Dendritic cell activation by Toll-like receptors (TLR) is crucial for the generation of protective immune responses. In addition to the common myeloid differentiation factor 88 (MyD88)-dependent signaling pathway, TLR4 engages the adaptor protein Toll/IL-1 receptor (TIR)-domain-containing adaptor inducing IFN-g (TRIF), leading to interferon regulatory factor 3 (IRF-3) activation and type I interferon production. Using microarray expression profiling we now identify TRIF as a major regulator of the TLR4-triggered activation program of dendritic cells. We show that the expression of 47% of the genes that are responsive to TLR4 stimulation in wild-type dendritic cells is significantly altered in cells carrying a loss-of-function mutation of TRIF. Specifically, expression of IL-12, IL-18, and IL-23 was impaired in the absence of functional TRIF, suggesting that TLR4-promoted Th1 responses are TRIFdependent. Furthermore, we provide evidence that TRIF regulates TLR4-mediated gene expression both by type I IFN-dependent and -independent mechanisms. Whereas dendritic cell production of CXCL10 and CCL12 was dependent on both TRIF and the type I interferon receptor, expression of IL-6 required TRIF but not type I interferon activity. Functional TRIF was also required for the normal induction of numerous genes considered important for host defense against diverse pathogens. Together, these data therefore identify TRIF as a crucial regulator of TLR4-dependent dendritic cell responses.

Although global MyD88 deficiency attenuates lethal inflammation in sepsis, cell-specific functions of MyD88 remain largely unknown. Using mice with selective expression of MyD88 in myeloid cells (Myd88MYEL), we show that, during polymicrobial septic peritonitis, both myeloid and nonmyeloid cells contribute to systemic inflammation, whereas myeloid cell MyD88 was sufficient to fully establish the peritoneal cytokine response. Importantly, Myd88MYEL mice developed markedly aggravated liver injury that was linked to impaired upregulation of cellular inhibitor of apoptosis protein 2 and an excessive production of TNF-α. Upregulation of inducible cAMP early repressor (ICER), a known transcriptional repressor of the Tnfa gene, was impaired in Myd88MYEL mice. Moreover, Myd88MYEL mice showed enhanced transcription of the Tnfa gene and an excessive production of CCL3, which is also negatively regulated by ICER, but they had normal levels of CXCL1, which is expressed in an ICER-independent manner. Together, these findings suggest a novel protective role for nonmyeloid cell MyD88 in attenuating liver injury during septic peritonitis.

Environmental signals shape the phenotype and function of activated macrophages. Here, we show that the neuropeptide calcitonin gene-related peptide (CGRP), which is released from sensory nerves, modulates the phenotype of TLR4-activated murine macrophages by enhancing expression of the regulatory macrophage markers IL-10, sphingosine kinase 1 (SPHK1), and LIGHT (lymphotoxin-like, exhibits inducible expression and competes with HSV glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytes). In contrast, CGRP inhibits production of cytokines characteristic of inflammatory macrophages and does not affect expression of wound-healing macrophage markers upon TLR4 engagement. In IL-4-stimulated macrophages, CGRP increased LIGHT expression, but failed to induce IL-10 and SPHK1. The stimulatory effect of CGRP on IL-10 production required activation of protein kinase A and was linked to prolonged phosphorylation of CREB and sustained nuclear accumulation of CRTC2 and CRTC3 (where CRTC is CREB-regulated transcriptional cofactor). CGRP enhanced expression of regulatory macrophage markers during the early, but not late, phase of LPS-stimulation and this effect was independent of autocrine type-I IFN activity. In contrast, autocrine type-I IFN activity and treatment of macrophages with IFN-β promoted late-phase IL-10 production, but had only minor influence on LIGHT and SPHK1 expression. Together, the results identify neuroimmunological communication through CGRP as a novel costimulatory pathway promoting the development of a regulatory phenotype of TLR4-stimulated macrophages. CGRP appears to act through a mechanism that involves sustained activation of CREB-dependent gene transcription. Keywords:Calcitonin gene-related peptide (CGRP) r IL-10 r Regulatory macrophages r CREB Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionSensory nerves containing calcitonin gene-related peptide (CGRP) are abundant in lymphoid organs and directly contact tissueresident immune cells [1][2][3][4][5]. During inflammation, tryptase secreted from inflammatory mast cells may stimulate the release Correspondence: Prof. Bernhard Holzmann e-mail: bernhard.holzmann@tum.de of CGRP by activating the proteinase-activated receptor 2 on sensory nerves [6]. The cellular receptor for CGRP is expressed on most immune cells and is generated by association of the type-I transmembrane protein receptor activity modifying protein-1 (RAMP1) with the seven-transmembrane domain protein calcitonin receptor-like receptor [7]. This heterodimeric receptor is * These authors contributed equally to this work. Anti-inflammatory effector mechanisms of CGRP include upregulation of the transcriptional repressor inducible cAMP early repressor, inhibition of NF-κB activity, and enhanced production of . Increased production of IL-10 in response to CGRP may dampen the release of IL-12 by PBMCs and DCs, reduce DC expression of CD86, and impair the capacity of Langerhans cells to ...

The signaling adapters MyD88 and TRIF are engaged by TLRs and/or receptors of the IL-1 family and are considered important for innate immune responses that combat bacterial infections. Here, the consequences of a combined MyD88 and TRIF deficiency for the innate immune response against severe septic peritonitis was examined. We demonstrate that Myd88 Ϫ/Ϫ ;Trif Lps2/Lps2