Regulation Of IL-6 Expression Research Articles

17\u03b2-estradiol upregulates IL6 expression through the ER\u03b2 pathway to promote lung adenocarcinoma progression

BackgroundIn non-small cell lung cancer (NSCLC), estrogen (E2) significantly promotes NSCLC cell growth via estrogen receptor beta (ERβ). Discovery and elucidation of the mechanism underlying estrogen-promoted NSCLC progression is critical for effective preventive interventions. IL6 has been demonstrated to be involved in the development, progression and metastasis in several cancers and IL6 overexpression is associated with poor prognosis in NSCLC. However, the exact role played by IL6 in estrogen-promoted NSCLC progress remain unknown. Here, we evaluated the expression and biological effects of IL6 in NSCLC cells when treated with E2 and explored the underlying mechanism of IL6 in E2-promoted NSCLC progression.MethodsExpression of ERβ/IL6 in 289 lung cancer samples was assessed by immunohistochemistry. Matched samples of metastatic lymph node and primary tumor tissues were used to quantify the expression of ERβ/IL6 by western blot. Expression levels of IL6 in NSCLC cells were quantified by western blotting, ELISA, and immunofluorescence staining. The effects of IL6 stimulated by E2 on cell malignancy were evaluated using CCK8, colony formation, wound healing and transwell. Furthermore, overexpression and knockdown ERβ constructs were constructed to measure the expression of IL6. The effects of IL6 stimulated by E2 on tumor growth were evaluated using a urethane-induced adenocarcinoma model. In addition, a xenograft mouse model was used to observe differences in ERβ subtype tumor growth with respect to IL6 expression.ResultsIL6/ERβ expression were significantly increased in lung cancer. Higher IL6/ERβ expression was associated with decreased differentiation or increased metastasis. IL6 was an independent prognostic factor for overall survival (OS), higher IL6 expression was associated with decreased OS. Furthermore, ERβ regulates IL6 expression via MAPK/ERK and PI3K/AKT pathways when stimulated by E2 and promotes cell malignancy in vitro and induced tumor growth in vivo. Finally we confirm that ERβ isolation 1/5 is essential for E2 promotion of IL6 expression, while ERβ2 not.ConclusionsOur findings demonstrate that E2 stimulates IL6 expression to promote lung adenocarcinoma progression through the ERβ pathway. We also clarify the difference in each ERβ subtype for E2 promoting IL6 expression, suggesting that ERβ/IL6 might be potential targets for prognostic assessment and therapeutic intervention in lung cancer.

Trim33 mediates the proinflammatory function of Th17 cells.

Transforming growth factor-β (TGF-β) regulates reciprocal regulatory T cell (T reg) and T helper 17 (Th17) differentiation, the underlying mechanism of which is still not understood. Here, we report that tripartite motif-containing 33 (Trim33), a modulator of TGF-β signaling that associates with Smad2, regulates the proinflammatory function of Th17 cells. Trim33 deficiency in T cells ameliorated an autoimmune disease in vivo. Trim33 was required for induction in vitro of Th17, but not T reg cells. Moreover, Smad4 and Trim33 play contrasting roles in the regulation of IL-10 expression; loss of Trim33 enhanced IL-10 production. Furthermore, Trim33 was recruited to the Il17a and Il10 gene loci, dependent on Smad2, and mediated their chromatin remodeling during Th17 differentiation. Trim33 thus promotes the proinflammatory function of Th17 cells by inducing IL-17 and suppressing IL-10 expression.

Group A Streptococcus M1T1 Intracellular Infection of Primary Tonsil Epithelial Cells Dampens Levels of Secreted IL-8 Through the Action of SpyCEP.

Streptococcus pyogenes (Group A Streptococcus; GAS) commonly causes pharyngitis in children and adults, with severe invasive disease and immune sequelae being an infrequent consequence. The ability of GAS to invade the host and establish infection likely involves subversion of host immune defenses. However, the signaling pathways and innate immune responses of epithelial cells to GAS are not well-understood. In this study, we utilized RNAseq to characterize the inflammatory responses of primary human tonsil epithelial (TEpi) cells to infection with the laboratory-adapted M6 strain JRS4 and the M1T1 clinical isolate 5448. Both strains induced the expression of genes encoding a wide range of inflammatory mediators, including IL-8. Pathway analysis revealed differentially expressed genes between mock and JRS4- or 5448-infected TEpi cells were enriched in transcription factor networks that regulate IL-8 expression, such as AP-1, ATF-2, and NFAT. While JRS4 infection resulted in high levels of secreted IL-8, 5448 infection did not, suggesting that 5448 may post-transcriptionally dampen IL-8 production. Infection with 5448ΔcepA, an isogenic mutant lacking the IL-8 protease SpyCEP, resulted in IL-8 secretion levels comparable to JRS4 infection. Complementation of 5448ΔcepA and JRS4 with a plasmid encoding 5448-derived SpyCEP significantly reduced IL-8 secretion by TEpi cells. Our results suggest that intracellular infection with the pathogenic GAS M1T1 clone induces a strong pro-inflammatory response in primary tonsil epithelial cells, but modulates this host response by selectively degrading the neutrophil-recruiting chemokine IL-8 to benefit infection.

Bhlhe40 is an essential repressor of IL-10 during Mycobacterium tuberculosis infection.

The cytokine IL-10 antagonizes pathways that control Mycobacterium tuberculosis (Mtb) infection. Nevertheless, the impact of IL-10 during Mtb infection has been difficult to decipher because loss-of-function studies in animal models have yielded only mild phenotypes. We have discovered that the transcription factor basic helix-loop-helix family member e40 (Bhlhe40) is required to repress Il10 expression during Mtb infection. Loss of Bhlhe40 in mice results in higher Il10 expression, higher bacterial burden, and early susceptibility similar to that observed in mice lacking IFN-γ. Deletion of Il10 in Bhlhe40-/- mice reverses these phenotypes. Bhlhe40 deletion in T cells or CD11c+ cells is sufficient to cause susceptibility to Mtb Bhlhe40 represents the first transcription factor found to be essential during Mtb infection to specifically regulate Il10 expression, revealing the importance of strict control of IL-10 production by innate and adaptive immune cells during infection. Our findings uncover a previously elusive but significant role for IL-10 in Mtb pathogenesis.

Sympathetic neural control of inflammation by ADRB2-mediated IL-10 secretion

Abstract The nervous and immune systems reciprocally regulate their functions through the release of chemical messengers. Norepinephrine (NE), a neurotransmitter released by catecholaminergic nerve endings, allows the sympathetic nervous system to communicate with immune cells through adrenergic receptors (ADR). We identified a novel role for the β2-ADR (ADRB2) in controlling inflammation. NE suppresses pro-inflammatory cytokine secretion from primary macrophages in response to multiple TLR agonists, and ADRB2 signaling enhances early induction of IL-10. We assessed the in vivo role of this pathway using a LPS endotoxemia model. ADRB2 −/− animals rapidly succumbed to a sub-lethal LPS challenge, which correlated with elevated serum levels of TNFα and reduced IL-10. The lethality in the ADRB2 −/− animals was rescued by administering exogenous IL-10. Additionally, the ADRB2-specific agonist salmeterol rescued wild-type animals from a lethal LPS challenge, which was reversed by neutralizing anti-IL-10 antibody. Additionally, ADRB2 −/− mice are unable to control inflammation models of infection and colitis. These observations suggest that ADRB2 signaling is critical for controlling inflammation through the rapid induction of IL-10. Through transcriptome analysis, the NR4A nuclear orphan family members were induced by NE, and the presence of several putative NR4A binding sites within the IL-10 promoter suggests that these factors may directly regulate IL-10 expression in response to ADRB2 signaling. Further studies will test the control of IL-10 expression by the NR4A family of transcription factors. Understanding this pathway will provide new insights into how the nervous and immune systems communicate through ADRB2 signaling.

Rs13293512 polymorphism located in the promoter region of let-7 is associated with increased risk of radiation enteritis in colorectal cancer.

RE (Radiation enteritis) has been characterized by the inflammation reaction, and in this study, we aim to explore inflammatory cytokines and underlying mechanism involved in the pathogenesis of RE. Luciferase assay was performed to explore whether polymorphism affected the expression of let-7, and also validated let-7 directly regulated f IL-6 expression. Then Elisa was performed to study the mechanism of rs13293512 polymorphism associated with enteritis occurrence. And Western-blot and real-time PCR were performed to verify the relationship between let-7 and IL-6. 380 colorectal cancer patients were recruited, and all participants were genotyped. We found that occurrence probability of enteritis patients carried CC genotype (32%) was much higher than that in TT and TC groups (15%). In addition, we showed that the presence of the minor (C) allele of the polymorphism in the promoter region of let-7 substantially reduced the transcription activity of let-7, furthermore, we validated that let-7 directly regulated IL-6 expression by using luciferase reporter system. Moreover, IL-6 was highly expressed in peripheral blood and colonic mucosa samples genotyped as CC compared to those in TT and TC groups, furthermore, IL-6 was highly expressed in peripheral blood and colonic mucosa samples from participants with enteritis than without enteritis, whereas let-7 was highly expressed in peripheral blood and colonic mucosa samples genotyped as TT and TC compared to those in CC groups. Let-7 polymorphism (rs13293512) was associated with risk of RE in the colorectal cancer patients who received radiotherapy.

Hypoxia-inducible factor-1\u03b1 is a critical transcription factor for IL-10-producing B cells in autoimmune disease

Hypoxia-inducible factors (HIFs) are key elements for controlling immune cell metabolism and functions. While HIFs are known to be involved in T cells and macrophages activation, their functions in B lymphocytes are poorly defined. Here, we show that hypoxia-inducible factor-1α (HIF-1α) contributes to IL-10 production by B cells. HIF-1α regulates IL-10 expression, and HIF-1α-dependent glycolysis facilitates CD1dhiCD5+ B cells expansion. Mice with B cell-specific deletion of Hif1a have reduced number of IL-10-producing B cells, which result in exacerbated collagen-induced arthritis and experimental autoimmune encephalomyelitis. Wild-type CD1dhiCD5+ B cells, but not Hif1a-deficient CD1dhiCD5+ B cells, protect recipient mice from autoimmune disease, while the protective function of Hif1a-deficient CD1dhiCD5+ B cells is restored when their defective IL-10 expression is genetically corrected. Taken together, this study demonstrates the key function of the hypoxia-associated transcription factor HIF-1α in driving IL-10 expression in CD1dhiCD5+ B cells, and in controlling their protective activity in autoimmune disease.

MUC16 C terminal-induced secretion of tumor-derived IL-6 contributes to tumor-associated Treg enrichment in pancreatic cancer

Pancreatic cancer is the most lethal tumor. CA125 (gene symbol MUC16) is an important serum marker for pancreatic cancer diagnosis and treatment. High serum CA125 is related to metabolic tumor burden and poor prognosis. The circulating Treg subset is another independent prognostic factor for pancreatic cancer. Our unpublished data indicated that the circulating Treg proportion might be related to the serum CA125 level. However, the potential relationship and underlying mechanism of MUC16 and Treg in pancreatic cancer tissues remain unclear. In this study, we found that pancreatic cancer tissues were positive for both MUC16 C terminal (MUC16c) and Foxp3 expression and that their expression was correlated. MUC16c released into the cytoplasm via EGF induction significantly increased IL-6 expression and secretion. The PI3K/AKT pathway may participate in the regulation of IL-6 expression and secretion. By treating CD4+ T cells with IL-6 or co-culturing the cells with pancreatic cancer cells, tumor-derived IL-6 was identified to promote Foxp3 expression and Treg differentiation, which was significantly inhibited by the JAK2 inhibitor AG-490. In sum, our study demonstrated that the relationship between the MUC16c level and Foxp3 expression in the local tumor environment was consistent with that of the serum CA125 level and circulating Treg proportion in the systemic environment. MUC16c promoted Foxp3 expression and tumor-associated Treg enrichment in tumor tissues through tumor-secreted IL-6 activation of the JAK2/STAT3 pathway. These findings may provide deeper insight into potential pancreatic cancer therapy approaches.

Palmitate Conditions Macrophages for Enhanced Responses toward Inflammatory Stimuli via JNK Activation.

Obesity is associated with low-grade inflammation and elevated levels of circulating saturated fatty acids, which trigger inflammatory responses by engaging pattern recognition receptors in macrophages. Because tissue homeostasis is maintained through an adequate balance of pro- and anti-inflammatory macrophages, we assessed the transcriptional and functional profile of M-CSF-dependent monocyte-derived human macrophages exposed to concentrations of saturated fatty acids found in obese individuals. We report that palmitate (C16:0, 200 μM) significantly modulates the macrophage gene signature, lowers the expression of transcription factors that positively regulate IL-10 expression (MAFB, AhR), and promotes a proinflammatory state whose acquisition requires JNK activation. Unlike LPS, palmitate exposure does not activate STAT1, and its transcriptional effects can be distinguished from those triggered by LPS, as both agents oppositely regulate the expression of CCL19 and TRIB3 Besides, palmitate conditions macrophages for exacerbated proinflammatory responses (lower IL-10 and CCL2, higher TNF-α, IL-6, and IL-1β) toward pathogenic stimuli, a process also mediated by JNK activation. All of these effects of palmitate are fatty acid specific because oleate (C18:1, 200 μM) does not modify the macrophage transcriptional and functional profiles. Therefore, pathologic palmitate concentrations promote the acquisition of a specific polarization state in human macrophages and condition macrophages for enhanced responses toward inflammatory stimuli, with both effects being dependent on JNK activation. Our results provide further insight into the macrophage contribution to obesity-associated inflammation.

The extracellular role of DNA damage repair protein APE1 in regulation of IL-6 expression

The human apurinic/apyrimidinic endonuclease 1 (APE1) is a pleiotropic nuclear protein with roles in DNA base excision repair pathway as well as in regulation of transcription. Recently, the presence of extracellular plasma APE1 was reported in endotoxemic rats. However, the biological significance and the extracellular function of APE1 remain unclear. In this study, we found that monocytes secrete APE1 upon inflammatory challenges. Challenging the monocytic cells with extracellular APE1 resulted in the increased expression and secretion of the pro-inflammatory cytokine IL-6. Additionally, the extracellular APE1 treatment activated the transcription factor NF-κB, followed by its increased occupancy at the IL-6 promoter, resulting in the induction of IL-6 expression. APE1-induced IL-6 further served to elicit autocrine and paracrine cellular responses. Moreover, the extracellular IL-6 promoted the secretion of APE1, thus indicating a functional feedforward loop in this pathway. Furthermore, we show that APE1 is secreted through extracellular vesicles formation via endosomal sorting complex required for transport (ESCRT)-dependent pathway. Together, our study demonstrates a novel role of extracellular APE1 in IL-6-dependent cellular responses.

Association of IL-10-Regulating MicroRNAs in Peripheral Blood Mononuclear Cells with the Pathogenesis of Autoimmune Thyroid Disease

ABSTRACTInterleukin (IL)-10 is known to suppress inflammation in autoimmune diseases. IL-10 can be regulated by miRNAs. To elucidate the involvement of miRNAs that regulate IL-10 expression with the pathogenesis of autoimmune thyroid disease (AITD), we examined the expression levels of hsa-miR-27a-3p, hsa-miR-98-5p, hsa-miR-106a-5p, and hsa-miR-223-3p in peripheral blood mononuclear cells (PBMCs) from 43 patients with Graves’ disease (GD), 38 patients with Hashimoto’s disease (HD), and 21 healthy volunteers. We evaluated the association between the expression levels of four miRNAs and intracellular expression of IL-10 in PBMCs from 11 healthy volunteers. We also genotyped MIR27A rs895819 G/A and MIR106A rs3747440 C/G polymorphisms, which may be related to the expression of these miRNAs in 141 patients with GD, 178 patients with HD, and 84 healthy volunteers. The expression level of hsa-miR-106a-5p was significantly higher in patients with intractable GD than in those with GD in remission (p = 0.0113). The expression level of hsa-miR-223-3p was significantly lower in GD than in HD and lower in patients with intractable GD than in healthy volunteers (p = 0.0094, 0.0340). We found a negative correlation between the expression levels of hsa-miR-98-5p and the proportions of IL-10+ cells in stimulated PBMCs from healthy volunteers (p = 0.0092). The G allele of the MIR27A polymorphism was significantly more frequent in patients with mild HD than in healthy volunteers (p = 0.0432). In conclusion, the expression levels of hsa-miR-106a-5p and hsa-miR-223-3p were associated with the pathogenesis of AITDs. hsa-miR-98-5p may negatively regulate the expression of IL-10. The functional polymorphism of MIR27A was associated with HD severity.

Abstract TMEM-016: DENDRITIC CELL VACCINATION AND INHIBITION OF TUMOR–ASSOCIATED IMMUNE SUPPRESSION

Abstract PURPOSE: Th17 cell infiltration correlates with markedly prolonged overall survival among ovarian cancer patients, prompting the question of whether Th17 cells could be induced or expanded to therapeutic advantage. To address this question, a phase I clinical trial to test the safety and immunogenicity of Th17-inducing dendritic cell (DC) vaccination for stage IIIC–IV ovarian cancer patients (Clinicaltrials.gov NCT02111941) is nearing its accrual goal (n = 22). Interim analyses show that 8 of 9 patients completing the induction phase of five DC vaccinations mounted Th17 and antibody responses to FRα target antigen. These results are encouraging, but we found that ovarian tumor-associated CD14+ macrophages strongly suppress Th17-inducing DC-stimulated tumor antigen-specific Th1/Th17 effector CD4+ T cells responses. The purpose of this project was to elucidate the mechanisms by which ovarian tumor ascites CD14+ cells suppress DC-stimulated T cell responses, and to explore innovative strategies for alleviation of CD14+ cell-mediated immune suppression, or redirection of CD14+ cells from a suppressive phenotype to a pro-inflammatory phenotype. EXPERIMENTAL PLAN: Primary ovarian ascites CD14+ myeloid cells were tested for their ability suppress DC-stimulated tumor antigen-specific CD4+ T cell responses in contact or transwell coculture assays. Ascites fluid and ascites CD14+ cells were screened for expression of key chemokines and cytokines associated with T cell migration and immune suppression. Ascites CD14+ cells were also screened for PD-L1 expression, indoleamine 2,3-dioxygenase (IDO) activity, and key signaling pathways that determine the balance between M1 and M2 function. We also tested the ability of small molecule inhibitors to block key functions and alleviate immune suppression. SUMMARY OF RESULTS: Ovarian ascites CD14+ cells suppress DC-stimulated CD4+ T cell responses at least in part through expression of IL-10 and IDO. PD-L1 and PD-1 are consistently expressed by ascites CD14+ cells. Flow cytometric phosphoprotein assays with ascites CD14+ cells (n = 9) revealed consistent activation of AktS473, ERK1/2T202/Y204, CREBS133 and STAT3S727. Ovarian tumor-associated CD14+ macrophages consistently show high activity of a non-canonical STAT3 signaling pathway revealed by phosphorylation of S727 rather than the canonical Y705 phosphorylation seen in the classical JAK-STAT3 pathway. This innovative finding is consistent for every patient sample tested to date - canonical STAT3Y705 is absent. Inhibition of p38T180/Y182 MAPK signaling reduced expression of IL-10, RANTES, MIP-1β and IP-10. CCL22 (MDC) expression was also limited by p38 inhibition, but markedly elevated by blockade of JAK1/2 signaling. Inhibition of c-Kit and PI3K/Akt signaling showed a trend towards reduced K/T ratios, whereas inhibition of STAT3 reduced kynurenine production, suggesting that IDO activity is regulated at least in part by non-canonical STAT3S727 signaling. CONCLUSIONS: Multiple pathways regulate mechanisms of immune suppression by ovarian tumor ascites CD14+ myeloid cells. The p38 MAPK pathway regulates IL-10 expression, consistent with our published work on DC function. The non-canonical STAT3S727 pathway may also play a central role in regulating immune function by myeloid cells in the tumor microenvironment. These studies are of fundamental importance for development of adjuvant treatments to enhance the clinical efficacy of Th-17-inducing DC vaccination for ovarian cancer. Citation Format: Martin J Cannon, PhD. DENDRITIC CELL VACCINATION AND INHIBITION OF TUMOR–ASSOCIATED IMMUNE SUPPRESSION [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-016.

Beta-2 adrenergic receptor signaling prevents hyperinflammation through early release of IL-10

Abstract Innervation of the lymphoid tissues by catecholaminergic nerve endings allow the sympathetic nervous system to communicate with immune cells through the release of norepinephrine (NE). NE signals through adrenergic receptors, which are widely expressed by immune cells. We have identified a novel pathway where NE can suppress pro-inflammatory cytokine secretion from bone marrow-derived macrophages in response to multiple TLR agonists. This pathway operates through the beta2-adrenergic receptor (ADRB2), and the suppressive effect of NE depends on the early induction of the anti-inflammatory cytokine IL-10. We assessed the in vivo role of this pathway using a LPS endotoxemia model and found that ADRB2 knockout (KO) animals rapidly succumbed to a sub-lethal LPS challenge. This sensitivity is correlated with elevated serum levels of TNFα and suppressed IL-10 in ADRB2 KO animals. The lethality in the ADRB2 KO animals was rescued by administering exogenous IL-10. Additionally, the ADRB2-specific agonist salmeterol rescued wild-type animals from a lethal LPS challenge, which was reversed by neutralizing anti-IL-10 antibody. These observations suggest that ADRB2 signaling is critical for controlling inflammation through the rapid induction of IL-10. Through transcriptome analysis, the NR4A nuclear orphan family members were induced by NE, and the presence of several putative NR4A binding sites within the IL-10 promoter suggests that these factors may directly regulate IL-10 expression in response to ADRB2 signaling. Further studies will directly test the control of IL-10 expression by NR4A factors. Understanding this pathway will provide new insights into how the nervous and immune systems communicate through ADRB2 signaling.

TNF Blockade Maintains an IL-10+ Phenotype in Human Effector CD4+ and CD8+ T Cells.

CD4+ and CD8+ effector T cell subpopulations can display regulatory potential characterized by expression of the prototypically anti-inflammatory cytokine IL-10. However, the underlying cellular mechanisms that regulate expression of IL-10 in different T cell subpopulations are not yet fully elucidated. We recently showed that TNF inhibitors (TNFi) promote IL-10 expression in human CD4+ T cells, including IL-17+ CD4+ T cells. Here, we further characterized the regulation of IL-10 expression via blockade of TNF signaling or other cytokine/co-stimulatory pathways, in human T cell subpopulations. Addition of the TNFi drug adalimumab to anti-CD3-stimulated human CD4+ T cell/monocyte cocultures led to increased percentages of IL-10+ cells in pro-inflammatory IL-17+, IFNγ+, TNFα+, GM-CSF+, and IL-4+ CD4+ T cell subpopulations. Conversely, exogenous TNFα strongly decreased IL-10+ cell frequencies. TNF blockade also regulated IL-10 expression in CD4+ T cells upon antigenic stimulation. Using time course experiments in whole peripheral blood mononuclear cell (PBMC) cultures, we show that TNF blockade maintained, rather than increased, IL-10+ cell frequencies in both CD4+ and CD8+ T cells following in vitro stimulation in a dose- and time-dependent manner. Blockade of IL-17, IFNγ, IL-6R, or CD80/CD86-mediated co-stimulation did not significantly regulate IL-10 expression within CD4+ or CD8+ T cell subpopulations. We show that TNF blockade acts directly on effector CD4+ T cells, in the absence of monocytes or CD4+ CD25highCD127low regulatory T cells and independently of IL-27, resulting in higher IL-10+ frequencies after 3 days in culture. IL-10/IL-10R blockade reduced the frequency of IL-10-expressing cells both in the presence and absence of TNF blockade. Addition of recombinant IL-10 alone was insufficient to drive an increase in IL-10+ CD4+ T cell frequencies in 3-day CD4+ T cell/monocyte cocultures, but resulted in increased IL-10 expression at later time points in whole PBMC cultures. Together, these data provide additional insights into the regulation of IL-10 expression in human T cells by TNF blockade. The maintenance of an IL-10+ phenotype across a broad range of effector T cell subsets may represent an underappreciated mechanism of action underlying this widely used therapeutic strategy.

Hemoglobin induced cell trauma indirectly influences endothelial TLR9 activity resulting in pulmonary vascular smooth muscle cell activation.

It is now well established that both inherited and acquired forms of hemolytic disease can promote pulmonary vascular disease consequent of free hemoglobin (Hb) induced NO scavenging, elevations in reactive oxygen species and lipid peroxidation. It has recently been reported that oxidative stress can activate NFkB through a toll-like receptor 9 (TLR9) mediated pathway; further, TLR9 can be activated by either nuclear or mitochondrial DNA liberated by stress induced cellular trauma. We hypothesis that Hb induced lipid peroxidation and subsequent endothelial cell trauma is linked to TLR9 activation, resulting in IL-6 mediated pulmonary smooth muscle cell proliferation. We examined the effects of Hb on rat pulmonary artery endothelial and smooth muscle cells (rPAEC and rPASMC, respectively), and then utilized TLR9 and IL6 inhibitors, as well as the Hb and heme binding proteins (haptoglobin (Hp) and hemopexin (Hpx), respectively) to further elucidate the aforementioned mediators. Further, we explored the effects of Hb in vivo utilizing endothelial cell (EC) specific myeloid differentiation primary response gene-88 (MyD88) and TLR9 null mice. Our data show that oxidized Hb induces lipid peroxidation, cellular toxicity (5.5 ± 1.7 fold; p≤0.04), increased TLR9 activation (60%; p = 0.01), and up regulated IL6 expression (1.75±0.3 fold; p = 0.04) in rPAEC. Rat PASMC exhibited a more proliferative state (13 ± 1%; p = 0.01) when co-cultured with Hb activated rPAEC. These effects were attenuated with the sequestration of Hb or heme by Hp and Hpx as well as with TLR9 an IL-6 inhibition. Moreover, in both EC-MyD88 and TLR9 null mice Hb-infusion resulted in less lung IL-6 expression compared to WT cohorts. These results demonstrate that Hb-induced lipid peroxidation can initiate a modest TLR9 mediated inflammatory response, subsequently generating an activated SMC phenotype.

IL-1α Up-Regulates IL-6 Expression in Bovine Granulosa Cells via MAPKs and NF-κB Signaling Pathways

Background/Aims: IL-6 is one of the main cytokines in regulating ovarian follicular development and ovulation. However, the factors that regulate IL-6 expression in follicles are still unclear. The aim of this study was to elucidate the mechanisms underlying the effect of IL-1α on IL-6 expression in granulosa cells. Methods: IL-6 expression after IL-1α with/without inhibitors treatment was analyzed by RT-qPCR and ELISA. The phosphorylation of proteins induced by IL-1α was analyzed by western blot. The intracellular cAMP level was assayed by immunoassay kit. Results: IL-1α has a dose-dependent effect on IL-6 expression in granulosa cells. This promoting effect can be significantly attenuated by Erk, c-Jun, p38 and IκB proteins inhibitors, respectively. Moreover, the phosphorylation levels of Erk, c-Jun, p38 and IκBα proteins were significantly increased after IL-1α treatment. In addition, we also found that IL-1α not only reversed the cAMP attenuated IL-6 expression， but also increased IL-1α mRNA expression in granulosa cells. Conclusion: The regulation of IL-1α on IL-6 expression is mediated by activation of MAPKs and NF-κB signaling pathways. Moreover，IL-1α may regulate the ovulation-related genes expression in granulosa cells by an autocrine and/or paracrine manner.

MicroRNA-142-3p and let-7g Negatively Regulates Augmented IL-6 Production in Neonatal Polymorphonuclear Leukocytes.

Neonatal PMN are qualitatively impaired in functions, yet they frequently reveal augmented inflammatory reactions during sepsis. Here, we hypothesized that PMN from newborns produce more IL-6 than those from adults under LPS stimulation, in which transcriptional or posttranscriptional regulation is involved in the altered expression. We found that neonatal PMN produced significantly higher IL-6 mRNA and protein than adult PMN. The higher IL-6 expression was not related to transcriptional but posttranscriptional regulation as the IL-6 expression was affected by the addition of cycloheximide but not actinomycin. To examine whether miRNA was involved in the IL-6 regulation of neonatal PMN, we surveyed differential displays of miRNAs that could potentially regulate IL-6 expression before and after LPS stimulation. Four miRNAs: hsa-miR-26a, hsa-miR-26b, hsa-miR-142-3p and hsa-let 7g decreased or increased after LPS treatment for 4 h. Further validation by qRT-PCR identified miR-26b, miR-142-3p and let-7g significantly changed in neonatal PMN after LPS stimulation. The functional verification by transfection of miR-142-3p and let-7g precursors into neonatal PMN significantly repressed the IL-6 mRNA and protein expression, suggesting that miR-142-3p and let-7g negatively regulate IL-6 expression in neonatal PMN. Modulation of miRNA expression may be used to regulate IL-6 production in newborns with altered inflammatory reactions.

Balancing the immune response in the brain: IL-10 and its regulation.

BackgroundThe inflammatory response is critical to fight insults, such as pathogen invasion or tissue damage, but if not resolved often becomes detrimental to the host. A growing body of evidence places non-resolved inflammation at the core of various pathologies, from cancer to neurodegenerative diseases. It is therefore not surprising that the immune system has evolved several regulatory mechanisms to achieve maximum protection in the absence of pathology.Main bodyThe production of the anti-inflammatory cytokine interleukin (IL)-10 is one of the most important mechanisms evolved by many immune cells to counteract damage driven by excessive inflammation. Innate immune cells of the central nervous system, notably microglia, are no exception and produce IL-10 downstream of pattern recognition receptors activation. However, whereas the molecular mechanisms regulating IL-10 expression by innate and acquired immune cells of the periphery have been extensively addressed, our knowledge on the modulation of IL-10 expression by central nervous cells is much scattered. This review addresses the current understanding on the molecular mechanisms regulating IL-10 expression by innate immune cells of the brain and the implications of IL-10 modulation in neurodegenerative disorders.ConclusionThe regulation of IL-10 production by central nervous cells remains a challenging field. Answering the many remaining outstanding questions will contribute to the design of targeted approaches aiming at controlling deleterious inflammation in the brain.

Social isolation impairs remyelination in mice through modulation of IL-6.

Recent studies have revealed that social experience affects myelination. These findings have important implications for disorders that feature abnormal myelination, such as multiple sclerosis (MS), as previous studies have shown that psychosocial stress exacerbates the pathobiology of MS. However, most studies have focused on psychosocial stress during the demyelination phase of MS and have not investigated the effects of social experience on remyelination. Thus, the current study sought to determine whether social experience can alter remyelination after myelin depletion. Myelin in the mouse medial prefrontal cortex was depleted with cuprizone, and the effects of subsequent social isolation on remyelination were evaluated. Remyelination was severely impaired in socially isolated mice. Social isolation also increased IL-6 levels in the medial prefrontal cortex, and administration of an IL-6 inhibitor (ND50 = 0.01-0.03 μg for 0.25 ng/ml IL-6) ameliorated remyelination impairments. Consistent with this result, IL-6 administration (ED50 = 0.02-0.06 ng/ml) disturbed remyelination. In addition, neuron-oligodendrocyte coculture experiments showed that IL-6 treatment (ED50 ≤ 0.02 ng/ml) markedly impeded myelination, which was recovered with IL-6 inhibitor administration (ND50 = 0.01-0.03 μg for 0.25 ng/ml IL-6). This study provides the first direct evidence, to our knowledge, that social experience influences remyelination via modulation of IL-6 expression. These findings indicate that psychosocial stress may disturb remyelination through regulation of IL-6 expression in patients with such demyelinating diseases that involve remyelination as MS.-Makinodan, M., Ikawa, D., Miyamoto, Y., Yamauchi, J., Yamamuro, K., Yamashita, Y., Toritsuka, M., Kimoto, S., Okumura, K., Yamauchi, T., Fukami, S., Yoshino, H., Wanaka, A., Kishimoto, T. Social isolation impairs remyelination in mice through modulation of IL-6.

Micro RNA-19a suppresses IL-10 in peripheral B cells from patients with atherosclerosis

Background and aimsThe interleukin (IL)-10-production B cells play an important role in the pathogenesis of atherosclerosis (Asro) with unknown mechanism. Micro RNA (miR)-17-92 cluster has strong immune regulatory activities. This study tests a hypothesis that miR-17-92 cluster suppresses IL-10 expression in B cells of Asro patients. MethodsPatients with Asro were recruited into this study. Peripheral blood samples were collected from the patients. B cells were isolated from the blood samples and analyzed to elucidate the role of miR-17-92 in the regulation of IL-10 expression. ResultsPeripheral B cells from patients with Asro show lower levels of IL-10 than that from healthy subjects. The IL-10 expression in the B cells is negatively correlated with the expression of miR-19a in the B cells. The serum levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-4 in Asro patients were higher than healthy subjects. Exposure to TNF-α or IFN-γ or IL-4 suppressed IL-10 expression in B cells via increasing the expression of miR-19a in B cells, which could be abolished by Inhibition of miR-19a. ConclusionsTNF-α or IFN-γ or IL-4 suppresses IL-10 in B cells via up regulating miR-19a expression.