Differential regulation of IFNα, IFNβ and IFNε gene expression in human cervical epithelial cells

Interferonε (IFNε) is a unique type I IFN that has distinct functions from IFNα/β. IFNε is constitutively expressed at mucosal tissues, including the female genital mucosa, and is reported to be modulated by estrogen and seminal plasma. However, its regulation by cytokines, including TNFα, IL-1β, IL-6, IL-8, IL-17, IL-22 and IFNα, which are commonly present in the female genital mucosa, is not well documented in freshly isolated primary cervical cells from tissues. We determined the effect of these cytokines on gene expression of type I IFNs in an immortalized endocervical epithelial cell line (A2EN) and in primary cervical epithelial cells. Several pro-inflammatory cytokines were found to induce IFNε, and TNFα induced the strongest response in both cell types. Pretreatment of cells with the IκB inhibitor, which blocks the NF-κB pathway, suppressed TNFα-mediated IFNε gene induction and promoter activation. Expression of IFNα, IFNβ, and IFNε was differentially regulated in response to various cytokines. Taken together, our results show that regulation of these IFNs depends on cell type, cytokine concentration, and incubation time, highlighting the complexity of the cytokine network in the cervical epithelium.     

Lymphotoxin α stimulates proliferation and pro-inflammatory cytokine secretion of rheumatoid arthritis synovial fibroblasts

ObjectiveTNFα plays a crucial role in rheumatoid arthritis (RA) by stimulating fibroblast-like synoviocytes (FLS). Lymphotoxin α (LTα) is a pro-inflammatory cytokine with significant homology to TNFα. We compared the effects of both cytokines on cultured RA FLS.MethodsReceptor expression on RA FLS was analyzed by FACS. Cells were stimulated with LTα or TNFα and proliferation was measured by [3H]thymidine incorporation and secretion of inflammatory cytokines and metalloproteinase 3 by ELISA. Activation of MAP kinases and Akt was analyzed by Western blotting. Nuclear translocation of NFκB was visualized by immunofluorescence.Results60–80% and 30–50% of the RA FLS tested expressed TNF receptors I and II, respectively, and 70–75% expressed HVEM. LTα induced RA FLS proliferation at the same level of TNFα, which was blocked by etanercept. Both LTα and TNFα induced activation of MAP kinases ERK1/2 and p38 as well as Akt. 95–98% of FLS showed nuclear translocation of NFκB after stimulation with either cytokines. LTα and TNFα were potent to induce secretion of IL-6, IL-8 and metalloproteinase 3 in FLS.ConclusionLTα is as effective as TNFα in stimulating RA FLS. Blocking both cytokines might allow a better control of inflammation and synovial proliferation in RA.     

IL-17A injury to retinal ganglion cells is mediated by retinal Müller cells in diabetic retinopathy

Diabetic retinopathy (DR), the most common and serious ocular complication, recently has been perceived as a neurovascular inflammatory disease. However, role of adaptive immune inflammation driven by T lymphocytes in DR is not yet well elucidated. Therefore, this study aimed to clarify the role of interleukin (IL)-17A, a proinflammatory cytokine mainly produced by T lymphocytes, in retinal pathophysiology particularly in retinal neuronal death during DR process. Ins2^Akita (Akita) diabetic mice 12 weeks after the onset of diabetes were used as a DR model. IL-17A-deficient diabetic mice were obtained by hybridization of IL-17A-knockout (IL-17A-KO) mouse with Akita mouse. Primarily cultured retinal Müller cells (RMCs) and retinal ganglion cells (RGCs) were treated with IL-17A in high-glucose (HG) condition. A transwell coculture of RGCs and RMCs whose IL-17 receptor A (IL-17RA) gene had been silenced with IL-17RA-shRNA was exposed to IL-17A in HG condition and the cocultured RGCs were assessed on their survival. Diabetic mice manifested increased retinal microvascular lesions, RMC activation and dysfunction, as well as RGC apoptosis. IL-17A-KO diabetic mice showed reduced retinal microvascular impairments, RMC abnormalities, and RGC apoptosis compared with diabetic mice. RMCs expressed IL-17RA. IL-17A exacerbated HG-induced RMC activation and dysfunction in vitro and silencing IL-17RA gene in RMCs abolished the IL-17A deleterious effects. In contrast, RGCs did not express IL-17RA and IL-17A did not further alter HG-induced RGC death. Notably, IL-17A aggravated HG-induced RGC death in the presence of intact RMCs but not in the presence of RMCs in which IL-17RA gene had been knocked down. These findings establish that IL-17A is actively involved in DR pathophysiology and particularly by RMC mediation it promotes RGC death. Collectively, we propose that antagonizing IL-17RA on RMCs may prevent retinal neuronal death and thereby slow down DR progression.Subject terms: Cell death, Medical research     

Dietary supplementation with α-tocopherol reduces neuroinflammation and neuronal degeneration in the rat brain after kainic acid-induced status epilepticus

Abstract

Vitamin E (as α-tocopherol, α-T) is proposed to alleviate glia-mediated inflammation in neurological diseases, but such a role in epilepsy is still elusive. This study investigated the effect of α-T supplementation on glial activation, neuronal cell death and oxidative stress of rat brain exposed to kainate-induced seizures. Animals were fed for 2 weeks with a α-T-enriched diet (estimated intake of 750 mg/kg/day) before undergoing status epilepticus. Compliance to supplementation was demonstrated by the remarkable increase in brain α-T. Four days after seizure, brain α-T returned to baseline and lipid peroxidation markers decreased as compared to non-supplemented rats. Status epilepticus induced a lower up-regulation of astrocytic and microglial antigens (GFAP and MHC II, respectively) and production of pro-inflammatory cytokines (IL-1β and TNF-α) in supplemented than in non-supplemented animals. This anti-inflammatory effect was associated with a lower neuronal cell death. In conclusion, α-T dietary supplementation prevents oxidative stress, neuroglial over-activation and cell death occurring after kainate-induced seizures. This evidence paves the way to an anti-inflammatory and neuroprotective role of α-T interventions in epilepsy.     

Stage-specific expression of TNFα regulates bad/bid-mediated apoptosis and RIP1/ROS-mediated secondary necrosis in Birnavirus-infected fish cells

Infectious pancreatic necrosis virus (IPNV) can induce Bad-mediated apoptosis followed by secondary necrosis in fish cells, but it is not known how these two types of cell death are regulated by IPNV. We found that IPNV infection can regulate Bad/Bid-mediated apoptotic and Rip1/ROS-mediated necrotic death pathways via the up-regulation of TNFα in zebrafish ZF4 cells. Using a DNA microarray and quantitative RT-PCR analyses, two major subsets of differentially expressed genes were characterized, including the innate immune response gene TNFα and the pro-apoptotic genes Bad and Bid. In the early replication stage (0-6 h post-infection, or p.i.), we observed that the pro-inflammatory cytokine TNFα underwent a rapid six-fold induction. Then, during the early-middle replication stages (6-12 h p.i.), TNFα level was eight-fold induction and the pro-apoptotic Bcl-2 family members Bad and Bid were up-regulated. Furthermore, specific inhibitors of TNFα expression (AG-126 or TNFα-specific siRNA) were used to block apoptotic and necrotic death signaling during the early or early-middle stages of IPNV infection. Inhibition of TNFα expression dramatically reduced the Bad/Bid-mediated apoptotic and Rip1/ROS-mediated necrotic cell death pathways and rescued host cell viability. Moreover, we used Rip1-specific inhibitors (Nec-1 and Rip1-specific siRNA) to block Rip1 expression. The Rip1/ROS-mediated secondary necrotic pathway appeared to be reduced in IPNV-infected fish cells during the middle-late stage of infection (12-18 h p.i.). Taken together, our results indicate that IPNV triggers two death pathways via up-stream induction of the pro-inflammatory cytokine TNFα, and these results may provide new insights into the pathogenesis of RNA viruses.     

Macrophage colony-stimulating factor (CSF-1) induces pro-inflammatory gene expression and enhances antimicrobial responses of goldfish (Carassius auratus L.) macrophages

We report on the regulation of pro-inflammatory functions of goldfish macrophages and induction of gene expression by recombinant goldfish CSF-1 (rgCSF-1). Recombinant goldfish TNFα-2 (rg TNFα-2), rgIFNγ but not rgTGFβ induced time-dependent increase of CSF-1 expression in macrophages. Treatment of goldfish macrophages with rgCSF-1 increased expression of several immune genes including CXCL-8 (= IL-8), CCL-1, TNFα-1, TNFα-2, IL-1β-1, IL-1β-2, IL-12-p35, IL-12-p40, IFN, IL-10 and iNOS A and B. The rgCSF-1 treatment did not significantly alter the mRNA levels of TGFβ and NRAMP in macrophages up to 48 h post treatment. However, at 72 h post treatment, the expression of TGFβ increased whereas that of NRAMP decreased. The treatment of macrophages with rgCSF-1 enhanced their respiratory burst and nitric oxide responses that were abrogated after addition of soluble CSF-1 receptor (sCSF-1R) to cell cultures. Macrophages exhibited a concentration-dependent chemotactic response toward rgCSF-1 as well as an increase in phagocytic activity that was abrogated after addition of sCSF-1R to cell cultures. Our results indicate that in addition to being an important growth factor of goldfish macrophages, rgCSF-1 also plays a central role in the regulation of their pro-inflammatory responses.     

Tumor necrosis factor inhibits glucocorticoid receptor function in mice: a strong signal toward lethal shock

As glucocorticoid resistance (GCR) and the concomitant burden pose a worldwide problem, there is an urgent need for a more effective glucocorticoid therapy, for which insights into the molecular mechanisms of GCR are essential. In this study, we addressed the hypothesis that TNFα, a strong pro-inflammatory mediator in numerous inflammatory diseases, compromises the protective function of the glucocorticoid receptor (GR) against TNFα-induced lethal inflammation. Indeed, protection of mice by dexamethasone against TNFα lethality was completely abolished when it was administered after TNFα stimulation, indicating compromised GR function upon TNFα challenge. TNFα-induced GCR was further demonstrated by impaired GR-dependent gene expression in the liver. Furthermore, TNFα down-regulates the levels of both GR mRNA and protein. However, this down-regulation seems to occur independently of GC production, as TNFα also resulted in down-regulation of GR levels in adrenalectomized mice. These findings suggest that the decreased amount of GR determines the GR response and outcome of TNFα-induced shock, as supported by our studies with GR heterozygous mice. We propose that by inducing GCR, TNFα inhibits a major brake on inflammation and thereby amplifies the pro-inflammatory response. Our findings might prove helpful in understanding GCR in inflammatory diseases in which TNFα is intimately involved.     

The Role of Reactive Oxygen Intermediates in the Regulation of Cytokine-Induced ICAM-1 Surface Expression on Endothelial Cells

ICAM-1 upregulation by endothelial cells plays a pivotal role in many disease processes, but signalling mechanisms leading to increased expression are poorly understood. In the current study we investigated the regulatory capacity of reactive oxygen intermediates (ROIs) in ICAM-1 activation by stimulating endothelial cells with the pro-inflammatory cytokines IL-1β, TNFα, IFNγ, IL-2, and IL-4 prior to antioxidant treatment. ICAM-1 was expressed constitutively and upregulated on ECV304 by IL1-β, IL2, and IFNγ and on SKHEP-1 by IFNγ, IL1-β, and TNFα. Phenanthroline (PHE) and disulfiram (DIS) showed the greatest ability to inhibit cytokine-stimulated ICAM-1 expression and in a dose-dependent manner. The α,α-diphenyl-β-picrylhydrazyl (DPPH) conversion assay showed that PHE and DIS had zero ability to scavenge free radicals and thus no known antioxidant activity. However, both are known metal chelators and our findings therefore suggest a unique role for metal ions in the control of cytokine-induced ICAM-1 expression on endothelial cells.