxia associated with severe morbidity to influenza virus infection, while viral clearance was affected moderately. Virus-induced morbidity was far more severe in Csf2 −/− mice lacking AM, as compared to Batf3-deficient mice lacking CD8α+ and CD103+ DCs. Csf2 −/− mice showed intact anti-viral CD8+ T cell responses despite slightly impaired CD103+ DC development. Importantly, selective reconstitution of AM development in Csf2rb −/− mice by neonatal transfer of wild-type AM progenitors prevented severe morbidity and mortality, demonstrating that absence of AM alone is responsible for disease severity in mice lacking GM-CSF or its receptor. In addition, CD11c-Cre/Pparg fl/fl mice with a defect in AM but normal adaptive immunity showed increased morbidity and lung failure to influenza virus. Taken together, our results suggest a superior role of AM compared to CD103+ DCs in protection from acute influenza and vaccinia virus infection-induced morbidity and mortality.

Influenza is a ssRNA virus that has been responsible for widespread morbidity and mortality; however, the innate immunological mechanisms that drive the adaptive anti-influenza immune response in vivo are yet to be fully elucidated. TLRs are pattern recognition receptors that bind evolutionarily conserved pathogen-associated molecular patterns, induce dendritic cell maturation, and consequently aid the development of effective immune responses. We have examined the role of TLRs in driving effective T and B cell responses against influenza virus. We found TLR3 and its associated adapter molecule, Toll/IL-R domain-containing adaptor-inducing IFN-β, did not play a role in the development of CD4+ or CD8+ T cell responses against influenza virus, nor did they influence influenza-specific B cell responses. Surprisingly, TLR7 and MyD88 also played negligible roles in T cell activation and effector function upon infection with influenza virus; however, their signaling was critical for regulating anti-influenza B cell Ab isotype switching. The induction of appropriate anti-influenza humoral responses involved stimulation of TLRs on B cells directly and TLR-induced production of IFN-α, which acted to reduce IgG1 and increase IgG2a/c class switching. Notably, direct TLR signaling on B cells or T cell help through the CD40-CD40L interaction was sufficient to support B cell proliferation and IgG1 production, whereas IFN-α was critical for fine-tuning the nature of the isotype switch. Taken together, these data reveal that TLR signaling is not required for anti-influenza T cell responses, but through both direct and indirect means orchestrates appropriate anti-influenza B cell responses.

Upon encounter of viral Ags in an inflammatory environment, dendritic cells up-regulate costimulatory molecules and the chemokine receptor CCR7, with the latter being pivotal for their migration to the lymph node. By utilizing mice deficient in CCR7, we have examined the requirement of dendritic cell-mediated Ag transport from the lung to the draining lymph node for the induction of anti-influenza immune responses in vivo. We found that CCR7-mediated migration of dendritic cells was more crucial for CD8+ T cell than CD4+ T cell responses. While no specific CD8+ T cell response could be detected in the airways or lymphoid tissues during the primary infection, prolonged infection in CCR7-deficient mice did result in a sustained inflammatory chemokine profile, which led to nonspecific CD8+ T cell recruitment to the airways. The recruitment of influenza-specific CD4+ T cells to the airways was also below levels of detection in the absence of CCR7 signaling, although a small influenza-specific CD4+ T cell population was detectable in the draining lymph node, which was sufficient for the generation of class-switched anti-influenza Abs and a normal CD4+ T cell memory population. Overall, our data show that CCR7-mediated active Ag transport is differentially required for CD4+ and CD8+ T cell expansion during influenza infection.

PI-3-kinases have been identified as key signaling proteins involved in many basic biological processes in health and disease. Transgenic animals have been essential tools to study the underlying molecular mechanisms in this context and have therefore been widely used to elucidate the role of these factors in many different settings. More specifically, PI3Kγ, a subunit highly expressed in the hematopoietic system, has been implicated to play an important role in many inflammatory diseases as well as cancer. Here we report identification of multiple additional previously unknown mutations in the genome of a widely used PI3Kγ-deficient mouse colony. These include a STOP-mutation in the GM-CSFRα chain leading to a complete and specific deficiency in GM-CSF signaling. PI3Kγ-deficient animals consequently lacked alveolar macrophages and succumbed rapidly to influenza virus infection. Furthermore, PI3Kγ-deficient mice carried an additional mutation that affects Mucin 2 (Muc2) transcripts. This protein is strongly involved in the regulation of colorectal cancer and indeed conflicting reports have indicated that PI3Kγ-deficient animals spontaneously develop colorectal tumors. Thus, we uncover previously unknown confounding factors present in a strain of PI3Kγ-deficient mice leading to additional deficiencies in important signaling pathways with potentially wide-ranging implications for the interpretation of previous studies. By separating the mutations, we established unique Csf2ra-/-mouse model that allows study the role of cell intrinsic GM-CSF receptor signaling in vivo without confounding variables introduced by defective IL-5 receptor and IL-3 receptor signaling in mice lacking the common β chain (Csf2rb).

Phosphoinositide-3-kinases have been shown to be involved in influenza virus pathogenesis. They are targeted directly by virus proteins and are essential for efficient viral replication in infected lung epithelial cells. However, to date the role of PI3K signaling in influenza infection in vivo has not been thoroughly addressed. Here we show that one of the PI3K subunits, p110γ, is in fact critically required for mediating the host’s antiviral response. PI3Kγ deficient animals exhibit a delayed viral clearance and increased morbidity during respiratory infection with influenza virus. We demonstrate that p110γ is required for the generation and maintenance of potent antiviral CD8+ T cell responses through the developmental regulation of pulmonary cross-presenting CD103+ dendritic cells under homeostatic and inflammatory conditions. The defect in lung dendritic cells leads to deficient CD8+ T cell priming, which is associated with higher viral titers and more severe disease course during the infection. We thus identify PI3Kγ as a novel key host protective factor in influenza virus infection and shed light on an unappreciated layer of complexity concerning the role of PI3K signaling in this context.