Advances in the Treatment of Implant-Associated Infections of the Pelvis: Eradication Rates, Recurrence of Infection, and Outcome.

INTRODUCTION: Surgical site infections after operative stabilization of pelvic and acetabular fractures are rare but serious complications. The treatment of these infections involves additional surgical procedures, high health care costs, a prolonged stay, and often a worse outcome. In this study, we focused on the impact of the different causing bacteria, negative microbiological results with wound closure, and recurrence rates of patients with implant-associated infections after pelvic surgery. MATERIAL AND METHODS: We retrospectively analyzed a study group of 43 patients with microbiologically proven surgical site infections (SSI) after surgery of the pelvic ring or the acetabulum treated in our clinic between 2009 and 2019. Epidemiological data, injury pattern, surgical approach, and microbiological data were analyzed and correlated with long-term follow-up and recurrence of infection. RESULTS: Almost two thirds of the patients presented with polymicrobial infections, with staphylococci being the most common causing agents. An average of 5.7 (±5.4) surgical procedures were performed until definitive wound closure. Negative microbiological swabs at time of wound closure were only achieved in 9 patients (21%). Long-term follow-up revealed a recurrence of infection in only seven patients (16%) with an average interval between revision surgery and recurrence of 4.7 months. There was no significant difference of recurrence rate for the groups of patients with positive/negative microbiology in the last operative revision (71% vs. 78%). A positive trend for a correlation with recurrent infection was only found for patients with a Morel-Lavallée lesion due to run-over injuries (30% vs. 5%). Identified causing bacteria did not influence the outcome and rate of recurrence. CONCLUSION: Recurrence rates after surgical revision of implant-associated infections of the pelvis and the acetabulum are low and neither the type of causing agent nor the microbiological status at the timepoint of wound closure has a significant impact on the recurrence rate.

Gut microbiota promote liver regeneration through hepatic membrane phospholipid biosynthesis.

BACKGROUND & AIMS: Hepatocyte growth and proliferation depends on membrane phospholipid biosynthesis. Short-chain fatty acids (SCFAs) generated by bacterial fermentation, delivered through the gut-liver axis, significantly contribute to lipid biosynthesis. We therefore hypothesized that dysbiotic insults like antibiotic treatment not only affect gut microbiota, but also impair hepatic lipid synthesis and liver regeneration. METHODS: Stable isotope labeling and 70% partial hepatectomy (PHx) was carried out in C57Bl/6J wild-type mice, in mice treated with broad-spectrum antibiotics, in germ-free mice and mice colonized with minimal microbiota. The microbiome was analyzed by 16S rRNA gene sequencing and microbial culture. Gut content, liver, blood and primary hepatocyte organoids were tested by mass spectrometry-based lipidomics, quantitative reverse-transcription PCR (qRT-PCR), immunoblot and immunohistochemistry for expression of proliferative and lipogenic markers. Matched biopsies from hyperplastic and hypoplastic liver tissue of patients subjected to surgical intervention to induce hyperplasia were analyzed by qRT-PCR for lipogenic enzymes. RESULTS: Three days of antibiotic treatment induced persistent dysbiosis with significantly decreased beta-diversity and richness, but a massive increase of Proteobacteria, accompanied by decreased colonic SCFAs. After PHx, antibiotic-treated mice showed delayed liver regeneration, increased mortality, impaired hepatocyte proliferation and decreased hepatic phospholipid synthesis. Expression of the lipogenic enzyme SCD1 was upregulated after PHx but delayed by antibiotic treatment. Germ-free mice essentially recapitulated the phenotype of antibiotic treatment. Phospholipid biosynthesis, hepatocyte proliferation, liver regeneration and survival were rescued in gnotobiotic mice colonized with a minimal SCFA-producing microbial community. SCFAs induced the growth of murine hepatocyte organoids and hepatic SCD1 expression in mice. Further, SCD1 was required for proliferation of human hepatoma cells and was associated with liver regeneration in human patients. CONCLUSION: Gut microbiota are pivotal for hepatic membrane phospholipid biosynthesis and liver regeneration. IMPACT AND IMPLICATIONS: Gut microbiota affect hepatic lipid metabolism through the gut-liver axis, but the underlying mechanisms are poorly understood. Perturbations of the gut microbiome, e.g. by antibiotics, impair the production of bacterial metabolites, which normally serve as building blocks for membrane lipids in liver cells. As a consequence, liver regeneration and survival after liver surgery is severely impaired. Even though this study is preclinical, its results might allow physicians in the future to improve patient outcomes after liver surgery, by modulation of gut microbiota or their metabolites.

Suppression of mutant Kirsten-RAS (KRASG12D)-driven pancreatic carcinogenesis by dual-specificity MAP kinase phosphatases 5 and 6.

The cytoplasmic phosphatase DUSP6 and its nuclear counterpart DUSP5 are negative regulators of RAS/ERK signalling. Here we use deletion of either Dusp5 or Dusp6 to explore the roles of these phosphatases in a murine model of KRASG12D-driven pancreatic cancer. By 56-days, loss of either DUSP5 or DUSP6 causes a significant increase in KRASG12D-driven pancreatic hyperplasia. This is accompanied by increased pancreatic acinar to ductal metaplasia (ADM) and the development of pre-neoplastic pancreatic intraepithelial neoplasia (PanINs). In contrast, by 100-days, pancreatic hyperplasia is reversed with significant atrophy of pancreatic tissue and weight loss observed in animals lacking either DUSP5 or DUSP6. On further ageing, Dusp6-/- mice display accelerated development of metastatic pancreatic ductal adenocarcinoma (PDAC), while in Dusp5-/- animals, although PDAC development is increased this process is attenuated by atrophy of pancreatic acinar tissue and severe weight loss in some animals before cancer could progress. Our data suggest that despite a common target in the ERK MAP kinase, DUSP5 and DUSP6 play partially non-redundant roles in suppressing oncogenic KRASG12D signalling, thus retarding both tumour initiation and progression. Our data suggest that loss of either DUSP5 or DUSP6, as observed in certain human tumours, including the pancreas, could promote carcinogenesis.

Toll-like receptor 3 expression in myeloid cells is essential for efficient regeneration after acute pancreatitis in mice.

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 (TLR3OFF ) or in mice re-expressing TLR3 exclusively in the myeloid cell lineage (TLR3Mye ). Compared to WT mice, TLR3OFF 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 TLR3OFF mice. Importantly, the phenotype of TLR3OFF mice was rescued in TLR3Mye 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.

Notch and TLR signaling coordinate monocyte cell fate and inflammation.

Conventional Ly6Chi monocytes have developmental plasticity for a spectrum of differentiated phagocytes. Here we show, using conditional deletion strategies in a mouse model of Toll-like receptor (TLR) 7-induced inflammation, that the spectrum of developmental cell fates of Ly6Chi monocytes, and the resultant inflammation, is coordinately regulated by TLR and Notch signaling. Cell-intrinsic Notch2 and TLR7-Myd88 pathways independently and synergistically promote Ly6Clo patrolling monocyte development from Ly6Chi monocytes under inflammatory conditions, while impairment in either signaling axis impairs Ly6Clo monocyte development. At the same time, TLR7 stimulation in the absence of functional Notch2 signaling promotes resident tissue macrophage gene expression signatures in monocytes in the blood and ectopic differentiation of Ly6Chi monocytes into macrophages and dendritic cells, which infiltrate the spleen and major blood vessels and are accompanied by aberrant systemic inflammation. Thus, Notch2 is a master regulator of Ly6Chi monocyte cell fate and inflammation in response to TLR signaling.

TLR3 promotes hepatocyte proliferation after partial hepatectomy by stimulating uPA expression and the release of tissue-bound HGF.

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.

Migration of murine intestinal dendritic cell subsets upon intrinsic and extrinsic TLR3 stimulation.

Initiation of adaptive immunity to particulate antigens in lymph nodes largely depends on their presentation by migratory dendritic cells (DCs). DC subsets differ in their capacity to induce specific types of immunity, allowing subset-specific DC-targeting to influence vaccination and therapy outcomes. Faithful drug design, however, requires exact understanding of subset-specific versus global activation mechanisms. cDC1, the subset of DCs that excel in supporting immunity toward viruses, intracellular bacteria, and tumors, express uniquely high levels of the pattern recognition receptor TLR3. Using various murine genetic models, we show here that both, the cDC1 and cDC2 subsets of cDCs are activated and migrate equally well in response to TLR3 stimulation in a cell extrinsic and TNF-α dependent manner, but that cDC1 show a unique requirement for type I interferon signaling. Our findings reveal common and differing pathways regulating DC subset migration, offering important insights for the design of DC-based vaccination and therapy approaches.

The CGRP receptor component RAMP1 links sensory innervation with YAP activity in the regenerating liver.

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.

A dual role for hepatocyte-intrinsic canonical NF-κB signaling in virus control.

BACKGROUND & AIMS: Hepatic innate immune control of viral infections has largely been attributed to Kupffer cells, the liver-resident macrophages. However, hepatocytes, the parenchymal cells of the liver, also possess potent immunological functions in addition to their known metabolic functions. Owing to their abundance in the liver and known immunological functions, we aimed to investigate the direct antiviral mechanisms employed by hepatocytes. METHODS: Using lymphocytic choriomeningitis virus (LCMV) as a model of liver infection, we first assessed the role of myeloid cells by depletion prior to infection. We investigated the role of hepatocyte-intrinsic innate immune signaling by infecting mice lacking canonical NF-κB signaling (IkkßΔHep) specifically in hepatocytes. In addition, mice lacking hepatocyte-specific interferon-α/ß signaling-(IfnarΔHep), or interferon-α/ß signaling in myeloid cells-(IfnarΔMyel) were infected. RESULTS: Here, we demonstrate that LCMV activates NF-κB signaling in hepatocytes. LCMV-triggered NF-κB activation in hepatocytes did not depend on Kupffer cells or TNFR1 signaling but rather on Toll-like receptor signaling. LCMV-infected IkkßΔHep livers displayed strongly elevated viral titers due to LCMV accumulation within hepatocytes, reduced interferon-stimulated gene (ISG) expression, delayed intrahepatic immune cell influx and delayed intrahepatic LCMV-specific CD8+ T cell responses. Notably, viral clearance and ISG expression were also reduced in LCMV-infected primary hepatocytes lacking IKKß, demonstrating a hepatocyte-intrinsic effect. Similar to livers of IkkßΔHep mice, enhanced hepatocytic LCMV accumulation was observed in livers of IfnarΔHep mice, whereas IfnarΔMyel mice were able to control LCMV infection. Hepatocytic NF-κB signaling was also required for efficient ISG induction in HDV-infected dHepaRG cells and interferon-α/ß-mediated inhibition of HBV replication in vitro. CONCLUSIONS: Together, these data show that hepatocyte-intrinsic NF-κB is a vital amplifier of interferon-α/ß signaling, which is pivotal for strong early ISG responses, immune cell infiltration and hepatic viral clearance. LAY SUMMARY: Innate immune cells have been ascribed a primary role in controlling viral clearance upon hepatic infections. We identified a novel dual role for NF-κB signaling in infected hepatocytes which was crucial for maximizing interferon responses and initiating adaptive immunity, thereby efficiently controlling hepatic virus replication.

The neuropeptide receptor subunit RAMP1 constrains the innate immune response during acute pancreatitis in mice.

OBJECTIVES: The importance of the Calcitonin-gene-related-peptide-pathway (CGRP) as neuronal modulator of innate immune responses in mice has been previously demonstrated. The CGRP-receptor is composed of two subunits: the receptor-activity-modifying-protein-1 (RAMP1) and the calcitonin-receptor-like-receptor (CLR). CGRP can influence immune cells and their capacity of producing inflammatory cytokines. Using a RAMP1 knockout-mouse (RAMP1-/-) we examined the role of the CGRP-receptor in the acute-phase of cerulein-induced pancreatitis. METHODS: Hourly cerulein-injections for a period of 8 h in RAMP1-/- and wild-type mice were performed. To compare severity and extent of inflammation in RAMP1-/- and wild-type mice, histological analyses were done and cytokine levels were assessed using qRT-PCR 8 h, 24 h, 2 days, and 7 days post-cerulein-treatment. Furthermore, serum activities of LDH and lipase were determined. RESULTS: After 8 h RAMP1-/- mice showed a higher pancreas-to-body-weight-ratio, increased tissue edema and immune cell infiltration with higher amount of F4/80-positive cells as compared to wild-type mice. Overall infiltration of immune cells at 24 h was increased in RAMP1-/- mice and composed predominantly of MPO-positive neutrophils. In addition, after 24 h RAMP1-/- mice presented a higher pancreas-to-body-weight-ratio, higher expression of Ccl3, Il6, and Il1b and increased number of cleaved caspase 3 positive cells. Serum lipase correlated with the extent of tissue damage in RAMP1-/- compared to wild-type mice 24 h post-cerulein treatment. CONCLUSION: Mice lacking RAMP1 showed increased inflammation, tissue edema, and pancreas injury particularly in the early phase of acute pancreatitis. This study highlights the essential role of CGRP for dampening the innate immune response in acute pancreatitis.

A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis through its ZnF7 ubiquitin-binding domain.

Deficiency in the deubiquitinating enzyme A20 causes severe inflammation in mice, and impaired A20 function is associated with human inflammatory diseases. A20 has been implicated in negatively regulating NF-κB signalling, cell death and inflammasome activation; however, the mechanisms by which A20 inhibits inflammation in vivo remain poorly understood. Genetic studies in mice revealed that its deubiquitinase activity is not essential for A20 anti-inflammatory function. Here we show that A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis and that this function depends on its zinc finger 7 (ZnF7). We provide genetic evidence that RIPK1 kinase-dependent, RIPK3-MLKL-mediated necroptosis drives inflammasome activation in A20-deficient macrophages and causes inflammatory arthritis in mice. Single-cell imaging revealed that RIPK3-dependent death caused inflammasome-dependent IL-1ß release from lipopolysaccharide-stimulated A20-deficient macrophages. Importantly, mutation of the A20 ZnF7 ubiquitin binding domain caused arthritis in mice, arguing that ZnF7-dependent inhibition of necroptosis is critical for A20 anti-inflammatory function in vivo.

Cell-type specific MyD88 signaling is required for intestinal tumor initiation and progression to malignancy.

The signal adapter MyD88, an essential component of Toll-like receptor (TLR) signaling, is important for gut-microbiome interactions. However, its contribution to cancer and its cell-type specific functions are controversially discussed. Therefore, we generated new tissue-specific mouse models and analyzed the clinical importance in human colorectal cancer. A gene-trap was inserted into the murine Myd88 gene (Myd88LSL), yielding MyD88-deficient background with Cre-mediated re-expression in myeloid (MYEL) or intestinal epithelial cells (IECs). These lines were bred with the Apc1638N model that develops invasive adenocarcinoma and analyzed at 12 months. Further, two patient collectives of colorectal cancer (n = 61, and n = 633) were analyzed for expression of Myd88 and TLRs. MyD88 expression was significantly increased in carcinomas, and increased intratumoral levels of MyD88 and TLR pathway components were associated with significantly shorter disease-free (P = .011), and overall survival (P < .0001). In accordance, fully MyD88-deficient mice showed highly significantly decreased tumor incidence, tumor numbers, increased survival, and, importantly, fully lacked malignant lesions. Thus, MyD88 is essential for tumorigenesis and especially progression to malignancy. Tissue-specific re-expression of MyD88 highly significantly increased tumor initiation by differing mechanisms. In intestinal epithelia, MyD88 enhanced epithelial turnover, whereas in myeloid cells, it led to increased production of tumor- and stemness-enhancing cytokines, significantly associated with altered expression of adaptive immune genes. However, neither re-expression of MyD88 in IECs or myeloid cells was sufficient for malignant progression to carcinoma. Thus, MyD88 crucially contributes to colorectal cancer initiation and progression with non-redundant and cell-type specific functions, constituting an attractive therapeutic target.

Cytosolic nucleic acid sensors of the innate immune system promote liver regeneration after partial hepatectomy.

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 G1-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-ß2 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.

MyD88 signaling in dendritic cells and the intestinal epithelium controls immunity against intestinal infection with C. rodentium.

MyD88-mediated signaling downstream of Toll-like receptors and the IL-1 receptor family is critically involved in the induction of protective host responses upon infections. Although it is known that MyD88-deficient mice are highly susceptible to a wide range of bacterial infections, the cell type-specific contribution of MyD88 in protecting the host against intestinal bacterial infection is only poorly understood. In order to investigate the importance of MyD88 in specific immune and nonimmune cell types during intestinal infection, we employed a novel murine knock-in model for MyD88 that enables the cell type-specific reactivation of functional MyD88 expression in otherwise MyD88-deficient mice. We report here that functional MyD88 signaling in CD11c+ cells was sufficient to activate intestinal dendritic cells (DC) and to induce the early group 3 innate lymphoid cell (ILC3) response as well as the development of colonic Th17/Th1 cells in response to infection with the intestinal pathogen C. rodentium. In contrast, restricting MyD88 signaling to several other cell types, including macrophages (MO), T cells or ILC3 did not induce efficient intestinal immune responses upon infection. However, we observed that the functional expression of MyD88 in intestinal epithelial cells (IEC) also partially protected the mice during intestinal infection, which was associated with enhanced epithelial barrier integrity and increased expression of the antimicrobial peptide RegIIIγ and the acute phase protein SAA1 by epithelial cells. Together, our data suggest that MyD88 signaling in DC and IEC is both essential and sufficient to induce a full spectrum of host responses upon intestinal infection with C. rodentium.

Requirement of MyD88 signaling in keratinocytes for Langerhans cell migration and initiation of atopic dermatitis-like symptoms in mice.

Atopic dermatitis (AD) is a chronic inflammatory disease controlled by the innate and adaptive immune system. To elucidate the impact of innate immune signaling in AD, we analyzed MyD88-deficient mice in a murine model of AD-like dermatitis by epicutaneous sensitization with ovalbumin (OVA). Global MyD88 deficiency led to reduced epidermal thickening and diminished accumulation of macrophages within the inflamed skin. In addition, we observed impaired emigration of Langerhans cells (LCs) out of the epidermis of MyD88-deficient mice. These findings indicate that MyD88 deficiency affects various skin-resident cell types in the AD model. Moreover, production of IFN-g, IL-17, and CCL17 was reduced in skin draining lymph node cells and OVA-specific immunoglobulin levels were lower in MyD88-deficient mice. We further investigated the role of MyD88 in keratinocytes, as keratinocytes contribute to AD pathology. Exclusive expression of MyD88 in epidermal keratinocytes partially restored LC emigration after AD induction and expression of CCL17 in skin draining lymph nodes (LNs), but did not promote epidermal thickening nor production of IL-17. Altogether, these data demonstrate that MyD88 signaling in keratinocytes is able to restore LC migration in an otherwise MyD88-deficient background, and significantly contributes to the development of AD-like dermatitis.

Procalcitonin ratio and on-demand relaparotomy for septic peritonitis: validation of the focus index (FI).

PURPOSE: Secondary peritonitis remains challenging to manage and some recent evidence suggests that on-demand relaparotomy is more appropriate than planned relaparotomy. This study was designed to validate the predictive power of postoperative procalcitonin (PCT) changes in relation to elimination of the septic abdominal focus. METHODS: In this prospective trial, postoperative PCT serum levels were monitored in 234 surgical patients with secondary peritonitis. The PCT ratio on postoperative days (PODs) 1 and 2 (focus index; FI) was calculated and correlated with the success of the operation. RESULTS: A cutoff value of 1.1 was calculated for the FI. Values below 1.1 indicated insufficient elimination of the focus and values above 1.1 correlated with effective treatment. The optimal time for first PCT sampling was found to be 12-24 h after the index operation. After the respective data cleanup, successful elimination of the intraabdominal focus could be confirmed, with a sensitivity of 93 % and a specificity of 71 %. CONCLUSIONS: The FI is a single parameter-based reliable predictor of successful surgical eradication and strengthens the on-demand relaparotomy concept as the method of choice to treat secondary peritonitis.

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.

Dendritic cells coordinate innate immunity via MyD88 signaling to control Listeria monocytogenes infection.

Listeria monocytogenes (LM), a facultative intracellular Gram-positive pathogen, can cause life-threatening infections in humans. In mice, the signaling cascade downstream of the myeloid differentiation factor 88 (MyD88) is essential for proper innate immune activation against LM, as MyD88-deficient mice succumb early to infection. Here, we show that MyD88 signaling in dendritic cells (DCs) is sufficient to mediate the protective innate response, including the production of proinflammatory cytokines, neutrophil infiltration, bacterial clearance, and full protection from lethal infection. We also demonstrate that MyD88 signaling by DCs controls the infection rates of CD8α(+) cDCs and thus limits the spread of LM to the T cell areas. Furthermore, in mice expressing MyD88 in DCs, inflammatory monocytes, which are required for bacterial clearance, are activated independently of intrinsic MyD88 signaling. In conclusion, CD11c(+) conventional DCs critically integrate pathogen-derived signals via MyD88 signaling during early infection with LM in vivo.

MyD88 signalling in myeloid cells is sufficient to prevent chronic mycobacterial infection.

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis that is responsible for almost 1.5 million deaths per year. Sensing of mycobacteria by the host's immune system relies on different families of receptors present on innate immune cells. Amongst them, several members of the TLR family are involved in the activation of immune cells by mycobacteria, yet the in vivo contribution of individual TLRs to the protective immune response remains controversial. On the contrary, MyD88, the adaptor molecule for most TLRs, plays a non-redundant role in the protection against tuberculosis and mice with a complete germline deletion of MyD88 succumb very early to infection. MyD88 is expressed in both immune and non-immune cells, but it is not clear whether control of mycobacteria requires ubiquitous or cell-type specific MyD88 expression. Therefore, using novel conditional switch-on mouse models, we aimed to investigate the importance of MyD88 signalling in DCs and macrophages for the induction of protective effector mechanisms against mycobacterial infection. We conclude that specific reactivation of MyD88 signalling in CD11c- or lysozyme M-expressing myeloid cells during Mycobacterium bovis Bacille Calmette-Guerin infection is sufficient to restore systemic and local inflammatory cytokine production and to control pathogen burden.