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.
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