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The liver is innervated by primary sensory nerve fibres releasing the neuropeptide calcitonin gene-related peptide (CGRP). Elevated plasma levels of CGRP have been found in patients with liver fibrosis or cirrhosis. We hypothesised that signalling of CGRP and its receptors might regulate liver fibrosis and propose a novel potential target for the treatment. In this study, hepatic expression of CGRP and its receptor component, the receptor activity-modifying protein 1 (RAMP1), was dramatically increased in diseased livers of patients. In a murine liver fibrosis model, deficiency of RAMP1 resulted in attenuated fibrogenesis characterized by less collagen deposition and decreased activity of hepatic stellate cells (HSC). Mechanistically, activity of the TGFβ1 signalling core component Smad2 was severely impaired in the absence of RAMP1, and Yes-associated protein (YAP) activity was found to be diminished in RAMP1-deficient liver parenchyma. In vitro, stimulation of the HSC line LX-2 cells with CGRP induces TGFβ1 production and downstream signalling as well as HSC activation documented by increased α-SMA expression and collagen synthesis. We further demonstrate in LX-2 cells that CGRP promotes YAP activation and its nuclear translocation subsequent to TGFβ1/Smad2 signals. These data support a promotive effect of CGRP signalling in liver fibrosis via stimulation of TGFβ1/Smad2 and YAP activity., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Stringent regulation of the inflammatory response is crucial for normal tissue regeneration. Here, we analyzed the role of Toll-like receptor 3 (TLR3) in pancreatic regeneration after acute pancreatitis (AP). AP was induced by caerulein treatment in mice with global TLR3 deficiency (TLR3 OFF ) or in mice re-expressing TLR3 exclusively in the myeloid cell lineage (TLR3 Mye ). Compared to WT mice, TLR3 OFF mice had a markedly increased formation of acinar-to-ductal metaplasia (ADM) that persisted until day 7 after initiation of AP. Pancreatic tissue of WT mice was completely regenerated after 5 days with no detectable ADM structures. The enhancing effect of TLR3-deficiency on ADM formation was closely linked with an increased and prolonged accumulation of macrophages in pancreata of TLR3 OFF mice. Importantly, the phenotype of TLR3 OFF mice was rescued in TLR3 Mye mice, demonstrating the causative role of myeloid cell selective TLR3 signaling. Moreover, in vitro stimulation of macrophages through TLR3 initiated cell death by a caspase-8-associated mechanism. Therefore, these findings provide evidence that TLR3 signaling in myeloid cells is sufficient to limit inflammation and ADM formation and to promote regeneration after AP. Notably, resolution of inflammation after AP was associated with macrophage sensitivity to TLR3-mediated cell death., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

TLR3 is implicated in anti-viral immune responses, but may also act as a sensor of tissue damage in the absence of infection. Here, we provide evidence for an essential role of TLR3 in liver regeneration after an acute loss of tissue due to partial hepatectomy. Mice lacking TLR3 had a severe and sustained defect in the restoration of liver tissue with reduced liver-to-body weight ratios even after an extended recovery period of 2 weeks. Hepatocyte cell cycle progression into S phase was impaired in TLR3-deficient mice. Mechanistic analyses revealed that TLR3-deficient mice had markedly reduced systemic levels of active HGF, but had increased amounts of inactive tissue-bound HGF. Importantly, expression of uPA, which orchestrates the processing and release of HGF from the hepatic extracellular matrix, was reduced in regenerating livers of TLR3-deficient mice. In addition, expression of the HGF maturation factor HGFAC was transiently diminished in TLR3-deficient mice. In vitro, engagement of TLR3 directly stimulated expression of uPA by hepatic stellate cells. Thus, TLR3 supports liver regeneration through upregulation of uPA, which promotes the release of preformed HGF from extracellular matrix stores., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

The effectiveness of liver regeneration limits surgical therapies of hepatic disorders and determines patient outcome. Here, we investigated the role of the neuropeptide calcitonin gene-related peptide (CGRP) for liver regeneration after acute or chronic injury. Mice deficient for the CGRP receptor component receptor activity-modifying protein 1 (RAMP1) were subjected to a 70% partial hepatectomy or repeated intraperitoneal injections of carbon tetrachloride. RAMP1 deficiency severely impaired recovery of organ mass and hepatocyte proliferation after both acute and chronic liver injury. Mechanistically, protein expression of the transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) was decreased in regenerating livers of RAMP1-deficient mice. Lack of RAMP1 was associated with hyperphosphorylation of YAP on Ser127 and Ser397, which regulates YAP functional activity and protein levels. Consequently, expression of various YAP-controlled cell cycle regulators and hepatocyte proliferation were severely reduced in the absence of RAMP1. In vitro, CGRP treatment caused increased YAP protein expression and a concomitant decline of YAP phosphorylation in liver tissue slice cultures of mouse and human origin and in primary human hepatocytes. Thus, our results indicate that sensory nerves represent a crucial control element of liver regeneration after acute and chronic injury acting through the CGRP-RAMP1 pathway, which stimulates YAP/TAZ expression and activity., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Stimulation of cytosolic nucleic acid sensors of innate immunity by pathogen-derived nucleic acids is important for antimicrobial defence, but stimulation through self-derived nucleic acids may contribute to autoinflammation and cancer. DNA sensing in the cytosol requires the stimulator of interferon genes (STING), while cytosolic RNA sensors use mitochondrial antiviral-signalling protein (MAVS). In a murine model of two-thirds hepatectomy, combined deficiency of MAVS and STING resulted in strongly impaired hepatocyte proliferation and delayed recovery of liver mass. Whereas lack of MAVS and STING did not influence upregulation of the G 1 -phase cyclins D1 and E1, it substantially reduced the hyperphosphorylation of retinoblastoma protein, attenuated the activation of cyclin-dependent kinase (CDK)-2, delayed upregulation of CDK1 and cyclins A2 and B1, and impaired S-phase entry of hepatocytes. Mechanistically, lack of cytosolic nucleic acid sensors strongly upregulated the anti-proliferative mediators TGF-β2 and activin A, which was associated with an increased expression of the cell cycle inhibitors p15 and p21. Partial hepatectomy was followed by the release of exosomes with abundant nucleic acid cargo, which may provide ligands for the MAVS and STING pathways. Together, these findings identify a previously unrecognised function of cytosolic nucleic acid sensors of innate immunity for promoting liver regeneration.

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.

It has been postulated that an early systemic inflammatory response syndrome (SIRS) and a subsequent compensatory anti-inflammatory response syndrome (CARS) occur sequentially in sepsis. Co-existence of both is referred to as mixed antagonist response syndrome (MARS). Pro- and anti-inflammatory cytokine production was investigated in patients with postoperative sepsis, a murine peritonitis model and in vitro to further delineate the interaction of hyper- and hypo-inflammation in sepsis. IL-6 and IL-10 were measured in serum samples from 80 patients on d1 and d2 of postoperative sepsis and were similarly determined at various time points after induction of septic peritonitis in mice. Cytokine production of RAW264 macrophages was stimulated in vitro using TLR agonists. IL-6 and IL-10 were measured in supernatants. All cytokine measurements were performed by ELISA. In patients, the initial phase of the immune response to sepsis was characterized by a concomitant elevation of serum IL-6 and IL-10 levels. IL-10 levels were correlated with IL-6 levels in an exponential manner (p<0.001), which could be confirmed in a mouse model of septic peritonitis. In vitro experiments revealed that the observed exponential correlation may occur as function of TLR signaling intensity. Early postoperative sepsis seems to be characterized by a primary MARS. Sepsis severity was positively correlated with a disproportionate elevation of the anti-inflammatory response relative to the pro-inflammatory response, a pattern reminiscent of TLR-driven responses. Detailed characterization of immune responses in sepsis may help to direct standard therapies and to develop effective immunomodulatory strategies., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

The adapter protein TRIF mediates signal transduction through TLR3 and TLR4, inducing production of type I IFNs and inflammatory cytokines. The present study investigates the mechanisms by which TRIF signaling controls TNF-alpha biosynthesis. We provide evidence that, in LPS-stimulated murine dendritic cells, TRIF stimulates TNF-alpha biosynthesis selectively at the posttranscriptional level by promoting mRNA translation. In the absence of functional TRIF, the production of TNF-alpha protein was severely impaired, whereas TNF-alpha mRNA levels and stability, as well as transcriptional activity of the Tnfa gene, were not affected. Similarly, TRIF was required for production of LPS-induced TNF-alpha protein, but not of mRNA, in bone marrow-derived macrophages. In peritoneal macrophages, however, TRIF was also required for normal induction of TNF-alpha mRNA, suggesting cell type-related functions of TRIF. The influence of TRIF on dendritic cell TNF-alpha production was independent of type I IFNs. TRIF was required for prolonged activation of MAPKs in LPS-stimulated dendritic cells but was dispensable for the activation of NF-kappaB. Inhibition of late p38 activity attenuated LPS-stimulated elevation of TNF-alpha protein but not mRNA levels. The p38 effector kinase MK2 was directly activated through the TRIF pathway of TLR4. Importantly, stimulation of Mk2(-/-) cells through TLR3 or TLR4 severely impaired TNF-alpha protein production but did not affect TNF-alpha mRNA induction. Together, these results indicate that the TRIF signaling pathway promotes TNF-alpha mRNA translation through activation of the protein kinase MK2.

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.

Communication between the nervous and immune systems involves the release of neuropeptides, such as calcitonin gene-related peptide (CGRP), from sensory nerves during inflammation. CGRP may inhibit the activities of both innate and adaptive immune cells, but the molecular pathways underlying this function are largely unknown. In this study, we identify CGRP as a potent inhibitor of TLR-stimulated production of inflammatory mediators, such as TNF-alpha and CCL4, by murine dendritic cells. Inhibition of TLR responses was independent of IL-10 and did not involve perturbation of canonical TLR signaling, including activation of MAPK and NF-kappaB. Instead, the inhibitory activity of CGRP was mediated by the cAMP/protein kinase A pathway leading to rapid up-regulation of the transcriptional repressor, inducible cAMP early repressor (ICER). Ectopically expressed ICER directly repressed the LPS-stimulated activity of a synthetic Tnf promoter, as well as TNF-alpha protein production driven by the endogenous promoter. Inhibition of dendritic cell gene expression by CGRP was associated with the presence of a composite cAMP response element/kappaB promoter element. In a murine model of endotoxemia, CGRP markedly attenuated serum TNF-alpha levels, and this effect was associated with the up-regulation of ICER. Together, these results establish a novel pathway for the negative regulation of TLR responses through the nervous system that critically involves induction of the transcriptional repressor ICER by the neuropeptide CGRP.

Interleukin-22 (IL-22) is a recently discovered proinflammatory cytokine, structurally related to IL-10. Since IL-22 is induced by lipopolysaccharide in vivo, we studied the role of IL-22 in a model of polymicrobial peritonitis. Quantitative real-time reverse transcription-PCR analysis showed marked induction of IL-22 and IL-22 receptor in spleen and kidney during the course of sepsis. The biological activity of IL-22 is modulated by IL-22-binding protein (IL-22BP), which is considered a natural antagonist of IL-22. To further analyze the role of IL-22 during septic peritonitis, mice were treated with recombinant IL-22BP generated as Fcgamma2a fusion protein. IL-22BP-Fc completely blocked IL-22-induced STAT3 activation in hepatocytes in vitro. Treatment of mice with IL-22BP-Fc 4 h before sepsis induction led to enhanced accumulation of neutrophils and mononuclear phagocytes and a reduced bacterial load at the site of infection. In addition, IL-22 blockade led to an enhanced bacterial clearance in liver and kidney and reduced kidney injury. These results imply an important proinflammatory role of IL-22 during septic peritonitis, contributing to bacterial spread and organ failure. IL-22 therefore appears to play an important role in the regulation of inflammatory processes in vivo.

Vincristine (VCR) accumulation in chronic lymphatic leukemia of B-cell origin (B-CLL) has recently been shown not to be inversely correlated to P-glycoprotein (PGP) levels. Therefore, we studied, in addition to PGP expression and accumulation of VCR, the cellular beta-tubulin content in quiescent and rhIL-2 activated B-CLL cells. VCR mediates cytotoxicity by binding to tubulin. Constitutive beta-tubulin levels in B-CLL cells varied considerably. Upon activation with rhIL-2, beta-tubulin expression increased significantly. Therefore, tubulin levels could be correlated over a wide range to VCR accumulation. When the PGP-mediated drug efflux was blocked by verapamil (VRP), tubulin levels correlated linearly to VCR accumulation. All B-CLL cases expressed PGP at different levels. There was no linear correlation between PGP expression and VCR accumulation. A modulation factor m was defined as a quotient of VCR accumulation in the presence and absence of VRP to define the extent by which VRP inhibited a steady-state accumulation of VCR. The factor allowed discrimination between B-CLLs expressing low versus high PGP, irrespective of the levels of tubulin. However, PGP and beta-tubulin levels together were predictive for VCR accumulation in steady state. There was no uniform-accumulation defect for VCR in B-cell CLL because beta-tubulin and PGP were expressed independently. Non PGP-mediated VCR transport seems to play a minor role in B-cell CLL. Leukemia-associated varying of cytoskeletal organization in B-cell CLL might be one reason for the diverse cellular responses to receptor-mediated signals.

Reduced drug accumulation may be one reason for intrinsic drug resistance in chronic lymphatic leukemia of the B-cell type (B-CLL). Immunophenotyped leukemic human B-cells from 38 cases of B-CLL were characterized for P-glycoprotein (PGP) content. In all, 30 cases of B-CLL were additionally analyzed for further parameters: accumulation of daunorubicin (DNR, n = 20) and rhodamine 123 (Rh123, n = 30) in both the presence and the absence of verapamil (VRP). Also, 16 cases of B-CLL were analyzed for vincristine (VCR) accumulation with or without VRP. Concerning the relative expression of PGP, these 16 cases of B-CLL were representative for the whole group of 30 cases. Spontaneous accumulation of Rh123 and VCR varied over a wide range: accumulation of Rh123, by a factor of 11.8; accumulation of VCR, by a factor of 9.7; and accumulation of DNR, by a factor of 3.6. VRP modulated the accumulation of RH123 in 16/30 cases (53%), that of VCR in 69% of cases, and that of DNR in 11% of cases. The maximal VRP-mediated increases in accumulation amounted to factors of 1.3 for DNR, 2.3 for Rh123, and 7.8 for VCR. Spontaneous drug accumulation did not correlate with the extent of chemomodulation. The amount of PGP in B-CLL cells (all cases studied were PGP-positive) did not correlate with drug accumulation or with the extent of VRP-mediated chemomodulation. Thus, high expression of PGP is only partially responsible for defective drug accumulation in B-CLL. Only the degree of chemomodulation by VRP is predictive for the extent of the PGP-related functional drug accumulation defect. Thus, in B-CLL, PGP-independent drug accumulation defects seem to be as important as those mediated by PGP. The extent of this drug accumulation defect varies for the different drugs in the following order VCR > Rh123 > DNR. The relevance of PGP-mediated and -independent drug accumulation defects in vivo may depend to a large extent on the drug being used and on the individual cell type. Both types of defect in drug accumulation are of high importance when regimens include VCR a drug commonly used in second-line chemotherapy of B-CLL. Both defects in drug accumulation may be responsible for intrinsic VCR resistance in B-CLL.