Tissue Necrosis Factor Research Articles

Abstract 158: Indoleamine 2,3-dioxygenase 1 is Expressed in Macrophages in Human Coronary Atherosclerotic Plaque and is Associated With Tissue Factor Expression in Activated THP-1 Macrophages

Background: Recently, several clinical studies have found that changes in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism are associated with cardiovascular events. Indoleamine 2,3-dioxygenase 1 (IDO1) is a rate-limiting enzyme of the Kyn pathway and is induced by cytokines, particularly IFNγ. However, the roles of the Kyn pathway on vascular wall thrombogenicity remain unknown. Objective: The present study aimed to localize IDO1 in human coronary atherosclerotic plaques from patients with stable angina pectoris (SAP) and unstable angina pectoris (UAP) and define its role in plaque thrombogenicity. Methods: Immunohistochemical methods were applied to localize IDO1 in coronary atherosclerotic plaques from patients with SAP and UAP. We examined the role of IDO1 in tissue factor (TF) expression in THP-1 macrophages activated by interferon (IFN)γ and tissue necrosis factor (TNF)α. Results: We localized IDO1 mainly in CD68-positive macrophages in atherosclerotic plaques, and in close association with TF. Areas that were immunopositive for IDO1, TF, and CD3-positive T lymphocytes were significantly larger in plaques from patients with UAP than SAP. THP-1 macrophages activated by IFNγ and TNFα upregulated IDO1 expression, increased the Kyn/Trp ratio and enhanced TF expression and activity, but not TF pathway inhibitor expression. The IDO1 inhibitor epacadostat significantly reduced the Kyn/Trp ratio, TF expression and activity, as well as NF-κB (p65) binding activity in activated THP-1 macrophages. Inhibition of the aryl hydrocarbon receptor (AHR) that binds to Kyn, also reduced Kyn-induced TF expression in activated THP-1 macrophages. Kynurenine dose-dependently increased TF expression under IDO1 inhibition whereas hydroxyanthranilic and quinolinic acids did not. Conclusion: The IDO1 is expressed in coronary atherosclerotic plaques and might contribute to thrombus formation through TF upregulation via NF-κB (p65) and AHR in activated macrophages.

Modulation of Vascular Endothelial Growth Factor (VEGF) and Tissue Necrosis Factor Alpha (TNFα) by the Ethanol Stem Bark Extract of Boswellia dalzielli H Attenuates Ethanol Induced Gastric Ulcer in Albino Rats.

Modulation of Vascular Endothelial Growth Factor (VEGF) and Tissue Necrosis Factor Alpha (TNFα) by the Ethanol Stem Bark Extract of Boswellia dalzielli H Attenuates Ethanol Induced Gastric Ulcer in Albino Rats.

Chloroquine prevents acute kidney injury induced by lipopolysaccharide in rats via inhibition of inflammatory factors

Purpose: To investigate the role of chloroquine (CQ) in lipopolysaccharide (LPS)-induced renal injury in rats. Methods: Rats were assigned to one of four groups ( n = 10). Control group was only given saline solution, whereas the model control, LPS + CQ, and LPS + yohimbine (YOH) + CQ groups were administered LPS intraperitoneally. At the end of the study, blood urea nitrogen (BUN) and creatinine (Cr) levels were determined. Results: CQ treatment significantly decreased the blood concentrations of tissue necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-18, BUN, and Cr in the model control rats. There were also significant decreases in the levels of high mobility group protein 1 and kidney injury molecule-1 in the renal injury rats compared to the model control group. However, the inhibitory effects of CQ in the LPS-treated rats were blocked by treatment with YOH, an α-2-adrenergic receptor antagonist. Conclusions: Treatment with CQ attenuates LPS-induced renal injury by inhibiting inflammatory response. Keywords: Creatinine, Chloroquine, Inflammatory reactions, Kidney injury, Lipopolysaccharide

Valproic acid increases NF-κB transcriptional activation despite decreasing DNA binding ability in P19 cells, which may play a role in VPA-initiated teratogenesis

The nuclear factor-kappa B (NF-κB) family of transcription factors regulate gene expression in response to diverse stimuli. We previously demonstrated that valproic acid (VPA) exposure in utero decreases total cellular protein expression of the NF-κB subunit p65 in CD-1 mouse embryos with a neural tube defect but not in phenotypically normal littermates. This study evaluated p65 mRNA and protein expression in P19 cells and determined the impact on DNA binding ability and activity. Exposure to 5mM VPA decreased p65 mRNA and total cellular protein expression however, nuclear p65 protein expression was unchanged. VPA reduced NF-κB DNA binding and nuclear protein of the p65 DNA-binding partner, p50. NF-κB transcriptional activity was increased with VPA alone, despite decreased phosphorylation of p65 at Ser276, and when combined with tissue necrosis factor α. These results demonstrate that VPA increases NF-κB transcriptional activity despite decreasing DNA binding, which may play a role in VPA-initiated teratogenesis.

The impact of spontaneous ventilation on non-operative lung injury in thoracic surgery: a randomized controlled rabbit model study.

One-lung ventilation (OLV) with general anaesthesia may increase adverse effects after thoracic surgery, specifically ventilator-induced lung injury. Spontaneous ventilation (SV) has no mechanical ventilation process, thus, we established a rabbit model to assess non-operative lung injury between OLV and SV. Thirty-six rabbits were randomly divided into 6 groups: OLV and SV (0, 2 and 4 h). Blood gas analysis was performed after thoracic surgery. Lung tissue and bronchoalveolar lavage fluid were obtained from the non-operative lung. Pathological injury score in lung tissue and tumour necrosis factor α (TNF-α) level in bronchoalveolar lavage fluid using enzyme-linked immunosorbent assay were determined. Moreover, messenger RNA and protein of TNF-α in lung tissue were also determined by quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. Compared with the OLV group, significantly higher partial pressure of carbon dioxide (47.78 ± 3.57 vs 38.95 ± 3.88 mmHg, P < 0.01) and partial pressure of oxygen (101.08 ± 13.1 vs 85.6 ± 11.07 mmHg, P < 0.01), as well as a significantly lower pathological injury score (6.83 ± 1.17 vs 8.83 ± 1.72, P < 0.05), TNF-α level in bronchoalveolar lavage fluid (290.32 ± 29.38 vs 368.43 ± 31.26 pg/ml, P < 0.01), TNF-α messenger RNA (6.31 ± 1.13 vs 8.6 ± 1.34, P < 0.01), immunostaining intensity in lung tissue were found at 4 h in the SV group. However, there are no significant differences between OLV and SV groups at 2 h (P > 0.05), except in TNF-α messenger RNA. Based on this rabbit model, SV for thoracic surgery is not inferior to OLV in terms of lung injury. Considering our results, when performing time-consuming thoracic procedures under OLV, surgeons should more closely examine patients for non-operative lung injury postoperatively.

An agent-based model of leukocyte transendothelial migration during atherogenesis.

A vast amount of work has been dedicated to the effects of hemodynamics and cytokines on leukocyte adhesion and trans-endothelial migration (TEM) and subsequent accumulation of leukocyte-derived foam cells in the artery wall. However, a comprehensive mechanobiological model to capture these spatiotemporal events and predict the growth and remodeling of an atherosclerotic artery is still lacking. Here, we present a multiscale model of leukocyte TEM and plaque evolution in the left anterior descending (LAD) coronary artery. The approach integrates cellular behaviors via agent-based modeling (ABM) and hemodynamic effects via computational fluid dynamics (CFD). In this computational framework, the ABM implements the diffusion kinetics of key biological proteins, namely Low Density Lipoprotein (LDL), Tissue Necrosis Factor alpha (TNF-α), Interlukin-10 (IL-10) and Interlukin-1 beta (IL-1β), to predict chemotactic driven leukocyte migration into and within the artery wall. The ABM also considers wall shear stress (WSS) dependent leukocyte TEM and compensatory arterial remodeling obeying Glagov’s phenomenon. Interestingly, using fully developed steady blood flow does not result in a representative number of leukocyte TEM as compared to pulsatile flow, whereas passing WSS at peak systole of the pulsatile flow waveform does. Moreover, using the model, we have found leukocyte TEM increases monotonically with decreases in luminal volume. At critical plaque shapes the WSS changes rapidly resulting in sudden increases in leukocyte TEM suggesting lumen volumes that will give rise to rapid plaque growth rates if left untreated. Overall this multi-scale and multi-physics approach appropriately captures and integrates the spatiotemporal events occurring at the cellular level in order to predict leukocyte transmigration and plaque evolution.

Microglial NFκB-TNFα hyperactivation induces obsessive–compulsive behavior in mouse models of progranulin-deficient frontotemporal dementia

Frontotemporal dementia (FTD) is the second most common dementia before 65 years of age. Haploinsufficiency in the progranulin (GRN) gene accounts for 10% of all cases of familial FTD. GRN mutation carriers have an increased risk of autoimmune disorders, accompanied by elevated levels of tissue necrosis factor (TNF) α. We examined behavioral alterations related to obsessive-compulsive disorder (OCD) and the role of TNFα and related signaling pathways in FTD patients with GRN mutations and in mice lacking progranulin (PGRN). We found that patients and mice with GRN mutations displayed OCD and self-grooming (an OCD-like behavior in mice), respectively. Furthermore, medium spiny neurons in the nucleus accumbens, an area implicated in development of OCD, display hyperexcitability in PGRN knockout mice. Reducing levels of TNFα in PGRN knockout mice abolished excessive self-grooming and the associated hyperexcitability of medium spiny neurons of the nucleus accumbens. In the brain, PGRN is highly expressed in microglia, which are a major source of TNFα. We therefore deleted PGRN specifically in microglia and found that it was sufficient to induce excessive grooming. Importantly, excessive grooming in these mice was prevented by inactivating nuclear factor κB (NF-κB) in microglia/myeloid cells. Our findings suggest that PGRN deficiency leads to excessive NF-κB activation in microglia and elevated TNFα signaling, which in turn lead to hyperexcitability of medium spiny neurons and OCD-like behavior.

Inter-Ethnic Variations in Association of TNF-Alpha G308a Single Nucleotide Polymorphism with Type 2 Diabetes Mellitus - A Review

According to World Health Organization, the prevalence of diabetes mellitus (DM) is on the rise. Tissue necrosis factor- alpha (TNF-α) is one of the inflammatory markers which play role in the pathogenesis of DM. Single nucleotide polymorphism (SNP) at 308G/A has been reported to be associated with type 2 DM. Frequency of TNF-α G308A polymorphism was determined in different ethnic groups and is found to be highly variable. Limited number of studies reported a positive association between type 2 DM and TNF-α SNP and many studies; including meta-analyses failed to find such association. TNF-α G308A polymorphism has been found to be associated with insulin resistance and BMI, although these findings are challenged by many studies. The probable explanation of higher BMI is the association of presence of TNF- α G308A polymorphism with higher rates of lipid synthesis, and suppression of FFA levels in obese persons. There is no single explanation for such highly variable results in different ethnic groups. Presence of yet unidentified gene polymorphism in linkage disequilibrium with TNF-α gene polymorphism could be responsible. The different results can also be contributed to the fact that the study groups differ in age, gender distribution, age of onset of disease, life style, degree of obesity and glucose tolerance. Comprehensive genetic studies of whole TNF-alpha promoter area are required to be done in large population samples in different ethnic groups. Polymorphisms are usually co-related in a complex manner and are co-inherited and conclusions are difficult to be drawn on small sample size.

Effects of resveratrol on diabetes-induced vascular tissue damage and inflammation in male rats

Abstract Objective The present study aims to investigate the short-term effects of resveratrol on histopathological characteristics and inflammatory cytokines of the heart and thoracic aorta tissues in animal models of streptozotocin (STZ)-induced diabetes. Methods Male Wistar rats were randomly divided into four groups; (1) control/vehicle, (2) control/20 mg/kg resveratrol, (3) diabetic/vehicle, (4) diabetic/20 mg/kg resveratrol. Heart and thoracic aorta were examined histopathologically and the levels of interleukin (IL)-1β, IL-18 and tissue necrosis factor (TNF)-α were analyzed by ELISA. Malondialdehyde (MDA) contents were determined with HPLC. Results Diabetes group had significantly higher vascular MDA content (p&lt;0.05) as compared with the control and resveratrol treated groups. Resveratrol significantly reduced vascular MDA level in diabetic animals (p&lt;0.05). Significant elevation in IL-1β and TNF-α contents in thoracic aorta and IL-18 contents in cardiac and arterial tissues with diabetes were almost normalized with resveratrol treatment. Additionally, diabetic animals demonstrated significant endothelial damage, irregularities in smooth muscle fibers and degeneration of elastic fibers in thoracic aortas together with significant irregularities and hypertrophy in cardiac muscle fibers. Resveratrol significantly improved most of these histopathological alterations. Conclusion Four-week-long intraperitoneal administration of resveratrol may restore the diabetes related inflammation and oxidative stress within the cardiovascular system.

Abstract 48: Administration of Gut Bacteria Expressing N-acyl Phosphatidylethanolamine Reduces Steatohepatitis in LDLR-/- Mice Fed a Western Diet

Background: The rise in obesity in the United States has led to a concomitant rise in prevalence of non-alcoholic fatty liver disease (NAFLD). The four stages of NAFLD include accumulation of triglyceride (hepatosteatosis), development of chronic inflammation (non-alcoholic steatohepatitis, NASH), fibrosis, and finally cirrhosis. Unlike wildtype C57BL6 mice, low density lipoprotein receptor (LDLR) -/- mouse fed a diet enriched in fat and cholesterol (Western Diet) progress to NASH and fibrotic stages of NAFLD. We showed that incorporating engineered bacteria expressing N-acyl phosphatidylethanolamine (NAPE) into the gut microbiota can inhibit development of obesity. NAPE is a precursor of N-acylethanolamines, which are bioactive lipids with anti-inflammatory functions. Here, we test the hypothesis that administering these NAPE-expressing bacteria inhibits development of NASH and fibrosis. Methods: NAPE-expressing E. coli Nissle 1917 (pNAPE-EcN, n=10), control Nissle 1917 (pEcN, n=10), or vehicle (veh, n=10) were given via drinking water to LDLR -/- mice fed a Western diet for 12 weeks. LDLR -/- mice fed a low fat diet (LFD) (n=10) were included for comparison. Results: pNAPE-EcN reduced adiposity by 26% compared with pEcN and veh (P&lt;0.05). pNAPE-EcN also dramatically reduced hepatic triglyceride levels by 45% (p&lt;0.05) and lipid droplet size, as well as the hepatic expressions of tissue necrosis factor α (TNFα, p&lt;0.05), chemokine receptor 2 (CCR2, p&lt;0.01), and tissue inhibitor of matrix metalloproteinase (TIMP1, p&lt;0.05), consistent with reduced NASH and fibrosis. Sirius Red staining of liver sections further demonstrated reduced fibrosis. Because fatty liver is associated with atherosclerosis, we checked to see if pNAPE-EcN was able to reduce the development of atherosclerotic lesions. While serum cholesterol was reduced by 23% with pNAPE-EcN treatment (p&lt;0.05), atherosclerotic lesion size in proximal or en face aortas only tended to be reduced (20%, 18.6%) but was not statistically significant. Conclusions: Our results demonstrate that incorporating therapeutically modified bacteria into the gut microbiota has potential to inhibit the development of NAFLD.

A Dysregulated Balance of Proinflammatory and Anti-Inflammatory Host Cytokine Response Early During Therapy Predicts Persistence and Mortality in Staphylococcus aureus Bacteremia.

The contribution of individual immune response to Staphylococcus aureus bacteremia on outcome has not been well studied. The objective was to relate the host cytokine response to outcome of Staphylococcus aureus bacteremia. Prospective observational study. Three U.S. university-affiliated medical centers. Adult patients infected with Staphylococcus aureus bacteremia hospitalized between July 2012 and August 2014. Blood specimens were obtained at Staphylococcus aureus bacteremia onset and 72 hours after therapy initiation. Levels of tissue necrosis factor, interleukin-6, interleukin-8, interleukin-17A, and interleukin-10 were measured by enzyme-linked immunosorbent assay at each time point and compared between those with persistent bacteremia (≥ 4 d) and resolving bacteremia. Primary outcome was persistent bacteremia after 4 days of effective therapy. Secondary outcomes were 30-day mortality and 30-day recurrence. A total of 196 patients were included (mean age, 59 yr); of them, 33% had methicillin-resistant Staphylococcus aureus bacteremia. Forty-seven percent of the methicillin-resistant Staphylococcus aureus strains were staphylococcal cassette chromosome mec IV. Persistent bacteremia occurred in 24% of patients (47/196); they were more likely to die than resolving bacteremia group (28% vs 5%; p < 0.001). Compared with resolving bacteremia group, persistent bacteremia patients had higher initial median levels of tissue necrosis factor (44.73 vs 21.68 pg/mL; p < 0.001), interleukin-8 (124.76 vs 47.48 pg/mL; p = 0.028), and interleukin-10 (104.31 vs 29.72 pg/mL; p < 0.001). Despite 72 hours of treatment, levels remained higher for the persistent bacteremia group than for the resolving bacteremia group (tissue necrosis factor: 26.95 vs 18.38 pg/mL, p = 0.02; interleukin-8: 70.75 vs 27.86 pg/mL, p = 0.002; interleukin-6: 67.50 vs 21.81 pg/mL, p = 0.005; and interleukin-10: 30.98 vs 12.60 pg/mL, p < 0.001). Interleukin-17A levels were similar between groups at both time points. After controlling for confounding variables by multivariate analysis, interleukin-10/tissue necrosis factor ratio at 72 hours most significantly predicted persistence (odds ratio, 2.98; 95% CI, 1.39-6.39; p = 0.005) and mortality (odds ratio, 9.87; 95% CI, 2.64-36.91; p < 0.001) at values more than 1.00 and more than 2.56, respectively. Sustained elevation of interleukin-10/tissue necrosis factor ratio at 72 hours suggests a dysregulated immune response and may be used to guide management to improve outcomes.

Effects of Bacillus subtilis on Epithelial Tight Junctions of Mice with Inflammatory Bowel Disease.

Intestinal mucosal barrier dysfunction associated with inflammatory bowel disease (IBD). Effects of Bacillus subtilis on epithelial tight junctions (TJs) and intrinsic regulatory mechanisms of the intestine were studied in pursuit of better treatments for IBD. Fifty Balb/c mice given 5% dextran sulfate sodium (DSS) in tap water ad libitum over a 7-day period (to induce colitis) were randomly assigned to 4 test groups [DSS, DSS+B. subtilis, DSS+5 amino salicylic acid (5ASA), and DSS+B. subtilis+5ASA] to compare with normal controls. In the test groups DSS was administered daily by oral gavage in normal saline (0.2 mL), adding B. subtilis (1 × 10(8) CFU), 5ASA (6 mg), or both for respective test groups. Defecation, body weight, colitis score, pathological features, epithelial TJs proteins [claudin-1, occludin, junctional adhesion molecule (JAM)-A, and zona occludens (ZO)-1], and various cytokines [interleukin (IL)-6, IL-17, IL-23, and tissue necrosis factor (TNF)-α] were evaluated. Relative to the DSS group, disease activity index scores, and graded histologic damage were all significantly reduced by B. subtilis intake. All parameters declined even further when B. subtilis and 5ASA were combined. Analytic testing (immunohistochemical, western blot, and PCR) revealed progressive increase in TJ protein (claudin-1, occludin, JAM-A, and ZO-1) expression in DSS, DSS+B. subtilis, DSS+5ASA, DSS+B. subtilis+5ASA, and normal control groups (P < 0.05), whereas cytokine (IL-6, IL-17, IL-23, and TNF-α) expression similarly declined (P < 0.05). B. subtilis intake upregulated expression of TJ proteins (claudin-1, occludin, JAM-A, and ZO-1), for improved barrier function, and downregulated cytokine expression (IL-6, IL-17, IL-23, and TNF-α) to reduce intestinal epithelial damage.

Effects of dantrolene on ischemia-reperfusion injury in animal models: a review of outcomes in heart, brain, liver, and kidney.

Ischemia-reperfusion (IR) is the restoration of blood flow to a tissue that was formerly deficient of blood flow. Tissue damage after IR is considered an IR injury (IRI). During IR, there is an increased level of cytosolic calcium ([Ca(2+)]i) due to the release of calcium from mitochondrial, sarcoendoplasmic reticulum, and nuclear organelles. Dantrolene sodium (dantrolene) is a 1-[[[5-(4-nitrophenol)-2-furanyl]methylene]amino]-2, 4-imidazolidinedione sodium salt with a nonspecific mechanism, inhibiting organelle release of Ca(2+) into the cytosol. This work reviews the outcomes of administering dantrolene in brain, heart, liver, and kidney animal models of IRI. An extensive PubMed, MEDLINE, and MEDLAR literature review during the last 30 years on the effect of dantrolene in IRI in animal models was analyzed to determine the clinical implications of this important study. Particular attention was given to dantrolene in heart, brain, liver, and kidney IRI. Heart: Nine studies of heart IRI were reviewed and include an in vivo dog model (n = 1), in vivo rabbit model (n = 1), isolated dog myocardial fibers (n = 1), and isolated rat hearts (n = 6). Four studies showed decreased infarct size and increased cardiac function after IRI. One in vivo rabbit study found no difference in infarct size or cardiac function after IRI versus controls. Dantrolene may be protective or inductive of post-IRI arrhythmias depending on preestablished myocyte cycling times. Brain: Nine studies of brain IRI were reviewed and include an in vivo dog model (n = 1), in vivo gerbil model (n = 2), and in vivo rat models (n = 6). Dantrolene shows protective decreases in apoptotic markers in 6 studies, but it shows no effect on the necrotic core and mixed effects on reduction of infarct volume. One study found increased mortality in the dantrolene group. Liver: One study of in vivo rat liver IRI found that dantrolene decreased liver function tests, tissue necrosis factor α, tissue necrosis, and increased interleukin 10. Kidney: One study of in vivo rat kidney IRI showed that dantrolene had no effect. Dantrolene shows protective effects in animal models of heart, brain, and potentially liver IRI, reinforcing the importance of calcium homeostasis during IRI. Variations of dose, timing of administration, route of administration, and outcomes between studies make definitive conclusions difficult. The nonspecific mechanism of action of dantrolene may also account for the variation among studies. Lack of studies in the liver and kidney makes any consensus in these organs premature, and thus, emphasis for this review was put on studies of the heart and brain.

Xanthii fructus inhibits inflammatory responses in LPS-stimulated RAW 264.7 macrophages through suppressing NF-κB and JNK/p38 MAPK

Ethnopharmacological relevanceXanthii fructus (XF) has long been used to treat a variety of inflammatory conditions in Korean traditional medicine, but the underlying mechanisms that could explain the anti-inflammatory actions of XF remain largely unknown. Aim of the studyThis study aimed to elucidate the anti-inflammatory effects of X. fructus (XF) and to examine its underlying molecular mechanisms in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Materials and methodsThe effect of XF on LPS-induced mRNA and protein expressions of inflammatory mediators and cytokines were determined. Moreover, the activation of the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways and the expression of heme oxygenase-1 (HO-1) were explored to elucidate the anti-inflammatory mechanisms. ResultsXF significantly inhibited LPS-induced production of inflammatory mediators, interleukin-6 (IL-6), nitric oxide (NO), and prostaglandin E2 (PGE2), without any cytotoxicity. However, it did not affect tissue necrosis factor (TNF)-α or IL-1β production in LPS-stimulated RAW 264.7 cells. Expression levels of inducible nitric oxide synthase (iNOS) mRNA and protein were inhibited dose-dependently by XF in LPS-stimulated RAW 264.7 cells, but there were no changes in cyclooxygenase-2 (COX-2) mRNA and protein. XF significantly attenuated LPS-induced phosphorylation and degradation of inhibitory kappa Bα (IκBα) and consequently reduced the nuclear translocation of p65 NF-κB. Pretreatment with XF also strongly inhibited the LPS-induced phosphorylation of p38 kinase and JNK, whereas the phosphorylation of ERK1/2 was not affected. In addition, XF led to an increase in HO-1 expression. ConclusionTaken together, our findings support that XF inhibits LPS-induced inflammatory responses by blocking NF-κB activation, inhibiting JNK/p38 MAPK phosphorylation, and enhancing HO-1 expression in macrophages, suggesting that it could be an attractive therapeutic candidate for various inflammatory diseases.

1,25-Dihydroxyvitamin D Protects Intestinal Epithelial Barrier by Regulating the Myosin Light Chain Kinase Signaling Pathway.

The myosin light chain kinase (MLCK) pathway controls intestinal epithelial barrier permeability by regulating the tight junction. 1,25-dihydroxyvitamin D (1,25(OH)2D3)-vitamin D receptor (VDR) signaling protects the epithelial barrier, but the molecular mechanism is incompletely understood. MLCK activation and barrier permeability were studied using monolayers of HCT116, Caco-2, and SW480 cells treated with tissue necrosis factor α with or without 1,25(OH)2D3. The MLCK pathway was analyzed in normal and inflamed colonic biopsies from patients with ulcerative colitis. Colonic mucosal barrier permeability and MLCK activation were also investigated using trinitrobenzene sulfonic acid-induced colitis models in vitamin D analog paricalcitol-treated wild-type mice and mice carrying VDR deletion in colonic epithelial cells. Tissue necrosis factor α increased cell monolayer permeability and induced long isoform of MLCK expression and myosin II regulatory light chain (MLC) phosphorylation, and 1,25(OH)2D3 blocked tissue necrosis factor α-induced increases in monolayer permeability and MLCK-MLC pathway activation by a VDR-dependent fashion. 1,25(OH)2D3 directly suppressed long MLCK expression by attenuating NF-κB activation, and chromatin immunoprecipitation assays confirmed that 1,25(OH)2D3 disrupted p65 binding to 3 κB sites in long MLCK gene promoter. In human ulcerative colitis biopsies, VDR reduction was associated with increases in long MLCK expression and MLC phosphorylation. In trinitrobenzene sulfonic acid colitis models, paricalcitol ameliorated colitis, attenuated the increase in mucosal barrier permeability, and inhibited long MLCK induction and MLC phosphorylation. In contrast, mice with colonic epithelial VDR deletion exhibited more robust increases in mucosal barrier permeability and MLCK activation compared with wild-type mice. These data demonstrate that 1,25(OH)2D3-VDR signaling preserves the mucosal barrier integrity by abrogating MLCK-dependent tight junction dysregulation during colonic inflammation.

Interleukin-23 Receptor Gene Polymorphism May Enhance Expression of the IL-23 Receptor, IL-17, TNF-α and IL-6 in Behcet's Disease.

PurposeRecent studies identified an association between Behcet’s disease (BD) and the IL-23R gene polymorphism (rs17375018) in different populations. This study examined whether this IL-23R gene polymorphism is associated with enhanced inflammatory responses.MethodsWe recruited 27 BD patients and 32 controls with three genotypes. Peripheral blood mononuclear cells (PBMCs) were seeded with or without anti-CD3 and CD28. Cells were incubated for 24 hours, and then supernatants were collected and stored at −20◦C until analyzed. Levels of interferon (IFN)-γ, tissue necrosis factor (TNF)-α, interleukin (IL)-17 and IL-6 were detected by ELISA. IL-23R expression was assessed by quantitative real-time polymerase chain reaction (RT-PCR).ResultsThe expression of IL-23R was significantly higher in both BD patients and healthy controls with the GG genotype compared to the AG and AA genotype with anti-CD3 and CD28 stimulation (all P-value < 0.05). Among the PBMCs cultured with anti-CD3 and CD28 stimulation, there was an elevated secretion of TNF-α, IL-6 and IL-17 in BD patients and healthy controls with the GG genotype. However, there was no significant change in secretion of IFN- γ in BD patients and healthy controls among the genotype of this IL-23R gene polymorphism.ConclusionsThe results suggest that the GG genotype of the rs17375018 variant in the IL-23R gene enhances pro-inflammatory cytokine responses.

Phagocytosed Clofazimine Biocrystals Can Modulate Innate Immune Signaling by Inhibiting TNFα and Boosting IL-1RA Secretion.

Clofazimine (CFZ) is an FDA-approved leprostatic and anti-inflammatory drug that massively accumulates in macrophages, forming insoluble, intracellular crystal-like drug inclusions (CLDIs) during long-term oral dosing. Interestingly, when added to cells in vitro, soluble CFZ is cytotoxic because it depolarizes mitochondria and induces apoptosis. Accordingly, we hypothesized that, in vivo, macrophages detoxify CFZ by sequestering it in CLDIs. To test this hypothesis, CLDIs of CFZ-treated mice were biochemically isolated and then incubated with macrophages in vitro. The cell biological effects of phagocytosed CLDIs were compared to those of soluble CFZ. Unlike soluble CFZ, phagocytosis of CLDIs did not lead to mitochondrial destabilization or apoptosis. Rather, CLDIs altered immune signaling response pathways downstream of Toll-like receptor (TLR) ligation, leading to enhanced interleukin-1 receptor antagonist (IL-1RA) production, dampened NF-κB activation and tissue necrosis factor alpha (TNFα) production, and ultimately decreased TLR expression levels. In aggregate, our results constitute evidence that macrophages detoxify soluble CFZ by sequestering it in a biocompatible, insoluble form. The altered cellular response to TLR ligation suggests that CLDI formation may also underlie CFZ's anti-inflammatory activity.

Glucosylceramide attenuates the inflammatory mediator expression in lipopolysaccharide-stimulated RAW264.7 cells

The positive effect of glucosylceramide (GlcCer) on skin conditions is well known. Recently, there has been increasing interest in the potential antiinflammatory effects of GlcCer due to its efficacy in relieving atopic skin symptoms. However, the role of GlcCer in inflammation has not been investigated completely. Thus, we hypothesized that GlcCer might exhibit the antiinflammatory effects through the inhibition of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling. To test this hypothesis, the antiinflammatory effects and signaling mechanisms of GlcCer were investigated in lipopolysaccharide (LPS)-induced RAW 264.7 cells. We report that GlcCer inhibited messenger RNA and protein expression of tissue necrosis factor α and interleukin 1β without cytotoxicity. However, it did not affect interleukin 6 production in LPS-stimulated RAW 264.7 macrophages. Glucosylceramide also suppressed prostaglandin E2 but not nitric oxide production, consistent with its inhibition of cyclooxygenase 2 but not of inducible nitric oxide synthase expression. The molecular mechanism of GlcCer-mediated inhibition of LPS-induced inflammation in RAW 264.7 cells is closely related to suppression of NF-κB p65 subunit nuclear translocation as well as to phosphorylation of extracellular signal-regulated kinase and, in particular, p38 MAPK. In addition, GlcCer did not affect c-Jun N-terminal kinase phosphorylation. In conclusion, GlcCer inhibits LPS-induced inflammation by blocking the nuclear translocation of NF-κB and inhibiting the phosphorylation of extracellular signal-regulated kinase/p38 MAPK pathways in macrophages, suggesting that it might be a promising potential drug candidate for various inflammatory diseases.

Cytokines, MMP-2, and MMP-9 levels in patients with a solitary cysticercus granuloma.

In patients with neurocysticercosis, perilesional inflammatory activity is thought to be responsible for seizures. This study was designed to evaluate the serum and cerebrospinal fluid (CSF) concentrations of cytokines as well as matrix metalloproteinase (MMP)-2 and MMP-9 in patients with a solitary cysticercus granuloma. The study included 47 patients suffering from seizures in whom a solitary cysticercus granuloma was detected on a computed tomography (CT) scan. The study also included 47 control subjects. Their serum and cerebrospinal fluid (CSF) was analysed for cytokines and MMP levels. A follow-up CT was performed after 6 months. The median levels of cytokines, interleukin (IL)-1β, IL-5, IL-6, IL-10 and tissue necrosis factor (TNF)-α, MMP-2 and MMP-9 were significantly elevated, both in the serum and CSF of patients having an intracerebral solitary cysticercus granuloma, in comparison to that of controls. The follow-up CT revealed that in 27 patients, the lesions were calcified and in 5 patients, there was complete resolution of the lesions. In 15 patients, the lesions remained unchanged. Higher baseline CSF MMP-2 and TNF-α level were seen in patients with persisting lesions. Higher serum baseline MMP-2, IL-6 and a low CSF IL-10 level were seen in patients with complete resolution of the granuloma. A high baseline IL-1β level was associated with a calcified lesion. Fourteen patients had recurrence of seizures. A high baseline serum TNF-α level was independently associated with seizure recurrence (P = 0.021, OR = 1.041, CI = 1.006 to 1.078). In patients with a solitary cysticercus granuloma, cytokines and matrix metalloproteinases in the CSF and serum are elevated. Different patterns of immunological changes were observed in patients following resolution or calcification of the lesion.

Adipose tissue remodeling in a novel domestic porcine model of diet-induced obesity

ObjectiveTo establish and characterize a novel domestic porcine model of obesity.Design and MethodsFourteen domestic pigs were fed normal (lean, n=7) or high-fat/high-fructose diet (obese, n=7) for 16 weeks. Subcutaneous abdominal adipose tissue biopsies were obtained after 8, 12 and 16 weeks of diet, and pericardial adipose tissue after 16 weeks, for assessments of adipocyte size, fibrosis, and inflammation. Adipose tissue volume and cardiac function were studied with multi-detector computed-tomography, and oxygenation with magnetic resonance imaging. Plasma lipids profiles, insulin resistance, and markers of inflammation were evaluated.ResultsCompared with lean, obese pigs had elevated cholesterol and triglycerides levels, blood pressure, and insulin resistance. Both abdominal and pericardial fat volume increased after 16 weeks of obese. In abdominal subcutaneous adipose tissue, adipocyte size and both tumor necrosis factor (TNF)-α expression progressively increased. Macrophage infiltration showed in both abdominal and pericardial adipose tissues. Circulating TNF-α increased in obese only at 16 weeks. Compared with Lean, obese pigs had similar global cardiac function, but myocardial perfusion and oxygenation were significantly impaired.ConclusionA high-fat/high-fructose diet induces in domestic pigs many characteristics of metabolic syndrome, which is useful to investigate the effects of the obesity.