Increased IL-6 mRNA Expression Research Articles

Mechanical Stress Induces Sodium Entry and Osmoprotective Responses in Murine Synovial Fibroblasts.

Osteoarthritis (OA) is a multifactorial disease depending on molecular, genetic, and environmental factors like mechanical strain. Next to the cartilage and the subchondral bone, OA also affects the synovium, which is critically involved in the maintenance of joint homeostasis. As there is a correlation between the extracellular sodium content in the knee joint and OA, this study investigates the impact of sodium on OA-associated processes like inflammation and bone remodeling without and with mechanical loading in synovial fibroblasts. For that purpose, murine synovial fibroblasts from the knee joint were exposed to three different extracellular sodium chloride concentrations (-20 mM, ±0 mM and +50 mM NaCl) in the absence or presence of compressive or intermittent tensile strain. In addition to the intracellular Na+ content and gene expression of the osmoprotective transcription factor nuclear factor of activated T cells 5 (Nfat5), the gene and protein expression of inflammatory mediators (interleukin-6 (IL6), prostaglandin endoperoxide synthase-2 (Ptgs2)/prostaglandin E2 (PGE2)), and factors involved in bone metabolism (receptor activator of NF-κB ligand (RANKL), osteoprotegerin (OPG)) were analyzed by qPCR and ELISA. Mechanical strain already increased intracellular Na+ and Nfat5 gene expression at standard salt conditions to levels obtained by exposure to increased extracellular Na+ content. Both high salt and compressive strain resulted in elevated IL6 and PGE2 release. Intermittent tensile strain did not increase Il6 mRNA expression or IL6 protein secretion but triggered Ptgs2 expression and PGE2 production. Increased extracellular Na+ levels and compressive strain increased RANKL expression. In contrast, intermittent tension suppressed RANKL expression without this response being subject to modification by extracellular sodium availability. OPG expression was only induced by compressive strain. Changes in extracellular Na+ levels modified the inflammatory response and altered the expression of mediators involved in bone metabolism in cells exposed to mechanical strain. These findings indicate that Na+ balance and Nfat5 are important players in synovial fibroblast responses to mechanical stress. The integration of Na+ and Na+-dependent signaling will help to improve the understanding of the pathogenesis of osteoarthritis and could lead to the establishment of new therapeutic targets.

Cytochrome P450 26A1 Contributes to the Maintenance of Neuropathic Pain

The cytochrome P450 proteins (CYP450s) have been implicated in catalyzing numerous important biological reactions and contribute to a variety of diseases. CYP26A1, a member of the CYP450 family, carries out the oxidative metabolism of retinoic acid (RA), the active metabolite of vitamin A. Here we report that CYP26A1 was dramatically upregulated in the spinal cord after spinal nerve ligation (SNL). CYP26A1 was mainly expressed in spinal neurons and astrocytes. HPLC analysis displayed that the content of all-trans-RA (at-RA), the substrate of CYP26A1, was reduced in the spinal cord on day 7 after SNL. Inhibition of CYP26A1 by siRNA or inhibition of CYP26A1-mediated at-RA catabolism by talarozole relieved the SNL-induced mechanical allodynia during the maintenance phase of neuropathic pain. Talarozole also reduced SNL-induced glial activation and proinflammatory cytokine production but increased anti-inflammatory cytokine (IL-10) production. The RA receptors RARα, RXRβ, and RXRγ were expressed in spinal neurons and glial cells. The promoter of Il-10 has several binding sites for RA receptors, and at-RA directly increased Il-10 mRNA expression in vitro. Finally, intrathecal IL-10 attenuated SNL-induced neuropathic pain and reduced the activation of astrocytes and microglia. Collectively, the inhibition of CYP26A1-mediated at-RA catabolism alleviates SNL-induced neuropathic pain by promoting the expression of IL-10 and suppressing glial activation. CYP26A1 may be a potential therapeutic target for the treatment of neuropathic pain.

Relative expression of IL-10 and TNF-α mRNA of kidney and spleen tissues of rat with and without mammary tumor after exposed to alternating current electric field

Background: Cancer therapy based on alternating current electric field exposure, Electro-Capacitive Cancer Therapy (ECCT), has been pre-clinically tested for its effectiveness in breast tumor inhibition. However, concerns regarding the safety of electric field (EF) exposure to vital organs have been raised. In the kidney and spleen, IL-10 and TNF-α play roles in the response to inflammation.
 Objective: This research aimed to evaluate the safety of electric field exposure to vital organs, by assessing the expression of IL-10 and TNF-α mRNA in the kidney and spleen of rats, both with and without breast tumors.
 Methods: Female rats were divided into four groups; non-induction-non-therapy (NINT), non-induction-therapy (NIT), induction-non-therapy (INT), and induction-therapy (IT). Rats were induced by 7,12-dimethylbenz[a]anthracene (DMBA) at a dosage 20 mg/Kg body weight and exposed to electric fields for 10 hours, followed by a 2-hour rest period. Total RNA from the kidney and spleen was isolated, cDNA was synthesized by reverse transcription PCR, and analysis of IL-10 and TNF-α mRNA expression was performed by RT-qPCR method. Data were analyzed using the Livak formula, GraphPad Prism 9.0 software, and significance test ANOVA (α = 0.05).
 Results: Alternating current electric field exposure had no effects on the expression of TNF-α and IL-10 mRNA in the kidney and spleen of rats. Specifically, no effect was observed on TNF-α mRNA expression in all groups, and there was no effect on IL-10 mRNA expression in the spleen. Only the INT group showed a significant increase in IL-10 mRNA expression in the kidney (p < 0.05).
 Conclusion: Exposure of an alternating current electric field did not affect the relative mRNA expression of TNF-α and IL-10 in the kidneys and spleen of rats.

Characteristics of Mycoplasma bovis, Mycoplasma arginini, and Mycoplasma californicum on immunological response of bovine synovial cells

Mycoplasma arthritis in calves caused by M. bovis exhibits joint swelling, lameness, and immobility. In contrast to M. bovis, M. arginini, and M. californicum which were similarly isolated from the affected joints, only induced mild inflammation. The changes in pathogenesis that depended on species, however, remained unknown. This investigation aims to examine the characteristics of immune responses to M. bovis, M. arginini, and M. californicum in synovial cells. Intracellular M. bovis was detected by gentamicin assay, but M. arginini and M. californicum were not detected. M. bovis-infected synovial cells were encouraged to proliferate and had their apoptosis suppressed. We suggest that M. bovis invaded and inhibited apoptosis of synovial to evade host immunity, which led to long term survival in joints. M. bovis infection significantly increased IL-6 mRNA expression compared to control, although M. arginini and M. californicum infection were comparable to control. We suggest that M. arginini and M. californicum have low abilities to induce inflammation in joints and therefore do not cause severe pathology. Our findings are the first to show the variations in synovial cell immune responses to M. bovis, M. arginini, and M. californicum, which are thought to be related to the pathogenicity of arthritis.

Cigarette smoke induces mitochondrial DNA damage and activates cGAS-STING pathway -Application to a biomarker for atherosclerosis.

Cigarette smoking is a major risk factor for atherosclerosis. We previously reported that DNA damage was accumulated in atherosclerotic plaque, and was increased in human mononuclear cells by smoking. As vascular endothelial cells are known to modulate inflammation, we investigated the mechanism by which smoking activates innate immunity in endothelial cells focusing on DNA damage. Further, we sought to characterize the plasma level of cell-free DNA (cfDNA), a result of mitochondrial and/or genomic DNA damage, as a biomarker for atherosclerosis. Cigarette smoke extract (CSE) increased DNA damage in the nucleus and mitochondria in human endothelial cells. Mitochondrial damage induced minority mitochondrial outer membrane permeabilization, which was insufficient for cell death but instead led to nuclear DNA damage. DNA fragments, derived from the nucleus and mitochondria, were accumulated in the cytosol, and caused a persistent increase in IL-6 mRNA expression via the cGAS-STING pathway. cfDNA, quantified with quantitative PCR in culture medium was increased by CSE. Consistent with in vitro results, plasma mitochondrial cfDNA (mt-cfDNA) and nuclear cfDNA (n-cfDNA) were increased in young healthy smokers compared to age-matched non-smokers. Additionally, both mt-cfDNA and n-cfDNA were significantly increased in patients with atherosclerosis compared to the normal controls. Our multivariate analysis revealed that only mt-cfDNA predicted the risk of atherosclerosis. In conclusion, accumulated cytosolic DNA caused by cigarette smoke and the resultant activation of the cGAS-STING pathway may be a mechanism of atherosclerosis development. The plasma level of mt-cfDNA, possibly as a result of DNA damage, may be a useful biomarker for atherosclerosis.

The Probable Protective Effect of Photobiomodulation on the Immunologic Factor's mRNA Expression Level in the Lung: An Extended COVID-19 Preclinical and Clinical Meta-analysis.

Different expression of cytokine genes in the body determines the type of immune response (Th1 or Th2), which can play an important role in the pathogenesis of the COVID-19 disease. This meta-analysis was conducted to evaluate the probable effect of photobiomodulation (PBMT) therapy on the cytokine's mRNA expression in the lung. We systematically searched indexing databases, including PubMed/Medline, ISI web of science, Scopus, EMBASE, and Cochrane central, using standard terms without language, study region or type restrictions. Studies on using PBM in lung injury modeling with samples collected from lung tissue to observe IL-1β, TNF-α, IL-10, and IL-6 mRNA expression were included. RevMan 5.3 software was used for data analysis and standardized mean difference as effect size. Of the 438 studies found through initial searches, 17 met the inclusion criteria. The main properties of 13 articles on 384 animals included in this meta-analysis with a wide range of species include rats (n = 10) and rabbits (n = 3). The analysis revealed that PBMT reduced the mRNA expression of TNFα (SMD: -3.70, 95% CI: -6.29, -1.11, P = .005,I 2 = 71%) and IL-1β (SMD: -5.85, 95% CI: -8.01, -3.69, P < .00001,I 2 = 37%) significantly, but no statistically significant reduction in IL-6 (SMD: -2.89, 95% CI: -5.79, 0.01, P = .05,I 2 = 88%) was observed compared with the model controls. Also, PBMT increased IL-10 mRNA expression significantly compared with the model controls (SMD: 1.04, 95% CI: 0.43, 1.64, P = .0008,I 2 = 17%). This meta-analysis revealed that the PBMT utilizes beneficial anti-inflammatory effects and modulation of the immune system on lung damage in animal models and clinical studies. However, animal models and clinical studies appear limited considering the evidence's quality; therefore, large clinical trials are still required.

L-amino acid oxidase-1 is involved in the gut-liver axis by regulating 5-aminolevulinic acid production in mice.

L-amino acid oxidase (LAAO) is a metabolic enzyme that converts L-amino acids into ketoacids, ammonia, and hydrogen peroxide (H2O2). The generated H2O2 has previously been shown to have antibacterial and gut microbiota-modulatory properties in LAO1 knock-out (KO) mice. Since most microbial metabolites reach the liver through the portal vein, we examined gut-liver interactions in LAO1 KO mice. We found lower total cholesterol levels, higher glutamic pyruvic transaminase (GPT) levels in the serum, and higher pro-inflammatory cytokine mRNA expression in the liver tissue. In wild-type (WT) mice, LAO1 was expressed in gut tissues (ileum and colon). Microbiome analysis revealed that the abundance of some bacteria was altered in LAO1 KO mice. However, short-chain fatty acid (SCFAs) levels in cecal feces and gut permeability did not change. Fecal microbiota transplantation (FMT) revealed that feces from LAO1 KO mice slightly stimulated pro-inflammatory cytokine expression in the liver. During metabolomic analysis, 5-Aminolevulinic acid (5-ALA) was the only metabolite found to be significantly upregulated in the portal and abdominal veins of the LAO1 KO mice. Intraperitoneal administration of 5-ALA to WT mice significantly increased IL-6 mRNA expression in the liver. These observations suggest that gut LAO1 plays a role in regulating 5-ALA production and that a high level of 5-ALA stimulates the liver to increase pro-inflammatory cytokine expression by disrupting LAO1 in mice.

The Human Milk Oligosaccharide 2'-Fucosyllactose Shows an Immune-Enhancing Effect in a Cyclophosphamide-Induced Mouse Model.

The 2'-fucosyllactose (2'-FL) is the richest components in a human milk oligosaccharide. Several studies have reported that 2'-FL has beneficial effects in infants. However, there are few studies on its immune-enhancing effects. This research aimed to examine the immune-enhancing effect of 2'-FL on immunosuppression by cyclophosphamide (CCP) in ICR mice. Mice were orally administered distilled water or 0.5 mg/kg B.W. 2'-FL for 14 days. An immunocompromised mouse model was induced using CCP 80 mg/kg B.W. at 12-14 days. Using the CCP had effects on reducing their body weight, organ weight, spleen index, natural killer (NK) cell activity, and cytokines concentration and expression. This study also used concanavalin A-mediated T-cell proliferation to verify the immune-enhancing effects in the sample. Body weight, spleen index, organ weight, and cytokine levels were measured to estimate the immune-enhancing effects. The body weight at 14 days tended to increase, and the spleen weight and index significantly increased in the 2'-FL group compared to the CCP group. The NK cell activity increased in the 2'-FL group compared to the CCP group, but there was no significant difference. The concentration of interleukin (IL)-2 tended to recover in the 2'-FL group compared to the CCP group. The 2'-FL group showed a significant increase of IL-10 and IFN-gamma concentration compared to the CCP group. In addition, there was a trend of increased IL-10 mRNA expression compared to the CCP group. These results revealed that 2'-FL improved CCP-induced immunosuppression, suggesting that 2'-FL may have the potential to enhance the immune system.

Omeprazole Increases Survival Through the Inhibition of Inflammatory Mediaters in Two Rat Sepsis Models.

ABSTRACTBackground:Omeprazole (OMZ) is a proton pump inhibitor that is used to reduce gastric acid secretion, but little is known about its possible liver protective effects. This study investigated whether OMZ has beneficial effects in rat septic models of LPS-induced liver injury after D-galactosamine (GalN) treatment and 70% hepatectomy (PH), and to determine the mechanisms of OMZ in an in vitro model of liver injury.Methods:In the in vivo models, the effects of OMZ were examined 1 h before treatments in both models on survival, nuclear factor (NF)-κB activation, histopathological analysis, and proinflammatory mediator expression in the liver and serum. In the in vitro model, primary cultured rat hepatocytes were treated with IL-1β in the presence or absence of OMZ. The influence of OMZ on nitric oxide (NO) product and inducible NO synthase (iNOS) induction and on the associated signaling pathway was analyzed.Results:OMZ increased survival and decreased tumor necrosis factor-alpha, iNOS, cytokine-induced neutrophil chemoattractant 1, IL-6, and IL-1β mRNA expression, and increased IL-10 mRNA expression in the livers of both GaIN/LPS- and PH/LPS-treated rats. Necrosis and apoptosis were inhibited by OMZ in GaIN/LPS rats, but OMZ had no effects on necrosis in PH/LPS rats. OMZ inhibited iNOS induction partially through suppression of NF-κB signaling in hepatocytes.Conclusions:OMZ inhibited the induction of several inflammatory mediators, resulting in the prevention of LPS-induced liver injury after GalN liver failure and PH, although OMZ showed different doses and mechanisms in the two models.

Isolation, Purification, Characterization, and Immunomodulatory Activity Analysis of α-Glucans from Spirulina platensis.

Crude polysaccharides from Spirulina platensis (SP) were isolated by maceration with a hot alkali solution and further fractionated by DEAE-52 cellulose and Sephadex G-100 chromatography into two purified fractions PSP-1 and PSP-2. The monosaccharide composition analysis indicated that SP was mainly composed of rhamnose and glucose, while PSP-1 and PSP-2 were composed only of glucose. The composition analysis of PSP-1 and PSP-2 by HPLC, FT-IR, and NMR showed that PSP-1 and PSP-2 were branching dextran, and their structures were (1 → 4)-linked-α-D-Glcp as the main chain, and C-6 replaced the single α-D-Glcp as the linear structure of the branch chain. The glucans (SP/PSP-1/PSP-2) can significantly improve the phagocytic ability of macrophages, enhance iNOS activity, promote NO production, and increase IL-6 mRNA expression, so they may possess certain immunomodulatory activity.

Kaempferol Alleviates Murine Experimental Colitis by Restoring Gut Microbiota and Inhibiting the LPS-TLR4-NF-κB Axis.

Intestinal microbiota dysbiosis is an established characteristic of ulcerative colitis (UC). Regulating the gut microbiota is an attractive alternative UC treatment strategy, considering the potential adverse effects of synthetic drugs used to treat UC. Kaempferol (Kae) is an anti-inflammatory and antioxidant flavonoid derived from a variety of medicinal plants. In this study, we determined the efficacy and mechanism of action of Kae as an anti-UC agent in dextran sulfate sodium (DSS)-induced colitis mice. DSS challenge in a mouse model of UC led to weight loss, diarrhea accompanied by mucous and blood, histological abnormalities, and shortening of the colon, all of which were significantly alleviated by pretreatment with Kae. In addition, intestinal permeability was shown to improve using fluorescein isothiocyanate (FITC)–dextran administration. DSS-induced destruction of the intestinal barrier was also significantly prevented by Kae administration via increases in the levels of ZO-1, occludin, and claudin-1. Furthermore, Kae pretreatment decreased the levels of IL-1β, IL-6, and TNF-α and downregulated transcription of an array of inflammatory signaling molecules, while it increased IL-10 mRNA expression. Notably, Kae reshaped the intestinal microbiome by elevating the Firmicutes to Bacteroidetes ratio; increasing the linear discriminant analysis scores of beneficial bacteria, such as Prevotellaceae and Ruminococcaceae; and reducing the richness of Proteobacteria in DSS-challenged mice. There was also an evident shift in the profile of fecal metabolites in the Kae treatment group. Serum LPS levels and downstream TLR4-NF-κB signaling were downregulated by Kae supplementation. Moreover, fecal microbiota transplantation from Kae-treated mice to the DSS-induced mice confirmed the effects of Kae on modulating the gut microbiota to alleviate UC. Therefore, Kae may exert protective effects against colitis mice through regulating the gut microbiota and TLR4-related signaling pathways. This study demonstrates the anti-UC effects of Kae and its potential therapeutic mechanisms, and offers novel insights into the prevention of inflammatory diseases using natural products.

391-P: The Nrf2 Activator Increases TFAM and Suppresses STING in Human Renal Proximal Tubular Cells

Recent study has demonstrated the Nrf2 (NF-E2-related factor 2) activator bardoxolone methyl improved renal function in diabetic kidney disease (DKD). On the other hand, mitochondrial transcription factor A (TFAM) activation inhibits mitochondrial DNA (mtDNA) release into the cytoplasm and suppresses stimulator of interferon genes (STING)-mediated inflammation in renal proximal tubular cells. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is recognized as activators of Nrf2. In this study, we investigated the effects of SFN on TFAM and STING expression and its mitochondrial protective and anti-inflammatory effects. Because mitochondrial damage caused by fatty acids bound to albumin is involved in pathogenesis of DKD due to increased albumin reabsorption, we treated human renal proximal tubular cells (Lonza) with palmitic acid (PA, 200µM). Total RNA, DNA and protein were extracted and analyzed by qRT-PCR and western blotting. To quantify the mitochondrial DNA released into the cytoplasm, DNA was extracted from cytoplasmic and nuclear fractions. SFN (10µM) induced Nrf2 downstream target gene, NAD(P)H:quinone oxidoreductase 1 (NQO-1) mRNA expression to 359% (p&amp;lt;0.05). SFN alone increased TFAM mRNA expression to 132% (p&amp;lt;0.05) and suppressed STING mRNA expression by 26% (p&amp;lt;0.05). Whereas, PA had no effect on TFAM mRNA expression but increased STING mRNA expression to 135% (p&amp;lt;0.05). SFN suppressed PA-induced STING mRNA expression by 34% (p&amp;lt;0.05) and STING protein expression by 48%. PA increased cytoplasmic mtDNA to 237% and SFN decreased PA-induced cytoplasmic mtDNA release by 41%. PA increased IL-6 mRNA expression to 139% (p&amp;lt;0.05) and MCP-1 mRNA expression to 138% (p&amp;lt;0.05). SFN suppressed PA-induced IL-6 mRNA expression by 33% (p&amp;lt;0.05) and MCP-1 mRNA expression by 47% (p&amp;lt;0.05). Together, the Nrf2 activators may have renoprotective effects through mitochondrial protection and anti-inflammatory effect by activating TFAM and inhibiting STING. Disclosure R. Bessho: None. T. Takiyama: None. S. Promsuwan: None. H. Kitsunai: None. K. Sawamoto: Other Relationship; Self; Abbott, Boehringer Ingelheim Pharmaceuticals, Inc., Merck &amp; Co., Inc., Ono Pharmaceutical Co., Ltd., Roche Diagnostics K. K., Taisho Pharmaceutical Co., Ltd. Y. Takeda: None. Y. Takiyama: Other Relationship; Self; Gilead Sciences, Inc., Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Mochida Pharmaceutical Co., Ltd., Roche Diabetes Care, Taisho Pharmaceutical Co., Ltd.

The Role of Obesity in the Development of Kidney and Glomerular Ultrafiltration Barrier Injuries: Leptin Contributions

Introduction Obesity is associated with multiple risk factors to the development of comorbidities such as kidney injuries. Lesions in the glomerular ultrafiltration barrier may cause impairment of the kidney filtration process. Nephrin is one of the main components of the glomerular ultrafiltration barrier, so changes in the expression of this protein indicate impaired renal function. Thus, the aim of this study is to investigate the role of obesity in the development of kidney and glomerular ultrafiltration barrier injuries. The contribution of leptin to kidney function in this context was also evaluated. Methods The protocols are in agreement with CEUA (nº 13/2017). Twenty-four mice aged 18 weeks were used: Six C57BL / 6J were assigned to the lean control group (CTL) and 18 ob/ob (provided by Prof. Dr. José Donato Junior from ICB-USP) were randomly assigned to obese control group (ob/ob) and obese treated with leptin group (ob/ob - lep). Leptin treatment was performed through osmotic mini-pump (model 1004, Alzet) insertion. Mini-pumps were filled with a solution containing leptin at a dose of 3.5 μg/day for 42 days. During the treatment period food intake was monitored daily. Weight gain and blood glucose were monitored weekly. At the end of the treatment period, the animals were kept in metabolic cages for 24 hours to evaluate water intake and urine collection. Ultimately, the animals were submitted to euthanasia by exsanguination. Renal function was assessed by plasma concentration of creatinine and urea, and urinary albumin excretion. Renal tissue was used to evaluate gene expression by qPCR, histological and immunofluorescence analysis. Results Plasma leptin measurements confirm that ob/ob animals did not produce leptin and that introduction of exogenous leptin elevated the levels of this hormone to values close to control. Ob/ob - lep group had a reduction in food intake and body weight when compared to ob/ob group, this reduction led to values of food intake and body weight similar to CTL group. There were no changes in water intake, urine flow and blood glucose among groups. There were also no changes in plasma creatinine values among groups, however, in the ob/ob group, there was an increase in serum urea and leptin treatment partially reversed this parameter to values closer to CTL. In ob/ob animals, there was an increase in urinary albumin excretion and leptin treatment lowered this parameter to values closer to CTL group. Inflammation of the renal tissue in obese animals was confirmed by increased IL-6 mRNA expression, leptin had no effect at this parameter. There was no significant difference in the expression of mRNA to MCP1 between groups. However, there is a tendency to increasing that gene's expression in obese animals and leptin treatment reduces that parameter back to control. In 42 days of protocol, there was no glomerular hypertrophy in the ob/ob group, however, there was a reduction in the expression of nephrin and leptin treatment did not reverse this parameter. Conclusion: Obesity impairs renal function, increases expression of inflammatory molecules and decreases nephrin expression. Leptin therapy was partially renoprotective since it reversed some of the observed parameters.

HSP90 inhibitors strengthen extracellular ATP-stimulated synthesis of interleukin-6 in osteoblasts: Amplification of p38 MAP kinase.

Heat shock protein 90 (HSP90) is expressed ubiquitously in a variety of cell types including osteoblasts. HSP90 acts as a key driver of proteostasis under pathophysiological conditions. Here, we investigated the involvement of HSP90 in extracellular ATP-stimulated interleukin (IL)-6 synthesis and HSP90 downstream signalling in osteoblast-like MC3T3-E1 cells. In osteoblasts, extracellular ATP stimulates the synthesis of IL-6, a bone-remodelling agent. Geldanamycin, 17-allylamino-17-demethoxy-geldanamycin (17-AAG) and onalespib, three different HSP90 inhibitors, amplified the ATP-stimulated IL-6 release. Geldanamycin increased IL-6 mRNA expression elicited by ATP. ATP enhanced the triiodothyronine-induced osteocalcin release, but HSP90 inhibitors suppressed the release. Extracellular ATP induced the phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK), p38 MAPK, c-Jun N-terminal kinase (JNK), p70 S6 kinase, Akt, and myosin phosphatase-targeting subunit (MYPT), a Rho-kinase substrate. SB203580, an inhibitor of p38 MAPK, suppressed ATP-stimulated IL-6 release. Inhibitors of MEK1/2 (PD98059), JNK (SP600125), upstream kinase of p70 S6 kinase (rapamycin) and Akt (deguelin), all increased IL-6 release. Y27632, a Rho-kinase inhibitor, failed to affect the IL-6 release stimulated by ATP. Geldanamycin and 17-AAG both amplified ATP-induced p38 MAPK phosphorylation, although geldanamycin inhibited the phosphorylation of Akt induced by ATP. In addition, SB203580 significantly reduced the amplification by geldanamycin of the IL-6 release. Taken together, our results strongly suggest that HSP90 inhibitors up-regulate extracellular ATP-stimulated IL-6 synthesis via amplification of p38 MAPK activation in osteoblasts. SIGNIFICANCE OF THE STUDY: Heat shock protein 90 (HSP90) acts as a key driver of proteostasis under pathophysiological conditions in a variety of cell types. We have previously shown that HSP90 is expressed at high levels in osteoblast-like MC3T3-E1 cells, even in their quiescent state, consistent with HSP90 performing an important physiological function in osteoblasts. In the present study, we investigated whether HSP90 is implicated in extracellular ATP-induced interleukin (IL)-6 synthesis in osteoblast-like MC3T3-E1 cells. Our results strongly suggest that HSP90 inhibitors up-regulate extracellular ATP-stimulated IL-6 synthesis via amplification of p38 mitogen-activated protein kinase activation in osteoblasts.

Effects of Colonization of Gnotobiotic Swiss Webster Mice with Helicobacter bilis.

Helicobacter bilis (Hb) causes hepatitis in some strains of inbred mice. The current study confirmed that Hb directly causes portal hepatitis in outbred gnotobiotic Swiss Webster (SW) mice, as we previously reported for conventional SW mice. Hbmonoassociated SW mice also developed mild enterocolitis, expanded gut-associated lymphoid tissue (GALT), and tertiary lymphoid tissue in the lower bowel. At 1 and 10 mo after infection, Hb-induced GALT hyperplasia exhibited well-organized, ectopic germinal centers with increased mononuclear cell apoptosis, MHC class II antigen presentation, and pronounced endothelial venule formation, consistent with features of tertiary lymphoid tissue. In the lower bowel, Hb induced mainly B220+ cells as well as CD4+ IL17+, CD4+ IFNγ+, and CD4+ FoxP3+ regulatory T cells and significantly increased IL10 mRNA expression. This gnotobiotic model confirmed that Hb causes portal hepatitis in outbred SW mice but stimulated GALT with an antiinflammatory bias. Because Hb had both anti- and proinflammatory effects on GALT, it should be considered a 'pathosymbiont provocateur' and merits further evaluation in mouse models of human disease.

Discriminative expression of CD39 and CD73 in Cerebrospinal fluid of patients with Multiple Sclerosis and Neuro-Behçet’s disease

Treg‐mediated immune suppression involves many molecular mechanisms including the cleavage of inflammatory extracellular ATP to adenosine by CD39 ectoenzyme. In the peripheral blood of Multiple Sclerosis (MS) patients, it has been suggested that CD39+ Treg cells have the potential to suppress pro-inflammatory IL-17 secreting cells. Herein, we studied cellular phenotype and mRNA expression of CD39 and CD73 ectoenzymes in the Cerebrospinal fluid (CSF) of MS patients and another neuro-inflammatory disease: the Neuro-behçet’s disease (NBD).Using qRT-PCR, we assessed mRNA expression of CD39 and CD73 as well as anti-inflammatory (IL-10) and pro-inflammatory (IL-6, TNF-α, IL-1β) cytokines in patients Peripheral blood mononuclear cells (PBMCs) and CSF of 28 relapsing-remitting multiple sclerosis (RRMS), 20 NBD and 22 controls with non inflammatory neurological disorders (NIND). The most substantial result in the CSF was the higher expression of CD39 in both RRMS and NBD patients compared to NIND. While, the expression of CD73 in CSF samples of NBD was low. In RRMS samples, we detected a significant positive correlation of both CD39 and CD73 with IL-10 expression. Moreover, results by flow cytometry revealed a high percentage of CD39 Treg cells in RRMS CSF. CD39 was preferentially expressed on B cells of NBD. Regarding inflammatory response, we showed a significant increase of IL-6 mRNA expression in NBD patients CSF while in RRMS this increase concerned TNF-α. These results bring evidence that CD39 correlates positively with an anti-inflammatory IL-10 response in RRMS. In contrast, no such association was observed in CSF of NBD patients and CD39 was preferentially expressed on B cells.

Expression of IL-10 in A.actinomycetemcomitans Induced Rat Treated by Purple Miana Leaves

Periodontitis therapy originating from local natural resources is still lacking so it needs to be researched and developed, one of the natural resources from Soppeng Regency in South Sulawesi, Indonesia is the purple miana leaf. It has not been reported before about the effect of purple miana leaf extract on periodontitis related to IL-10 mRNA expression. This study aims to determine the effect of purple miana leaf extract on IL-10 mRNA expression in rat induced by Aggregatibacter actinomycetemcomitans. Rats were divided into three groups, purple miana leaf extract (PMLE), negative control (aquades), antibiotic (levofloxacin nine mg/kg body weight) as positive control. Rat blood was drawn before (H1) and after induction of 3x108 cfu/ml Aggregatibacter actinomycetemcomitans in the gingival sulcus of the mandibular anterior teeth/after periodontitis (H8) and seven days after intervention (H15). IL 10 mRNA expression was measured by Real-time PCR. The results obtained are processed using SPSS. There were no significant differences in H1-H8 in all groups. IL-10 mRNA expression on H8-H15 has a different pattern between PMLE, negative control and positive control. In the negative control, there was a decrease in IL-10 mRNA expression in H8-H15. In PMLE and positive control, an increase in IL-10 mRNA expression. The treatment of purple miana leaf extract in rat induced with Aggregatibacter actinomycetemcomitans significantly had the same effect as levofloxacin on IL-10 mRNA expression.

Losartan does not inhibit cigarette smoke-induced lung inflammation in mice

Chronic Obstructive Pulmonary Disease (COPD) is a progressive lung disease largely caused by cigarette smoking (CS) and is characterized by lung inflammation and airflow limitation that is not fully reversible. Approximately 50% of people with COPD die of a cardiovascular comorbidity and current pharmacological strategies provide little benefit. Therefore, drugs that target the lung and the cardiovascular system concurrently may be an advantageous therapeutic strategy. The aim of this study was to see whether losartan, an angiotensin-II AT1a receptor antagonist widely used to treat hypertension associated with cardiovascular disease, protects against CS-induced lung inflammation in mice. Male BALB/c mice were exposed to CS for 8 weeks and treated with either losartan (30 mg/kg) or vehicle daily. Mice were euthanized and bronchoalveolar lavage fluid (BALF) inflammation, and whole lung cytokine, chemokine and protease mRNA expression assessed. CS caused significant increases in BALF total cells, macrophages, neutrophils and whole lung IL-6, TNF-α, CXCL-1, IL-17A and MMP12 mRNA expression compared to sham-exposed mice. However, losartan only reduced CS-induced increases in IL-6 mRNA expression. Angiotensin-II receptor expression was reduced in lung tissue from CS-exposed mice. In conclusion, losartan did not inhibit CS-induced BALF cellularity despite reducing whole lung IL-6 mRNA and Ang-II receptor expression.

Direct effects of antipsychotic drugs on insulin, energy sensing and inflammatory pathways in hypothalamic mouse neurons

IntroductionSecond-generation antipsychotics cause serious metabolic side effects, but the mechanisms behind these effects remain largely unknown. However, emerging evidence supports that antipsychotics may act upon the hypothalamus, the primary brain region understood to regulate energy homeostasis. We have recently reported that the antipsychotics olanzapine, clozapine, and aripiprazole can directly act on hypothalamic rat neurons (rHypoE-19) to impair insulin, energy sensing, and modulate inflammatory pathways. In the current paper, we sought to replicate these findings to a mouse neuronal model. MethodsThe mouse hypothalamic neuronal cell line, mHypoE-46, was treated with olanzapine, clozapine, or aripiprazole. Western blots were used to measure the energy sensing protein AMPK, components of the insulin signalling pathway (AKT, GSK3β), and components of the MAPK pathway (ERK1/2, JNK, p38), the latter linked to inflammation. RT-qPCR was used to measure mRNA expression of the inflammatory mediators IL-6, IL-10, and BDNF, well as putative receptors in the mHypoE-46 (current) and the rHypoE-19 (previously studied) cell lines. ResultsIn the mHypoE-46 neurons, olanzapine and aripiprazole increased AMPK phosphorylation, while clozapine and aripiprazole inhibited insulin-induced phosphorylation of AKT. Clozapine increased JNK and aripiprazole decreased ERK1/2 phosphorylation. Olanzapine also decreased IL-6 mRNA expression, while olanzapine and clozapine increased IL-10 mRNA expression. The rHypoE-19 neurons expressed the H1, 5 H T2A, and M3 receptors, while the mHypoE-46 neurons expressed the 5 H T2A, D2, and M3 receptors. Neither cell line expressed the 5 H T2C receptor. ConclusionSimilar to observed effects of these agents in rat neurons, induction of AMPK by aripiprazole and olanzapine suggests impaired energy sensing, while suppression of insulin-induced pAKT by clozapine and aripiprazole suggests impaired insulin signalling, seen across both rodent derived hypothalamic cell lines. Conversely, olanzapine-induced suppression of pro-inflammatory IL-6, alongside olanzapine and clozapine-induced IL-10, demonstrate anti-inflammatory effects, which do not corroborate with our prior observations in the rat neuronal line. The different findings between cell lines could be explained by differential expression of neurotransmitters receptors and/or reflect genetic heterogeneity across the rat and mouse lines. However, overall, our findings support direct effects of antipsychotics to impact insulin, energy sensing, and inflammatory pathways in hypothalamic rodent neurons.

Interaction of the immune-inflammatory and the kynurenine pathways in rats resistant to antidepressant treatment in model of depression

The kynurenine pathway (KP), a major route of tryptophan catabolism, may be associated with the pathophysiology of depressive disorders. KP is responsible for ca. 99% of brain tryptophan metabolism via its degradation to kynurenine (KYN) catalyzed by indoleamine 2,3-dioxygenase (IDO). Some cytokines, such as interferon-γ (IFN-γ) and interleukin (IL)-6 are potent inducers of IDO. KYN is further converted by kynurenine aminotransferase (KAT) to the more neuroprotective kynurenic acid or by kynurenine 3-monooxygenase (KMO) to neurotoxic 3-hydroxykynurenine. The aim of the present study was to delineate whether the administration of imipramine (IMI) to rats subjected to chronic mild stress (CMS) may reverse behavioral changes induced by CMS in association with changes in immune-inflammatory markers and KP. We confirmed that the CMS procedure modeled one of the main symptoms of depression, i.e. anhedonia, and administration of IMI for 5 weeks resulted in a significant reduction in anhedonia in a majority of animals (CMS IMI-R animals), whereas 20% of animals did not respond to IMI treatment (CMS IMI-NR animals). We established that CMS procedure increased IFN-γ and IDO mRNA and decreased KAT II mRNA expression in the rat cortex. In the cortex and hippocampus, IMI treatment and non-responsiveness to IMI (in CMS IMI-NR animals) were associated with increased IL-6 mRNA expression. In the spleen, CMS increased production of IFN-γ and IL-6 proteins, while these cytokines were decreased by IMI in CMS IMI-R animals. Chronic IMI administration to CMS rats decreased IDO and KMO mRNA and protein expression and increased KAT II/KMO mRNA and protein ratio in IMI responders (CMS IMI-R) in comparison to CMS rats. In CMS IMI-NR rats, a significant increase in IDO mRNA expression and protein level in comparison with IMI responders was observed. Our findings indicate that resistance to therapeutic action of IMI could be explained by a deficiency of the inhibitory properties of IMI on IDO, KMO and KYN synthesis in the cortex. We conclude that the antidepressant activity of IMI may, at least in part, be explained by modulatory activities on the KAT II/KMO ratio in brain areas.