Absence Of Interleukin Research Articles

Interleukin-6 deficiency reduces neuroinflammation by inhibiting the STAT3-cGAS-STING pathway in Alzheimer’s disease mice

BackgroundThe Interleukin-6 (IL-6)-signal transducer and activator of transcription 3 (STAT3) pathway, along with the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, are critical contributors to neuroinflammation in Alzheimer’s disease (AD). Although previous research outside the context of AD has indicated that the IL-6-STAT3 pathway may regulate the cGAS-STING pathway, the exact molecular mechanisms through which IL-6-STAT3 influences cGAS-STING in AD are still not well understood.MethodsThe activation of the IL-6-STAT3 and cGAS-STING pathways in the hippocampus of 5×FAD and WT mice was analyzed using WB and qRT-PCR. To explore the effects of IL-6 deficiency, Il6+/− mice were crossed with 5×FAD mice, and the subsequent impact on hippocampal STAT3 pathway activity, cGAS-STING pathway activation, amyloid pathology, neuroinflammation, and cognitive function was evaluated through WB, qRT-PCR, immunohistochemistry, ThS staining, ELISA, and behavioral tests. The regulatory role of STAT3 in the transcription of the Cgas and Sting genes was further validated using ChIP-seq and ChIP-qPCR on hippocampal tissue from 5×FAD and Il6−/−: 5×FAD mice. Additionally, in the BV2 microglial cell line, the impact of STAT3 activation on the transcriptional regulation of Cgas and Sting genes, as well as the production of inflammatory mediators, was examined through WB and qRT-PCR.ResultsWe observed marked activation of the IL-6-STAT3 and cGAS-STING pathways in the hippocampus of AD mice, which was attenuated in the absence of IL-6. IL-6 deficiency reduced beta-amyloid deposition and neuroinflammation in the hippocampus of AD mice, contributing to cognitive improvements. Further analysis revealed that STAT3 directly regulates the transcription of both the Cgas and Sting genes. These findings suggest a potential mechanism involving the STAT3-cGAS-STING pathway, wherein IL-6 deficiency mitigates neuroinflammation in AD mice by modulating this pathway.ConclusionThese findings indicate that the STAT3-cGAS-STING pathway is critical in mediating neuroinflammation associated with AD and may represent a potential therapeutic target for modulating this inflammatory process in AD.

Targeting of the IL-5 pathway in severe asthma reduces mast cell progenitors

BackgroundTherapies targeting interleukin-5 (IL-5) or its receptor (IL-5Rα) are currently used to treat patients with severe eosinophilic asthma. ObjectiveTo investigate the impact of anti-IL-5 and anti-IL-5Rα biological therapies on mast cells (MCs) and their progenitors. MethodsSurface IL-5Rα expression was investigated on MCs and their progenitors in mouse lungs and bone marrow, and in human lungs and blood. Isolated human MC progenitors cultured in the presence or absence of IL-5 were analyzed in vitro. Circulating MC progenitors were quantified in patients with severe asthma, before and after anti-IL-5 (mepolizumab) or anti-IL-5Rα (benralizumab) therapy. Gene expression analysis of MC progenitors was performed before and after anti-IL-5Rα therapy. ResultsApproximately 50% of the human primary lung MCs, and 30% of the human MC progenitors from individuals with allergic asthma expressed IL-5Rα. In patients with mild-to-moderate allergic asthma and mice with acute allergic airway inflammation, the fraction of IL-5Rα + MC progenitors was elevated. Additionally, IL-5 promoted the proliferation and/or survival of isolated human MC progenitors. Further, patients with severe asthma from two independent cohorts demonstrated a reduction of blood MC progenitors after anti-IL-5 or anti-IL-5Rα treatment. This was associated with improved asthma control, as well as a decline in both blood eosinophils and Th2 cells. Finally, the blood MC progenitors remaining after anti-IL-5Rα (benralizumab) treatment exhibited a downregulated expression of genes involved in growth and proliferation. ConclusionThis study introduces the possibility that the clinical effects of targeting IL-5/IL-5Rα in severe asthma also may involve reduction of MC populations.

Neutrophil-derived IL-10 increases CVB3-induced acute pancreatitis pathology via suppressing CD8+T cell activation while increasing macrophage STAT3-IL-6 cascade

Acute pancreatitis (AP) is a lethal inflammatory disease of the pancreas. Its pathogenesis remains obscure and specific treatments are lacking. An increase in Interleukin-10 (IL-10) in the early stage of AP patients is closely related to AP severity. In Coxsackievirus B3 (CVB3) induced murine AP model, we found early IL-10 increased viral replication and pancreatic inflammation, yet the cellular source of IL-10 and the immunomodulatory role of neutrophils during viral infection remains unknown. Here we show that CVB3 infection enhanced neutrophil infiltration and IL-10 expression in the pancreas at day3 post infection (p.i.). Neutrophils served as an important early source of pancreatic IL-10 at the initiation of infection. Day3 pancreas extracts (D3P) also induced bone-marrow derived neutrophils (BMneu) to secrete IL-10. Adoptive transfer of D3P-pretreated BMneu into IL-10 KO mice increased viral replication and pancreas histopathology, which effect was blunted by the absence of IL-10 in BMneu. Mechanically, IL-10+ neutrophil increased IL-10R1 expression on MΦs and activated STAT3-IL-6/IL-10 signaling cascade while decreased IL-12 and MHC II expression in MΦs, thus impairing IFN-γ+Granzyme B+CD8+T cell activation and viral clearance. Adoptive transferring infected mice with activated CD8+T cells 4 days p.i. attenuated viral load and AP pathology indicating an AP-protective effect. Our findings document a novel immunoregulatory function of neutrophils in acute CVB3 infection, in which neutrophil-derived IL-10 impairs anti-viral CD8+T activation, and amplifies intrapancreatic inflammation via activating MΦ STAT3-IL-6 signaling cascade. An IL-10-targeting option is suggested for the future treatment of viral AP.

IL-10 constrains sphingolipid metabolism to limit inflammation

Interleukin-10 (IL-10) is a key anti-inflammatory cytokine that can limit immune cell activation and cytokine production in innate immune cell types1. Loss of IL-10 signalling results in life-threatening inflammatory bowel disease in humans and mice—however, the exact mechanism by which IL-10 signalling subdues inflammation remains unclear2–5. Here we find that increased saturated very long chain (VLC) ceramides are critical for the heightened inflammatory gene expression that is a hallmark of IL-10 deficiency. Accordingly, genetic deletion of ceramide synthase 2 (encoded by Cers2), the enzyme responsible for VLC ceramide production, limited the exacerbated inflammatory gene expression programme associated with IL-10 deficiency both in vitro and in vivo. The accumulation of saturated VLC ceramides was regulated by a decrease in metabolic flux through the de novo mono-unsaturated fatty acid synthesis pathway. Restoring mono-unsaturated fatty acid availability to cells deficient in IL-10 signalling limited saturated VLC ceramide production and the associated inflammation. Mechanistically, we find that persistent inflammation mediated by VLC ceramides is largely dependent on sustained activity of REL, an immuno-modulatory transcription factor. Together, these data indicate that an IL-10-driven fatty acid desaturation programme rewires VLC ceramide accumulation and aberrant activation of REL. These studies support the idea that fatty acid homeostasis in innate immune cells serves as a key regulatory node to control pathologic inflammation and suggests that ‘metabolic correction’ of VLC homeostasis could be an important strategy to normalize dysregulated inflammation caused by the absence of IL-10.

S100A9 Regulated M1/M2 Macrophage Polarization in Interleukin-10-Induced Promotion of Malignant Pleural Effusion.

Interleukin-10 (IL-10) promotes the formation and development of malignant pleural effusion (MPE). Previous studies have elucidated the pathogenesis from the view of the immune-regulation function of CD4+ T-cells. However, the underlying mechanism is still not fully understood. In this study, our results showed that IL-10 deficiency reduced the percentage of macrophages in mouse MPE and regulated M1/M2 polarization in vivo and in vitro. The migration capacity of tumor cells was suppressed, and apoptosis was promoted when tumor cells were cocultured with MPE macrophages in the absence of IL-10. Messenger RNA sequencing of MPE macrophages showed that S100A9 was downregulated in IL-10-/- mice. Bone marrow-derived macrophages obtained from wild-type mice transfected with S100A9-specific small interfering RNAs (siRNAs) also showed less M2 and more M1 polarization than those from the siRNA control group. Furthermore, downregulation of S100A9 using S100A9-specific siRNA suppressed MPE development, decreased macrophages, and modulated macrophage polarization in MPE in vivo. In conclusion, S100A9 plays a vital role in the process of IL-10 deficiency-mediated MPE suppression by regulating M1/M2 polarization, thus influencing the tumor-migration capacity and apoptosis. This could result in clinically applicable strategies to inhibit the formation of MPE by regulating the polarization of MPE macrophages.

Pentosan Polysulfate Affords Pleotropic Protection to Multiple Cells and Tissues.

Pentosan polysulfate (PPS), a small semi-synthetic highly sulfated heparan sulfate (HS)-like molecule, shares many of the interactive properties of HS. The aim of this review was to outline the potential of PPS as an interventional therapeutic protective agent in physiological processes affecting pathological tissues. PPS is a multifunctional molecule with diverse therapeutic actions against many disease processes. PPS has been used for decades in the treatment of interstitial cystitis and painful bowel disease, it has tissue-protective properties as a protease inhibitor in cartilage, tendon and IVD, and it has been used as a cell-directive component in bioscaffolds in tissue engineering applications. PPS regulates complement activation, coagulation, fibrinolysis and thrombocytopenia, and it promotes the synthesis of hyaluronan. Nerve growth factor production in osteocytes is inhibited by PPS, reducing bone pain in osteoarthritis and rheumatoid arthritis (OA/RA). PPS also removes fatty compounds from lipid-engorged subchondral blood vessels in OA/RA cartilage, reducing joint pain. PPS regulates cytokine and inflammatory mediator production and is also an anti-tumor agent that promotes the proliferation and differentiation of mesenchymal stem cells and the development of progenitor cell lineages that have proven to be useful in strategies designed to effect repair of the degenerate intervertebral disc (IVD) and OA cartilage. PPS stimulates proteoglycan synthesis by chondrocytes in the presence or absence of interleukin (IL)-1, and stimulates hyaluronan production by synoviocytes. PPS is thus a multifunctional tissue-protective molecule of potential therapeutic application for a diverse range of disease processes.

Current Understanding of the Role of Eosinophils in CRSwNP and Implications for Treatment with Mepolizumab and Benralizumab.

International consensus statements now subdivide chronic rhinosinusitis (CRS) into several phenotypes and endotypes, including the presence of polyps (CRSwNP) and eosinophilia (eCRSwNP). Biological treatments aimed at blocking eosinophilic inflammation in CRSwNP via interleukin 5 (IL5) or the interleukin 5 receptor (IL5R) have demonstrated limited efficacy thus far. To review the pathophysiology of eCRSwNP, the evidence for mepolizumab (anti-IL5) and benralizumab (anti-IL5R) in CRSwNP, and to highlight areas for future research and therapeutic intervention. Primary and secondary literature search. Clinical trials on mepolizumab and benralizumab in CRSwNP are limited and restricted by trial design which prevents direct comparison with other interventions, including surgery. Both agents would appear to provide some benefit in reducing nasal polyp size but limited clinical patient benefit. Molecular biological research highlights that eCRSwNP can occur in the absence of IL5 and that other cells/cytokines play an important part in the disease's pathophysiology. Blockade of IL5/IL5R alone would appear to provide limited "real life" clinical benefit in patients with CRSwNP due to the complexities of the pathophysiology of the condition. Therapy aimed at several simultaneous cytokine targets has logic but well-designed trials are unlikely to be forthcoming in the short term due to the financial cost and commercial conflicts of interest.

Functional characterization of interleukin 4 and retinoic acid signaling crosstalk during alternative macrophage activation

Retinoic acid possesses potent immunomodulatory properties in various cell types, including macrophages. In this study, we first investigated the effects at the transcriptional and functional levels of exogenous retinoic acid in murine bone marrow-derived macrophages (BMDMs) in the presence or absence of interleukin 4 (IL4), a cytokine with potent anti-inflammatory properties. We examined the effect of IL4 on vitamin A homeostasis in macrophages by quantifying retinoid synthesis and secretion. Our RNAseq data show that exogenous retinoic acid synergizes with IL4 to regulate anti-inflammatory pathways such as oxidative phosphorylation and phagocytosis. Efferocytosis and lysosomal degradation assays validated gene expression changes at the functional level. IL4 treatment altered the expression of several genes involved in vitamin A transport and conversion to retinoic acid. Radiolabeling experiments and mass spectrometry assays revealed that IL4 stimulates retinoic acid production and secretion in a signal transducer and activator of transcription 6 (STAT6)-dependent manner. In summary, our studies highlight the key role of exogenous and endogenous retinoic acid in shaping the anti-inflammatory response of macrophages.

Novel genetic regulators of fibrinogen synthesis identified by an in vitro experimental platform

BackgroundFibrinogen has an established, essential role in both coagulation and inflammatory pathways, and these processes are deeply intertwined in the development of thrombotic and atherosclerotic diseases. Previous studies aimed to better understand the (patho) physiological actions of fibrinogen by characterizing the genomic contribution to circulating fibrinogen levels. ObjectivesEstablish an in vitro approach to define functional roles between genes within these loci and fibrinogen synthesis. MethodsCandidate genes were selected on the basis of their proximity to genetic variants associated with fibrinogen levels and expression in hepatocytes and HepG2 cells. HepG2 cells were transfected with small interfering RNAs targeting candidate genes and cultured in the absence or presence of the proinflammatory cytokine interleukin-6. Effects on fibrinogen protein production, gene expression, and cell growth were assessed by immunoblotting, real-time polymerase chain reaction, and cell counts, respectively. ResultsHepG2 cells secreted fibrinogen, and stimulation with interleukin-6 increased fibrinogen production by 3.4 ± 1.2 fold. In the absence of interleukin-6, small interfering RNA knockdown of FGA, IL6R, or EEPD1 decreased fibrinogen production, and knockdown of LEPR, PDIA5, PLEC, SHANK3, or CPS1 increased production. In the presence of interleukin-6, knockdown of FGA, IL6R, or ATXN2L decreased fibrinogen production. Knockdown of FGA, IL6R, EEPD1, LEPR, PDIA5, PLEC, or CPS1 altered transcription of one or more fibrinogen genes. Knocking down ATXN2L suppressed inducible but not basal fibrinogen production via a post-transcriptional mechanism. ConclusionsWe established an in vitro platform to define the impact of select gene products on fibrinogen production. Genes identified in our screen may reveal cellular mechanisms that drive fibrinogen production as well as fibrin(ogen)-mediated (patho)physiological mechanisms.

Chicken CSF2 and IL-4-, and CSF2-dependent bone marrow cultures differentiate into macrophages over time.

Chicken bone marrow-derived macrophages (BMMΦ) and dendritic cells (BMDC) are utilized as models to study the mononuclear phagocytic system (MPS). A widely used method to generate macrophages and DC in vitro is to culture bone marrow cells in the presence of colony-stimulating factor-1 (CSF1) to differentiate BMMΦ and granulocyte-macrophage-CSF (GM-CSF, CSF2) and interleukin-4 (IL-4) to differentiate BMDC, while CSF2 alone can lead to the development of granulocyte-macrophage-CSF-derived DC (GMDC). However, in chickens, the MPS cell lineages and their functions represented by these cultures are poorly understood. Here, we decipher the phenotypical, functional and transcriptional differences between chicken BMMΦ and BMDC along with examining differences in DC cultures grown in the absence of IL-4 on days 2, 4, 6 and 8 of culture. BMMΦ cultures develop into a morphologically homogenous cell population in contrast to the BMDC and GMDC cultures, which produce morphologically heterogeneous cell cultures. At a phenotypical level, all cultures contained similar cell percentages and expression levels of MHCII, CD11c and CSF1R-transgene, whilst MRC1L-B expression decreased over time in BMMΦ. All cultures were efficiently able to uptake 0.5 µm beads, but poorly phagocytosed 1 µm beads. Little difference was observed in the kinetics of phagosomal acidification across the cultures on each day of analysis. Temporal transcriptomic analysis indicated that all cultures expressed high levels of CSF3R, MERTK, SEPP1, SPI1 and TLR4, genes associated with macrophages in mammals. In contrast, low levels of FLT3, XCR1 and CAMD1, genes associated with DC, were expressed at day 2 in BMDC and GMDC after which expression levels decreased. Collectively, chicken CSF2 + IL-4- and CSF2-dependent BM cultures represent cells of the macrophage lineage rather than inducing conventional DC.

Regulation of endothelial DNA methylation by IL‐6 signaling

Interleukin 6 (IL‐6) is a major mediator of the septic cytokine storm. It activates the transcription factor STAT3, resulting in pathological vascular leakage, leukocyte adhesion, and coagulation cascade activation, leading to acute multiorgan dysfunction. Septic shock survivors have a high risk of developing post‐intensive care syndrome (PICS), a condition of chronic cardiovascular disease, cognitive impairment, and reduced overall survival. The causes for this syndrome are not fully understood. We hypothesize that acute STAT3‐mediated endotheliopathy promotes changes in the endothelial DNA methylation landscape, leading to the long‐lasting effects of PICS. To test this in vitro, HUVEC were treated with a combination of IL‐6 and sIL‐6Rα (IL‐6+R) or saline for 6h, 24h, or 72h, and DNA methylation was profiled using Infinium Methylation EPIC arrays after bisulfite conversion. Unbiased clustering showed a separation between treated and control cells only after 72h. 360 CpG sites were significantly modified (FDR‐adjusted p=<0.05). Of these, 329 CpG sites were hypomethylated and 31 were hypermethylated. Gene ontology analysis of the differentially methylated genes showed enrichment of pathways directly linked with the platelet and leukocyte interaction, cell proliferation and angiogenesis, and signaling. Consistent with the demethylation of the LAMP3 locus, a 72h treatment with IL‐6+R induced a dramatic increase in LAMP3 expression. The same treatment induced an inhibition of ADAM19 and MYO10 gene expression, consistent with their hypermethylation status. These changes were not associated with differences in the expression of DNA methyltransferases and TET enzymes. The IL‐6+R‐induced increase in LAMP3 expression was further increased in cells that had been previously treated with IL‐6+R for 72h and then incubated in the absence of IL‐6+R for another 96h, compared to saline‐treated cells, suggesting a long‐lasting effect of a prior IL‐6+R treatment via demethylation of the promoter. In contrast, SOCS3 expression (a gene directly activated by STAT3) was similar in cells that were pretreated with IL‐6+R or not. In summary, persistent activation of IL‐6 leads to endothelial DNA methylation changes and corresponding gene expression differences. These differences may regulate pathways that can be associated with reduced long‐term survival and prolonged organ damage as observed in PICS.

Interleukin-6 absence triggers intestinal microbiota dysbiosis and mucosal immunity in mice

Interleukin-6 (IL-6) in mucosal immune cells is involved in post-injury intestinal regeneration, inflammation responses, and gastric homeostasis. However, the interaction between IL-6 and the dynamic balance of gut microbiota (GM) remains unexplored. Intestinal pathology was assessed by hematoxylin and eosin and periodic acid–Schiff staining in wild-type (WT) and IL-6 gene knockout (KO) C57BL/6J mice. GM profiles were established via high-throughput sequencing of the fecal bacterial 16S rRNA gene. Intestinal α- and β-defensins were measured by quantitative real-time PCR; further, flow cytometry was performed to analyze isolated intraepithelial lymphocytes (IELs). Compared with the WT, IL-6 KO did not obviously change gut structures, but significantly reduced GM diversity, resulting in reduced metabolic pathways with decreased gram-positive but elevated gram-negative bacteria. More taxa alterations included differences at the phyla (e.g., increased Verrucomicrobia and decreased Firmicutes) and genera (e.g., increased Akkermansia and decreased Lactobacillus) levels. Absence of IL-6 also significantly increased intestinal expression of defensins α3 and α4 (Defa3 and Defa4) and the percentage of natural TCRγδ+ IELs, providing a molecular basis for triggering mucosal immune response. Therefore, IL-6 loss remodels GM composition and alters IEL maintenance, identifying IL-6 as a crucial cytokine for GM dysbiosis and mucosal immunity.

Systems analysis of human T helper17 cell differentiation uncovers distinct time-regulated transcriptional modules

SummaryT helper (Th) 17 cells protect from infections and are pathogenic in autoimmunity. While human Th17 cell differentiation has been defined, the global and stepwise transcriptional changes accompanying this process remain uncharacterized. Herein, by performing transcriptome analysis of human Th17 cells, we uncovered three time-regulated modules: early, involving exclusively “signaling pathways” genes; late, characterized by response to infections; and persistent, involving effector immune functions. To assign them an inflammatory or regulatory potential, we compared Th17 cells differentiated in presence or absence of interleukin (IL)-1β, respectively. Most inflammatory genes belong to the persistent module, whereas regulatory genes are lately or persistently induced. Among inflammatory genes, we identified the effector molecules IL17A, IL17F, IL26, IL6, interferon (IFN)G, IFNK, LTA, IL1A, platelet-derived growth factor (PDGF) A and the transcriptional regulators homeodomain-only protein homeobox (HOPX) and sex-determining-region-Y-box (SOX)2, whose expression was independently validated. This study provides an integrative representation of the stepwise human Th17 differentiation program and offers new perspectives toward therapeutic targeting of Th17-related autoimmune diseases.

Absence of interleukin-10 reduces progression of shiga toxin-induced hemolytic uremic syndrome.

Hemolytic Uremic Syndrome (HUS), a disease triggered by Shiga toxin (Stx), is characterized by hemolytic anemia, thrombocytopenia and renal failure. The inflammatory response mediated by polymorphonuclear neutrophils (PMNs) and monocytes is essential to HUS onset. Still, the role of anti-inflammatory cytokines is less clear. The deficiency of IL-10, an anti-inflammatory cytokine, leads to severe pathology in bacterial infections but also to beneficial effects in models of sterile injury. The aim of this work was to analyze the role of IL-10 during HUS. Control and IL-10 lacking mice (IL-10-/-) were intravenously injected with Stx type 2 (Stx2) and survival rate was evaluated. PMN and circulating and renal pro- and anti-inflammatory factors were analyzed by FACS and enzyme-linked immunosorbent assay (ELISA) respectively. IL-10-/- mice showed a higher survival associated with lower renal damage reflected by reduced plasma urea and creatinine levels than control mice. Circulating PMN increased at 72 h in both mouse strains accompanied by an up-regulation of CD11b in control mice. In parallel, renal PMN were significantly increased only in control mice after toxin. Plasma TNF-α, IL-6 and corticosterone levels were higher increased in IL-10-/- than control mice. Simultaneously renal TNF-α raised constantly but was accompanied by increased TGF-β levels in IL-10-/- mice. These results demonstrate that the profile of circulating and renal cytokines after Stx2 differed between strains suggesting that balance of these factors could participate in renal protection. We conclude that IL-10 absence has a protective role in an experimental model of HUS by reducing PMN recruitment into kidney and renal damage, and increasing mice survival.

Dysregulated IL-6/GP130/JAK Signaling in Calreticulin Mutated Myeloproliferative Neoplasms (MPN)

Introduction. Frameshift mutations in exon 9 of calreticulin (CALR) gene represent the second most frequent alteration in MPN. More than 30 types of CALR mutations are described; the most frequent are a 52bp deletion (Type1, T1) and a 5bp insertion (T2). All mutations cause a recurrent frameshift that originates a novel C-terminal peptide lacking the KDEL Endoplasmic Reticulum-retention sequence. Recent data described the interaction of mutated CALR (CALRm) with the thrombopoietin receptor MPL and the resulting activation of JAK/STATs as well MAPK and PI3K pathways (Araki M et al & Chachoua I et al, Blood 2016; Elf S et al, Blood 2018). However, full characterization of the molecular mechanisms linking CALRm with MPN remains to be fully accomplished. Methods. By using CRISPR/Cas9 technology we generated CALR knock out (KO) and T1 variants from the GM-CSF cytokine dependent UT7 cell line; primary CD34+ cells from CALRm pts were also analyzed. Results. Introduction of CALR T1 mutation or generation of KO status did not appreciably modified the proliferation rate, cell cycle and apoptosis of UT7 cells under standard conditions. However, UT7 T1 and KO cells were similarly able to grow in the absence of GM-CSF, otherwise necessary for WT cells; in these cytokine-depleted conditions, apoptosis was significantly increased in WT cells compared both T1 and KO (4- and 8-fold higher; p<0.01) suggesting acquisition of cytokine independency. Exploring the mechanisms leading to cytokine independence we found that signal transducer and activation of transcription 3 (STAT3) was the main constitutively phosphorylated STAT in T1 and KO cells. Among a panel of STAT3 activation candidates assessed by RT-PCR, we found that mRNA levels of interleukin 6 (IL6), which induces STAT3 phosphorylation and activation through the glycoprotein 130/Janus kinase (GP130/JAK) pathway, were significantly increased in T1 and KO compared to parental cells (2- and 3.5-fold higher; p<0.01). The levels of IL6 released in cell culture medium were also significantly increased (1.5- and 2-fold higher; p<0.05). Blocking GP130 with SC144 or IL6 receptor (IL6R) with Tocilizumab (TCZ), in cytokine-depleted conditions, led to cell growth arrest in T1 and KO with no effects on WT cells. SC144 treatment induced a marked dephosphorylation of STAT3 in T1 and KO cells. Also, the membrane expression of GP130 and IL6R resulted higher in KO and T1 compared to WT cells, whilst intracellular content was similar. Co-immunoprecipitation showed that wild type calreticulin, unlike the mutated T1, formed complexes with GP130 and IL6R suggesting that CALRm lost the capacity to negatively regulate their membrane exposure resulting in downstream increased IL6 synthesis and release. The role of IL6 pathway in CALRm patients was evaluated by assessing megakaryocyte (Mk) colony generation in plasma clot cultures of CD34+ cells in the absence/presence of IL6, as part of cytokine cocktail optimally supporting CFU-Mk growth. We found that CFU-Mk generation by CALRm CD34+ cells was largely independent of exogenously added IL6, unlike control CFU-MK whose number was halved in the absence of IL6 (p<0.05). Moreover, inhibition of GP130 and IL6R by SC144 and TCZ, respectively, significantly reduced CFU-Mk by CALRm CD34+ cells (2.9- and 3.2-fold; p<0.05), with no effect in control cultures. As anticipated, JAK inhibitors ruxolitinib and momelotinib reduced CFU-Mk formation by CALRm CD34+ cells (2- and 3.2-fold, respectively; p<0.01), particularly evident in exogenous IL6-free conditions (5.8- and 4.8-fold, respectively; p<0.01). The combination of either ruxolitinib or momelotinib with TCZ on CFU-Mk formation was also assessed. Both combinations proved synergistic activity in reducing CFU-Mk from CALRm CD34+ cells (4.3- and 5-fold vs control cultures, respectively; p<0.01; combination compared to single agent, 2.8- fold vs ruxolitinib, 4.5-fold vs momelotinib and 4-fold vs TCZ, p<0.05 for all). Conclusions. Overall, our data suggest a novel model of action of mutant CALR resulting from the loss of its capability to complex GP130 and IL6R and regulate their cell membrane expression. Augmented expression of IL6 receptor complex on CALRm CD34+ cells on turn results in deregulated IL6-dependent signaling and augmented megakaryocyte generation. Blockade of IL-6/GP130/JAK signaling pathway might represent a new therapeutic target in pts with CALR mutation. Disclosures Vannucchi: Italfarmaco: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Consequences of the Lack of IL-10 in Different Endotoxin Effects and its Relationship With Glucocorticoids.

Sepsis constitutes one of the major causes of death in ICUs. In sepsis induced by gram-negative, although lipopolysaccharide (LPS) initially induces an exacerbated secretion of proinflammatory cytokines leading to endotoxic shock and death resembling a septic shock, it is also capable of inducing refractoriness to subsequent challenge with LPS, a state known as endotoxin tolerance, which is considered the initial step of the immunosuppression found in septic patients. As we previously demonstrated the importance of glucocorticoids in endotoxin tolerance, the aim of this study was to evaluate the contribution of Interleukin-10 (IL-10) both in the endotoxic shock and in the development of the tolerance and its relationship with glucocorticoids. Our results show that, upon LPS challenge, IL-10 knockout mice (KO) mice had an enhanced LPS sensitivity, along with elevated levels of proinflammatory cytokines as tumor necrosis factor-α, IL-12 and interferon-γ, and enhanced tissue damage, despite the high levels of glucocorticoids. This effect may be because, in part, of the higher expression of tumor necrosis factor receptors in IL-10 KO mice. Further, the injection of dexamethasone did not protect IL-10 KO mice from a LPS lethal challenge. Although tolerance was achieved in the absence of IL-10, it was weaker and the elevated levels of glucocorticoids were not able to reverse the high sensitivity of IL-10 KO mice to LPS. Nevertheless, glucocorticoids would play a pivotal role in the establishment and maintenance of this partial tolerance in IL-10 KO mice. Finally, our results show that IL-10 and glucocorticoids could act in a bidirectional way influencing the inflammatory and anti-inflammatory periods.

Oral administration of EPA-rich oil impairs collagen reorganization due to elevated production of IL-10 during skin wound healing in mice

Wound healing is an essential process for organism survival. Some fatty acids have been described as modulators of wound healing. However, the role of omega-3 fatty acids is unclear. In the present work, we investigate the effects of oral administration of eicosapentaenoic acid (EPA)-rich oil on wound healing in mice. After 4 weeks of EPA-rich oil supplementation (2 g/kg of body weight), mice had increased serum concentrations of EPA (20:5ω-3) (6-fold) and docosahexaenoic acid (DHA; 22:6ω-3) (33%) in relation to control mice. Omega-3 fatty acids were also incorporated into skin in the EPA fed mice. The wound healing process was delayed at the 3rd and 7th days after wounding in mice that received EPA-rich oil when compared to control mice but there was no effect on the total time required for wound closure. Collagen reorganization, that impacts the quality of the wound tissue, was impaired after EPA-rich oil supplementation. These effects were associated with an increase of M2 macrophages (twice in relation to control animals) and interleukin-10 (IL-10) concentrations in tissue in the initial stages of wound healing. In the absence of IL-10 (IL-10−/− mice), wound closure and organization of collagen were normalized even when EPA was fed, supporting that the deleterious effects of EPA-rich oil supplementation were due to the excessive production of IL-10. In conclusion, oral administration of EPA-rich oil impairs the quality of wound healing without affecting the wound closure time likely due to an elevation of the anti-inflammatory cytokine IL-10.

GPER and ERα mediate estradiol enhancement of mitochondrial function in inflamed adipocytes through a PKA dependent mechanism

Obesity is associated with inflammation, dysregulated adipokine secretion, and disrupted adipose tissue mitochondrial function. Estradiol (E2) has been previously reported to increase mitochondrial function and biogenesis in several cell lines, but neither the type of oestrogen receptor (ERα, ERβ and GPER) involved nor the mechanism whereby such effects are exerted have been fully described. Considering the anti-inflammatory activity of E2 as well as its effects in enhancing mitochondrial biogenesis, the aim of this study was to investigate the contribution of ERα, ERβ, and GPER signaling to the E2-mediated enhancement of adipocyte mitochondrial function in a pro-inflammatory situation.3T3-L1 cells were treated for 24 h with ER agonists (PPT, DPN, and G1) and antagonists (MPP, PHTPP, and G15) in the presence or absence of interleukin 6 (IL6), as a pro-inflammatory stimulus. Inflammation, mitochondrial function and biogenesis markers were analyzed. To confirm the involvement of the PKA pathway, cells were treated with a GPER agonist, a PKA inhibitor, and IL6. Mitochondrial function markers were analyzed.Our results showed that activation of ERα and GPER, but not ERβ, was able to counteract the proinflammatory effects of IL6 treatment, as well as mitochondrial biogenesis and function indicators. Inhibition of PKA prevented the E2- and G1-associated increase in mitochondrial function markers.In conclusion E2 prevents IL6 induced inflammation in adipocytes and promotes mitochondrial function through the combined activation of both GPER and ERα. These findings expand our understanding of ER interactions under inflammatory conditions in female rodent white adipose tissue.

Activation of IL5R and CRTH2 on Human Eosinophils Elicit a Similar Molecular Response and Reveal a Synergistic Effect

Eosinophils represent a key cell driving inflammatory processes in diseases such as asthma and eosinophilic esophagitis. The entire process of migration and activation of eosinophils is orchestrated by a myriad of molecules, among which interleukin 5 (IL5) plays a crucial role. Another receptor expressed on eosinophils is chemo-attractant receptor like expressed on Th2 cells (CRTH2). CRTH2 activation has a prominent role in stimulating Th2 and type 2 innate lymphoid cells to produce IL5 in disease tissue, but also induces eosinophils migration and adhesion. Prostaglandin D2 is the endogenous ligand of CRTH2, predominately released by activated mast cells, another key cell involved in eosinophilic diseases. Consequently, the IL5 and CRTH2 pathway are likely triggered simultaneously in eosinophils in disease tissue. Understanding the molecular effects of CRTH2 and IL5 receptor (IL5R) activated eosinophils might provide new insights for efficient eosinophils blocking. We performed gene expression profiling with isolated human eosinophils to compare the molecular signatures of CRTH2 and IL5R activation. The identified genes were confirmed using an orthogonal technology, and simultaneous IL5R and CRTH2 activation was investigated on the protein level. This study reveals that IL5R and CRTH2 activation trigger a similar molecular response in eosinophils with stronger IL5R effects. C-C chemokine ligand 2 was produced synergistically upon IL5R and CRTH2 stimulation and might contribute to monocyte and macrophage recruitment to disease tissue. The effect on C-C chemokine receptor 1 suggests that eosinophils maturation in the tissue might even occur in absence of IL5 via the CRTH2 pathway.

Effects of local Interleukin‐6 on Skeletal Muscle Mitochondrial Physiology

Skeletal muscle is vital to metabolic health and physical function. Skeletal muscle mitochondria have been extensively interrogated as organelles whose function and dysfunction are closely tied with chronic diseases such as diabetes and sarcopenia. There is accumulating evidence suggesting that chronic inflammation that is prevalent with aging and obesity is associated with skeletal muscle abnormalities such as functional impairments in mitochondria. Despite compelling links between inflammation, oxidative stress, and skeletal muscle dysfunction, the local effects of inflammatory cytokines on skeletal muscle mitochondrial physiology are currently not well‐understood. The purpose of this study was to determine how the inflammatory cytokine interleukin‐6 (IL‐6) influences skeletal muscle mitochondrial function. To determine the acute effects of IL‐6, mitochondria were isolated from skeletal muscle of healthy male mice (C57BL6) and were exposed to IL‐6 (IL6+,50 ng/ml) or vehicle (CTRL) for one hour. Multiplexed measurements of mitochondrial respiration (high‐resolution respirometry) and reactive oxygen species production (ROS, fluorometry) revealed that IL‐6 significantly increased ROS production as a percent of oxygen (4.14%±0.40) compared to CTRL (2.74%±0.34). Similarly, IL‐6 increased ROS production in differentiated C2C12 myotubes that were incubated for 18 hrs with 50 ng/mL IL‐6 (4.71%±.57) compared to CTRL (2.21%±.34). To evaluate the possibility that IL‐6 triggers ROS production by stimulating the mitochondrial transition pore (mPTP), isolated mitochondria were ale treated with cyclosporin A (CsA), a mPTP inhibitor in the presence or absence of IL‐6. Inhibition of mPTP attenuated IL‐6 induced ROS production (IL6+ (4.14%±0.40) vs. IL6+/CsA (2.78%±0.58). These results suggest that IL‐6 triggers an increase in mitochondrial ROS production that appears to be mediated by opening of the mPTP. Prolonged opening of the mPTP caused by chronically elevated IL‐6 may contribute to increased oxidative stress that is evident in skeletal muscle with aging and obesity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.