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Influenza A virus (IAV) is a leading cause of respiratory infections, with increased risk of severe illness and death in the very young, aged, and immunocompromised individuals. In both mice and humans, IAV-specific T cell responses are protective during primary as well as homologous and heterologous challenge infections. Many mouse studies have focused on CD4 T cells specific for a single, known model or IAV antigen. However, studies have demonstrated that the IAV-specific CD4 T cell response comprises many epitopes spread across multiple viral proteins. Therefore, herein we track the antigen-experienced CD4 T cell response using the surrogate markers CD49d and CD11a. This novel surrogate marker method allows us to characterize the full IAV-specific CD4 T cell response without the potential bias that could occur when examining an individual Ag-specificity. Our findings demonstrate that the immunodominant I-A -binding NP epitope often used in studies of IAV-specific CD4 T cells represents only about 5% of the total IAV-specific CD4 T cell response. Further, we find that the kinetics of the full pulmonary CD4 T cell response is similar to that of NP -specific T cells and that the full CD4 T cell response in the lungs is predominantly composed of cells expressing the Th1 transcription factor T-bet, with smaller but significant portions of the response expressing the Treg and Tfh associated transcription factors Foxp3 and Bcl-6, respectively. Interestingly, although Th1 cells are the most abundant Th subset in the lungs of both BALB/c and C57Bl/6 mice following IAV, the relative abundance of Treg and Tfh is reversed in the different mouse strains. In BALB/c mice, Foxp3 cells are more abundant than Bcl6 cells, whereas in C57Bl/6 mice, there are more Bcl6 cells. As a whole, these data highlight the diversity of the endogenous CD4 T cell response to a primary IAV infection, providing an important context for past and future studies of the IAV-specific CD4 T cell response.

The inbred mouse strain C57BL/6J is widely used in models of immunological and infectious diseases. Here we show that C57BL/6J mice have a defect in neutrophil recruitment to a range of inflammatory stimuli compared with the related C57BL/6N substrain. This immune perturbation is associated with a missense mutation in Nlrp12 in C57BL/6J mice. Both C57BL/6J and NLRP12-deficient mice have increased susceptibility to bacterial infection that correlates with defective neutrophil migration. C57BL/6J and NLRP12-deficient macrophages have impaired CXCL1 production and the neutrophil defect observed in C57BL/6J and NLRP12-deficient mice is rescued by restoration of macrophage NLRP12. These results demonstrate that C57BL/6J mice have a functional defect in NLRP12 and that macrophages require NLRP12 expression for effective recruitment of neutrophils to inflammatory sites. The role of NLRP12 in immunity to bacterial infection is controversial as varied and contrasting results have been published using C57BL/6 mice. Here the authors shed light on this issue, showing that unlike C57BL/6N mice, C57BL/6J mice have a missense point mutation in NLRP12 that is associated with defective neutrophil recruitment.

The nuclear lamina represents a protein network required for nuclear structure and function. One family of lamina proteins is defined by an ∼40-aa LAP2, Emerin, and MAN1 (LEM) domain (LEM-D) that binds the nonspecific DNA-binding protein, barrier-to-autointegration factor (BAF). Through interactions with BAF, LEM-D proteins serve as a bridge between chromosomes and the nuclear envelope. Mutations in genes encoding LEM-D proteins cause human laminopathies that are associated with tissue-restricted pathologies. Drosophila has five genes that encode proteins with LEM homology. Using yeast two-hybrid analyses, we demonstrate that four encode proteins that bind Drosophila (d)BAF. In addition to dBAF, dMAN1 associates with lamins, the LEM-D protein Bocksbeutel, and the receptor-regulated Smads, demonstrating parallel protein interactions with vertebrate homologs. P-element mobilization was used to generate null dMAN1 alleles. These mutants showed decreased viability, with surviving adults displaying male sterility, decreased female fertility, wing patterning and positioning defects, flightlessness, and locomotion difficulties that became more severe with age. Increased phospho-Smad staining in dMAN1 mutant wing discs is consistent with a role in transforming growth factor (TGF)-β/bone morphogenic protein (BMP) signaling. The tissue-specific, age-enhanced dMAN1 mutant phenotypes are reminiscent of human laminopathies, suggesting that studies in Drosophila will provide insights into lamina dysfunction associated with disease.

Paraoxonases (PON) are a family of proteins (PON1, 2 and 3) with multiple enzymatic activities. PON1 interferes with homoserine lactone-mediated quorum sensing in bacteria and with reactive oxygen species (ROS) in humans and mice. PON1 gene mutations have been linked to multiple traits, including aging, and diseases of the cardiovascular, nervous and gastrointestinal system. The overlapping enzymatic activities in the PON family members and high linkage disequilibrium rates within their polymorphisms confound animal and human studies of PON1 function. In contrast, arthropods such as Drosophila melanogaster have no PON homologs, resulting in an ideal model to study interactions between PON genotype and host phenotypes. We hypothesized that expression of PON1 in D. melanogaster would alter ROS. We found that PON1 alters expression of multiple oxidative stress genes and decreases superoxide anion levels in normal and germ-free D. melanogaster. We also found differences in the composition of the gut microbiota, with a remarkable increase in levels of Lactobacillus plantarum and associated changes in expression of antimicrobial and cuticle-related genes. PON1 expression directly decreased superoxide anion levels and altered bacterial colonization of the gut and its gene expression profile, highlighting the complex nature of the interaction between host genotype and gut microbiota. We speculate that the interaction between some genotypes and human diseases may be mediated by the presence of certain gut bacteria that can induce specific immune responses in the gut and other host tissues.

Members of the NLR family can assemble inflammasome complexes with the adaptor protein ASC and caspase-1 that result in the activation of caspase-1 and the release of IL-1β and IL-18. Although the NLRC4 inflammasome is known to have a protective role in tumorigenesis, there is an increased appreciation for the inflammasome-independent actions of NLRC4. Here, we utilized a syngeneic subcutaneous murine model of B16F10 melanoma to explore the role of NLRC4 in tumor suppression. We found that NLRC4-deficient mice exhibited enhanced tumor growth that was independent of the inflammasome components ASC and caspase-1. Nlrc4 expression was critical for cytokine and chemokine production in tumor-associated macrophages and was necessary for the generation of protective IFN-γ-producing CD4+ and CD8+ T cells. Tumor progression was diminished when WT or caspase-1-deficient, but not NLRC4-deficient, macrophages were coinjected with B16F10 tumor cells in NLRC4-deficient mice. Finally, examination of human primary melanomas revealed the extensive presence of NLRC4+ tumor-associated macrophages. In contrast, there was a paucity of NLRC4+ tumor-associated macrophages observed in human metastatic melanoma, supporting the concept that NLRC4 expression controls tumor growth. These results reveal a critical role for NLRC4 in suppressing tumor growth in an inflammasome-independent manner.

Influenza A virus (IAV)-specific T cell responses are important correlates of protection during primary and subsequent infections. Generation and maintenance of robust IAV-specific T cell responses relies on T cell interactions with dendritic cells (DCs). In this study, we explore the role of nucleotide-binding domain leucine-rich repeat containing receptor family member NLRC4 in modulating the DC phenotype during IAV infection. Nlrc4-/- mice had worsened survival and increased viral titers during infection, normal innate immune cell recruitment and IAV-specific CD8 T cell responses, but severely blunted IAV-specific CD4 T cell responses compared to wild-type mice. The defect in the pulmonary IAV-specific CD4 T cell response was not a result of defective priming or migration of these cells in Nlrc4-/- mice but was instead due to an increase in FasL+ DCs, resulting in IAV-specific CD4 T cell death. Together, our data support a novel role for NLRC4 in regulating the phenotype of lung DCs during a respiratory viral infection, and thereby influencing the magnitude of protective T cell responses.

Influenza A virus-specific (IAV-specific) T cell responses are important correlates of protection during primary and subsequent infections. The generation and maintenance of robust IAV-specific T cell responses relies on T cell interactions with dendritic cells (DCs). In this study, we explore the role of the nucleotide-binding domain leucine-rich repeat- containing receptor family member NLRC4 in modulating the DC phenotype during IAV infection. [Nlrc4.sup.-/-] mice had worsened survival and increased viral titers during infection, normal innate immune cell recruitment, and IAV-specific [CD8.sup.+] T cell responses, but severely blunted IAV-specific [CD4.sup.+] T cell responses compared with WT mice. The defect in the pulmonary IAV-specific [CD4.sup.+] T cell response was not a result of defective priming or migration of these cells in [Nlrc4.sup.-/-] mice but was instead due to an increase in [FasL.sup.+] DCs, resulting in IAV- specific [CD4.sup.+] T cell death. Together, our data support a role for NLRC4 in regulating the phenotype of lung DCs during a respiratory viral infection and thereby influencing the magnitude of protective T cell responses.

Members of the NLR family can assemble inflammasome complexes with the adaptor protein ASC and caspase-1 that result in the activation of caspase-1 and the release of IL-1β and IL-18. Although the NLRC4 inflammasome is known to have a protective role in tumorigenesis, there is an increased appreciation for the inflammasome-independent actions of NLRC4. Here, we utilized a syngeneic subcutaneous murine model of B16F10 melanoma to explore the role of NLRC4 in tumor suppression. We found that NLRC4-deficient mice exhibited enhanced tumor growth that was independent of the inflammasome components ASC and caspase-1. Nlrc4 expression was critical for cytokine and chemokine production in tumor-associated macrophages and was necessary for the generation of protective IFN-γ-producing [CD4.sup.+] and [CD8.sup.+] T cells. Tumor progression was diminished when WT or caspase-1-deficient, but not NLRC4-deficient, macrophages were coinjected with B16F10 tumor cells in NLRC4-deficient mice. Finally, examination of human primary melanomas revealed the extensive presence of [NLRC4.sup.+] tumor-associated macrophages. In contrast, there was a paucity of [NLRC4.sup.+] tumor-associated macrophages observed in human metastatic melanoma, supporting the concept that NLRC4 expression controls tumor growth. These results reveal a critical role for NLRC4 in suppressing tumor growth in an inflammasome-independent manner.

Pattern recognition receptors, including members of the NBD and LLR-containing (NLR) family, are key sensors of infection and injury. Early sensing of pathogen invasion and subsequent activation of pro-inflammatory signaling cascades is essential for controlling infection. However, signaling pathways activated upon pathogen recognition can also contribute to inflammation-mediated tissue damage. The studies detailed in Chapters 3 and 4 are primarily concerned with the roles of two NLR family members, Nlrp12 and Nlrc4, during influenza A virus (IAV) infection. While IAV itself is cytopathic, the immune response is responsible for a great deal of the tissue damage during infection in some contexts. NLR family members are involved in both pathogen sensing and modulation of pro-inflammatory signaling, thus they are perfectly situated to shift the balance between pathogen clearance and immunopathology. Nlrp12 has been implicated in regulation of pro-inflammatory signaling through NFκB family members. In Chapter 3 we report that during IAV infection, we find no differences in those pathways, and instead we describe a novel role for Nlrp12 in regulating transcript stability. Previous work has shown that one of the key differences between lethal and sublethal IAV infections is the early and exaggerated recruitment of neutrophils. Previous studies in our laboratory had established a role for Nlrp12 in CXCL1-mediated neutrophil recruitment during respiratory bacterial infections. We therefore hypothesized that Nlrp12-/- mice would be protected from pathogenic neutrophil recruitment during lethal IAV infection due to decreased CXCL1 production. In Chapter 3 we show that indeed, Nlrp12-/- mice have improved survival, decreased pulmonary microvascular permeability, and decreased necrosis and hemorrhage in their airways compared to WT mice. Nlrp12-/- mice also have fewer neutrophils in their lungs, due to decreased production of CXCL1 by neutrophils, DCs and macrophages. Our data showing decreased Cxcl1 transcript stability in R848-treated Nlrp12 -/- BMDCs strongly suggest that the reduction in CXCL1 production by DCs in the Nlrp12-/- lungs is a result of decreased Cxcl1 transcript stability. Nlrc4 is a best known as a member of the Nlrc4 inflammasome, which is activated upon sensing of Gram-negative bacterial pathogens. However, a recent study from our laboratory showed an inflammasome-independent role for Nlrc4 in supporting critical anti-tumor T cell responses. Given that T cells are also critical for successful resolution of IAV infection, we hypothesized that during IAV infection, Nlrc4-/- mice would have compromised IAV-specific T cell responses and therefore poorer survival. Indeed, our studies in Chapter 4 show that in IAV-infected Nlrc4 -/- mice, the pulmonary IAV-specific CD4 T cell response is significantly diminished and mortality is significantly increased compared to WT mice. During IAV infection, the blunted CD4 T cell response is a result of increased death of the CD4 T cells, perhaps due to increased expression of FasL on CD11c + cells in the Nlrc4-/- lung environment.

TNF receptor-associated factor 3 (TRAF3) is an adapter protein that inhibits many signals that promote B cell survival and activation. Mice with a B cell-specific TRAF3 deficiency and humans with a rare haploinsufficiency in TRAF3 have enhanced development of BCLs as they age. Loss-of-function mutations in TRAF3 are common in B cell malignancies. Recent studies show that pharmacological inhibition of the enzyme glycogen synthase kinase 3 (GSK3), which regulates cellular growth, survival, and metabolism, inhibits growth and survival of BCL-derived B cells. In this study, we found that TRAF3 and GSK3 associated in B cells. The relative levels of TRAF3 in BCL cell lines correlated positively with the ratio of inactive to total GSK3β, and negatively correlated with susceptibility to GSK3 inhibition by the GSK3 inhibitory drug 9-ING-41, currently in clinical trials. Uniquely in BCLs with low TRAF3, GSK3 inhibition caused increased loss of the TRAF3-regulated, anti-apoptotic protein Mcl-1. GSK3 inhibition also blocked hyperresponsiveness to IL-6 receptor signaling in TRAF3-deficient BCL cells. Together, these results support the utility of 9-ING-41 as a treatment for BCL, and suggest that a decrease or loss of TRAF3 in BCLs could act as a biomarker for increased susceptibility to GSK3 inhibitor treatment.