Neuroimmunological communication via CGRP promotes the development of a regulatory phenotype in TLR4-stimulated macrophages.

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-ß 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.

Deficiency of the CGRP receptor component RAMP1 attenuates immunosuppression during the early phase of septic peritonitis.

The neuropeptide CGRP contributes to the control of excessive cytokine production in endotoxemia models. However, the function of CGRP in sepsis caused by infection with viable pathogens is unknown. Here, we show that mice deficient for the CGRP receptor component RAMP1 have an improved anti-bacterial defense during the early, but not late, phase of polymicrobial septic peritonitis. The protective effect of Ramp1-deficiency was associated with reduced levels of IL-10 in plasma and peritoneal lavage fluid. Consistent with these findings, CGRP markedly increased IL-10 production of peritoneal and bone marrow-derived macrophages in response to short term stimulation with LPS in vitro. In addition, the lack of an intact CGRP receptor resulted in an increased recruitment and activation of neutrophils and caused an enhanced release of defensin-α1 in the peritoneal cavity. Considered together, our results identify the neuropeptide CGRP as a crucial immunosuppressive mediator impairing host defense during the early, but not late, phase of septic peritonitis.

Cutting edge: Divergent cell-specific functions of MyD88 for inflammatory responses and organ injury in septic peritonitis.

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 (Myd88(MYEL)), 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, Myd88(MYEL) 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 Myd88(MYEL) mice. Moreover, Myd88(MYEL) 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.

Improved host defense against septic peritonitis in mice lacking MyD88 and TRIF is linked to a normal interferon response.

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(-/-);Trif(Lps2/Lps2) mice had markedly reduced bacterial numbers in the peritoneal cavity and peripheral blood, indicating that bacterial clearance in this model is improved in the absence of MyD88/TRIF signals. Survival of Myd88(-/-); Trif(Lps2/Lps2) mice was improved significantly. The lack of MyD88/TRIF signaling prevented the excessive production of inflammatory cytokines and of IL-10. In contrast, Ifng mRNA was expressed at WT levels, and induction of Ifnb mRNA was reduced only by one-half. Consistent with these findings, numerous IFN-regulated genes, including p47 and p65 GTPases, as well as CXCL10, were expressed in a MyD88/TRIF-independent manner. In support of the in vivo data, Myd88(-/-); Trif(Lps2/Lps2) macrophages stimulated with live intestinal bacteria produced normal amounts of CXCL10. The production of p47 GTPases and CXCL10 in septic peritonitis was found to be dependent on the presence of IFNAR1, but not IFN-γ, indicating a normal induction of the type I IFN response in Myd88(-/-);Trif(Lps2/Lps2) mice, despite attenuated IFN-ß production. Together, these results provide evidence that in severe septic peritonitis, the absence of MyD88 and TRIF balances the innate immune response in a favorable manner by attenuating deleterious responses such as excessive cytokine release, while leaving intact protective IFN responses.

The neuropeptide calcitonin gene-related peptide causes repression of tumor necrosis factor-alpha transcription and suppression of ATF-2 promoter recruitment in Toll-like receptor-stimulated dendritic cells.

Sensory nerves may dampen inflammatory processes through the release of the neuropeptide calcitonin gene-related peptide (CGRP). CGRP mediates immunosuppressive activities through up-regulation of interleukin-10 or, alternatively, through an interleukin-10-independent pathway that is associated with rapid induction of the transcriptional inducible cAMP early repressor (ICER). In this work, we further investigated the molecular mechanisms of immune modulation by CGRP. Using TLR2-stimulated dendritic cells, we show that inhibition of tumor necrosis factor-alpha production by CGRP is dependent on up-regulation of endogenous ICER. Dendritic cell expression of ICER was selectively induced by CGRP and elevation of cellular cAMP levels but not by numerous pro- and anti-inflammatory cytokines. Treatment of dendritic cells with CGRP did not interfere with the induction of Tnfa gene expression but caused premature repression of TLR2-induced transcriptional activity. ATF-2 was rapidly phosphorylated and recruited to the Tnfa promoter following ligation of TLR2. Concomitant administration of CGRP completely prevented binding of ATF-2 to the Tnfa promoter, whereas recruitment of ICER was markedly elevated. In contrast, CGRP did not influence TLR2-stimulated binding of NF-kappaB p65. Together, these results are consistent with a model suggesting that CGRP causes rapid up-regulation of ICER, which in turn competes with ATF-2 for binding to the Tnfa promoter, leading to repression of gene expression.

Organ-specific role of MyD88 for gene regulation during polymicrobial peritonitis.

Sepsis leads to the rapid induction of proinflammatory signaling cascades by activation of the innate immune system through Toll-like receptors (TLR). To characterize the role of TLR signaling through MyD88 for sepsis-induced transcriptional activation, we investigated gene expression during polymicrobial septic peritonitis by microarray analysis. Comparison of gene expression profiles for spleens and livers from septic wild-type and MyD88-deficient mice revealed striking organ-specific differences. Whereas MyD88 deficiency strongly reduced sepsis-induced gene expression in the liver, gene expression in the spleen was largely independent of MyD88, indicating organ-specific transcriptional regulation during polymicrobial sepsis. In addition to genes regulated by MyD88 in an organ-dependent manner, we also identified genes that exhibited an organ-independent influence of MyD88 and mostly encoded cytokines and chemokines. Notably, the expression of interferon (IFN)-regulated genes was markedly increased in septic MyD88-deficient mice compared to that in septic wild-type controls. Expression of IFN-regulated genes was dependent on the adapter protein TRIF. These results suggest that the influence of MyD88 on gene expression during sepsis strongly depends on the organ compartment affected by inflammation and that the lack of MyD88 may lead to disbalance of the expression of IFN-regulated genes.

Identification of a TLR4- and TRIF-dependent activation program of dendritic cells.

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-beta (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 TRIF-dependent. 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.