Browsing by Subject "Toll-Like Receptors"
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Item B-Cell Adapter for Phosphoinositide 3-Kinase Is a Signaling Adapter in the Toll-Like Receptor/Interleukin-1 Receptor Superfamily(2014-02-17) Troutman, Ty Dale; van Oers, Nicolai S. C.; Pasare, Chandrashekhar; Hooper, Lora V.; Chen, Zhijian J.; Krämer, HelmutToll-like receptor (TLR)/Interleukin-1 receptor (IL1R) superfamily members share signaling components and (with the exception of TLR3) depend on the adapter myeloid differentiation primary response gene 88 (MyD88) for engagement of downstream pathways. Signals from the receptor to the adapter are transmitted through homotypic interaction of TIR (Toll-Interleukin-1 receptor) homology domains found in all TLR/IL1R family members and their adapters. The present work defines a novel TLR/IL1R signaling adapter, termed BCAP (B-cell adapter for PI3K), which was identified based on the presence of a cryptic N-terminal TIR domain. I show here that BCAP (B-cell adapter for PI3K) contains a functional TIR domain enabling its participation in the TLR signaling pathway. Through its TIR domain, BCAP associates with the TLR/IL1R signaling adapter MyD88, as well as the TLR signaling adapter toll-interleukin 1 receptor domain containing adapter protein (TIRAP). Importantly, BCAP plays an obligate role in linking TLRs to activation of phosphoinositide 3-kinase (PI3K) through recruitment of PI3K to the signaling complex and relief of inhibitory influences on PI3K activity. Importantly, BCAP selectively mediates TLR signaling towards the PI3K branch without affecting signaling to NFκB nor MAP kinases. In this capacity, BCAP inhibits secretion of inflammatory cytokines and regulates susceptibility to inflammatory colitis. Because the TLR/IL1R family shares signaling components, BCAP may also function in IL1R family signaling. To test this hypothesis, T cells were chosen as a model cell type responding to IL1R family signals. T helper cells utilize IL18 and IL1 (which engage the IL18R or the IL1R respectively, both IL1R family members) cytokines provided by myeloid cells to achieve optimal Th1 and Th17 effector capacities. I show here that BCAP intrinsically regulates differentiation of naïve T cells towards Th1 and Th17 effector lineages by participation in the IL1R family signaling pathways. Further, BCAP intrinsically regulates both T cell proliferation and survival during priming. The significance of this work lies in the revelation of a TLR signaling adapter serving as a node connecting TLRs to PI3K. Further, the findings here will increase the understanding of key signaling pathways involved in disease and inflammation.Item A Chemically Induced Colitis Screen Reveals the Necessity for Membrane Traffic in Intestinal Homeostasis(2019-03-21) McAlpine, William Elliott; Winter, Sebastian E.; Beutler, Bruce; Burstein, Ezra; Schmid, Sandra; Scherer, PhilippInflammatory bowel disease is most commonly a complex disorder caused by the interaction of environmental and genetic aberrations. Under normal conditions, a genetic program actively prevents inflammatory bowel disease, preventing invasion of microbes without permitting severe inflammation of the gut. To identify genes that maintain this balance, we performed a sensitized screen of 49,420 third generation (G3) germline mutant mice derived from N-ethyl-N-nitrosourea-mutagenized grandsires, bearing 104,658 coding/splicing mutations. We induced mild mucosal damage in these mice by orally administering dextran sodium sulfate (DSS) and found mutations that led to diarrhea and weight loss under these conditions. Causative mutations were mapped concurrently with screening using an automated mapping procedure. Among 114 DSS phenotypes identified and mapped, 36 have been validated by CRISPR/Cas9 targeting. Three vesicle trafficking genes, Myo1d, Smcr8, and Tvp23b, were selected for mechanistic evaluation. MYO1D is a class I myosin that binds both actin and lipid. MYO1D localizes to the basolateral membrane of enterocytes and functions in the intestinal epithelium to protect against colitis. SMCR8, along with C9ORF72 and WDR41, is a member of a tripartite complex that functions as a guanine exchange factor. SMCR8 localizes to the lysosome, and its absence results in perturbations to endocytic and phagocytic pathways. Hyperactivation of endosomal Toll-like receptors in Smcr8-/- mice causes spontaneous inflammation, and hyperactivation of multiple pathways contributes to DSS susceptibility. TVP23B is a trans-Golgi protein that binds YIPF6. Both TVP23B and YIPF6 are necessary for the formation of secretory granules in goblet and Paneth cells of the intestinal epithelium. These studies reveal non-redundant molecules required for the return of normal physiologic balance within the intestine after DSS insult.Item Neutrophil-Derived IFN-γ in Toxoplasma gondii Infection and Innate Immunity(2014-11-19) Sturge, Carolyn Rowena; van Oers, Nicolai S. C.; Yuan, Dorothy; Zhang, Chengcheng "Alec"; Koh, Andrew Y.; Yarovinsky, FelixInterferon-gamma (IFN-γ) is a major cytokine that is critical for host resistance to a broad range of intracellular pathogens. Production of IFN-γ by Natural Killer (NK) and T cells is initiated by the recognition of pathogens through Toll-like receptors (TLRs). In an experimental model of toxoplasmosis we have identified the presence of a non-lymphoid source of IFN-γ that was particularly evident in the absence of TLR-mediated recognition of Toxoplasma gondii. Flow-cytometry and morphological examinations of non-NK/non-T IFN-γ-positive cells identified neutrophils as the cell type capable of producing IFN-γ. Selective elimination of neutrophils in TLR11-/- mice infected with the parasite resulted in acute susceptibility similar to that observed in IFN-γ-deficient mice. These data show that neutrophils are a biologically significant source of IFN-γ during T. gondii infection. Additionally, we investigated the role of neutrophil IFN-γ in another intracellular infection, Salmonella typhimurium, and found that neutrophils were also IFN-γ-positive. Examination of neutrophils in different locations in a mouse model revealed that they all expressed low amounts of IFN-γ regardless of infection status. In particular, the bone marrow niche contained an IFN-γ+ population that was negative for the Ly-6G marker characteristic of mature neutrophils in peripheral tissues. Recent work defining neutrophil developmental stages by flow-cytometry allowed us to discern that precursor neutrophils at the promyelocyte stage (Ly-6G negative) were positive for IFN-γ. Furthermore, neutrophil-derived IFN-γ was prestored in granules during neutrophil lineage development although the mechanisms behind this phenomena are not yet understood. This work, combined with the recent work of other laboratories, suggests that neutrophils can have defined phenotypes and cytokine production similar to that of T cells or Innate Lymphoid cells (ILCs). These findings have broad implications for all disease states where neutrophils are the first responders to infections.Item Regulation of Reparative Macrophage Transition by the B-cell Adapter for PI3K (BCAP)(2021-05-01T05:00:00.000Z) Irizarry-Caro, Ricardo A.; Satterthwaite, Anne B.; Pasare, Chandrashekhar; Street, Nancy E.; Tagliabracci, Vincent S.; Zaki, HasanMacrophages respond to microbial ligands and various noxious cues by initiating an inflammatory response aimed at eliminating the original pathogenic insult. Transition of macrophages from a pro-inflammatory state to a reparative state, however, is vital for resolution of inflammation and return to homeostasis. The molecular players governing this transition remain poorly defined. Here, we find that the reparative macrophage transition is dictated by B-cell adapter for PI3K (BCAP). Mice harboring a macrophage specific deletion of BCAP fail to recover from and succumb to DSS-induced colitis due to prolonged intestinal inflammation and impaired tissue repair. Following microbial stimulation, gene expression in WT macrophages switches from an early inflammatory signature to a late reparative signature, a process that is hampered in BCAP deficient macrophages. We find that absence of BCAP hinders inactivation of FOXO1 and GSK3b that contributes to their enhanced inflammatory state. BCAP deficiency also results in defective aerobic glycolysis and reduced lactate production. This translates into reduced histone lactylation and decreased expression of reparative macrophage genes. Thus, our results reveal BCAP to be critical cell intrinsic switch that regulates transition of inflammatory macrophages to reparative macrophages by imprinting epigenetic changes.Item Searching for Genes of Host Defense(2013-01-22) SoRella, Jeffrey A.; Shi, He-Xin; Wang, Ying; Beutler, Bruce A.Through random mutation of the mouse genome and phenotypic screening of the mutated mice, genes can be identified that are associated with dysfunction in the innate immune system. The strategy proposed works under the knowledge that many genes are involved in the immune system and that random mutation could lead to a change in their genetic code. This mutation can present as a phenotypically abnormal immune system. Once a phenotype is identified, the genome can be analyzed in an attempt to trace the mutated gene responsible for the weakened immune system. One of the elegant aspects of this genetic method is that it does not rely on a hypothesis about how the immune response works. This leads to an unbiased approach where interpretation errors are rarely made. A forward genetic approach is used to create abnormal phenotypes of the innate immune system and then determine the genetic cause. The normal mutation rate is accelerated by the widely used germline mutagen N-ethyl-N-nitrosourea (ENU) to produce an average of 3,000 single nucleotide changes per host leading to an average of 60 coding changes. To produce homozygotes, males of the G1 generation are bred with normal mice of the same strain to yield the G2 generation. Recessive mutations can be found in the G3 generation by a backcross of G2 females with the G1 father. Screening 6 G3 progeny should capture 50% of the mutations in the homozygous form. Phenotypic screening was performed on peritoneal macrophages ex vivo by stimulation with the following toll-like receptor agonists: lipopolysaccharide (TLR4), double stranded RNA (TLR3), triacylated lipoprotein (TLR 1/2), diacylated lipoprotein (TLR 2/6), resiquimod (TLR 7), and unmethylated DNA (TLR 9). The inflammasome pathway was probed by lipopolysaccharide priming followed by stimulation with either nigericin (K+ efflux) or ATP. The secreted TNF-alpha (TLR screen) and IL-1beta (inflammasome screen) were measured by ELISA to determine phenovariance. This research can lead to a deeper understanding of how we combat infection. The study can lead to the development of mutations involved in both the innate and adaptive immune system so autoimmune diseases can also be studied. A long term goal is to identify genes that would render an individual resistant to infection and to study the interaction of these genes.Item Toll Like Receptor Dependent Control of T Helper 17 Cell Differentiation and Inflammasome Activation(2014-04-14) Hu, Wei; Niederkorn, Jerry Y.; Pasare, Chandrashekhar; van Oers, Nicolai S. C.; Fontoura, BeatrizActivation of pattern recognition receptors on dendritic cells (DCs) and macrophages leads to secretion of cytokines that control activation and differentiation of CD4+ T cells. While IL-12 is critical for Th1 lineage development, IL-6 and TGF-b play an important role in differentiation of Th17 cells in vitro. IL-1 has also been implicated in regulating Th17 differentiation. In this dissertation, we examined the relative contribution of IL-6 and IL-1 for in vivo Th17 differentiation, and found that requirements for Th17 polarization depend entirely on the site of priming. While IL-6 plays a critical role in Th17 lineage priming in the skin and the mucosal tissue such as the gut and the lung, it is completely dispensable for Th17 priming in the spleen. IL-1R signaling in T cells is however necessary for Th17 priming in all tissues. Moreover, the differential cytokine requirements for Th17 lineage commitment are guided by differential population of DCs resident in different tissues. These results reveal fundamental differences by which the systemic, mucosal, and cutaneous immune systems guide Th17 cell lineage commitment. Pathogenic infections and tissue injuries trigger the assembly of inflammasomes, cytosolic protein complexes that activate caspase-1 leading to cleavage of pro-IL-1β/pro-IL-18, and pyroptosis, a pro-inflammatory cell death program. Although microbial recognition by Toll-like receptors (TLR) induces synthesis of pro-IL-1β and pro-IL-18, its role in inflammasome activation is insufficiently understood. In this dissertation, we have discovered that simultaneous activation of TLRs and NLRP3, which likely mimics natural infection by pathogenic microbes, triggers rapid caspase-1 cleavage, release of alarmins and pyroptosis. This acute caspase-1 activation is independent of new protein synthesis and depends on the TLR signaling molecule IRAK-1 and its kinase activity. Importantly, Listeria monocytogenes induces NLRP3-dependent rapid caspase-1 activation and pyroptosis, both of which depend on IRAK-1. These results demonstrate that simultaneous sensing of microbial ligands and virulence factors by TLRs and NLRP3 respectively leads to a rapid TLR- and IRAK-1-dependent assembly of the NLRP3 inflammasome, and such activation is important for release of alarmins, pyroptosis, and early IFN-γ production by memory T cells, all of which could be critical for early host defense.