Role of Inflammation, Innate Resistance, and Immunity in Carcinogenesis

Progress has been made in several of the aims of the project, in particular on the role of TLR signaling in regulating proliferation and survival of fibroblasts and dendritic cells, on the regulation of cytokine production from human DC, on the role of plasmacytoid DC in regulating oral tolerance, on the effect of DC and NK cells on Th1 differentiation in Toxoplasma Gondii infection, role of TNF, MyD88 and IL-18 in colitis-dependent carcinogenesis.Hepatic plasmacytoid dendritic cells contribute to orally induced CD8+ T cell tolerance (Immunity. 2008; 9:464-75).W have shown that the liver is a site of oral antigen presentation and that hepatic dendritic cells (DC) can tolerize mice to subsequent CD8+ T cell priming, in a model of hapten-specific contact sensitivity (CHS). The tolerogenic potential of liver DC is confined to plasmacytoid DC (pDC), is enhanced by exposure to hapten, and requires CD4+ T cells. Finally, in vivo depletion of pDC abrogated oral tolerance and restored hapten-specific CD8+ T cell and CHS responses. Thus, pDC are tolerogenic and play an essential role in oral tolerance. <P>These studies are now being extended to analyze the role of liver pDC in other model of tolerance and in particular in testing whether pDC are require to induce TNBS tolerance in a model of colitis and carcinogenesis. We have generatd a mouse expressing the human Dyptheria Toxin Receptor and the Green Fluorescent protein specifically on pDC. These mice can be used for the identication and in vivo depletion of pDC and they will be used in experiments aimed to identify and deplete pDCs in vivo. In addition to the models of oral tolerance we have established that TLR9-activated pDC in the intestinal lamina propria prevent the conversion of naive CD4 T cells in FoxP3+ Treg and we are estending our studies to other models in which pDCs are involved in immunoregulation. Toxoplasma gondii infected TAP1 deficient mice display impaired NK cell IFN-gamma production leading to defective CD4+ T cell priming and increased mortality (J Exp Med. 2007, 204:2591-602).To investigate if TAP1 is required for CD8+ T cell mediated control of Toxoplasma gondii in vivo, we compared the resistance of TAP1-/-, CD8-/- and wild-type (WT) mice to infection with the parasite. Surprisingly TAP1-/- mice displayed greater susceptibility than either CD8-/- or WT mice to infection with an avirulent parasite strain. The decreased resistance of the TAP1-/- mice correlated with a reduction in the frequency of activated and IFN-gamma-producing CD4+ T cells. Interestingly, infected TAP1-/- mice showed a reduced frequency of IFN-gamma producing natural killer (NK) cells relative to that of WT controls, and after NK cell-depletion both CD8-/- and WT mice succumbed to infection with the same kinetics as TAP1-/- animals and displayed impaired CD4+T cell IFN-gamma responses. Together, these results reveal a previously unappreciated role for TAP1 in the induction of IFN-gamma producing NK cells and provide the first demonstration of the function of this cell population in the priming of CD4+ T lymphocyte responses to T. gondii infection. <P>We are completing the analysis of the cell types responsible for IL-12 production in peritoneal T. gondii infection. It has been known for long time that the major DC population able to produce IL-12 in response to T. gondii soluble extract, or the molecule profilin contained in it, are CD8alpha+ DC that are able to respond to profilin through TLR11 in an IFN-gamma independent fashion . However, we determined that during peritoneal infection with T. gondii, the dominant IL-12 producing cells are CD11b+ CD11c+ DC derived from peripheral monocytes that require priming by IFN-gamma produced by resident NK cells. Cell transfer experiments have allowed us to demonstrate that upon infection, the major peritoneal macrophage populations disappear while NK cells remain in the peritoneal cavity and are activated to produce IFN-gamma that primes monocyte-derived DC immigrating from the peripheral circulation for IL-12 production.Innate resistance and pro-inflammatory cytokines in carcinogenesis.A very extensive investigation has been initiated to study the role of inflammatory receptors and cytokines in skin and colon chemical carcinogenesis. Signaling through the adaptor protein MyD88 promotes carcinogenesis in several cancer models. In contrast, MyD88 signaling has a protective role in the development of azoxymethane (AOM)/ dextran sodium sulfate (DSS) colitis-associated cancer (CAC). The inability of Myd88-/- mice to heal ulcers generated upon injury creates an altered inflammatory environment that induces early alterations in expression of genes encoding pro-inflammatory factors as well as pathways regulating cell proliferation, apoptosis, and DNA repair resulting in a dramatic increase in adenoma formation and progression to infiltrating adenocarcinomas with frequent clonal mutations in the beta-catenin gene. <P>Others have reported that Tlr4 deficient mice have a similar susceptibility to colitis as Myd88 deficient mice but, unlike the latter, are resistant to CAC. We have observed that mice deficient for Tlr2 or IlL1Rr do not show a differential susceptibility to colitis or CAC. However, upon AOM/DSS treatment Il18-/- and Il18r1-/- mice were more susceptible to colitis and polyp formation than wild type mice, suggesting that the phenotype of Myd88 -/- mice is in part due to their inability to signal through the IL-18 receptor. This study revealed a previously unknown level of complexity surrounding MyD88 activities downstream of different receptors that impact tissue homeostasis and carcinogenesis.Constitutively active RAS plays a central role in the development of skin cancer in the classical two stage skin carcinogenesis in mice and in a number of human cancers. Ras mediated tumor formation is commonly associated with upregulation of cytokines and chemokines that mediate an inflammatory response considered relevant to oncogenesis. MyD88 is a crucial intermediate in the expression of multiple innate immune responders through signaling from the Toll-like/IL-1R family. <P>We report that mice ablated for MyD88 or the IL-1R are resistant to topical skin carcinogenesis, and cultured MyD88-/- keratinocytes transduced with an oncogenic ras vector form only a few small tumors in orthotopic grafts. Initiated keratinocytes arising from oncogenic activation of Ras mimic the phenotype of in vivo induced skin tumors and are hyperproliferative but also resist signals for induced differentiation and upregulate cytokine, chemokine, and metalloprotease genes that in vivo mediate an inflammatory response considered relevant to oncogenesis. Oncogenic Ras transduced MyD88-/- keratinocytes are also hyperproliferative but the differentiation response is intact and pro-inflammatory genes are not upregulated. Using both genetic and pharmacological approaches, we find that in keratinocytes, the differentiation and pro-inflammatory functions mediated by oncogenic ras require the establishment of an autocrine loop through IL-1alpha and its receptor leading to NF-kB activation. In the absence of MyD88 or IL-1R, this loop cannot be established.<P> Further, blocking the IL-1alpha mediated NF-kB activation in ras-transduced wildtype keratinocytes corrects the defect in differentiation response and inhibits proinflammatory gene expression. Collectively, these results demonstrate that Ras activation converts normal keratinocytes to an initiated phenotype through a series of potentially reversible feedback signals that provide therapeutic opportunities through inhibition of IL-1 signaling.