Role of inflammation, innate resistance, and immunity in carcinogenesis.

该项目的几个目标已经取得了进展，特别是TLR信号在调节成纤维细胞和树突状细胞增殖和存活中的作用，人类DC细胞因子产生的调节，浆细胞样DC在调节口服耐受性中的作用，DC和NK细胞在弓形虫感染中对Th1分化的影响，TNF， MyD88和IL-18在结肠炎依赖性癌变中的作用。肝浆细胞样树突状细胞有助于口服诱导CD8+ T细胞耐受（免疫。2008;9:464-75）。在半抗原特异性接触敏感性（CHS）模型中，我们已经证明肝脏是一个口腔抗原呈递的部位，并且肝树突状细胞（DC）可以耐受小鼠随后的CD8+ T细胞启动。肝DC的耐受性潜能局限于浆细胞样DC (pDC)，通过暴露于半抗原而增强，并且需要CD4+ T细胞。最后，体内pDC的消耗消除了口服耐受性，恢复了半抗原特异性CD8+ T细胞和CHS反应。因此，pDC具有耐受性，在口腔耐受性中起重要作用。这些研究目前正在扩展，以分析肝pDC在其他耐受模型中的作用，特别是在结肠炎和癌变模型中是否需要pDC来诱导TNBS耐受。我们培育了在pDC上特异性表达人白喉毒素受体和绿色荧光蛋白的小鼠。这些小鼠可用于pDC的鉴定和体内消耗，并将用于pDC的体内鉴定和消耗实验。除了口服耐受模型外，我们已经建立了肠固有层中tlr9激活的pDC阻止未成熟CD4 T细胞在FoxP3+ Treg中的转化，我们正在将我们的研究扩展到其他pDCs参与免疫调节的模型。刚地弓形虫感染TAP1基因缺陷小鼠的NK细胞ifn - γ产生受损，导致CD4+ T细胞启动缺陷和死亡率升高[J] .中华检验医学杂志，2007,24(4):591- 592。为了研究CD8+ T细胞介导的弓形虫体内控制是否需要TAP1，我们比较了TAP1-/-、CD8-/-和野生型（WT）小鼠对弓形虫感染的抗性。令人惊讶的是，TAP1-/-小鼠比CD8-/-或WT小鼠更容易感染一种无毒的寄生虫菌株。TAP1-/-小鼠的抗性降低与激活和产生ifn - γ的CD4+ T细胞频率降低相关。有趣的是，与WT对照组相比，感染TAP1-/-的小鼠表现出产生ifn - γ的自然杀伤（NK）细胞的频率降低，NK细胞消耗后，CD8-/-和WT小鼠都以与TAP1-/-动物相同的动力学屈服于感染，并表现出受损的CD4+T细胞ifn - γ反应。总之，这些结果揭示了TAP1在诱导产生ifn - γ的NK细胞中的作用，并首次证明了该细胞群在启动CD4+ T淋巴细胞对弓形虫感染的反应中的功能。我们正在完成对腹膜弓形虫感染中产生IL-12的细胞类型的分析。长期以来，人们已经知道，能够产生IL-12以响应弓形虫可溶性提取物或其所含分子profilin的主要DC群体是CD8alpha+ DC，它们能够以不依赖ifn - γ的方式通过TLR11对profilin作出反应。然而，我们确定在弓形虫腹膜感染期间，主要产生IL-12的细胞是来自外周单核细胞的CD11b+ CD11c+ DC，这些细胞需要由常驻NK细胞产生的ifn - γ启动。细胞转移实验证明，感染后，主要的腹膜巨噬细胞群消失，而NK细胞留在腹膜腔中，被激活产生ifn - γ，引发单核细胞来源的DC从外周循环迁移，产生IL-12。先天抵抗和促炎性细胞因子在癌变中的作用。一项非常广泛的研究已经开始研究炎症受体和细胞因子在皮肤和结肠化学致癌中的作用。在几种癌症模型中，通过接头蛋白MyD88的信号传导促进了癌变。相反，MyD88信号在偶氮氧甲烷(AOM)/葡聚糖硫酸钠（DSS）结肠炎相关癌症（CAC）的发展中具有保护作用。Myd88-/-小鼠无法愈合损伤后产生的溃疡，导致炎症环境改变，导致编码促炎因子的基因表达早期改变，以及调节细胞增殖、凋亡和DNA修复的途径，导致腺瘤形成和浸润性腺癌的急剧增加，β -连环蛋白基因频繁发生克隆突变。其他研究报道，Tlr4缺陷小鼠对结肠炎的易感性与Myd88缺陷小鼠相似，但与后者不同的是，Tlr4缺陷小鼠对CAC具有耐药性。我们观察到，缺乏Tlr2或IlL1Rr的小鼠对结肠炎或CAC的易感性没有差异。然而，在AOM/DSS治疗后，Il18-/-和Il18r1-/-小鼠比野生型小鼠更容易发生结肠炎和息肉形成，这表明Myd88 -/-小鼠的表型部分是由于它们无法通过IL-18受体发出信号。这项研究揭示了MyD88下游不同受体影响组织稳态和癌变的复杂程度。在小鼠和许多人类癌症的经典两期皮肤癌变中，组成型活性RAS在皮肤癌的发展中起着核心作用。Ras介导的肿瘤形成通常与细胞因子和趋化因子的上调有关，这些细胞因子和趋化因子介导的炎症反应被认为与肿瘤发生有关。MyD88是通过toll样/IL-1R家族信号表达多种先天免疫应答的关键中间体。我们报道，切除MyD88或IL-1R的小鼠对局部皮肤癌变具有抗性，并且用致癌ras载体转导的培养MyD88-/-角化细胞在原位移植物中仅形成少数小肿瘤。Ras的致癌激活引起的初始角质形成细胞模仿体内诱导的皮肤肿瘤的表型，具有高增殖性，但也会抵抗诱导分化的信号，并上调细胞因子、趋化因子和金属蛋白酶基因，这些基因在体内介导被认为与肿瘤发生相关的炎症反应。致癌Ras转导的MyD88-/-角化细胞也具有高增殖性，但分化反应是完整的，促炎基因没有上调。通过遗传学和药理学方法，我们发现在角质形成细胞中，由致癌ras介导的分化和促炎功能需要通过il -1 α及其受体建立一个自分泌环，导致NF-kB活化。在没有MyD88或IL-1R的情况下，该循环不能建立。此外，阻断ras转导的野生型角质形成细胞中il -1 α介导的NF-kB活化可以纠正分化反应中的缺陷，并抑制促炎基因的表达。总的来说，这些结果表明Ras激活通过一系列潜在可逆的反馈信号将正常角质形成细胞转化为初始表型，这些反馈信号通过抑制IL-1信号提供治疗机会。

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. 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. 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. 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. 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. 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.