MAP3K8 in immunoregluation, host defense and autoimmunity

细胞因子是调节细胞生长和分化的分泌蛋白质。这些因子在调节免疫和炎症反应、调节淋巴样细胞的发育和分化方面尤为重要。细胞因子还调节免疫稳态、耐受性和记忆力。毫不奇怪，细胞因子在类风湿性关节炎、系统性红斑狼疮、炎症性肠病和牛皮癣等自身免疫性疾病的发病机制中起着关键作用。了解细胞因子作用的分子基础有助于深入了解免疫介导性疾病的发病机制，并提供新的治疗靶点。 细胞因子受体与Janus家族激酶(JAKs)相关，后者启动信号传递(见AR041106-14项目)。在JAKs激活后，信号传递的下一步是激活一系列称为STAT(信号转导和转录激活子)的转录因子(见AR041159-01项目)。 为了更好地了解细胞因子的分子作用，我们对白介素12激活的小鼠和人T细胞进行了转录图谱分析。我们鉴定了大量的诱导基因，其中一些以前被认为是IL-12诱导的。其中一个这样的基因是编码丝氨酸/苏氨酸激酶Cot/Tpl2的基因。该激酶可被IL-12直接诱导，但被IL-4抑制。由于IL-12激活了转录因子STAT4，我们还评估了STAT4缺陷小鼠可诱导的MAP3K8表达的依赖性。此外，通过染色质免疫沉淀，我们发现MAP3K8是STAT4的直接靶标。为了确定MAP3K8的作用，我们研究了MAP3K8基因敲除小鼠。我们发现MAP3K8-/-小鼠Therper1分化明显不足。我们进一步发现，这与未能上调转录因子STAT4和Tbox21(T-bet)有关，从而解释了未能正确产生IFNG。用弓形虫攻击MAP3K8基因缺陷小鼠表明，这些小鼠对这种模型病原体的敏感性增加，这与产生IFNG的能力受损是一致的。相反，在模拟多发性硬化症、实验性自身免疫性脑脊髓炎或EAE的自身免疫性疾病模型中，MAP3K8基因敲除小鼠的疾病严重程度降低。我们进一步证实，OVA免疫的Tpl2(-/-)小鼠在鼻腔注射OVA时，比Tpl2(+/+)对照组小鼠表达高水平的IgE，并发展成更严重的支气管肺泡嗜酸性炎症。免疫的Tpl2(-/-)小鼠的支气管肺泡渗出物和经OVA刺激的脾细胞上清液表达高水平的IL-4和IL-5，提示Tpl2消融促进了T细胞应答的Th2极化。对野生型和Tpl2基因敲除小鼠的CD4(+)T细胞的抗CD3刺激表明，Tpl2去除导致了细胞自主T细胞缺陷，这是T细胞对抗原反应的Th2极化的主要原因。Tpl2(+/+)/OT2或Tpl2(-/-)/OT2小鼠的CD4(+)T细胞和Tpl2(+/+)或Tpl2(-/-)小鼠的树突状细胞在OVA刺激的混合培养中Th1和Th2细胞因子的表达进一步支持了这一观察结果。进一步的研究表明，Th1细胞比Th2细胞表达更高水平的Tpl2。因此，Tpl2(-/-)Th1细胞在抗CD3激活ERK方面表现出比Th2细胞更强的缺陷，并且表达低水平的T-bet。鉴于Th1和Th2细胞的发育依赖于T细胞自身的正反馈信号，Tpl2(-/-)Th1细胞的功能缺陷为Tpl2(-/-)小鼠T细胞自主Th2极化提供了机制解释。 我们还研究了MAP3K8在先天免疫细胞中的作用。为了解决这个问题，我们用模型病原体李斯特菌感染了Tpl2(-/-)小鼠。我们发现Tpl2(-/-)小鼠被静脉注射感染。与野生型小鼠相比，单核细胞增多性李斯特氏菌增加了病原菌的负担，并迅速死亡。增强的敏感性与通过TLR2和核苷酸结合寡聚化结构域2的信号受损相关，这两个受体先前被证明介导李斯特菌识别。令人惊讶的是，尽管Tpl2参与了对肿瘤坏死因子的调节，但感染时产生的肿瘤坏死因子并没有受到显著的影响。我们发现，Tpl2的作用在调节肿瘤坏死因子方面具有细胞类型的特异性效应，并传递来自部分但不是全部的模式识别受体(PRR)的信号。与肿瘤坏死因子的细胞类型和受体特异性调节不同，我们发现Tpl2对巨噬细胞和树突状细胞产生IL-1β是必不可少的。这些研究表明，Tpl2是模式识别受体与危险相关分子模式协作的重要媒介，以诱导肿瘤坏死因子和白介素1β的产生，并优化宿主防御。

Cytokines are secreted proteins that regulate cell growth and differentiation. These factors are especially important in regulating immune and inflammatory responses, regulating lymphoid development and differentiation. Cytokines also regulate immune homeostasis, tolerance, and memory. Not surprisingly, cytokines are critical in the pathogenesis of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease and psoriasis. Understanding the molecular basis of cytokine action provides important insights into the pathogenesis of immune-mediated disease and offers new therapeutic targets. Cytokine receptors are associated with Janus family kinases (Jaks), which initiate signaling (see project AR041106-14). Following activation of Jaks, the next step in signaling is the activation of a family of transcription factors called Stats (signal transducers and activators of transcription) (see project AR041159-01). To better understand the molecular actions of cytokines, we performed transcriptional profiling of mouse and human T cells activated by interleukin (IL)-12. We identified a large number of inducible genes, some of which had been previously recognized as being IL-12-inducible. One such gene was the gene encoding the serine/threonine kinase Cot/Tpl2. This kinase was directly inducible by IL-12 and inhibited by IL-4. As IL-12 activates the transcription factor Stat4, we also assessed the dependence of inducible MAP3K8 expression in Stat4-deficient mice. Furthermore, using chromatin immunoprecipitation, we found that MAP3K8 was a direct Stat4 target. To define the role of MAP3K8, we studied MAP3K8 knockout mice. We found that Thelper1 differentiation of MAP3K8-/- mice was markedly deficient. We further found that this was associated with failure to upregulate the transcription factors Stat4 and Tbox21 (T-bet), thus explaining the failure to properly generate IFNg. Challenge of MAP3K8-deficient mice with Toxoplasma gondii showed that these mice had increased susceptibility to this model pathogen, consistent with the impaired ability to generate IFNg. Conversely, in the model of autoimmune disease that mimics multiple sclerosis, experimental autoimmune encephalomyelitis or EAE, MAP3K8-knockout mice had reduced severity of disease. We further established that OVA-immunized Tpl2(-/-) mice express high levels of IgE and develop more severe bronchoalveolar eosinophilic inflammation than Tpl2(+/+) controls, when challenged with OVA intranasally. Bronchoalveolar exudates and supernatants of OVA-stimulated splenocytes from immunized Tpl2(-/-) mice express elevated levels of IL-4 and IL-5, suggesting that Tpl2 ablation promotes the Th2 polarization of the T cell response. Anti-CD3 stimulation of CD4(+) T cells of wild-type and Tpl2 knockout mice revealed that Tpl2 ablation gives rise to a cell autonomous T cell defect that is primarily responsible for the Th2 polarization of the T cell response to Ag. This observation was further supported by experiments addressing the expression of Th1 and Th2 cytokines in OVA-stimulated mixed cultures of CD4(+) T cells from Tpl2(+/+)/OT2 or Tpl2(-/-)/OT2 mice and dendritic cells from Tpl2(+/+) or Tpl2(-/-) mice. Further studies revealed that Th1 cells express significantly higher levels of Tpl2 than Th2 cells. As a result, Tpl2(-/-) Th1 cells exhibit a stronger defect in ERK activation by anti-CD3 than Th2 cells and express low levels of T-bet. Given that the development of Th1 and Th2 cells depends on positive feedback signals from the T cells, themselves, the functional defect of the Tpl2(-/-) Th1 cells provides a mechanistic explanation for the T cell autonomous Th2 polarization in Tpl2(-/-) mice. We also investigated the role of MAP3K8 in innate immune cells. To address this issue, we infected Tpl2(-/-) mice with the model pathogen Listeria monocytogenes. We found that Tpl2(-/-) mice infected i.v. with L. monocytogenes had increased pathogen burdens compared with wild-type mice and rapidly succumbed to infection. Enhanced susceptibility correlated with impaired signaling through TLR2 and nucleotide-binding oligomerization domain 2, two receptors previously shown to mediate Listeria recognition. Surprisingly, TNF production in response to infection was not significantly impaired, even though Tpl2 has been implicated in the regulation of TNF. We found that the role of Tpl2 has cell-type specific effects in regulating TNF and transduces signals from some, but not all, pattern recognition receptors (PRR). In contrast to the cell-type- and receptor-specific regulation of TNF, we found that Tpl2 is essential for IL-1beta production from both macrophages and dendritic cells. These studies implicate Tpl2 as an important mediator for collaboration of pattern recognition receptors with danger-associated molecular patterns to induce TNF and IL-1beta production and optimal host defense.