Regulation of human dendritic cell activation

toll样受体（TLR）配体的识别导致宿主免疫的机制仍然不清楚。现在认为，为了诱导有效的免疫应答，微生物必须刺激TLR家族内外的复杂的模式识别受体。这些不同受体的联合激活可产生互补、协同或拮抗作用，从而调节先天免疫和适应性免疫。因此，要全面了解tlr在宿主抗感染中的作用，需要“解码”这些多受体相互作用。我们过去的工作已经揭示了不同T细胞受体配体在树突状细胞活化中的重要协同作用，以及I型干扰素在树突状细胞、组织和肿瘤细胞中调节TLR下游信号传导的作用。特别是，我们发现细胞对TLR配体的反应不仅是促炎介质的产生，而且还参与组织稳态的控制，调节细胞分化、增殖和凋亡。MyD88和TRIF信号和I型IFN产生之间的平衡决定了组织和肿瘤细胞的增殖与凋亡以及树突状细胞的活化与存活。我们还发现，β -葡聚糖（酵母和真菌的一种成分）通过dectin 1受体刺激树突状细胞，促进了人类Th17反应的诱导，并与TLR受体刺激协同激活树突状细胞和促炎细胞因子的产生。在去年，我们发现通过dectin 1（一种细胞质部分具有itam样基元的受体）的信号传导只会诱导少量早期应答细胞因子，如il -1 β、Il-6和TNF，并且只会非常适度地激活NF-kappaB。然而，β -葡聚糖刺激诱导的il -1 β（并通过炎性体加工，也通过dectin-1刺激激活）发挥了强有力的正反馈机制，这是优化NF-kappaB激活和产生晚反应细胞因子如IL-12和IL-23所必需的。我们现在计划扩展IL-1家族细胞因子在含有itam的受体激活树突状细胞和其他免疫细胞中的研究。我们特别想了解这些受体激活NF-kappaB的能力在多大程度上是直接通过BCL10激活的，还是依赖于内源性IL-1提供的放大信号。我们还想了解不同的含itam受体正或负调节树突状细胞和髓细胞对TLR配体的反应性的分子机制。我们特别想了解Akt/mTOR通路的作用以及I型干扰素对其的调节。观察到β -葡聚糖刺激而非LPS刺激诱导人单核dc分泌高水平的il -1 β(6)，我们研究il -1 β是否在β -葡聚糖反应中对细胞因子的分泌起反馈积极作用。IL-1的产生依赖于SYK介导的NALP3的激活，而NALP3反过来激活促IL-1剪接和分泌所需的caspase-1。用IL-1RA（或用il -1 β抗体或阻断caspase-1）阻断内源性il -1 β的作用，早期基因的诱导不受影响，但几个晚期基因的表达几乎完全被消除。诱导基因可分为三大类；早期，il -1独立基因（在4 h诱导良好，但在12 h诱导较差）；晚期il -1依赖性基因（12 h诱导效果好于4 h）；和晚期非il -1基因。总的来说，我们的分析确定了β -葡聚糖诱导的基因亚群严格依赖内源性IL-1来维持单dc的表达，这些基因包括许多免疫相关基因，如IL-12、IL-23、IL-10和TNF。这些基因中的许多也被LPS诱导，但大多数只具有短暂的动力学。在LPS中加入外源性IL-1 β维持了这些基因的表达，这表明IL-1R和TLR同样可以为这些基因的转录发出信号，但它们的长期表达需要内源性IL-1的正反馈。TNF对所有NF-kB依赖基因的基因表达也有一定的正反馈作用，但对IL-1依赖基因的作用是适度的，在缺乏IL-1的情况下，TNF的作用不足以维持这些基因对β -葡聚糖或LPS的表达。这两种配体都诱导I型IFN依赖基因的早期表达，当内源性I型IFN信号被中和抗体阻止时，它被阻断。然而，LPS对ifn - β的诱导是适度的，β -葡聚糖几乎检测不到。这些研究清楚地揭示了炎症和体内平衡基因调控的新机制，这些机制可能在炎症、免疫和癌症中发挥重要作用。白细胞介素-1和干扰素- γ通过IkB-zeta差异程序β -葡聚糖激活的树突状细胞（已提交发表）：调节树突状细胞（DC）对toll样受体（TLR）配体反应的分子网络已经进行了详细的研究，但itam信号凝集素受体引发的分子网络尚未得到很好的理解。在这里，我们使用基因表达/扰动方法分析了β -葡聚糖（Dectin-1的配体）对人类单核细胞来源的树突状细胞（dc）的激活。我们发现β -葡聚糖通过IL-1 - β介导的正反馈，通过维持转录辅助因子IkB-zeta的表达，转录激活晚期诱导基因，如编码免疫调节细胞因子IL -6、IL-12、IL-23和IL-10的基因。我们证明，除了已知的作用于T细胞的能力外，il -1 β还编程dc以促进Th17反应。两种il -1 β活性都依赖于myd88介导的IkB-zeta的诱导。干扰素(IFN)- γ启动β -葡聚糖激活的dc干扰IL-1/IkB-zeta轴，导致细胞因子产生促进Th1而不是Th17反应的模式。因此，内源性il -1 β和外源性ifn - γ差异调节DC编程相关基因的表达，并可能在病原体激活itam相关受体引起的炎症环境中微调免疫反应中发挥重要作用。树突状细胞、巨噬细胞和其他髓细胞在调节对感染的适应性和先天免疫反应以及炎症的调节和失调中发挥作用，因为它与感染、退行性和自身免疫性疾病和癌症有关。通过这些机制调节树突状细胞和骨髓细胞的功能在免疫治疗（如疫苗、癌症免疫治疗）的规划中以及在疾病治疗和癌症预防/治疗中靶向炎症病理中是重要的。

The mechanisms by which the recognition of Toll-like receptor (TLR) ligands leads to host immunity remain poorly defined. It is now thought that to induce an effective immune response, microorganisms must stimulate complex sets of pattern-recognition receptors, both within and outside of the TLR family. The combined activation of these different receptors can result in complementary, synergistic or antagonistic effects that modulate innate and adaptive immunity. Therefore, a complete understanding of the role of TLRs in host resistance to infection requires 'decoding' of these multiple receptor interactions. Our past work has uncovered the significant synergy in dendritic cell activation between ligands of different T cell receptors and the role of type I interferon in regulating TLR downstream signaling in both dendritic cells and tissue and tumor cells. In particular we showed that the cellular response to TLR ligands is not only production of pro-inflammatory mediators but they are also involved in control of tissue homeostasis and regulate cellular differentiation, proliferation, and apoptosis. The balance between MyD88 and TRIF signaling and the production of type I IFN determine proliferation versus apoptosis in tissue and tumor cells and activation versus survival in dendritic cells. We also have found that the stimulation of the dendritic cells by beta-glucan (a component of yeast and fungi) through the dectin 1 receptor facilitate the induction of an Th17 response in human and synergize with TLR receptor stimulation for activation of dendritic cells and pro-inflammatory cytokine production. In the last year we have discovered that the signaling through dectin 1, a receptor with an ITAM-like motif in its cytoplasmic portion, results in the induction of only a small number of early responding cytokine such as Il-1beta, Il-6, and TNF and in only a very modest activation of NF-kappaB. However, IL-1beta induced by beta-glucan stimulation (and processed through the inflammasome also activated through dectin-1 stimulation) exert a potent positive feedback mechanisms that it is necessary for optimal NF-kappaB activation and production of late responsive cytokines such as IL-12 and IL-23. We now plan to extend the study of the IL-1 family of cytokines in the activation of dendritic cells and other immune cells by ITAM-containing receptors. In particular we want to understand how much the ability of these receptors to activate NF-kappaB is direct through BCL10 activation or it is dependent through the amplifying signal provided by endogenous IL-1. We also want to understand the molecular mechanisms by which different ITAM-containing receptors positively or negatively regulate the responsiveness of dendritic cells and myeloid cells to TLR ligands. In particular we want to undersatnd the role of the Akt/mTOR pathway and its modulation by type I interferon. The observation that beta-glucan stimulation but not LPS stimulation induced high levels of IL-1beta secretion by human mono-DC (6) led us to investigate whether IL-1beta plays a feedback positive role in the secretion of cytokines in response to beta-glucan. IL-1 production was dependent on SYK mediated activation of NALP3 that in turn activated caspase-1 required for pro-IL-1beta splicing and secretion. Blocking the effect of endogenous IL-1beta with IL-1RA (or with antibodies to IL-1beta or by blocking caspase-1) the early gene induction was not affected but the expression of several of the late genes was almost completely abolished. Induced genes could be divided into three major groups; early, IL-1-independent genes (induced well at 4 h but less so at 12 h); late IL-1-dependent genes (induced better at 12 than at 4 h); and late IL-1-independent genes. Overall our analysis established that a subset of genes induced by beta-glucan is strictly dependent on endogenous IL-1 for maintained expression in mono-DC and these genes include many immunologically relevant genes such as IL-12, IL-23, IL-10, and TNF. Many of these genes are also induced by LPS but most of them with only a transient kinetics. Addition of exogenous IL-1beta to LPS maintains the expression of these genes, suggesting that the IL-1R and TLR can similarly signal for the transcription of these genes but that their long term expression requires the positive feedback of endogenous IL-1. TNF also has some positive feedback effects on gene expression of all NF-kB dependent genes but on the IL-1-dependent gene the effect of TNF is modest and insufficient in the absence of IL-1 to maintain the expression of these genes in response to either beta-glucan or LPS. Both ligands induce the early expression of type I IFN dependent genes that it is blocked when endogenous type I IFN signaling is prevented by neutralizing antibodies. However, the induction of IFN-beta is modest with LPS and almost undetectable with beta-glucan. These studies are clearly unveiling new mechanisms of inflammatory and homeostatic gene regulation that are likely to play an important role in inflammation, immunity, and cancer. Interleukin-1 and Interferon-gamma differentially Program beta-Glucan-Activated Dendritic Cells via IkB-zeta (submitted for publication): The molecular networks regulating the dendritic cell (DC) response to Toll-like receptor (TLR) ligands have been studied in great details but those elicited by ITAM-signaling lectin receptors are not as well understood. Here, we analyzed the activation of human monocyte-derived dendritic cells (DCs) by beta-glucan, a ligand for Dectin-1, using a gene expression/perturbation approach. We show that beta-glucan transcriptionally activates late induced genes such as those encoding the immunoregulatory cytokines interleukin (IL)-6, IL-12, IL-23, and IL-10 via an IL-1beta-mediated positive feedback that acts by maintaining the expression of the transcriptional cofactor IkB-zeta. We demonstrate that in addition to its known ability to act on T cells, IL-1beta also programs DCs to promote Th17 responses. Both IL-1beta activities are dependent on the MyD88-mediated induction of IkB-zeta. Interferon (IFN)-gamma priming of beta-glucan-activated DCs interfered with the IL-1/IkB-zeta axis and resulted in a pattern of cytokine production that promoted Th1 rather than Th17 responses. Thus, endogenous IL-1beta and exogenous IFN-gamma differentially regulate the expression of genes relevant for DC programming and are likely to play a major role in fine tuning the immune response in the inflammatory environment caused by pathogens activating ITAM-associated receptors. Dendritic cells, macrophages, and other myeloid cells play a role both in the regulation of the adaptive and innate immune response to infection as well as in the regulation and dysregulation of inflammation as it relates to infections, degenerative and autoimmune diseases and cancer. Regulating through these mechanisms dendritic cell and myeloid cell functions is important in the planning of immunotherapy (e.g. vaccine, cancer immunotherapy) as well in targeting inflammation-based pathology in disease therapy and cancer prevention/treatment.