Evidence for the prevention of enthesitis in HLA‐B27/hβ2m transgenic rats treated with a monoclonal antibody against TNF‐α

Transgenic rats with high expression of HLA‐B27 and human β₂‐microglobulin (B27TR) develop a multisystem inflammatory disease resembling human inflammatory bowel disease (IBD) and spondyloarthropaties (SpA). Tumour necrosis factor α (TNF‐α) has a crucial role in sustaining chronic inflammation in the gut and joints. The aim of this work was to evaluate whether TNF‐α blockade could prevent or reduce the inflammation of peripheral joints in B27TR. A first group of 9‐week‐old B27TR received an anti‐TNF‐α monoclonal antibody (mAb) or an isotypic IgG2a,k up to the age of 18 weeks. An untreated group was monitored up to the age of 18 weeks and then randomly assigned to a 9‐week treatment with anti‐TNF‐α mAb or IgG2a,k. Each rat was monitored for clinical IBD and peripheral joint manifestations. After sacrifice the colon and hind paws were examined for macroscopical and microscopical pathological changes. Early TNF‐α blockade prevented, and late treatment improved IBD signs in B27TR. Erythema, oedema, inflammatory infiltrate close to the tendons and enthesis, proliferating chondrocyte‐like cells, signs of new endochondral bone ossification and bone erosion were observed in peripheral joints of four out of six IgG2a,k‐treated B27TR, both at 18 and 27 weeks. Immunopositivity for phosphorylated Smad1/5/8 indicated that the process of joint remodelling was activated in B27TR. Some entheses showed chondroid nodules. Anti‐TNF‐α treatment reduced inflammation and preserved the enthesis organization in most animals. Occasional and transient erythema and oedema were still present in three of six of the late anti‐TNF‐α‐treated animals. Smad1/5/8 signalling was not inhibited by late anti‐TNF‐α treatment. In B27TR, articular involvement follows IBD onset and develops at entheses. Early TNF‐α blockade prevents the onset of IBD and consequently the development of enthesitis in peripheral joints in the B27TR model of human SpA.     

TNF-α inhibits aquaporin 5 expression in human salivary gland acinar cells via suppression of histone H4 acetylation

Sjögren's syndrome is a systemic autoimmune disease characterized by reductions in salivary and lacrimal secretions. The mechanisms underlying these reductions remain unclear. We have previously shown that TNF‐α plays an important role in the destruction of acinar structures. Here we examined TNF‐α's function in the expression of aquaporin (AQP) 5 in human salivary gland acinar cells. Immortalized human salivary gland acinar (NS‐SV‐AC) cells were treated with TNF‐α, and then the expression levels of AQP5 mRNA and protein were analysed. In addition, the mechanisms underlying the reduction of AQP5 expression by TNF‐α treatment were investigated. TNF‐α‐treatment of NS‐SV‐AC cells significantly suppressed the expression levels of AQP5 mRNA and protein, and reduced the net fluid secretion rate. We examined the expression and activation levels of DNA methyltransferases (Dnmts) in NS‐SV‐AC cells treated with TNF‐α. However, no significant changes were observed in the expression or activation levels of Dnmt1, Dnmt3a or Dnmt3b. Although we also investigated the role of NF‐κB activity in the TNF‐α‐induced suppression of AQP5 expression in NS‐SV‐AC cells, we detected similar TNF‐α suppression of AQP5 expression in non‐transfected cells and in a super‐repressor form of IκBα cDNA‐transfected cell clones. However, interestingly, chromatin immunoprecipitation analysis demonstrated a remarkable decrease in levels of acetylated histone H4 associated with the AQP5 gene promoter after treatment with TNF‐α in NS‐SV‐AC cells. Therefore, our results may indicate that TNF‐α inhibition of AQP5 expression in human salivary gland acinar cells is due to the epigenetic mechanism by suppression of acetylation of histone H4.     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.     

Integrin‐linked kinase is involved in TNF‐α‐induced inducible nitric‐oxide synthase expression in myoblasts

Tumor necrosis factor‐α (TNF‐α) is a pleiotropic cytokine produced by activated macrophages. Nitric oxide (NO) is a highly reactive nitrogen radical implicated in inflammatory responses. We investigated the signaling pathway involved in inducible nitric oxide synthase (iNOS) expression and NO production stimulated by TNF‐α in cultured myoblasts. TNF‐α stimulation caused iNOS expression and NO production in myoblasts (G7 cells). TNF‐α‐mediated iNOS expression was attenuated by integrin‐linked kinase (ILK) inhibitor (KP392) and siRNA. Pretreatment with Akt inhibitor, mammalian target of rapamycin (mTOR) inhibitor (rapamycin), NF‐κB inhibitor (PDTC), and IκB protease inhibitor (TPCK) also inhibited the potentiating action of TNF‐α. Stimulation of cells with TNF‐α increased ILK kinase activity. TNF‐α also increased the Akt and mTOR phosphorylation. TNF‐α mediated an increase of NF‐κB‐specific DNA–protein complex formation, p65 translocation into nucleus, NF‐κB‐luciferase activity was inhibited by KP392, Akt inhibitor, and rapamycin. Our results suggest that TNF‐α increased iNOS expression and NO production in myoblasts via the ILK/Akt/mTOR and NF‐κB signaling pathway. J. Cell. Biochem. 109: 1244–1253, 2010. © 2010 Wiley‐Liss, Inc.     

Apoptosis signal-regulating kinase 1 is mediated in TNF-α-induced CCL2 expression in human synovial fibroblasts

Tumor necrosis factor‐α (TNF‐α), a pro‐inflammatory cytokine with a critical role in osteoarthritis (OA), was primarily produced by monocytes/macrophages and plays a crucial role in the inflammatory response. Here, we investigated the intracellular signaling pathways involved in TNF‐α‐induced monocyte chemoattractant protein 1 (MCP‐1)/CCL2 expression in human synovial fibroblast cells. Stimulation of synovial fibroblasts (OASF) with TNF‐α induced concentration‐ and time‐dependent increases in CCL2 expression. TNF‐α‐mediated CCL2 production was attenuated by TNFR1 monoclonal antibody (Ab). Pretreatment with an apoptosis signal‐regulating kinase 1 (ASK1) inhibitor (thioredoxin), JNK inhibitor (SP600125), p38 inhibitor (SB203580), or AP‐1 inhibitor (curcumin or tanshinone IIA) also blocked the potentiating action of TNF‐α. Stimulation of cells with TNF‐α enhanced ASK1, JNK, and p38 activation. Treatment of OASF with TNF‐α also increased the accumulation of phosphorylated c‐Jun in the nucleus, AP‐1‐luciferase activity, and c‐Jun binding to the AP‐1 element on the CCL2 promoter. TNF‐α‐mediated AP‐1‐luciferase activity and c‐Jun binding to the AP‐1 element were inhibited by TNFR1 Ab, thioredoxin, SP600125, and SB203580. Our results suggest that the interaction between TNF‐α and TNFR1 increases CCL2 expression in human synovial fibroblasts via the ASK1, JNK/p38, c‐Jun, and AP‐1 signaling pathway. J. Cell. Biochem. 113: 3509–3519, 2012. © 2012 Wiley Periodicals, Inc.     

miR‐124a inhibits the proliferation and inflammation in rheumatoid arthritis fibroblast‐like synoviocytes via targeting PIK3/NF‐κB pathway

Abnormal hyperplasia of fibroblast‐like synoviocytes (FLS) leads to the progression of rheumatoid arthritis (RA). This study aimed to investigate the role of miR‐124a in the pathogenesis of RA. The viability and cell cycle of FLS in rheumatoid arthritis (RAFLS) were evaluated by Cell Counting Kit 8 and flow cytometry assay. The expression of PIK3CA, Akt, and NF‐κB in RAFLS was examined by real‐time PCR and Western blot analysis. The production of tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 was detected by ELISA. The joint swelling and inflammation in collagen‐induced arthritis (CIA) mice were examined by histological and immunohistochemical analysis. We found that miR‐124a suppressed the viability and proliferation of RAFLS and increased the percentage of cells in the G1 phase. miR‐124a suppressed PIK3CA 3'UTR luciferase reporter activity and decreased the expression of PIK3CA at mRNA and protein levels. Furthermore, miR‐124a inhibited the expression of the key components of the PIK3/Akt/NF‐κB signal pathway and inhibited the expression of pro‐inflammatory factors TNF‐α and IL‐6. Local overexpression of miR‐124a in the joints of CIA mice inhibited inflammation and promoted apoptosis in FLS by decreasing PIK3CA expression. In conclusion, miR‐124a inhibits the proliferation and inflammation in RAFLS via targeting PIK3/NF‐κB pathway. miR‐124a is a promising therapeutic target for RA.     

Cortisol is associated with low frequency of interleukin 10–producing B cells in patients with atherosclerosis

It is accepted that inflammation plays a critical role in the development of atherosclerosis; the pathogenesis is not clear. B‐cell–produced interleukin (IL) 10 is an immune regulatory cytokine that can inhibit immune inflammation. This study tests a hypothesis that a psychological stress hormone, cortisol, suppresses IL‐10 expression in peripheral B cells of patients with atherosclerosis. Peripheral blood samples were collected from patients with coronary artery atherosclerosis. B cells were isolated from the blood samples to be analyzed for the expression of IL‐10 and micro RNA (miR) 98 by real‐time polymerase chain reaction. We observed that the frequency of IL‐10⁺ B cell was less in patients with atherosclerosis than healthy controls. The serum cortisol levels were higher in the patients than that in healthy controls. Peripheral B‐cell frequency was negatively correlated with the serum cortisol levels. Exposure of B cells to cortisol increased the expression of miR‐98 in B cells. Cortisol also inhibited the expression of IL‐10 in B cells, in which miR‐98 played a critical role. Treating B cells from atherosclerosis patients with anti–miR‐98 liposomes reversed the ability of expression of IL‐10 in the cells. The expression of IL‐10 is suppressed in peripheral B cells, which can be up regulated by anti–miR‐98 liposomes.     

Novel role of the clustered miR‐23b‐3p and miR‐27b‐3p in enhanced expression of fibrosis‐associated genes by targeting TGFBR3 in atrial fibroblasts

Atrial fibrillation (AF) is the most common type of arrhythmia in cardiovascular diseases. Atrial fibrosis is an important pathophysiological contributor to AF. This study aimed to investigate the role of the clustered miR‐23b‐3p and miR‐27b‐3p in atrial fibrosis. Human atrial fibroblasts (HAFs) were isolated from atrial appendage tissue of patients with sinus rhythm. A cell model of atrial fibrosis was achieved in Ang‐II‐induced HAFs. Cell proliferation and migration were detected. We found that miR‐23b‐3p and miR‐27b‐3p were markedly increased in atrial appendage tissues of AF patients and in Ang‐II‐treated HAFs. Overexpression of miR‐23b‐3p and miR‐27b‐3p enhanced the expression of collagen, type I, alpha 1 (COL1A1), COL3A1 and ACTA2 in HAFs without significant effects on their proliferation and migration. Luciferase assay showed that miR‐23b‐3p and miR‐27b‐3p targeted two different sites in 3?‐UTR of transforming growth factor (TGF)‐β1 receptor 3 (TGFBR3) respectively. Consistently, TGFBR3 siRNA could increase fibrosis‐related genes expression, along with the Smad1 inactivation and Smad3 activation in HAFs. Additionally, overexpression of TGFBR3 could alleviate the increase of COL1A1, COL3A1 and ACTA2 in HAFs after transfection with miR‐23b‐3p and miR‐27b‐3p respectively. Moreover, Smad3 was activated in HAFs in response to Ang‐II treatment and inactivation of Smad3 attenuated up‐regulation of miR‐23b‐3p and miR‐27b‐3p in Ang‐II‐treated HAFs. Taken together, these results suggest that the clustered miR‐23b‐3p and miR‐27b‐3p consistently promote atrial fibrosis by targeting TGFBR3 to activate Smad3 signalling in HAFs, suggesting that miR‐23b‐3p and miR‐27b‐3p are potential therapeutic targets for atrial fibrosis.     

SIRT3, PP2A and TTP protein stability in the presence of TNF‐α on vincristine‐induced apoptosis of leukaemia cells

The contribution of vincristine (VCR)‐induced microtubule destabilization to evoke apoptosis in cancer cells remains to be resolved. Thus, we investigated the cytotoxic mechanism of VCR on U937 and HL‐60 human leukaemia cell lines. We discovered that VCR treatment resulted in the up‐regulation of TNF‐α expression and activation of the death receptor pathway, which evoked apoptosis of U937 cells. Moreover, VCR induced microtubule destabilization and mitotic arrest. VCR treatment down‐regulated SIRT3, and such down‐regulation caused mitochondrial ROS to initiate phosphorylation of p38 MAPK. p38 MAPK suppressed MID1‐modulated degradation of the protein phosphatase 2A (PP2A) catalytic subunit. The SIRT3‐ROS‐p38 MAPK‐PP2A axis inhibited tristetraprolin (TTP)‐controlled TNF‐α mRNA degradation, consequently, up‐regulating TNF‐α expression. Restoration of SIRT3 and TTP expression, or inhibition of the ROS‐p38 MAPK axis increased the survival of VCR‐treated cells and repressed TNF‐α up‐regulation. In contrast to suppression of the ROS‐p38 MAPK axis, overexpression of SIRT3 modestly inhibited the effect of VCR on microtubule destabilization and mitotic arrest in U937 cells. Apoptosis of HL‐60 cells, similarly, went through the same pathway. Collectively, our data indicate that the SIRT3‐ROS‐p38 MAPK‐PP2A‐TTP axis modulates TNF‐α expression, which triggers apoptosis of VCR‐treated U937 and HL‐60 cells. We also demonstrate that the apoptotic signalling is not affected by VCR‐elicited microtubule destabilization.     

Rac1 activation induces tumour necrosis factor‐α expression and cardiac dysfunction in endotoxemia

Induction of tumour necrosis factor‐α (TNF‐α) expression leads to myocardial depression during sepsis. However, the underlying molecular mechanisms are not fully understood. The aim of this study was to investigate the role of Rac1 in TNF‐α expression and cardiac dysfunction during endotoxemia and to determine the involvement of phosphoinositide‐3 kinase (PI3K) in lipopolysaccharide (LPS)‐induced Rac1 activation. Our results showed that LPS‐induced Rac1 activation and TNF‐α expression in cultured neonatal mouse cardiomyocytes. The response was inhibited in Rac1 deficient cardiomyocytes or by a dominant‐negative Rac1 (Rac1N17). To determine whether PI3K regulates Rac1 activation, cardiomyocytes were treated with LY294002, a PI3K selective inhibitor. Treatment with LY294002 decreased Rac1 activity as well as TNF‐α expression stimulated by LPS. Furthermore, inhibition of PI3K and Rac1 activity decreased LPS‐induced superoxide generation which was associated with a significant reduction in ERK1/2 phosphorylation. To investigate the role of Rac1 in myocardial depression during endotoxemia in vivo, wild‐type and cardiomyocyte‐specific Rac1 deficient mice were treated with LPS (2 mg/kg, i.p.). Deficiency in Rac1 significantly decreased myocardial TNF‐α expression and improved cardiac function during endotoxemia. We conclude that PI3K‐mediated Rac1 activation is required for induction of TNF‐α expression in cardiomyocytes and cardiac dysfunction during endotoxemia. The effect of Rac1 on TNF‐α expression seems to be mediated by increased NADPH oxidase activity and ERK1/2 phosphorylation.     

Soluble CXCL16 promotes TNF‐α‐induced apoptosis in DLBCL via the AMAD10‐NF‐κB regulatory feedback loop

We had previously identified that the co‐expression of transmembrane CXCL16 (TM‐CXCL16) and its receptor CXCR6 is an independent risk factor for poor survival in patients with diffuse large B‐cell lymphoma (DLBCL). However, the impact of the soluble form of CXCL16 (sCXCL16) on the pathogenesis of DLBCL remains unknown. In the present study, the synergistic effect of sCXCL16 and tumor necrosis factor α (TNF‐α) on apoptosis in DLBCL cell lines (OCI‐LY8 and OCI‐LY10) was investigated in vitro. sCXCL16 reinforced TNF‐α‐mediated inhibition of DLBCL cell proliferation, as determined by the cell counting kit‐8 assay. The results of annexin V staining showed that sCXCL16 enhanced TNF‐α‐induced apoptosis in OCI‐LY8 and OCI‐LY10 cells through a death receptor‐caspase signaling pathway. The results of gene microarray suggested a significant upregulation of differentially expressed genes in the TNF signaling pathway. sCXCL16 increased the concentration of extracellular TNF‐α by binding to CXCR6 to activate the nuclear factor‐κB (NF‐κB) signaling pathway. TNF‐α also induced the secretion of sCXCL16 by increasing the expression of ADAM10, which is known to cleave TM‐CXCL16 to yield sCXCL16. Moreover, bioinformatics analysis revealed that elevated TNF‐α and ADAM10 expression levels in tumor tissues predicted better survival in patients with DLBCL. Thus, our study suggests that sCXCL16 enhances TNF‐α‐induced apoptosis of DLBCL cells, which may involve a positive feedback loop consisting of TNF‐α, ADAM10, sCXCL16, and members of the NF‐κB pathway. sCXCL16 and TNF‐α may be used as prognostic markers in the clinic, and their combinational use is a promising approach in the context of DLBCL therapy.     

Tumour necrosis factor‐α promotes liver ischaemia‐reperfusion injury through the PGC‐1α/Mfn2 pathway

Tumour necrosis factor (TNF)‐α has been considered to induce ischaemia‐reperfusion injury (IRI) of liver which is characterized by energy dysmetabolism. Peroxisome proliferator–activated receptor‐γ co‐activator (PGC)‐1α and mitofusion2 (Mfn2) are reported to be involved in the regulation of mitochondrial function. However, whether PGC‐1α and Mfn2 form a pathway that mediates liver IRI, and if so, what the underlying involvement is in that pathway remain unclear. In this study, L02 cells administered recombinant human TNF‐α had increased TNF‐α levels and resulted in down‐regulation of PGC‐1α and Mfn2 in a rat liver IRI model. This was associated with hepatic mitochondrial swelling, decreased adenosine triphosphate (ATP) production, and increased levels of reactive oxygen species (ROS) and alanine aminotransferase (ALT) activity as well as cell apoptosis. Inhibition of TNF‐α by neutralizing antibody reversed PGC‐1α and Mfn2 expression, and decreased hepatic injury and cell apoptosis both in cell culture and in animals. Treatment by rosiglitazone sustained PGC‐1α and Mfn2 expression both in IR livers, and L02 cells treated with TNF‐α as indicated by increased hepatic mitochondrial integrity and ATP production, reduced ROS and ALT activity as well as decreased cell apoptosis. Overexpression of Mfn2 by lentiviral‐Mfn2 transfection decreased hepatic injury in IR livers and L02 cells treated with TNF‐α. However, there was no up‐regulation of PGC‐1α. These findings suggest that PGC‐1α and Mfn2 constitute a regulatory pathway, and play a critical role in TNF‐α‐induced hepatic IRI. Inhibition of the TNF‐α or PGC‐1α/Mfn2 pathways may represent novel therapeutic interventions for hepatic IRI.