Frontline Science: Coincidental null mutation of Csf2rα in a colony of PI3Kγ-/- mice causes alveolar macrophage deficiency and fatal respiratory viral infection.

PI3Ks have been identified as key signaling proteins involved in many basic biologic processes in health and disease. Transgenic animals have been essential tools to study the underlying molecular mechanisms in this context and therefore, have 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 (PI3Kγ-/-) mouse colony. These include a STOP mutation in the GM-CSFRα chain, leading to a complete and specific deficiency in GM-CSF signaling. PI3Kγ-/- animals consequently lacked alveolar macrophages (AMs) and succumbed rapidly to influenza virus infection. Furthermore, PI3Kγ-/- 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-/- animals spontaneously develop colorectal tumors. Thus, we uncover previously unknown, confounding factors present in a strain of PI3Kγ-/- 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 a unique Csf2ra-/- mouse model that allows us to study the role of cell intrinsic GM-CSFR signaling in vivo without confounding variables introduced by defective IL-5R and IL-3R signaling in mice lacking the common ß chain (Csf2rb).

Alveolar macrophages are essential for protection from respiratory failure and associated morbidity following influenza virus infection.

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/Ppargfl/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.

CD4+ and CD8+ T cells exhibit differential requirements for CCR7-mediated antigen transport during influenza infection.

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.

TLR signaling fine-tunes anti-influenza B cell responses without regulating effector T cell responses.

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-beta, 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-alpha, 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-alpha 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.