Increase In IL-10 Production Research Articles

JAK2V617F Mutation Promoted IL-6 Production and Glycolysis via Mediating PKM1 Stabilization in Macrophages.

A substitution mutation of valine to phenylalanine at codon encoding position 617 of the Janus kinase 2 (JAK2) gene (JAK2V617F) has been detected in myeloid cells of some individuals with higher levels of proinflammatory cytokine production such as interleukin (IL)-6. However, the mechanisms by which JAK2V617F mutation mediating those cytokines remain unclear. We, therefore, established JAK2V617F-expressing murine macrophages (JAK2V617F macrophages) and found that the levels of p-STAT3 were markedly elevated in JAK2V617F macrophages in association with an increase in IL-6 production. However, inhibition of STAT3 by C188-9 significantly decreased the production of IL-6. Furthermore, the JAK2V617F mutation endowed macrophages with an elevated glycolytic phenotype in parallel with aberrant expression of PKM1. Interestingly, silencing of PKM1 inactivated STAT3 in parallel with reduced IL-6 production. In contrast, ectopic expression of PKM1 elevated IL-6 production via STAT3 activation. Importantly, the JAK2V617F mutation contributed to PKM1 protein stabilization via blockade of lysosomal-dependent degradation via chaperone-mediated autophagy (CMA), indicating that the JAK2V617F mutation could protect PKM1 from CMA-mediated degradation, leading to activation of STAT3 and promoting IL-6 production.

Pregnancy-Induced Changes in microRNA Expression in Multiple Sclerosis.

Pregnancy affects the disease course in multiple sclerosis (MS), particularly in the third trimester, where the relapse rate is reduced by as much as two thirds. This study aimed at identifying changes in microRNA (miRNA) and immune cell phenotypes in pregnant MS patients. Discovery and validation studies to detect differentially expressed miRNAs were performed with quantitative real-time PCR on peripheral blood mononuclear cells (PBMC). Flow cytometry analysis was performed on PBMC stained with antibodies directed against surface markers of antigen presenting cells (APCs), NK-cells, NKT cells, CD4+ and CD8+ T cells and subsets of these cell types, including PDL1 and PDL2 expressing subsets. RNA was extracted from whole blood, monocytes, and NK-cells to investigate expression and correlation between regulated miRNAs and mRNAs. In total, 15 miRNAs were validated to be differentially expressed between third trimester pregnant and postpartum MS patients (Benjamini-Hochberg false discovery rate from p = 0.03–0.00004). Of these, 12 miRNAs were downregulated in pregnancy and 6 of the 15 miRNAs were altered by more than ±2-fold (+2.99- to -6.38-fold). Pregnant MS patients had a highly significant increase in the percentage of monocytes and a decrease of NK-cells and myeloid dendritic cells compared to non-pregnant MS patients. We confirm previous reports of a relative increase in CD56-bright NK-cells and a decrease in CD56-dim NK-cells in third trimester of pregnancy and report an increase in non-committed follicular helper cells. PDL1 and PDL2 expression was increased in pregnant patients together with IL10. Also, in monocytes IL10, PDL1, and PDL2 were upregulated whereas miR-1, miR-20a, miR-28, miR-95, miR-146a, miR-335, and miR-625 were downregulated between pregnant and untreated MS patients. IL10, PDL1, and PDL2 were predicted targets of MS pregnancy-changed miRNAs, further supported by their negative correlations. Additionally, previously identified pregnancy-regulated mRNAs were identified as predicted targets of the miRNAs. PDL1 and PDL2 bind PD-1 expressed on T cells with an inhibitory effect on T-cell proliferation and increase in IL10 production. These results indicate that some of the effects behind the disease-ameliorating third trimester of pregnancy might be caused by changed expression of miRNAs and immunoregulatory molecules in monocytes.

Effects of spinal manipulative therapy on inflammatory mediators in patients with non-specific low back pain: a non-randomized controlled clinical trial

BackgroundThe inflammatory profiles of patients with acute and chronic nonspecific low back pain (LBP) patients are distinct. Spinal manipulative therapy (SMT) has been shown to modulate the production of nociceptive chemokines differently in these patient cohorts. The present study further investigates the effect(s) of SMT on other inflammatory mediators in the same LBP patient cohorts.MethodsAcute (n = 22) and chronic (n = 25) LBP patients with minimum pain scores of 3 on a 10-point numeric scale, and asymptomatic controls (n = 24) were recruited according to stringent exclusion criteria. Blood samples were obtained at baseline and after 2 weeks during which patients received 6 SMTs in the lumbar or lumbosacral region. The in vitro production of tumor necrosis factor (TNFα), interleukin-1 β (IL-1β), IL-6, IL-2, interferon ɣ (IFNɣ), IL-1 receptor antagonist (IL-1RA), TNF soluble receptor type 2 (sTNFR2) and IL-10 was determined by specific immunoassays. Parametric as well as non-parametric statistics (PAST 3.18 beta software) was used to determine significance of differences between and within study groups prior and post-SMT. Effect size (ES) estimates were obtained using Cohen’s d.ResultsCompared with asymptomatic controls, SMT-related change scores were significant (P = 0.03–0.01) in reducing the production levels of TNFα in both patient cohorts and those of IL-6, IFNɣ and sTNFR2 (P = 0.001–0.02) in patients with chronic LBP. Above-moderate to large ES (d > 0.6–1.4) was observed for these mediators. Compared with respective baselines, a significant post-SMT reduction (P = 0.01) of IL-6 production was detected only in patients with chronic LBP while a significant increase of IL-2 production (P = 0.001 vs. control, and P = 0.004 vs. chronic LBP group) and a large ES (d = 0.87) were observed in patients with acute LBP. Pain and disability scores declined significantly (P < 0.001) in all LBP patients, and were positively correlated (P = 0.03) with IFNɣ and IL-2 levels in the acute LBP cohort.ConclusionThe short course of SMT treatments of non-specific LBP patients resulted in significant albeit limited and diverse alterations in the production of several of the mediators investigated in this study. This exploratory study highlights the potential of SMT to modulate the production of inflammatory components in acute and chronic non-specific LBP patients and suggests a need for further, randomized controlled clinical trials in this area.Trial registrationThis study was prospectively registered April 2012 with Clinical Trials.gov (#NCT01766141).https://register.clinicaltrials.gov/prs/app/action/SelectProtocol?sid=S0003ZIL&selectaction=Edit&uid=U0001V74&ts=2&cx=-axvqtg

Aurka loss in CD19+ B cells promotes megakaryocytopoiesis via IL-6/STAT3 signaling-mediated thrombopoietin production.

Rationale: Aurora kinase A (Aurora-A), which is required for mitosis, is a therapeutic target in various tumors. Targeting Aurora-A led to an increase in the differentiation and polyploidization of megakaryocytes both in vivo and in vitro. However, the mechanisms involved in controlling megakaryocyte differentiation have not been fully elucidated.Methods: Conditional Aurka knockout mice were generated. B cell development, platelet development and function were subsequently examined. Proplatelet formation, in vivo response to mTPO, post-transfusion experiment, colony assay, immunofluorescence staining and quantification, and ChIP assay were conducted to gain insights into the mechanisms of Aurka loss in megakaryocytopoiesis.Results: Loss of Aurka in CD19+ B cells impaired B cell development in association with an increase in the number of platelets in peripheral blood (PB). Surprisingly, thrombopoietin (TPO) production and IL-6 were elevated in the plasma in parallel with an increase in the number of differentiated megakaryocytes in the bone marrow (BM) of Aurkaf/f;Cd19Cre/+ mice. Interestingly, compared with that of the Aurkaf/f mice, a higher number of CD19+ B cells close to megakaryocytes was observed in the BM of the Aurkaf/f;Cd19Cre/+ mice. Moreover, Aurka loss in CD19+ B cells induced signal transducer and activator of transcription-3 (STAT3) activation. Inhibition of STAT3 reduced the Tpo mRNA levels. ChIP assays revealed that STAT3 bound to the TPO promoter. Additionally, STAT3-mediated TPO transcription was an autocrine effect provoked by IL-6, at least partially.Conclusions: Deletion of Aurka in CD19+ B cells led to an increase in IL-6 production, promoting STAT3 activation, which in turn contributed to TPO transcription and megakaryocytopoiesis.

Peanut protein acts as a TH2 adjuvant by inducing RALDH2 in human antigen-presenting cells

Peanut is a potent inducer of proallergenic TH2 responses in susceptible individuals. Antigen-presenting cells (APCs) including dendritic cells and monocytes instruct naive Tcells to differentiate into various effector cells, determining immune responses such as allergy and tolerance. We sought to detect peanut protein (PN)-induced changes in gene expression in human myeloid dendritic cells (mDCs) and monocytes, identify signaling receptors that mediate these changes, and assess how PN-induced genes in mDCs impact their ability to promote T-cell differentiation. mDCs, monocytes, and naive CD4+ T cells were isolated from blood bank donors and peanut-allergic patients. APCs were incubated with PN and other stimulants, and gene expression was measured using microarray and RT quantitative PCR. To assess T-cell differentiation, mDCs were cocultured with naive TH cells. PN induced a unique gene expression profile in mDCs, including the gene that encodes retinaldehyde dehydrogenase 2 (RALDH2), a rate-limiting enzyme in the retinoic acid (RA)-producing pathway. Stimulation of mDCs with PN also induced a 7-fold increase in the enzymatic activity of RALDH2. Blocking antibodies against Toll-like receptor (TLR)1/TLR2, as well as small interfering RNA targeting TLR1/TLR2, reduced the expression of RALDH2 in PN-stimulated APCs by 70%. Naive TH cells cocultured with PN-stimulated mDCs showed an RA-dependent 4-fold increase in production of IL-5 and expression of integrin α4β7. PN induces RALDH2 in human APCs by signaling through the TLR1/TLR2 heterodimer. This leads to production of RA, which acts on TH cells to induce IL-5 and gut-homing integrin. RALDH2 induction by PN in APCs and RA-promoted TH2 differentiation could be an important factor determining allergic responses to peanut.

Hsp65-Producing Lactococcocus lactis Prevents Antigen-Induced Arthritis in Mice.

Oral tolerance is the physiological process that enables the immune system to differentiate between harmless dietary and microbiota antigens from pathogen derived antigens. It develops at the mucosal surfaces and can result in local and systemic regulatory and anti-inflammatory effects. Translation of these benefits to the clinical practice faces limitations involving specificity and doses of antigen as well as regimens of feeding. To circumvent these problems, we developed a recombinant Hsp65 delivered by the acid lactic bacteria Lactococcus lactis NCDO 2118 directy in the intestinal mucosa. Hsp65 is a ubiquitous protein overexpressed in inflamed tissues and capable of inducing immunoregulatory mechanisms. L. lactis has probiotic properties and is commonly and safely used in dairy products. In this study, we showed that continuous delivery of HSP65 in the gut mucosa by L. lactis is a potent tolerogenic stimulus inducing regulatory CD4+LAP+ T cells that prevented collagen-induced and methylated bovine serum albumin-induced arthritis in mice. Clinical and histological signs of arthritis were inhibited as well as levels of inflammatory cytokines such as IL-17 and IFN-γ, serum titers of anti-collagen antibodies and rheumatoid factor. Oral administration of L. lactis induced alterations in microbiota composition toward an increased abundance of anaerobic bacteria such as Bifidobacterium and Lactobacillus. Tolerance to HSP65 and arthritis prevention induced by the recombinant L. lactis was associated with increase in IL-10 production by B cells and it was dependent on LAP+ T cells, IL-10 and TLR2 signaling. Therefore, HSP65-producing treatment induced effective tolerance and prevented arthritis development suggesting it can be used as a therapeutic tool for autoimmune diseases.

Developing immune-regulatory materials using immobilized monosaccharides with immune-instructive properties.

New strategies for immune modulation have shown real promise in regenerative medicine as well as the fight against autoimmune diseases, allergies, and cancer. Dendritic cells (DCs) are gatekeepers of the immune system and their ability in shaping the adaptive immune responses makes DCs ideal targets for immune modulation. Carbohydrates are abundant in different biological systems and are known to modulate DC phenotype and function. However, how simple monosaccharides instruct DC function is less well understood. In this study, we used a combinatorial array of immobilized monosaccharides to investigate how they modulate DC phenotype and function and crucially the impact of such changes on downstream adaptive immune responses. Our data show that a selection of monosaccharides significantly suppress lipopolysaccharide-induced DC activation as evidenced by a reduction in CD40 expression, IL-12 production, and indoleamine 2,3-dioxygenase activity, while inducing a significant increase in IL-10 production. These changes are indicative of the induction of an anti-inflammatory or regulatory phenotype in DCs, which was further confirmed in DC–T cell co-cultures where DCs cultured on the ‘regulatory’ monosaccharide-coated surfaces were shown to induce naïve T cell polarization toward regulatory phenotype. Our data also highlighted a selection of monosaccharides that are able to promote mixed Treg and Th17 cell differentiation, a T cell phenotype expected to be highly immune suppressive. These data show the potential immunomodulatory effects of immobilized monosaccharides in priming DCs and skewing T cell differentiation toward an immune-regulatory phenotype. The ability to fine-tune immune responses using these simple carbohydrate combinations (e.g. as coatings for existing materials) can be utilized as novel tools for immune modulation with potential applications in regenerative medicine, implantable medical devices, and wound healing where reduction of inflammatory responses and maintaining immune homeostasis are desirable.

Abstract A07: A marker to optimize BCG dosing schedules for immunotherapy

Abstract Background: BCG immunotherapy is the standard for non-muscle invasive bladder cancer (NMIBC) and there has been no change in therapy schedules, though some patients cannot tolerate repeated dosing. This study evaluates the potential of the GST theta 2 (GSTT2) gene that modulates BCG survival in bladder cancer cell lines to modulate patient response to therapy. Our hypothesis is that better intracellular BCG survival should correlate with better immune activation. GSTT2, a member of the glutathione-S-transferase family, lies on chromosome 22q. In man, a 37kb deletion removes a pseudogene (GSTT2B) and the 5' flanking region of the GSTT2 gene, resulting in low/no expression of GSTT2. This deletion is a reliable marker of GSTT2 gene expression. GSTT2B is deleted in 34% of Caucasians and 29.2% of Asians. Our aim was to genotype NMIBC patients who had received BCG immunotherapy to determine if their response correlates with their GSTT2B status and to evaluate the role of GSTT2 in immune activation. Methods: The GSTT2B deletion and the promoter region of GSTT2 were analyzed by PCR in patients (n=201) and controls (n=150). The impact of BCG instillation on recurrence was analyzed. GSTT2 overexpression was performed in U937 macrophages (GSTT2B homozygous deleted) and bacterial survival determined. GSTT2 knockout mice were generated. Bone marrow-derived dendritic cells (DC) from GSTT2 knockout and wild-type mice were exposed to BCG, and immune activation in terms of cytokine production was examined. Data were analyzed using SPSS 23.0 and p&amp;lt;0.05 was taken to be significant. The institutional animal care and use committee approved animal work and the institutional review board approved patient sample analysis (NHG DSRB: 2012/00475). Results: Overexpressing GSTT2 in U937 cells decreased BCG survival at 2h (p&amp;lt;0.05). SNPs in the GSTT2 promoter were not associated with response to BCG except for the GSTT2B deletion. Patients were segregated by the number of BCG instillations they received, namely 9 or more BCG instillations (n=135) or 8 or fewer BCG instillations (n=39). Patients with the GSTT2B homozygous deletion who received 8 or fewer BCG instillations (74% received 6 instillations), were 90% recurrence free (p&amp;lt;0.05). For those who received 9 or more BCG instillations, the recurrence incidence was 35.5% and recurrence occurred within 2 years. Patients with GSTT2B full-length genotype did better with more BCG instillations. DC from GSTT2 knockout mice produced a 3-fold increase in IL6 production but similar TNFα and IL10 levels as wild-type DC in response to BCG. Conclusions: GSTT2B deletion was associated with lower likelihood of recurrence for patients who received 8 or fewer BCG instillations, while for GSTT2B full-length patients, those who received 9 or more BCG instillations had reduced recurrence compared to those who received fewer instillations. GSTT2 expression modulates immune activation by modulating BCG survival. Hence, GSTT2B absence may be a marker for patients who might do well with less BCG therapy. Citation Format: Juwita N. Rahmat, Kah Wei Tan, Ting Li Ong, Sin Mun Tham, Chong Wen Tsung, Yiong Huak Chan, Lata Raman, Edmund Chiong, Kesavan Esuvaranathan, Ratha Mahendran. A marker to optimize BCG dosing schedules for immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A07.

CXCR4/MIF axis amplifies tumor growth and epithelial-mesenchymal interaction in non-small cell lung cancer

Overexpression of C-X-C chemokine receptor type 4 (CXCR4) has been shown in several cancers, including non-small cell lung cancer (NSCLC) and is linked to early metastasis and worse prognosis. The crosstalk between cancer cells and tumor stroma promotes the growth and metastasis and CXCR4 signaling is a key element of this crosstalk.To test the effects of CXCR4 overexpression (CXCR4-OE), we transduced the human NSCLC cell line A549 by using a lentiviral vector. A 3D cell culture model showed generations of tumorspheres and the effects derived by the co-culturing of lung fibroblasts. Using a xenograft mouse model, we also studied the effects of CXCR4-OE in pulmonary cell engraftment and tumor burden in vivo.Our data indicate that CXCR4-OE leads to increased tumorsphere formation and epithelial-mesenchymal transition (EMT). CXCR4-OE by A549 cells resulted in a significant increase in the production of the CXCR4-ligand macrophage migration inhibitory factor (MIF) compared to those transduced with an empty vector (EV) or in which the CXCR4 expression was deleted (KO). In our in vitro system, we did not detect any production of the canonical CXCR4 ligand CXCL12. Autocrine MIF production and CXCR4 signaling are part of a self-perpetuating loop that amplifies tumor growth and EMT. Co-culture with lung fibroblasts further increased tumorsphere formation, partially driven by an increase in IL-6 production. When A549 cells were injected into murine lungs, we observed more abundant and significantly larger tumor lesions in recipients of CXCR4-OE A549 cells compared to those receiving EV or KO cells, consistent with our in vitro findings. Treatment of mice with the MIF antagonist ISO-1 resulted in significantly less tumor burden.In conclusion, our data highlight the role of the CXCR4-OE/MIF/IL-6 axis in epithelial mesenchymal crosstalk and NSCLC progression.

Isolation, culture, and downstream characterization of primary microglia and astrocytes from adult rodent brain and spinal cord

BackgroundNeuroimmunologists aspire to understand the interactions between neurons, microglia, and astrocytes in the CNS. To study these cells, researchers work with either immortalized cell lines or primary cells acquired from animal tissue. Primary cells reflect in vivo characteristics and functionality compared to immortalized cells; however, they are challenging to acquire and maintain. New MethodEstablished protocols to harvest primary glia use neonatal rodents, here we provide a method for simultaneously isolating microglia and astrocytes from brain and/or spinal cord from adult rodents. We utilized a discontinuous percoll density gradient enabling easy discrimination of these cell populations without enzymatic digestion or complex sorting techniques. ResultsWe found cells isolated from the percoll interface between 70 %−50 % were microglia, as they express ionizing calcium-binding adaptor molecule 1 (Iba1) in immunocytochemistry and CD11bhi and CD45lo using flow cytometry. Isolated cells from the 50 %−30 % interface were astrocytes as they express glial fibrillary acidic protein (GFAP) in immunocytochemistry and Glutamate aspartate transporter (GLAST)-1 using flow cytometry. Cultured microglia and astrocytes showed a functional increase in IL-6 production after treatment of lipopolysaccharide (LPS). Comparison with Existing MethodsOur method allows for rapid isolation of both microglia and astrocytes in one protocol with relatively few resources, preserves cellular phenotype, and yields high cell numbers without magnetic or antibody sorting. ConclusionHere we show a novel, single protocol to isolate microglia and astrocytes from brain and spinal cord tissue, allowing for culturing and other downstream applications from the cells of animals of various ages, which will be useful for researchers investigating these two major glial cell types from the brain or spinal cord of the same rodent.

In vitro immunomodulatory effect of Bifidobacterium bifidum and Lactobacillus reuteri cell free extracts

Recent studies have shown that alterations of the immune response in the gastrointestinal mucosa are key components of the mechanism of the probiotic action of beneficial bacteria. Most of the beneficial effects of probiotics are due to the action of their structural components and metabolites. Macrophages are first-line defense cells of the immune system, which not only participate in the detection, phagocytosis and destruction of harmful microorganisms, but also determine the nature of the subsequent immune response by presenting antigens to T-cells and initiating inflammation by releasing cytokines. We researched the effect of two types of cell-free extracts (CFEs) containing probiotic derivatives (structural components and metabolites of bacteria) Bifidobacterium bifidum 1 (BbCFE) and Lactobacillus reuteri DSM 17938 (LrCFE) on the activity of mouse peritoneal macrophages and on the ability of peripheral human blood mononuclear cells to produce cytokines. CFEs were obtained by culturing probiotics in their own disintegrates and then removing cells and cell debris by centrifugation and filtration. Peritoneal macrophages were isolated from mice. Some of them were infected in vitro by Salmonella thyphimurium. Uninfected and infected macrophages were incubated in culture medium containing (30% vol) or not containing CFEs at 37 °С in a microaerobic atmosphere (5% СО2) for 18 hours. After incubation, peritoneal macrophages were lysed. The obtained suspensions were centrifuged and supernatants were carefully collected. Macrophages activity was assessed by the nitrites level, superoxide dismutase (SOD), lactate dehydrogenase (LDH) activity and antiinflammatory cytokines levels in supernatants using spectrophotometric method. Peripheral mononuclear cells were isolated from the blood of healthy volunteers. The ability of peripheral mononuclear blood cells to produce antiinflammatory cytokines was evaluated after cell stimulation with lipopolysaccharide (LPS) and incubation with or without CFEs. Cytokine levels in supernatants were determined using enzyme-linked immunosorbent assay (ELISA). After infection with S. thyphimurium in macrophages, nitrite levels increased 5.5-fold, SOD activity 4.8-fold, and LDH 2-fold. Both studied CFEs exerted a similar effect on the macrophages’ activity. Addition of BbCFE to the incubation medium of infected macrophages resulted in a 4-fold decrease in nitrite levels, and the addition of LrCFE was accompanied by a decrease in nitrite levels to levels in intact cells. Under the influence of both CFEs, the activity of SOD and LDH was significantly reduced and did not differ significantly from the activity of these enzymes in intact cells. BbCFE and LrCFE did not have a significant effect on nitrite levels, SOD and LDH activity in intact macrophages. Under the influence of BbCFE, there was a 2-fold decrease in the production of TNF, a 2-fold increase in IL10 production, and a 30% increase in IL6 production by mononuclear cells. LrCFE caused a decrease in TNF production by 26.7% and IL6 by 36%, and IL10 by 1.9 times. Thus, the studied CFEs normalized the nitrite levels in peritoneal macrophages infected with S. thyphymurium and infection-induced activation of SOD and LDH enzymes. This demonstrates their ability to modulate oxidative processes in macrophages. In addition, under the influence of the investigated CFEs, there was a decrease in the production of pro-inflammatory cytokines (TNFα and IL-6) and increased production of anti-inflammatory cytokine (IL-10) by human peripheral mononuclear cells. The results of the study indicate the ability of CFEs by influencing the functions of innate immunity cells to restrict the inflammatory response and oxidative stress. Based on this, CFEs can be considered as promising agents for the treatment of inflammatory diseases.

Nexus Between Immune Responses and Oxidative Stress: The Role of Dietary Hydrolyzed Lignin in ex vivo Bovine Peripheral Blood Mononuclear Cell Response.

The control of immune responses is particularly critical when an increase of oxidative stress occurs, causing an impairment of immune cell response and a condition of systemic inflammation, named oxinflammation. Nutritional strategies based on the use in the diet of phytochemicals extracted from plants, rich in antioxidants, could help restore the antioxidant/oxidant balance and obtain a modulation of immune response. Lignin represents a valuable resource of phenolic compounds, which are characterized by a corroborated antioxidant effect. To date, there are no studies reporting the effects of lignin in the diet on immune responses and oxidative stress in ruminants. The objective of the present experiment was the evaluation of the dietary inclusion of Pinus taeda hydrolyzed lignin on the ex vivo immune responses and oxidative stress biomarkers by peripheral blood mononuclear cells (PBMCs) isolated from beef steers. In order to test the effect during oxidative stress exposition, cells were treated with hydrogen peroxide (H2O2). The proliferation test and the viability assay were carried out on cells, whereas, on supernatants, the cytokine profile and the oxidative stress biomarkers were evaluated. The dietary inclusion with P. taeda hydrolyzed lignin resulted in cytoprotection after H2O2 exposition, increasing the number of viable monocytes and decreasing the reactive oxygen/nitrogen species production in supernatants. The cytokine profile indicated the modulatory role of hydrolyzed lignin on immune response, with a concomitant decrease of TNF-α and increase of IL-8 production, which are strictly connected with monocyte activation and antioxidant response pathway. These results demonstrated that hydrolyzed lignin may provide a modulation of oxidative stress and inflammatory response in PBMCs; thus, the P. taeda hydrolyzed lignin could be suggested as an innovative phytochemical in ruminant feed.

The Immunomodulatory Properties of the Human Amnion-Derived Mesenchymal Stromal/Stem Cells Are Induced by INF-γ Produced by Activated Lymphomonocytes and Are Mediated by Cell-To-Cell Contact and Soluble Factors.

Human mesenchymal stromal/stem cells (MSCs), being immunoprivileged and having immunomodulatory ability, represent a promising tool to be applied in the field of regenerative medicine. Based on numerous in vitro evidences, the immunological effects of MSCs on immune cells could depend on different mechanisms as cell-to-cell contact and paracrine signals. Furthermore, recent studies have shown that the immunomodulatory activity of MSCs is initiated by activated immune cells; thus, their interaction represents a potential homeostatic mechanism by which MSCs regulate the immune response. MSCs also release exosomes able to give different effects, in a paracrine manner, by influencing inflammatory processes. In this study, we aimed to establish the potential role of human amnion-derived MSCs (hAMSCs), in immunomodulation. We found that the immunosuppressive properties of hAMSCs are not constitutive, but require “supportive signals” capable of promoting these properties. Indeed, we observed that hAMSCs alone are not able to produce an adequate amount of soluble immunomodulatory factors. Here, we studied, in depth, the strong immunomodulatory licensing signal deriving from the direct interaction between hAMSCs and stimulated peripheral blood mononuclear cells. We found that the immunomodulatory effect of hAMSCs also depends on cell-to-cell contact through the contribution of the PDL-1/PD-1 axis. We then investigated the IFN-γ priming of hAMSCs (γ-hAMSCs), which induce the increase of PDL-1 expression, high production of IDO, and upregulation of different immunomodulatory exosome-derived miRNAs. Our miRNA–target network analysis revealed that nine of the deregulated miRNAs are involved in the regulation of key proteins that control both T cell activation/anergy and monocyte differentiation pathways. Finally, we observed that γ-hAMSCs induce in monocytes both M2-like phenotype and the increase of IL-10 production. The extensive implications of MSCs in modulating different aspects of the immune system make these cells attractive candidates to be employed in therapeutic application in immune-based diseases. For these reasons, we aimed, with this study, to shed light on the potential of hAMSCs, and how they could become a useful tool for treating different inflammatory diseases, including end-stage pathologies or adverse effects in transplanted patients.

DOP70 High-dimensional analysis reveals a novel signature for IL-17 producing CD8 T cells in inflammatory bowel disease

Abstract Background The pathogenesis of IBD has previously mainly been associated with a dysregulation of CD4 T-cell responses, and active disease is linked to induction of pathogenic IL-17 producing T helper cells. Importantly, CD8 T cells can also produce IL-17 and demonstrate a significant increase in active IBD. However, it remains unclear whether this is due to conventional or unconventional T-cell responses and whether these CD8 responses could play a role as novel biomarkers or therapeutic targets of active IBD. Methods We isolated lymphocytes from the peripheral blood and intestinal tissue of IBD patients (n = 56) and performed a detailed analysis of CD8 T-cell phenotype and function using high-parametric flow cytometry and mass cytometry in combination with algorithm-aided bioinformatic analysis. Results We observe a significant increase in IL-17 production by CD8 T cells in active IBD, primarily produced by conventional CD8 T cells. Unconventional T-cell subsets (e.g. MAIT cells, γδ T cells and NKT cells) represented only ~30% (peripheral blood) or ~25% (intestinal tissue) of IL-17 producing CD8 T cells (Tc17). The mass cytometric analysis identified Tc17 cells as a distinct cell population within the intestinal CD8 T-cell compartment that can be further subdivided into 3 subsets which share expression of phenotypic markers such as CD6, CD39, CD69 and PD1 and a low expression of CD27. This novel signature was validated in a separate cohort of IBD patients. Moreover, at initial IBD diagnosis, the IL-17 signature is associated with flare-free survival in a retrospective cohort analysis based on published transcriptome data. Conclusion Our data indicate that conventional IL-17 producing CD8 T cells are a very distinct cell population that is linked to IBD activity. The identification of a novel IL-17 CD8 signature may help guide treatment decisions as a biomarker and for immunotherapeutic approaches.

Cytokine signaling in the modulation of post-acute and chronic systemic inflammation: a review of the influence of exercise and certain drugs

Acute inflammation in response to stimuli such as infection can be of deleterious amplitude and/or duration in some individuals and often tends towards chronicity in older adults. This inflammatory pattern appears to be causally linked to higher all-cause mortality and other adverse outcomes such as frailty, sarcopenia, mood disorders and impaired cognitive function. Patients in this clinical state have a persistent pro-inflammatory cytokine profile. Exercise has been shown to shift baseline levels of tumor necrosis factor (TNF), interleukin-1 (IL-1) and other cytokines to a less inflamed setting, with interleukin-6 (IL-6) playing a key modulating role. Drugs can also modulate innate immune cells and their biochemical networks with a shift to a surveillance pattern. Theophylline and chloroquine are examples of drugs that could have clinical value as immune modulators. For example, theophylline induces a 20 percent fall in TNF and around 200 percent increase in IL-10 production by blood-harvested mononuclear cells, and a fall of about 50 percent in interferon-gamma (IF-γ) release. Pharmacological activity in that domain could be exploited in clinical practice, with the aim of establishing a less pro-inflammatory innate immune milieu after provocations such as infection, trauma or major surgery.

Hepatic syndromes and cytokine indices in patients with hepatic cirrhosis

Aim. To analyze the clinical manifestations of hepatic cirrhosis (HC) of different etiology and their correlation with proinflammatory cytokine level interleukin-6 and tumor necrosis factor-alpha (TNF-).&#x0D; Materials and methods. Fifty patients with viral hepatic cirrhosis (as a result of chronic hepatitis C), 20 patients with alcoholic HC, 16 persons with mixed etiology HC (viral and alcoholic) with the mean age 53 13.01 years were examined. Hepatic cirrhosis was diagnosed using complex data of clinicolaboratory and instrumental studies. The levels of IL-6 and TNF- were determined with the kits of VectorBest company (Russia, Novosibirsk) using the method of enzyme immunoassay and registration of results on photometer Stat-Fax 2100 (USA).&#x0D; Results. No reliable differences in clinical manifestations and cytokine level in HC of different etiology were detected. The levels of cytokine concentration median significantly elevated as HC progressed. Interleukin-6 demonstrated moderate correlations with lethargy and hepatocellular insufficiency as a whole (Ki = 0.673 and Ki = 0.607, respectively).&#x0D; Conclusions. Clinical syndromes of HC practically do not depend on etiology of disease. Increase in production of IL-6 and TNF-a demonstrates pathogenetic significance of proinflammatory cytokines in the lesion of the liver. IL-6 level correlates with the severity of liver lesion and, thus, objectifies clinical symptoms in HC patients, especially those based on patients complaints.

FOXO3 Transcription Factor Regulates IL-10 Expression in Mycobacteria-Infected Macrophages, Tuning Their Polarization and the Subsequent Adaptive Immune Response.

Alveolar Macrophages play a key role in the development of a robust adaptive immune response against the agent of Tuberculosis (TB), Mycobacterium tuberculosis (M.tb). However, macrophage response is often hampered by the production of IL-10, a potent suppressor of the host immune response. The secretion of IL-10 correlates with TB pathogenesis and persistence in host tissues. Concordantly, inhibition of IL-10 signaling, during BCG vaccination, confers higher protection against M.tb through a sustained Th1 and Th17 responses. Therefore, uncovering host effectors, underlying mycobacteria-induced expression of IL-10, may be beneficial toward the development of IL-10-blocking tools to be used either as adjuvants in preventive vaccination or as adjunct during standard treatment of TB. Here, we investigated the role of FOXO3 transcription factor in mycobacteria-induced secretion of IL-10. We observed that PI3K/Akt/FOXO3 axis regulates IL-10 expression in human macrophages. Knocking down of FOXO3 expression resulted in an increase of IL-10 production in BCG-infected macrophages. The gene reporter assay further confirmed the transcriptional regulation of IL-10 by FOXO3. In silico analysis identified four Forkhead binding motifs on the human IL-10 promoter, from which the typical FOXO3 one at position −203 was the major target as assessed by mutagenesis and CHIP binding assays. Further, we also observed a decrease in gene expression levels of the M1 typical markers (i.e., CD80 and CD86) in SiFOXO3-transfected macrophages while activation of FOXO3 led to the increase in the expression of CD86, MHCI, and MHCII. Finally, co-culture of human lymphocytes with siFOXO3-transfected macrophages, loaded with mycobacterial antigens, showed decreased expression of Th1/Th17 specific markers and a simultaneous increase in expression of IL-4 and IL-10. Taken together, we report for the first time that FOXO3 modulates IL-10 secretion in mycobacteria-infected macrophage, driving their polarization and the subsequent adaptive immune response. This work proposes FOXO3 as a potential target for the development of host-directed strategies for better treatment or prevention of TB.

Mechanistic Insights into the Inhibition of T Regulatory Cells By Dasatinib May Predict Immunostimulatory Effects in CML Patients

Background Dasatinib potently inhibits the Src family kinase Lck at therapeutic concentrations. Lck plays a critical role in signalling from the T cell receptor with immediate downstream targets including ZAP70 and LAT. The cell surface scaffolding protein LAT has been previously shown to play a central role in normal Treg development. STAT5, the downstream target of IL-2, also plays a critical role in Treg differentiation and maintenance of FOXP3 expression through its binding of the promoter region of the FOXP3 gene. The proportion of Tregs has been shown to inversely correlate with improved molecular response in patients on dasatinib and a subset of patients taking dasatinib will develop a large granular lymphocytosis (LGL) which is associated with inflammatory toxicity and improved outcome (Mustjoki et al. Leukaemia 2009). We hypothesised that a reduction in Treg frequency and function would correlate with the expansion of a clonal LGL population in certain patients taking dasatinib. To investigate this, we performed ex-vivo analysis of the phosphorylation of key cell signalling proteins and intracellular cytokine production in Tregs and T effectors from CML patients and healthy controls. Methods We first performed phosphoflow cytometry in Tregs and T effectors to assess the effect of dasatinib on signalling downstream from the TCR, including activated/phosphorylated ZAP70 (pZAP70), LAT (pLAT) and STAT5 (pSTAT5). 11-colour flow cytometry was performed after cells were activated with H2O2 for 15 minutes, due to its activity as a potent phosphatase inhibitor. A gating strategy of CD3+/CD4+/CD25+/FOXP3+/CD127lo cells was used for identification of Tregs. We then performed 10-colour intracellular flow cytometry assessing the impact of dasatinib on cellular cytokine production including TNF, IFN gamma, IL-2, IL-4 and IL-10 after stimulation with OKT3. Results 8 patients with CML and 2 healthy controls were recruited. Of the CML patients 5 were taking dasatinib, 100mg daily, two were taking imatinib and one nilotinib. Patients on dasatinib had lower proportion of Tregs compared with the non dasatinib group with a mean proportion of CD3+ cells of 1.4 vs 2.1. Patients on dasatinib had significantly reduced phosphorylation of ZAP70 compared with the non dasatinib group in CD3+ cells, CD4+ cells and Tregs, following H2O2 stimulation. The mean increase in median fluorescence intensity (MFI) of pZAP70 was 1.7 vs 3.6 in CD3+ cells, 1.4 vs 3.5 in CD4+ cells and 1.5 vs 2.8 in Tregs (p=0.006, p=0.004, p=0.045). Similarly, pLAT showed a lower increase in phosphorylation in the dasatinib group in all T cell subsets evaluated, with mean increase in MFI of 4.3 vs 11.9 in CD3+ cells, 4 vs 12.3 in CD4+ cells and 4.3 vs 8.2 in Tregs. STAT5 also showed reduced phosphorylation in the dasatinib group with a mean increase in MFI of 6.4 vs 21.2 in CD3+ cells, 5.7 vs 20.3 in CD4+ cells and 5.9 vs 19.7 in Tregs (p=0.007, p=0.003, p=0.01). Two patients on dasatinib had reversal of normal CD4:CD8 ratio, with one of these also having absolute lymphocytosis, in keeping with likely development of clonal LGL populations. These patients had lower increase in MFI of pZAP70, pLAT and pSTAT5 following stimulation within isolated Tregs compared to patients on dasatinib with normal CD4:CD8 (Figure 1A). Within CD4+ cells the mean proportional increase in IL-2 production was lower in the dasatinib group at 0.4 vs 5 (p=0.003). Using an additional panel, a single patient was also evaluated and shown to have strikingly reduced production of TNF within isolated Tregs and Il-2 within CD4+cells compared to control (Figure 1B). Conclusion Dasatinib inhibits phosphorylation of ZAP70, LAT and STAT5 within T cell subsets including Tregs, with the strongest effect seen against STAT5. In addition, dasatinib causes a reduction in pro-inflammatory cytokine production within CD4+ cells, with the most significant inhibitory effect seen against IL-2. Tregs have abundant expression of the IL-2 receptor on the cell surface and binding leads to STAT5 signalling resulting in transcription of FOXP3. A more pronounced inhibition of phosphorylation of ZAP70, LAT and STAT5 is seen in two patients on dasatinib with reversal of CD4:CD8 when compared with other patients on dasatinib, suggestive of a more potent reduction of Treg function. Reduction in number and function of Tregs might explain the immunostimulatory effects seen in patients who develop a monoclonal LGL population. Disclosures Harrington: Bristol-Myers Squibb: Research Funding. Dillon:TEVA: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria. Rousselot:Incyte: Research Funding; Pfizer: Research Funding. Radia:Blueprint Medicines: Consultancy; Novartis: Consultancy, Speakers Bureau. Harrison:Promedior: Honoraria; Gilead: Speakers Bureau; Sierra Oncology: Honoraria; Roche: Honoraria; Celgene: Honoraria, Speakers Bureau; CTI: Speakers Bureau; Shire: Speakers Bureau; AOP: Honoraria; Janssen: Speakers Bureau; Incyte: Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Kordasti:Celgene: Research Funding; Novartis: Research Funding; Boston Biomed: Consultancy; API: Consultancy. De Lavallade:Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding.

Cytokine Profile and Endothelial Status in Patients with Coronary Artery Disease and Different Outcomes of Coronary Artery Bypass Grafting

Aim.To assess the cytokine profile and the level of endothelial dysfunction in patients with coronary artery disease (CAD) and different outcomes of coronary artery bypass grafting (CABG).Materials and methods.The study included 95 people, with 22 of them being practically healthy (without CAD). The CAD patients (73) were retrospectively divided into 2 subgroups according to the presence of complications within the first month after the CABG. The first subgroup consisted of 25 patients suffering from complications after CABG, with 48 people making up the second subgroup (no complications after CABG). CABG was performed on a beating heart; bypass grafts were attached during revascularisation. The serum levels of IL-4 and IFN-γ were determined through the enzyme-linked immunosorbent assay (ELISA) using appropriate test systems manufactured by BCM Diagnostics and a Zenyth 340 microplate photometer (Biochrom Ltd). In order to determine the content of endothelin-1 in blood serum, ELISA test systems manufactured by Biomedica were used. The statistical analysis of study results was performed employing the descriptive statistics module of Statistica 12.0 (StatSoft, USA). The mean (M), its error (m), median (Me) and the interquartile range [Q25; Q75] were calculated. In cases when p ≤ 0.05, the results were considered statistically significant.Results.It was revealed that, in the preoperative period, CAD patients from both subgroups exhibited increased levels of endothelin-1 (p = 0.0001) and IFN-γ (p = 0.0001), whereas an increase in IL-4 production was noted only in the first subgroup (p = 0.0001), as compared to the control group. The follow-up revealed that CAD patients with complications after CABG exhibited stable high levels of IL-4 (p &gt; 0.05) and IFN-γ (p &gt; 0.05) during the entire observation period, as compared to the initial values. The content of endothelin-1 in both subgroups increased significantly on the 4th day of observation (p &gt; 0.05) and returned to its initial high values after 1 month.Conclusion.Immune response in CAD patients suffering from complications after CABG develops mainly along the pro-inflammatory pathway and is accompanied by endothelial dysfunction.

Nitrated food proteins induce a regulatory immune response associated with allergy prevention after oral exposure in a Balb/c mouse food allergy model

BackgroundFood allergy is associated with a high personal health and economic burden. For immunomodulation toward tolerance, food compounds could be chemically modified, for example, by posttranslational protein nitration, which also occurs via diet‐derived nitrating agents in the gastrointestinal tract.ObjectiveWe sought to analyze the effect of pretreatment with nitrated food proteins on the immune response in a mouse food allergy model and on human monocyte‐derived dendritic cells (moDCs) and PBMCs.MethodsThe model allergen ovalbumin (OVA) was nitrated in different nitration degrees, and the secondary structures of proteins were determined by circular dichroism (CD). Allergy‐preventive treatment with OVA, nitrated OVA (nOVA), and maximally nitrated OVA (nOVAmax) were performed before mice were immunized with or without gastric acid‐suppression medication. Antibody levels, regulatory T‐cell (Treg) numbers, and cytokine levels were evaluated. Human moDCs or PBMCs were incubated with proteins and evaluated for expression of surface markers, cytokine production, and proliferation of Th2 as well as Tregs.ResultsIn contrast to OVA and nOVA, the conformation of nOVAmax was substantially changed. nOVAmax pretreated mice had decreased IgE as well as IgG1 and IgG2a levels and Treg numbers were significantly elevated, while cytokine levels remained at baseline level. nOVAmax induced a regulatory DC phenotype evidenced by a decrease of the activation marker CD86 and an increase in IL‐10 production and was associated with a higher proliferation of memory Tregs.ConclusionOral pretreatment with highly nitrated proteins induces a tolerogenic immune response in the food allergy model and in human immune cells.