Mast cell-derived interleukin-4 mediates activation of dendritic cell during toll-like receptor 2-mediated inflammation.

Introduction: During an innate inflammation, immune cells form distinct pro- and anti-inflammatory regions around pathogen-containing core-regions. Mast cells are localized in an anti-inflammatory microenvironment during the resolution of an innate inflammation, suggesting antiinflammatory roles of these cells. Methods: High-content imaging was used to investigated mast cell-dependent changes in the regional distribution of immune cells during an inflammation, induced by the toll-like receptor (TLR)-2 agonist zymosan. Results: The distance between the zymosan-containing core-region and the anti-inflammatory region, described by M2-like macrophages, increased in mast cell-deficient mice. Absence of mast cells abolished dendritic cell (DC) activation, as determined by CD86-expression and localized the DCs in greater distance to zymosan particles. The CD86- DCs had a higher expression of the pro-inflammatory interleukins (IL)-1ß and IL-12/23p40 as compared to activated CD86+ DCs. IL-4 administration restored CD86 expression, cytokine expression profile and localization of the DCs in mast cell-deficient mice. The IL-4 effects were mast cell-specific, since IL-4 reduction by eosinophil depletion did not affect activation of DCs. Discussion: We found that mast cells induce DC activation selectively at the site of inflammation and thereby determine their localization within the inflammation. Overall, mast cells have antiinflammatory functions in this inflammation model and limit the size of the pro-inflammatory region surrounding the zymosan-containing core region.

BI-3231, an enzymatic inhibitor of HSD17B13, reduces lipotoxic effects induced by palmitic acid in murine and human hepatocytes.

17-ß-hydroxysteroid dehydrogenase 13 (HSD17B13), a lipid droplet-associated enzyme, is primarily expressed in the liver and plays an important role in lipid metabolism. Targeted inhibition of enzymatic function is a potential therapeutic strategy for treating steatotic liver disease (SLD). The present study is aimed at investigating the effects of the first selective HSD17B13 inhibitor, BI-3231, in a model of hepatocellular lipotoxicity using human cell lines and primary mouse hepatocytes in vitro. Lipotoxicity was induced with palmitic acid in HepG2 cells and freshly isolated mouse hepatocytes and the cells were coincubated with BI-3231 to assess the protective effects. Under lipotoxic stress, triglyceride (TG) accumulation was significantly decreased in the BI-3231-treated cells compared with that of the control untreated human and mouse hepatocytes. In addition, treatment with BI-3231 led to considerable improvement in hepatocyte proliferation, cell differentiation, and lipid homeostasis. Mechanistically, BI-3231 increased the mitochondrial respiratory function without affecting ß-oxidation. BI-3231 inhibited the lipotoxic effects of palmitic acid in hepatocytes, highlighting the potential of targeting HSD17B13 as a specific therapeutic approach in steatotic liver disease.NEW & NOTEWORTHY 17-ß-Hydroxysteroid dehydrogenase 13 (HSD17B13) is a lipid droplet protein primarily expressed in the liver hepatocytes. HSD17B13 is associated with the clinical outcome of chronic liver diseases and is therefore a target for the development of drugs. Here, we demonstrate the promising therapeutic effect of BI-3231 as a potent inhibitor of HSD17B13 based on its ability to inhibit triglyceride accumulation in lipid droplets (LDs), restore lipid metabolism and homeostasis, and increase mitochondrial activity in vitro.

Meloxicam treatment disrupts the regional structure of innate inflammation sites by targeting the pro-inflammatory effects of prostanoids.

BACKGROUND AND PURPOSE: Non-steroidal anti-inflammatory drugs (NSAIDs) are the most widely prescribed drugs in the world due to their analgesic, antipyretic and anti-inflammatory effects. However, NSAIDs inhibit prostanoid synthesis, interfering with their pro-inflammatory and anti-inflammatory functions and potentially prolonging acute inflammation. EXPERIMENTAL APPROACH: We used high-content immunohistochemistry to define the impact of meloxicam treatment on spatially separated pro-inflammatory and anti-inflammatory processes during innate inflammation in mice induced by zymosan. This allowed us to determine the effect of meloxicam treatment on the organization of pro-inflammatory and anti-inflammatory microenvironments, thereby identifying relevant changes in immune cell localization, recruitment and activation. KEY RESULTS: Meloxicam treatment reduced zymosan-induced thermal hypersensitivity at early time points but delayed its resolution. High-content immunohistochemistry revealed that the pro-inflammatory area was smaller after treatment, diminishing neutrophil recruitment, M1-like macrophage polarization, and especially phagocytosis by neutrophils and macrophages. The polarization of macrophages towards the M2-like anti-inflammatory phenotype was unaffected, and the number of anti-inflammatory eosinophils actually increased. CONCLUSION AND IMPLICATIONS: High-content immunohistochemistry was able to identify relevant meloxicam-mediated effects on inflammatory processes based on alterations in the regional structure of inflammation sites. Meloxicam delayed the clearance of pathogens by inhibiting pro-inflammatory processes, causing prolonged inflammation. Our data suggest that the prescription of NSAIDs as a treatment during an acute pathogen-driven inflammation should be reconsidered in patients with compromised immune systems.

Ocular surface changes in mice with streptozotocin-induced diabetes and diabetic polyneuropathy.

PURPOSE: Diabetes mellitus (DM) is a leading risk factor for corneal neuropathy and dry eye disease (DED). Another common consequence of DM is diabetic peripheral polyneuropathy (DPN). Both complications affect around 50 % of the DM patients but the relationship between DM, DED and DPN remains unclear. METHODS: In this study, we examined mice with early onset of DM and PN after streptozotocin (STZ)-induced diabetes (DPN). We compared the early morphological changes of the sciatic nerve, dorsal root and trigeminal ganglia with the changes in the ocular surface, including tear proteomic and we also investigated respective changes in the gene expressions and morphological alterations in the eye tissues involved in tear production. RESULTS: The lacrimal gland, conjunctival goblet cells and cornea showed morphological changes along with alterations in tear proteins without any obvious signs of ocular surface inflammation. The gene expression for respectively altered tear proteins i.e., of Clusterin in cornea, Car6, Adh3a1, and Eef1a1 in eyelids, and Pigr in the lacrimal gland also showed significant changes compared to control mice. In the trigeminal ganglia like in the dorsal root ganglia neuronal cells showed swollen mitochondria and, in the latter, there was a significant increase of NADPH oxidases and MMP9 suggestive of oxidative and neuronal stress. In the dorsal root ganglia and the sciatic nerve, there was an upregulation of a number of pro-inflammatory cytokines and pain-mediating chemokines. CONCLUSION: The early ocular changes in DM Mice only affect the lacrimal gland. Which, is reflected in the tear film composition of DPN mice. Due to the high protein concentration in tear fluid in humans, proteomic analysis in addition to noninvasive investigation of goblet cells and cornea can serve as a tools for the early diagnosis of DPN, DED in clinical practice. Early treatment could delay or even prevent the ocular complications of DM such as DED and PN.

Application of clinical and molecular profiling data to improve patient outcomes in psoriatic arthritis.

Achieving a good outcome for a person with Psoriatic Arthritis (PsA) is made difficult by late diagnosis, heterogenous clinical disease expression and in many cases, failure to adequately suppress inflammatory disease features. Single-centre studies have certainly contributed to our understanding of disease pathogenesis, but to adequately address the major areas of unmet need, multi-partner, collaborative research programmes are now required. HIPPOCRATES is a 5-year, Innovative Medicines Initiative (IMI) programme which includes 17 European academic centres experienced in PsA research, 5 pharmaceutical industry partners, 3 small-/medium-sized industry partners and 2 patient-representative organizations. In this review, the ambitious programme of work to be undertaken by HIPPOCRATES is outlined and common approaches and challenges are identified. It is expected that, when completed, the results will ultimately allow for changes in the approaches to diagnosing, managing and treating PsA allowing for better short-term and long-term outcomes.

Improving outcomes in Psoriatic Arthritis Psoriatic Arthritis (PsA) is a form of arthritis which is found in approximately 30% of people who have the skin condition, Psoriasis. Frequently debilitating and progressive, achieving a good outcome for a person with PsA is made difficult by late diagnosis, disease clinical features and in many cases, failure to adequately control features of inflammation. Research studies from individual centres have certainly contributed to our understanding of why people develop PsA but to adequately address the major areas of unmet need, multi-centre, collaborative research programmes are now required. HIPPOCRATES is a 5-year, Innovative Medicines Initiative (IMI) programme which includes 17 European academic centres experienced in PsA research, 5 pharmaceutical industry partners, 3 small-/medium-sized industry partners and 2 patient representative organisations (see appendix). In this review, the ambitious programme of work to be undertaken by HIPPOCRATES is outlined and common approaches and challenges are identified. The participation of patient research partners in all stages of the work of HIPPOCRATES is highlighted. It is expected that, when completed, the results will ultimately allow for changes in the approaches to diagnosing, managing and treating PsA allowing for improvements in short-term and long-term outcomes.

Pathogenetic mechanisms and therapeutic approaches of acute-to-chronic liver failure.

Liver cirrhosis is the end stage of all chronic liver diseases and contributes significantly to overall mortality of 2% globally. The age-standardized mortality from liver cirrhosis in Europe is between 10 and 20% and can be explained by not only the development of liver cancer but also the acute deterioration in the patient's overall condition. The development of complications including accumulation of fluid in the abdomen (ascites), bleeding in the gastrointestinal tract (variceal bleeding), bacterial infections, or a decrease in brain function (hepatic encephalopathy) define an acute decompensation that requires therapy and often leads to acute-on-chronic liver failure (ACLF) by different precipitating events. However, due to its complexity and organ-spanning nature, the pathogenesis of ACLF is poorly understood, and the common underlying mechanisms leading to the development of organ dysfunction or failure in ACLF are still elusive. Apart from general intensive care interventions, there are no specific therapy options for ACLF. Liver transplantation is often not possible in these patients due to contraindications and a lack of prioritization. In this review, we describe the framework of the ACLF-I project consortium funded by the Hessian Ministry of Higher Education, Research and the Arts (HMWK) based on existing findings and will provide answers to these open questions.

Differential Lipidomics, Metabolomics and Immunological Analysis of Alcoholic and Non-Alcoholic Steatohepatitis in Mice.

Non-alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH) are the leading causes of liver disease worldwide. To identify disease-specific pathomechanisms, we analyzed the lipidome, metabolome and immune cell recruitment in livers in both diseases. Mice harboring ASH or NASH had comparable disease severities regarding mortality rate, neurological behavior, expression of fibrosis marker and albumin levels. Lipid droplet size was higher in NASH than ASH and qualitative differences in the lipidome were mainly based on incorporation of diet-specific fatty acids into triglycerides, phosphatidylcholines and lysophosphatidylcholines. Metabolomic analysis showed downregulated nucleoside levels in both models. Here, the corresponding uremic metabolites were only upregulated in NASH suggesting stronger cellular senescence, which was supported by lower antioxidant levels in NASH as compared to ASH. While altered urea cycle metabolites suggest increased nitric oxide synthesis in both models, in ASH, this depended on increased L-homoarginine levels indicating a cardiovascular response mechanism. Interestingly, only in NASH were the levels of tryptophan and its anti-inflammatory metabolite kynurenine upregulated. Fittingly, high-content immunohistochemistry showed a decreased macrophage recruitment and an increased polarization towards M2-like macrophages in NASH. In conclusion, with comparable disease severity in both models, higher lipid storage, oxidative stress and tryptophan/kynurenine levels were seen in NASH, leading to distinct immune responses.

Eosinophil-derived IL-4 is necessary to establish the inflammatory structure in innate inflammation.

Pathogen-induced inflammation comprises pro- and anti-inflammatory processes, which ensure pathogen removal and containment of the proinflammatory activities. Here, we aimed to identify the development of inflammatory microenvironments and their maintenance throughout the course of a toll-like receptor 2-mediated paw inflammation. Within 24 h after pathogen-injection, the immune cells were organized in three zones, which comprised a pathogen-containing "core-region", a bordering proinflammatory (PI)-region and an outer anti-inflammatory (AI)-region. Eosinophils were present in all three inflammatory regions and adapted their cytokine profile according to their localization. Eosinophil depletion reduced IL-4 levels and increased edema formation as well as mechanical and thermal hypersensitivities during resolution of inflammation. Also, in the absence of eosinophils PI- and AI-regions could not be determined anymore, neutrophil numbers increased, and efferocytosis as well as M2-macrophage polarization were reduced. IL-4 administration restored in eosinophil-depleted mice PI- and AI-regions, normalized neutrophil numbers, efferocytosis, M2-macrophage polarization as well as resolution of zymosan-induced hypersensitivity. In conclusion, IL-4-expressing eosinophils support the resolution of inflammation by enabling the development of an anti-inflammatory framework, which encloses proinflammatory regions.

Omics and Multi-Omics Analysis for the Early Identification and Improved Outcome of Patients with Psoriatic Arthritis.

The definitive diagnosis and early treatment of many immune-mediated inflammatory diseases (IMIDs) is hindered by variable and overlapping clinical manifestations. Psoriatic arthritis (PsA), which develops in ~30% of people with psoriasis, is a key example. This mixed-pattern IMID is apparent in entheseal and synovial musculoskeletal structures, but a definitive diagnosis often can only be made by clinical experts or when an extensive progressive disease state is apparent. As with other IMIDs, the detection of multimodal molecular biomarkers offers some hope for the early diagnosis of PsA and the initiation of effective management and treatment strategies. However, specific biomarkers are not yet available for PsA. The assessment of new markers by genomic and epigenomic profiling, or the analysis of blood and synovial fluid/tissue samples using proteomics, metabolomics and lipidomics, provides hope that complex molecular biomarker profiles could be developed to diagnose PsA. Importantly, the integration of these markers with high-throughput histology, imaging and standardized clinical assessment data provides an important opportunity to develop molecular profiles that could improve the diagnosis of PsA, predict its occurrence in cohorts of individuals with psoriasis, differentiate PsA from other IMIDs, and improve therapeutic responses. In this review, we consider the technologies that are currently deployed in the EU IMI2 project HIPPOCRATES to define biomarker profiles specific for PsA and discuss the advantages of combining multi-omics data to improve the outcome of PsA patients.

Behind the Wall-Compartment-Specific Neovascularisation during Post-Stroke Recovery in Mice.

Ischemic stroke is a highly prevalent vascular disease leading to oxygen- and glucose deprivation in the brain. In response, ischemia-induced neovascularization occurs, which is supported by circulating CD34+ endothelial progenitor cells. Here, we used the transient middle cerebral artery occlusion (tMCAO) mouse model to characterize the spatio-temporal alterations within the ischemic core from the acute to the chronic phase using multiple-epitope-ligand cartography (MELC) for sequential immunohistochemistry. We found that around 14 days post-stroke, significant angiogenesis occurs in the ischemic core, as determined by the presence of CD31+/CD34+ double-positive endothelial cells. This neovascularization was accompanied by the recruitment of CD4+ T-cells and dendritic cells as well as IBA1+ and IBA1- microglia. Neighborhood analysis identified, besides pericytes only for T-cells and dendritic cells, a statistically significant distribution as direct neighbors of CD31+/CD34+ endothelial cells, suggesting a role for these cells in aiding angiogenesis. This process was distinct from neovascularization of the peri-infarct area as it was separated by a broad astroglial scar. At day 28 post-stroke, the scar had emerged towards the cortical periphery, which seems to give rise to a neuronal regeneration within the peri-infarct area. Meanwhile, the ischemic core has condensed to a highly vascularized subpial region adjacent to the leptomeningeal compartment. In conclusion, in the course of chronic post-stroke regeneration, the astroglial scar serves as a seal between two immunologically active compartments-the peri-infarct area and the ischemic core-which exhibit distinct processes of neovascularization as a central feature of post-stroke tissue remodeling. Based on our findings, we propose that neovascularization of the ischemic core comprises arteriogenesis as well as angiogenesis originating from the leptomenigeal vasculature.

T-Cell-Specific CerS4 Depletion Prolonged Inflammation and Enhanced Tumor Burden in the AOM/DSS-Induced CAC Model.

To better understand the role of sphingolipids in the multifactorial process of inflammatory bowel disease (IBD), we elucidated the role of CerS4 in colitis and colitis-associated cancer (CAC). For this, we utilized the azoxymethane/dextran sodium sulphate (AOM/DSS)-induced colitis model in global CerS4 knockout (CerS4 KO), intestinal epithelial (CerS4 Vil/Cre), or T-cell restricted knockout (CerS4 LCK/Cre) mice. CerS4 KO mice were highly sensitive to the toxic effect of AOM/DSS, leading to a high mortality rate. CerS4 Vil/Cre mice had smaller tumors than WT mice. In contrast, CerS4 LCK/Cre mice frequently suffered from pancolitis and developed more colon tumors. In vitro, CerS4-depleted CD8+ T-cells isolated from the thymi of CerS4 LCK/Cre mice showed impaired proliferation and prolonged cytokine production after stimulation in comparison with T-cells from WT mice. Depletion of CerS4 in human Jurkat T-cells led to a constitutively activated T-cell receptor and NF-κB signaling pathway. In conclusion, the deficiency of CerS4 in T-cells led to an enduring active status of these cells and prevents the resolution of inflammation, leading to a higher tumor burden in the CAC mouse model. In contrast, CerS4 deficiency in epithelial cells resulted in smaller colon tumors and seemed to be beneficial. The higher tumor incidence in CerS4 LCK/Cre mice and the toxic effect of AOM/DSS in CerS4 KO mice exhibited the importance of CerS4 in other tissues and revealed the complexity of general targeting CerS4.

GPR40 Activation Abolishes Diabetes-Induced Painful Neuropathy by Suppressing VEGF-A Expression.

G-protein-coupled receptor 40 (GPR40) is a promising target to support glucose-induced insulin release in patients with type 2 diabetes. We studied the role of GPR40 in the regulation of blood-nerve barrier integrity and its involvement in diabetes-induced neuropathies. Because GPR40 modulates insulin release, we used the streptozotocin model for type 1 diabetes, in which GPR40 functions can be investigated independently of its effects on insulin release. Diabetic wild-type mice exhibited increased vascular endothelial permeability and showed epineural microlesions in sciatic nerves, which were also observed in naïve GPR40-/- mice. Fittingly, expression of vascular endothelial growth factor-A (VEGF-A), an inducer of vascular permeability, was increased in diabetic wild-type and naïve GPR40-/- mice. GPR40 antagonists increased VEGF-A expression in murine and human endothelial cells as well as permeability of transendothelial barriers. In contrast, GPR40 agonists suppressed VEGF-A release and mRNA expression. The VEGF receptor inhibitor axitinib prevented diabetes-induced hypersensitivities and reduced endothelial and epineural permeability. Importantly, the GPR40 agonist GW9508 reverted established diabetes-induced hypersensitivity, an effect that was blocked by VEGF-A administration. Thus, GPR40 activation suppresses VEGF-A expression, thereby reducing diabetes-induced blood-nerve barrier permeability and reverting diabetes-induced hypersensitivities.

Lipidomic analysis of local inflammation models shows a specific systemic acute phase response to lipopolysaccharides.

Toll-like receptors (TLR) are crucial for recognizing bacterial, viral or fungal pathogens and to orchestrate the appropriate immune response. The widely expressed TLR2 and TLR4 differentially recognize various pathogens to initiate partly overlapping immune cascades. To better understand the physiological consequences of both immune responses, we performed comparative lipidomic analyses of local paw inflammation in mice induced by the TLR2 and TLR4 agonists, zymosan and lipopolysaccharide (LPS), respectively, which are commonly used in models for inflammation and inflammatory pain. Doses for both agonists were chosen to cause mechanical hypersensitivity with identical strength and duration. Lipidomic analysis showed 5 h after LPS or zymosan injection in both models an increase of ether-phosphatidylcholines (PC O) and their corresponding lyso species with additional lipids being increased only in response to LPS. However, zymosan induced stronger immune cell recruitment and edema formation as compared to LPS. Importantly, only in LPS-induced inflammation the lipid profile in the contralateral paw was altered. Fittingly, the plasma level of various cytokines and chemokines, including IL-1ß and IL-6, were significantly increased only in LPS-treated mice. Accordingly LPS induced distinct changes in the lipid profiles of ipsilateral and contralateral paws. Here, oxydized fatty acids, phosphatidylcholines and phosphatidylethanolamines were uniquely upregulated on the contralateral side. Thus, both models cause increased levels of PC O and lyso-PC O lipids at the site of inflammation pointing at a common role in inflammation. Also, LPS initiates systemic changes, which can be detected by changes in the lipid profiles.

Lysophospholipids Contribute to Oxaliplatin-Induced Acute Peripheral Pain.

Oxaliplatin, a platinum-based chemotherapeutic drug, which is used as first-line treatment for some types of colorectal carcinoma, causes peripheral neuropathic pain in patients. In addition, an acute peripheral pain syndrome develop in almost 90% of patients immediately after oxaliplatin treatment, which is poorly understood mechanistically but correlates with incidence and severity of the later-occurring neuropathy. Here we investigated the effects of acute oxaliplatin treatment in a murine model, showing that male and female mice develop mechanical hypersensitivity 24 h after oxaliplatin treatment. Interestingly, we found that the levels of several lipids were significantly altered in nervous tissue during oxaliplatin-induced acute pain. Specifically, the linoleic acid metabolite 9,10-EpOME (epoxide of linoleic acid) as well as the lysophospholipids lysophosphatidylcholine (LPC) 18:1 and LPC 16:0 were significantly increased 24 h after oxaliplatin treatment in sciatic nerve, DRGs, or spinal cord tissue as revealed by untargeted and targeted lipidomics. In contrast, inflammatory markers including cytokines and chemokines, ROS markers, and growth factors are unchanged in the respective nervous system tissues. Importantly, LPC 18:1 and LPC 16:0 can induce Ca2+ transients in primary sensory neurons, and we identify LPC 18:1 as a previously unknown endogenous activator of the ligand-gated calcium channels transient receptor potential V1 and M8 (transient receptor potential vanilloid 1 and transient receptor potential melastatin 8) in primary sensory neurons using both pharmacological inhibition and genetic knockout. Additionally, a peripheral LPC 18:1 injection was sufficient to induce mechanical hypersensitivity in naive mice. Hence, targeting signaling lipid pathways may ameliorate oxaliplatin-induced acute peripheral pain and the subsequent long-lasting neuropathy.SIGNIFICANCE STATEMENT The first-line cytostatic drug oxaliplatin can cause acute peripheral pain and chronic neuropathic pain. The former is causally connected with the chronic neuropathic pain, but its mechanisms are poorly understood. Here, we performed a broad unbiased analysis of cytokines, chemokines, growth factors, and ∼200 lipids in nervous system tissues 24 h after oxaliplatin treatment, which revealed a crucial role of lysophospholipids lysophosphatidylcholine (LPC) 18:1, LPC 16:0, and 9,10-EpOME in oxaliplatin-induced acute pain. We demonstrate for the first time that LPC 18:1 contributes to the activation of the ion channels transient receptor potential vanilloid 1 and transient receptor potential melastatin 8 in sensory neurons and causes mechanical hypersensitivity after peripheral injection in vivo These findings suggest that the LPC-mediated lipid signaling is involved in oxaliplatin-induced acute peripheral pain.

The Lipid Receptor G2A (GPR132) Mediates Macrophage Migration in Nerve Injury-Induced Neuropathic Pain.

Nerve injury-induced neuropathic pain is difficult to treat and mechanistically characterized by strong neuroimmune interactions, involving signaling lipids that act via specific G-protein coupled receptors. Here, we investigated the role of the signaling lipid receptor G2A (GPR132) in nerve injury-induced neuropathic pain using the robust spared nerve injury (SNI) mouse model. We found that the concentrations of the G2A agonist 9-HODE (9-Hydroxyoctadecadienoic acid) are strongly increased at the site of nerve injury during neuropathic pain. Moreover, G2A-deficient mice show a strong reduction of mechanical hypersensitivity after nerve injury. This phenotype is accompanied by a massive reduction of invading macrophages and neutrophils in G2A-deficient mice and a strongly reduced release of the proalgesic mediators TNFα, IL-6 and VEGF at the site of injury. Using a global proteome analysis to identify the underlying signaling pathways, we found that G2A activation in macrophages initiates MyD88-PI3K-AKT signaling and transient MMP9 release to trigger cytoskeleton remodeling and migration. We conclude that G2A-deficiency reduces inflammatory responses by decreasing the number of immune cells and the release of proinflammatory cytokines and growth factors at the site of nerve injury. Inhibiting the G2A receptor after nerve injury may reduce immune cell-mediated peripheral sensitization and may thus ameliorate neuropathic pain.

Tumors Provoke Inflammation and Perineural Microlesions at Adjacent Peripheral Nerves.

Cancer-induced pain occurs frequently in patients when tumors or their metastases grow in the proximity of nerves. Although this cancer-induced pain states poses an important therapeutical problem, the underlying pathomechanisms are not understood. Here, we implanted adenocarcinoma, fibrosarcoma and melanoma tumor cells in proximity of the sciatic nerve. All three tumor types caused mechanical hypersensitivity, thermal hyposensitivity and neuronal damage. Surprisingly the onset of the hypersensitivity was independent of physical contact of the nerve with the tumors and did not depend on infiltration of cancer cells in the sciatic nerve. However, macrophages and dendritic cells appeared on the outside of the sciatic nerves with the onset of the hypersensitivity. At the same time point downregulation of perineural tight junction proteins was observed, which was later followed by the appearance of microlesions. Fitting to the changes in the epi-/perineurium, a dramatic decrease of triglycerides and acylcarnitines in the sciatic nerves as well as an altered localization and appearance of epineural adipocytes was seen. In summary, the data show an inflammation at the sciatic nerves as well as an increased perineural and epineural permeability. Thus, interventions aiming to suppress inflammatory processes at the sciatic nerve or preserving peri- and epineural integrity may present new approaches for the treatment of tumor-induced pain.

The omega-3 lipid 17,18-EEQ sensitizes TRPV1 and TRPA1 in sensory neurons through the prostacyclin receptor (IP).

Oxidized lipids play an important role in pain processing by modulation of the activity of sensory neurons. However, the role of many signalling lipids that do not belong to the classical group of eicosanoids, especially of oxidized omega-3 lipids in pain processing is unclear. Here we investigated the role of the endogenously produced omega-3 lipids 17,18-EEQ and 19,20-EDP in modulating the activity of sensory neurons. We found that 17,18-EEQ but not 19,20-EDP can sensitize the transient receptor potential vanilloid 1 and ankyrin 1 ion channels (TRPV1 and TRPA1) in sensory neurons, which depends on activation of a Gs-coupled receptor and PKA activation. Screening of different Gs-coupled lipid receptor-deficient mice, identified the prostacyclin receptor IP as putative receptor for 17,18-EEQ. Since 17,18-EEQ is synthesized by the Cytochrome-P450-Epoxygenase CYP2J2, we established a cellular mass spectrometry-based screening assay to identify substances that can suppress 17,18-EEQ concentrations. Using this assay, we identify the antidepressant venlafaxine and the antihypertensive drug telmisartan as potent inhibitors of CYP2J2-dependent 17,18-EEQ synthesis. These findings identify 17,18-EEQ as first omega-3-derived lipid mediator that acts via the IP receptor and sensitizes the TRPV1 channel in sensory neurons. Moreover, the results give a mechanistic explanation for the antinociceptive effects of venlafaxine, which are still not well understood. Like telmisartan, venlafaxine may reduce neuronal activity by blocking CYP2J2 and 17,18-EEQ synthesis and by inhibiting the IP receptor-PKA-TRPV1 axis in sensory neurons.

Bacterial and Fungal Toll-Like Receptor Activation Elicits Type I IFN Responses in Mast Cells.

Next to their role in IgE-mediated allergic diseases and in promoting inflammation, mast cells also have antiinflammatory functions. They release pro- as well as antiinflammatory mediators, depending on the biological setting. Here we aimed to better understand the role of mast cells during the resolution phase of a local inflammation induced with the Toll-like receptor (TLR)-2 agonist zymosan. Multiple sequential immunohistology combined with a statistical neighborhood analysis showed that mast cells are located in a predominantly antiinflammatory microenvironment during resolution of inflammation and that mast cell-deficiency causes decreased efferocytosis in the resolution phase. Accordingly, FACS analysis showed decreased phagocytosis of zymosan and neutrophils by macrophages in mast cell-deficient mice. mRNA sequencing using zymosan-induced bone marrow-derived mast cells (BMMC) revealed a strong type I interferon (IFN) response, which is known to enhance phagocytosis by macrophages. Both, zymosan and lipopolysaccharides (LPS) induced IFN-ß synthesis in BMMCs in similar amounts as in bone marrow derived macrophages. IFN-ß was expressed by mast cells in paws from naïve mice and during zymosan-induced inflammation. As described for macrophages the release of type I IFNs from mast cells depended on TLR internalization and endosome acidification. In conclusion, mast cells are able to produce several mediators including IFN-ß, which are alone or in combination with each other able to regulate the phagocytotic activity of macrophages during resolution of inflammation.

5-Amino-1-ß-D-Ribofuranosyl-Imidazole-4-Carboxamide (AICAR) Reduces Peripheral Inflammation by Macrophage Phenotype Shift.

The stimulation of the AMP-activated kinase (AMPK) by 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) has been associated with antihyperalgesia and the inhibition of nociceptive signaling in the spinal cord in models of paw inflammation. The attenuated nociception comes along with a strongly reduced paw edema, indicating that peripheral antiinflammatory mechanisms contribute to antinociception. In this study, we investigated the impact of AICAR on the immune cell composition in inflamed paws, as well as the regulation of inflammatory and resolving markers in macrophages. By using fluorescence-activated cell sorting (FACS) analysis and immunofluorescence, we found a significantly increased fraction of proresolving M2 macrophages and anti-inflammatory interleukin (IL)-10 in inflamed tissue, while M1 macrophages and proinflammatory cytokines such as IL-1 were decreased by AICAR in wild type mice. In AMPKα2 knock-out mice, the M2 polarization of macrophages in the paw was missing. The results were supported by experiments in primary macrophage cultures which also showed a shift to a proresolving phenotype with decreased levels of proinflammatory mediators and increased levels of antiinflammatory mediators. However, in the cell cultures, we did not observe differences between the AMPKα2+/+ and -/- cells, thus indicating that the AICAR-induced effects are at least partially AMPK-independent. In summary, our results indicate that AICAR has potent antiinflammatory and proresolving properties in inflammation which are contributing to a reduction of inflammatory edema and antinociception.

Cancer-induced inflammation and inflammation-induced cancer in colon: a role for S1P lyase.

A role of sphingolipids for inflammatory bowel disease and cancer is evident. However, the relative and separate contribution of sphingolipid deterioration in inflammation versus carcinogenesis for the pathophysiology of colitis-associated colon cancer (CAC) was unknown and therefore examined in this study. We performed isogenic bone marrow transplantation of inducible sphingosine-1-phosphate (S1P) lyase knockout mice to specifically modulate sphingolipids and associated genes and proteins in a compartment-specific way in a DSS/AOM mediated CAC model. 3D organoid cultures were used in vitro. S1P lyase (SGPL1) knockout in either immune cells or tissue, caused local sphingolipid accumulation leading to a dichotomic development of CAC: Immune cell SGPL1 knockout (I-SGPL-/-) augmented massive immune cell infiltration initiating colitis with lesions and calprotectin increase. Pathological crypt remodeling plus extracellular S1P-signaling caused delayed tumor formation characterized by S1P receptor 1, STAT3 mRNA increase, as well as programmed cell death ligand 1 expression, accompanied by a putatively counter regulatory STAT1S727 phosphorylation. In contrast, tissue SGPL1 knockout (T-SGPL-/-) provoked immediate occurrence of epithelial-driven tumors with upregulated sphingosine kinase 1, S1P receptor 2 and epidermal growth factor receptor. Here, progressing carcinogenesis was accompanied by an IL-12 to IL-23 shift with a consecutive development of a Th2/GATA3-driven, tumor-favoring microenvironment. Moreover, the knockout models showed distinct lymphopenia and neutrophilia, different from the full SGPL1 knockout. This study shows that depending on the initiating cellular S1P source, the pathophysiology of inflammation-induced cancer versus cancer-induced inflammation develops through separate, discernible molecular steps.