Expression Of Tumor Necrosis Factor Receptor Research Articles

Restraint stress of male mice triggers apoptosis in spermatozoa and spermatogenic cells via activating the TNF-α system.

Studies have indicated that psychological stress impairs human fertility and that various stressors can induce apoptosis of testicular cells. However, the mechanisms by which psychological stress on males reduces semen quality and stressors induce apoptosis in testicular cells are largely unclear. Using a psychological (restraint) stress mouse model, we tested whether male psychological stress triggers apoptosis of spermatozoa and spermatogenic cells through activating tumour necrosis factor (TNF)-α signalling. Wild-type or TNF-α-/- male mice were restrained for 48 h before examination for apoptosis and expression of TNF-α and TNF receptor 1 (TNFR1) in spermatozoa, epididymis, seminiferous tubules and spermatogenic cells. The results showed that male restraint significantly decreased fertilization rate and mitochondrial membrane potential, while increasing levels of malondialdehyde, active caspase-3, TNF-α and TNFR1 in spermatozoa. Male restraint also increased apoptosis and expression of TNF-α and TNFR1 in caudae epididymides, seminiferous tubules and spermatogenic cells. Sperm quality was also significantly impaired when spermatozoa were recovered 35 days after male restraint. The restraint-induced damage to spermatozoa, epididymis and seminiferous tubules was significantly ameliorated in TNF-α-/- mice. Furthermore, incubation with soluble TNF-α significantly reduced sperm motility and fertilizing potential. Taken together, the results demonstrated that male psychological stress induces apoptosis in spermatozoa and spermatogenic cells through activating the TNF-α system and that the stress-induced apoptosis in spermatogenic cells can be translated into impaired quality in future spermatozoa.

Involvement of the TNF-α Pathway in TKI Resistance and Suggestion of TNFR1 as a Predictive Biomarker for TKI Responsiveness in Clear Cell Renal Cell Carcinoma.

BackgroundMechanism and predictive biomarkers for tyrosine kinase inhibitor (TKI) resistance of advanced clear cell renal cell carcinoma (ccRCC) have not been fully evaluated.MethodsWe performed gene expression profiling on samples from an acquired TKI resistance cohort that consisted of 10 cases of TKI-treated ccRCC patients with matched tumor tissues harvested at pre-treatment and TKI-resistant post-treatment periods. In addition, a public microarray dataset from patient-derived xenograft model for TKI-treated ccRCC (GSE76068) was retrieved. Commonly altered pathways between the datasets were investigated by Ingenuity Pathway Analysis using commonly regulated differently expressed genes (DEGs). The significance of candidate DEG on intrinsic TKI resistance was assessed through immunohistochemistry in a separate cohort of 101 TKI-treated ccRCC cases.ResultsTNFRSF1A gene expression and tumor necrosis factor (TNF)-α pathway were upregulated in ccRCCs with acquired TKI resistance in both microarray datasets. Also, high expression (> 10% of labeled tumor cells) of TNF receptor 1 (TNFR1), the protein product of TNFRSF1A gene, was correlated with sarcomatoid dedifferentiation and was an independent predictive factor of clinically unfavorable response and shorter survivals in separated TKI-treated ccRCC cohort.ConclusionTNF-α signaling may play a role in TKI resistance, and TNFR1 expression may serve as a predictive biomarker for clinically unfavorable TKI responses in ccRCC.

Tumor Necrosis Factor (TNF) Receptor Expression Determines Keratinocyte Fate upon Stimulation with TNF-Like Weak Inducer of Apoptosis.

The interaction between tumor necrosis factor- (TNF-) like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible 14 (Fn14) regulates the fate of keratinocytes, depending on the relative expression of TNF receptor (TNFR) 1 or TNFR2. However, the precise mechanism underlying this TWEAK-mediated regulation remains unclear. The aim of this study was to provide comprehensive insight into the roles of Fn14, TNFR1/2, and other relevant molecules in the fate of keratinocytes. Further, we sought to elucidate the structural basis for the interaction of TWEAK and Fn14 in regulating cellular outcomes. Normal keratinocytes (mainly expressing TNFR1) and TNFR2-overexpressing keratinocytes were stimulated with TWEAK. Through immunoprecipitation and Western blotting of keratinocyte lysates, we elucidated the associations between Fn14, TNFR-associated factor 2 (TRAF2), cellular inhibitor of apoptosis protein 1 (cIAP1), and TNFR1/2 molecules. Additionally, we found that TRAF2 exhibited binding to Fn14, cIAP1, and TNFR1/2. Our data suggest that TWEAK induces apoptosis in normal keratinocytes and proliferation in TNFR2-overexpressing keratinocytes in a TNF-α-independent manner; however, inhibition of TRAF2 appears to reverse this effect. Interestingly, the interaction between TWEAK and Fn14 increased TNFR1-associated death domain protein and caspase-8 expression in normal keratinocytes and promoted cytoplasmic import of cIAP1 in TNFR2-overexpressing keratinocytes. In conclusion, we found that the Fn14-TRAF2-TNFR signaling axis mediates TWEAK's regulation of the fate of keratinocytes, possibly in a manner involving the TNF-α-independent TNFR signal transduction.

A personalised approach for anti-GITR-based immunotherapy in pre-clinical models of pancreatic ductal adenocarcinoma

Abstract Background Patient outcomes with pancreatic ductal adenocarcinoma (PDA) remain dismal, despite the introduction of combination chemotherapies. Response to therapy is hampered by tumour stroma, which is highly heterogeneous but poorly characterised. The clinical success of immunotherapy in certain cancer types justifies investigation in PDA using a personalised approach of selecting pre-clinical models. Methods Patient PDA samples (n = 70; International Cancer Genome Consortium) were subjected to hierarchical clustering using a gene set representing CD8 and regulatory T cells to identify sub-groups. Syngeneic mouse models recapitulating these sub-groups (cross-species analysis) were developed using cell lines from genetically engineered mouse (GEM) models. Models bearing subcutaneous tumours were treated with anti-GITR agonist (DTA-1; 10mg/kg) or isotype (IgG2b) and tissues were collected for 12-color fluorescence activated cell sorting (FACS), NanoString and immunohistochemistry (IHC). Results Using clustering analysis, we identified four distinct patient PDA immune sub-groups with differential expression of T cell genes. These sub-groups showed differential expression of glucocorticoid-induced TNF receptor (GITR) and its associated genes. Hence, we hypothesised that these groups may respond differently to anti-GITR therapy in vivo. As expected, the three syngeneic models showed differential response to anti-GITR treatment. Model with increased GITR expression (>2-fold; NanoString and immunoblot) showed a significant (p = 0.02) survival benefit and a modest decrease in tumour burden upon anti-GITR treatment (n = 10) compared to the isotype control (n = 7) and the other two models. We observed anti-GITR mediated increase in GITR+ CD8+ cells and no change in GITR+ FOXP3+ cells (FACS, NanoString and IHC) along with increased gene expression of Pdcd1, Lag3, Spn and Itk. Conclusion Our models recapitulate the immunological gene expression profiles observed in patient PDA samples making them a highly useful resource to study personalized immunotherapies. This integrated study highlights the importance of patient selection to maximize therapy benefit and spare immune related adverse events. Legal entity responsible for the study The authors. Funding Bristol-Myers Squibb. Disclosure D. Cunningham: Research grant / Funding (self): Amgen; Research grant / Funding (self): Sanofi; Research grant / Funding (self): Merrimack; Research grant / Funding (self): AstraZeneca; Research grant / Funding (self): Celgene; Research grant / Funding (self): MedImmune; Research grant / Funding (self): Bayer; Research grant / Funding (self): 4SC; Research grant / Funding (self): Clovis; Research grant / Funding (self): Eli Lilly; Research grant / Funding (self): Janssen; Research grant / Funding (self): Merck. N. Starling: Research grant / Funding (self): AstraZeneca; Research grant / Funding (self): Bristol-Myers Squibb; Research grant / Funding (self): Pfizer; Travel / Accommodation / Expenses: AstraZeneca; Travel / Accommodation / Expenses: Bristol-Myers Squibb; Travel / Accommodation / Expenses: Eli Lilly; Travel / Accommodation / Expenses: Merck; Travel / Accommodation / Expenses: Roche; Honoraria (self): Eli Lilly; Honoraria (self): Merck Serono; Honoraria (self): MSD Oncology; Honoraria (self): Pierre Fabre; Advisory / Consultancy: Pfizer; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Servier. A. Sadanandam: Research grant / Funding (self): Bristol-Myers Squibb; Research grant / Funding (self): Merck KGaa; Research grant / Funding (self): Pierre Fabre. All other authors have declared no conflicts of interest.

IFNγ potentiates TNFα/TNFR1 signaling to induce FAT10 expression in macrophages

IntroductionThe tight regulation of the cytokine network during macrophage activation is of prime importance to enable a fast and potent innate immune response against exogenous pathogens. The inflammation mediating ubiquitin-like protein HLA-F adjacent transcript number 10 (FAT10) was shown to be transcriptionally regulated by and also regulate the nuclear factor-κB (NFκB) signaling pathway. However, very little is known about the regulation of FAT10 gene expression during macrophage activation. ResultsRNA sequencing of interferon (IFN)γ-stimulated mouse peritoneal macrophages analyzed by ingenuity pathway analysis revealed significant involvement of tumor necrosis factor receptor 1 (TNFR1) signaling in addition to IFNγ signaling. Subsequently, IFNγ robustly upregulated FAT10 expression compared to a milder induction seen with TNFα or lipopolysaccharide (LPS) stimulation. While low dose IFNγ with TNFα synergistically elevated FAT10 expression, preincubation of macrophages with IFNγ strongly augmented TNFα-induced FAT10 expression. Moreover, a short preincubation with IFNγ, which did not elevate FAT10, was sufficient to potentiate the induction of FAT10 by TNFα. A double augmentation mechanism of TNFα signaling was demonstrated, where IFNγ rapidly induced the expression of TNFα and TNFR1, which further augmented the induction of TNFα and TNFR1 expression by TNFα. Importantly, the induction of FAT10 by IFNγ in macrophages from TNFα-deficient or TNFR1-deficient mice was completely inhibited compared to macrophages from wild type (WT) mice. Finally, we show that TNFα-induced FAT10 expression is dependent on NFκB signaling. ConclusionIFNγ potentiates the TNFα/TNFR1 signaling pathway to induce FAT10 expression in mouse macrophages, mediated through NFκB network.

CD3+ Macrophages Deliver Proinflammatory Cytokines by a CD3- and Transmembrane TNF-Dependent Pathway and Are Increased at the BCG-Infection Site.

Macrophages are essential cells of the innate immune response against microbial infections, and they have the ability to adapt under both pro- and anti-inflammatory conditions and develop different functions. A growing body of evidence regarding a novel macrophage subpopulation that expresses CD3 has recently emerged. Here, we explain that human circulating monocytes can be differentiated into CD3+TCRαβ+ and CD3+TCRαβ− macrophages. Both cell subpopulations express on their cell surface HLA family molecules, but only the CD3+TCRαβ+ macrophage subpopulation co-express CD1 family molecules and transmembrane TNF (tmTNF). CD3+TCRαβ+ macrophages secrete IL-1β, IL-6 IP-10, and MCP-1 by both tmTNF- and CD3-dependent pathways, while CD3+TCRαβ− macrophages specifically produce IFN-γ, TNF, and MIP-1β by a CD3-dependent pathway. In this study, we also used a mouse model of BCG-induced pleurisy and demonstrated that CD3+ myeloid cells (TCRαβ+ and TCRαβ− cells) are increased at the infection sites during the acute phase (2 weeks post-infection). Interestingly, cell increment was mediated by tmTNF, and the soluble form of TNF was dispensable. BCG-infection also induced the expression of TNF receptor 2 on CD3+ myeloid cells, which increased after BCG-infection, suggesting that the tmTNF/TNFRs axis plays an important role in the presence or function of these cells in tuberculosis.

Herbs-partitioned moxibustion alleviates aberrant intestinal epithelial cell apoptosis by upregulating A20 expression in a mouse model of Crohn's disease.

BACKGROUNDA20 inhibits intestinal epithelial cell apoptosis in Crohn’s disease, and herbs-partitioned moxibustion (HPM) has been demonstrated to be an effective treatment for Crohn’s disease. However, the mechanism by which HPM reduces intestinal epithelial cell apoptosis in Crohn’s disease has not been thoroughly elucidated to date.AIMTo elucidate whether HPM exerts its effects by upregulating A20 to affect intestinal epithelial cell apoptosis in a Crohn’s disease mouse model.METHODSIn this study, mice with A20 deletion in intestinal epithelial cells (A20IEC-KO) were utilized to establish a Crohn’s disease mouse model with 2,4,6-trinitrobenzene sulfonic acid (TNBS) administration, as well as wild-type mice. Mice were randomly divided into normal control (NC), model control (MC), mesalazine (MESA), and HPM groups. The morphology of the colonic mucosa was observed by hematoxylin-eosin staining, and serum endotoxin and apoptosis of epithelial cells were evaluated by enzyme-linked immunosorbent assay and terminal dUTP nick-end labeling assay accordingly. The protein expression levels of A20 and tumor necrosis factor receptor 1 (TNFR1)-related signaling molecules were evaluated by Western blot, and co-expression of A20 and TNFR1-associated death domain (TRADD) and co-expression of A20 and receptor-interacting protein 1 (RIP1) were observed by double immunofluorescence staining.RESULTSThe intestinal epithelial barrier was noted to have an improvement in the HPM group of wild-type (WT) mice compared with that in A20IEC-KO mice. Compared with A20 IEC-KO HPM mice, serum endotoxin levels and apoptosis percentages were decreased (P < 0.01), A20 expression levels were increased (P < 0.01), and expression of TNFR1, TRADDD, and RIP1 was decreased in the HPM group of WT mice (PTNFR1 < 0.05, PTRADD < 0.01, PRIP1 < 0.01). Both of the co-expression of A20/TRADD and A20/RIP1 showed a predominantly yellow fluorescence in the HPM group of WT mice, while a predominantly red fluorescence was noted in the HPM group of A20IEC-KO mice.CONCLUSIONOur findings suggest that HPM in treating Crohn’s disease functions possibly via upregulation of the A20 expression level, resulting in downregulation of TNFR1, TRADD, and RIP1 to alleviate increased cell apoptosis in the intestinal epithelial barrier in Crohn's disease.

Modulation of cathelicidins, IFNβ and TNFα by bovine alpha-herpesviruses is dependent on the stage of the infectious cycle

Production of antimicrobial peptides cathelicidins, interferons and cytokines is an important feature in airway epithelial host defense. The innate immune response to alpha-herpesvirus infection at the sites of primary replication has not been fully studied. Thus, the aim of this study was to determine the expression of innate immune components, cathelicidins, IFNβ, TNFα and TNF receptors (TNFRI and TNFRII) during acute infection and reactivation of bovine herpesvirus type 1 (BoHV-1) and 5 (BoHV-5) in the respiratory tract and lymphoid tissue of their natural host. We found that BoHV infection modulates mainly the expression of BMAP28, a key cathelicidin in cattle. It was downregulated by both viruses in retropharyngeal lymph nodes of acutely infected-calves, and it was accompanied by a lower expression of IFNβ, TNFα and TNFRI. BoHV-5 showed a pronounced role in the downregulation of BMAP28, even in nasal mucosa and lung. However, during reactivation, BoHV-5 upregulated both BMAP28 and IFNβ in retropharyngeal lymph nodes. Acute replication induced also TNFα mRNA and protein synthesis, and expression of TNFRI and II was positively regulated during both acute infection and reactivation, particularly in the trachea. Moreover, BMAP27 was detected during BoHV-1 reactivation suggesting a potential role at this stage. Thus, cathelicidins are implicated in alpha-herpesvirus infections of the bovine respiratory system and the response is distinct during BoHV-1 and BoHV-5 acute infection and reactivation. This demonstrates that these viruses modulate differentially the components of innate immune response, possibly influencing their pathogenesis. This study provides an initial pilot analysis of factors that might be implicated in alpha-herpesvirus infection of the bovine respiratory system.

Preliminary Data on the Effect of High Laminar Shear Stress on TNFR1 and Adaptor Proteins Expression in Endothelial Cells

BackgroundTumor Necrosis Factor (TNF) is an inflammatory cytokine that targets the vascular endothelial cells and initiates a cascade of events that leads to endothelial dysfunction. TNF‐induced activation of NF‐κB and MAP kinases increases the expression of adhesion molecules and chemokines that cause leukocytes recruitment and activation consequently amplifying inflammation. Evidence suggests that high laminar shear stress (HLSS) mitigates inflammation and induces atheroprotective effects. Thus, the purpose of this preliminary study was to investigate the effect of HLSS on the gene expression of TNF Receptor 1 (TNFR1) and two associated adaptor proteins, TNF receptor‐associated factor‐2 (TRAF2) and lymphocyte adaptor protein (Lnk).MethodsHuman umbilical vein endothelial cells (HUVECs) from two donors were cultured and incubated with TNF (10 ng/mL) for 4hr, exposed to HLSS (20 dynes/cm2) for 24hr, or pre‐exposed to HLSS for 24hr and then incubated with TNF (10 ng/ml). The experiment was repeated 2–3 times.ResultsWe found a significant decrease in TNFR1 expression in response to TNF incubation (p = .001, Fig. 1). TNFR1 expression was further decreased with HLSS (p = .04, Fig. 1). TNF incubation and HLSS also significantly reduced TRAF2 expression by about 50% (p &lt;.01, Fig. 2). In response to HLSS, Lnk expression was significantly reduced (p &lt;.001, Fig 3). However, this reduction was blunted by TNF incubation for 4hr (p = .02 Fig. 3).ConclusionThe results suggest that HLSS acts as an exercise memetic to significantly affect TNF signaling proteins. HLSS may help as a novel preventive and therapeutic approach to modulate TNF signaling and improve vascular function.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TNFR2 but not TNFR1 is the main TNFR expressed by B and T lymphocytes in breast cancer draining lymph nodes

Tumor necrosis factor-α (TNF-α) is a key cytokine in inflammation and a driving force for leukocyte migration and recruitment. However, it may exert contrasting effects on the immune responses depend on its differential binding to its receptors (TNFR1 or TNFR2). The expression of TNF receptors by lymphocytes in the tumor draining lymph nodes (TDLNs) has not been thoroughly investigated. Herein, the expression of TNFRs on lymphocytes in the breast TDLNs was assessed. 40 axillary LN samples were obtained from breast cancer patients. Mononuclear cells were isolated using Ficoll-Hypaque gradient centrifugation and stained with anti-CD3, CD4, CD8, CD19, TNFR1 and TNFR2 and subjected to flow cytometry. Results showed that TNFR2 was detected on unstimulated B or T cells in the breast TDLNs while TNFR1 was nearly absent on these cells. Short or long term activation did not increase the expression of TNFR1. The percentage of TNFR2+ cells was significantly higher in CD4+ compared to CD19+ or CD8+ cells. No significant association was observed between the percentage of TNFR2 expressing T cells and prognostic indicators. However, the percentage of TNFR2+ B cells in the metastatic LNs had negative associations with tumor grade and the number of involved LNs (P = 0.009 and P = 0.04, respectively). Collectively, TNFR2 was the main TNFR expressed by unstimulated B or T lymphocytes in the breast TDLNs and the frequency of TNFR2+ B cells was connected to good prognosticators. The effects of TNFR2 expression by lymphocytes of breast TDLNs on their functions requires more functional studies.

First trimester placental endothelial cells from pregnancies with abnormal uterine artery Doppler are more sensitive to apoptotic stimuli

Failure of the placental capillary network to develop normally is associated with early onset fetal growth restriction (FGR) and pre-eclampsia (PE). Although the symptoms are observed at term, the problem begins in the first trimester. However, investigations at this clinically relevant time are hindered by difficulties in identifying earlystage pregnancies that are at risk of developing FGR/PE. Using uterine artery Doppler ultrasound in the first trimester as a proxy measure of poor placentation, we have identified pregnancies at increased risk of developing early onset FGR/PE. Placental endothelial cells (PEC) isolated from pregnancies at increased risk of developing FGR/PE grew more slowly and their basal rate of apoptosis was significantly higher than that seen in the normal group. The pro-apoptotic stimulus, TNFα, induced apoptosis in cells from both groups but this was significantly greater in the high risk group. TNF receptor expression was unaffected. Inhibition of nitric oxide (NO) production significantly increased the sensitivity of cells from the normal pregnancies to TNFα but not in the high risk group establishing a functional role for NO in this system. In conclusion, first trimester PEC from pregnancies at increased risk of developing early onset FGR/PE were inherently more sensitive to apoptotic stimuli and this was functionally linked to the synthesis of NO. This may contribute to the poor placental vascular development seen in on going pregnancies.

Size-Dependent Effect of Silver Nanoparticles on the Tumor Necrosis Factor α-Induced DNA Damage Response.

Silver nanoparticles (AgNPs) are widely used in many consumer products due to their anti-inflammatory properties. Therefore, the effect of exposure to AgNPs should be investigated in diseased states in addition to healthy ones. Tumor necrosis factor-α (TNFα) is a major cytokine that is highly expressed in many diseased conditions, such as inflammatory diseases, sepsis, and cancer. We investigated the effects of two different sizes of AgNPs on the TNFα-induced DNA damage response. Cells were exposed to 10 and 200 nm AgNPs separately and the results showed that the 200 nm AgNPs had a lower cytotoxic effect with a higher percent of cellular uptake compared to the 10 nm AgNPs. Moreover, analysis of reactive oxygen species (ROS) generation and DNA damage indicated that TNFα-induced ROS-mediated DNA damage was reduced by 200 nm AgNPs, but not by 10 nm AgNPs. Tumor necrosis factor receptor 1 (TNFR1) was localized on the cell surface after TNFα exposure with or without 10 nm AgNPs. In contrast, the expression of TNFR1 on the cell surface was reduced by the 200 nm AgNPs. These results suggested that exposure of cells to 200 nm AgNPs reduces the TNFα-induced DNA damage response via reducing the surface expression of TNFR1, thus reducing the signal transduction of TNFα.

TNF Induces the Production of Type 2 Cytokines in Human Group 2 Innate Lymphoid Cells

Group 2 innate lymphoid cells (ILC2s) play an important role in the initiation and amplification of type 2 inflammation by producing type 2 cytokines, including IL-5 and IL-13, and are elevated in type 2 inflammatory diseases. Recently, several studies, including our own, found that members of the TNF superfamily, TL1A and RANKL, induced production of type 2 cytokines in ILC2s. However, whether TNF itself induces type 2 cytokines in ILC2s has not been elucidated. We investigated the expression of TNF receptor II (TNFRII) on human ILC2s by flow cytometry. Purified human ILC2s sorted from either peripheral blood or nasal polyps (NPs) were stimulated with recombinant TNF and thymic stromal lymphopoietin (TSLP) in the presence or absence of inhibitors. The production of type 2 cytokines from ILC2s was evaluated by Luminex. Flow cytometric analysis revealed the expression of TNFRII on human ILC2 (n=6). TNF dose-dependently induced production of IL-5 and IL-13 in human blood ILC2s. We also found that TNF enhanced production of type 2 cytokines in NP-derived ILC2s. Interestingly, TSLP synergistically enhanced TNF mediated production of type 2 cytokines in blood ILC2s (3-fold, n=10, p<0.01). We also found that TNF-mediated production of type 2 cytokines was significantly inhibited by dexamethasone and IMD-0354 (an IKKβ inhibitor), indicating that NF-κB is a key transcription factor controlling TNF mediated production of type 2 cytokines in ILC2s. Local elevation of TNF and TSLP in tissues undergoing accumulation of ILC2s may lead to ILC2 activation and play an important role in type 2 inflammatory diseases.

Homeostatic Control of Sebaceous Glands by Innate Lymphoid Cells Regulates Commensal Bacteria Equilibrium

Immune cells and epithelium form sophisticated barrier systems in symbiotic relationships with microbiota. Evidence suggests that immune cells can sense microbes through intact barriers, but regulation of microbial commensalism remain largely unexplored. Here, we uncovered spatial compartmentalization of skin-resident innate lymphoid cells (ILCs) and modulation of sebaceous glands by a subset of RORγt+ ILCs residing within hair follicles in close proximity to sebaceous glands. Their persistence in skin required IL-7and thymic stromal lymphopoietin, and localization was dependent on the chemokine receptor CCR6. ILC subsets expressed TNF receptor ligands, which limited sebocyte growth by repressing Notch signaling pathway. Consequently, loss of ILCs resulted in sebaceous hyperplasia with increased production of antimicrobial lipids and restricted commensalismofGram-positive bacterial communities. Thus, epithelia-derived signals maintain skin-resident ILCs that regulate microbial commensalism through sebaceous gland-mediated tuning of the barrier surface, highlighting an immune-epithelia circuitry that facilitates host-microbe symbiosis.

Synergy between Probiotic Lactobacillus casei and Milk to Maintain Barrier Integrity of Intestinal Epithelial Cells.

We hypothesized that Lactobacillus casei BL23 and milk work synergistically to prevent damage to epithelial barrier integrity induced by pro-inflammatory cytokines. To test this, barrier disruption was induced in polarized Caco-2 monolayers by sequential, basolateral treatment with IFN-γ and TNF-α. Apical application of either 25% v/v reconstituted skim milk (RSM) or ultra high temperature (UHT) milk (2% fat) prior to cytokine exposure reduced losses to transepithelial electrical resistance (TER). Permeability to fluorescein isothiocyanate-dextran (FD-4; 4 kDa) was also significantly reduced in the presence of 25% v/v UHT milk ( P < 0.05) but not RSM. Protection against increases in paracellular permeability was even greater when cell-free preparations of L. casei BL23 fermented UHT milk or fermented RSM were applied. The permeability coefficients of cells incubated with BL23 fermented UHT milk were equivalent to the untreated controls ( P = 0.12) and those cells also produced 247.6 ± 35.5 pg/mL IL-8, quantities significantly lower than found for cytokine-treated controls (353.9 ± 40.0 pg/mL). The benefits of the fermented milk were also confirmed by the reduced expression of TNF receptor 2 (TNFR2), myosin light-chain kinase (MLCK), and claudin-encoding genes relative to the controls. By comparison, apical application of viable L. casei onto the Caco-2 cells did not result in protection from the barrier-disruptive actions of IFN-γ and TNF-α. These results indicate that milk can maintain intestinal barrier integrity during pro-inflammatory cytokine exposure and that this is enhanced by modifications to milk matrix caused by prior incubation with L. casei BL23.

In vivo and in vitro Approach to Anti-arthritic and Anti-inflammatory Effect of Crocetin by Alteration of Nuclear Factor-E2-Related Factor 2/hem Oxygenase (HO)-1 and NF-κB Expression.

Crocetin (apo carotenoid dicarboxylic acid) is a common constituent of saffron. Its importance is well documented in Chinese medicine. Some studies have reported the inhibitory effect on inflammation in rats. The aim of the current experimental investigation to scrutinize the anti-inflammatory effect of Crocetin using the lipo polysaccharide (LPS) induced mouse macrophages (RAW 264.7) in vitro and complete Freund’s adjuvant-induced arthritis model and to explore in vivo possible mechanism of action. RAW 264.7 macrophages were used for estimation of the effect of crocetin on the cyclooxygenase (COX-2), nitric oxide (NO)production, anti-inflammatory and along with pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10). Single intraperitoneal injection of complete freund’s adjuvant (CFA) was used to induce arthritis. The rats were divided into different group and received the oral administration of crocetin in a dose-dependent manner with indomethacin till 28 days. The paw edema and body weight was estimated at regular interval of time. The biochemical parameters, hematological and pro-inflammatory cytokines such as tumor necrosis factor receptor 1 (TNF-R1), IL-6, and IL-1β, Vascular endothelial growth factor (VEGF); heme oxygenase-1/nuclear factor erythroid 2–related factor 2 (HO-1/Nrf-2) expression were estimated at end of the experimental study. Crocetin inhibited the COX-2 catalyzed prostaglandin (PGE2) and inducible nitric oxide synthase catalyzed NO production on RAW 264.7. The paw edema and body weight was significantly (P < 0.001) modulated by the Crocetin in a dose-dependent manner. Crocetin treatment increased the level of red blood cells (RBC), hemoglobin (Hb) and decreased level of white blood cells (WBC), erythrocyte sedimentation rate (ESR), alkaline phosphatase (ALP), serum glutamic pyruvic transaminase (SGPT), and serum glutamic-oxaloacetic transaminase (SGOT) parameters, with reduction of TNF-α, IL-6, and IL-1β.The protective effect of crocetin was substantiated with a reduction in expression of IL-6, IL-1β, VEGF, and TNF-R1, respectively. Crocetin also increased the HO-1/Nrf-2 and decreased the nuclear factor kappa-B (NF-κB) mRNA, protein expression. On the basis of the result, we can conclude that the reduction of HO-1/Nrf-2 expression, as well as inflammatory mediators, may be involved in the protective effect of Crocetin in the CFA model.

Chinese medicine CGA formula ameliorates liver fibrosis induced by carbon tetrachloride involving inhibition of hepatic apoptosis in rats

Ethnopharmacological revelvanceCGA consisting of Cordyceps sinensis mycelia polysaccharide, gypenosides and amygdalin, was demonstrated to be the effective components formula in Fuzheng Huayu (FZHY) capsule, a traditional Chinese medicine approved by China food and drug administration for treatment of liver fibrosis and to inhibit transforming growth factor-β1 (TGF-β1) signaling, previously. Aim of the studyTo evaluate the effects of CGA on hepatic apoptosis in liver fibrosis induced by carbon tetrachloride (CCl4). Materials and methodsThe hepatic injury and histology was detected by serum biomarker assay and hematoxylin-eosin staining. The hepatic collagen was illustrated by Sirius red staining and hydroxyproline (Hyp) concentration. The hepatic stellate cells (HSCs) activation and hepatic apoptosis was visualized by immunohistochemical analysis of α-smooth muscle actin (α-SMA) and terminal deoxynucleotidyl transferase-mediated dUPT nick-end labeling (TUNEL) assay respectively. The protein expression of collagen type I (Col-I), α-SMA, TGF-β1, Fas, tumor necrosis factor receptor 1 (TNF-R1), cleaved-caspase-8, cleaved-caspase-10, cleaved-caspase-9, cleaved-caspase-3, mitochondrial Bcl-2, Bcl-2 associated X protein (Bax), Bcl-2 homologous antagonist/killer (Bak), cytochrome C and cytoplasmic cytochrome C was detected by western-blot. ResultsCGA or FZHY ameliorated liver histological changes, decreasing serum alanine aminotransferase, aspartate aminotransferase, hepatic Hyp, TUNEL positive-stained area, and down-regulated the protein expression of α-SMA, TGF-β1, Col-I, Fas, TNF-R1, cleaved-caspase-8, cleaved-caspase-10, cleaved-caspase-9, and cleaved-caspase-3, mitochondrial Bax, Bak, and cytoplasmic cytochrome C, while restored the expression of mitochondrial Bcl-2 and cytochrome C. ConclusionCGA formula ameliorates liver fibrosis induced by CCl4, which is correlated to its inhibition on hepatic apoptosis.

Berberine alleviates amyloid β25-35-induced inflammatory response in human neuroblastoma cells by inhibiting proinflammatory factors.

The present study investigated the effect of berberine (BBR) on amyloid β 25-35 (Aβ25-35)-induced inflammatory response in human neuroblastoma cells. To model the inflammatory response observed in the central nervous system of patients with Alzheimer's disease, SH-SY5Y and SK-N-SH neuroblastoma cells were induced by Aβ25-35 (5 µM) for 24 h. Subsequently, cells were treated with BBR or indomethacin for 2 h. The cell survival rate was determined by the MTT assay. The activity of lactate dehydrogenase (LDH) in the cell culture medium was examined by spectrophotometry. The expression levels of inflammatory factors prostaglandin E2 (PGE2) and tumor necrosis factor-α (TNF-α) were determined by ELISA assays. The mRNA and protein expression levels of interleukin (IL)-1β, cyclooxygenase 2 (COX-2) and tumor necrosis factor receptor 1 (TNFR1) were measured by reverse-transcription-quantitative polymerase chain reaction and Western blotting, respectively. The results indicated that, treatment with Aβ25-35 increased the expression levels of PGE2 and TNF-α, increased the activity of LDH, and up-regulated the mRNA and protein expression of COX-2, IL-1β, and TNFR1. Treatment with BBR down-regulated the expression levels of PGE2 and TNF-α, decreased the activity of LDH, and downregulated the mRNA and protein expression of COX-2, IL-1β, and TNFR1. Taken together, the present results suggested that BBR suppressed the inflammatory response induced by Aβ25-35 in neuroblastoma cells. The mechanism of action may be associated with the inhibition of proinflammatory factors.

Hyperoside suppresses tumor necrosis factor α-mediated vascular inflammatory responses by downregulating mitogen-activated protein kinases and nuclear factor-κB signaling

Vascular inflammation has been suggested to play a key role in the initiation and progression of atherosclerosis. Hyperoside (HPS) is a plant-derived quercetin 3-d-galactoside reported to have anti-inflammatory, anti-oxidant, anti-cancer, anti-hyperglycemic, anti-coagulant, and cardioprotective activities. However, the effects of HPS on vascular inflammation have not been studied. Therefore, in this study, we investigated the suppressive effect of HPS on tumor necrosis factor-α (TNFα)-dependent inflammatory responses in MOVAS-1 cells, a murine vascular smooth muscle cell (VSMC) line. HPS did not show any significant cytotoxicity up to 10 μg/mL over 24 h. TNFα challenge of VSMCs significantly increased the mRNA (3-fold) and protein expression (20-fold) of vascular cell adhesion molecule-1 (VCAM-1). However, these increases were abolished in the presence of HPS. Additionally, HPS significantly decreased monocyte adhesion to TNFα–stimulated VSMCs in a dose-dependent manner. Further, TNFα challenge induced activation of mitogen-activated protein kinases (MAPKs), such as p38 MAPK (38.0 ± 3.08 fold), JNK (51.6 ± 2.26 fold), and ERK (14.1 ± 0.77 fold); expression of nuclear factor-κB (NF-κB; ≅ 4-fold) and TNF receptor 1 (TNFR1; 2.7 ± 0.198 fold) were also increased. Notably, the TNFα-induced expression of these molecules was also significantly inhibited by the presence of HPS. Given that p38 MAPK, JNK, ERK, NF-κB, and TNFR1 all play regulatory roles in the expression of VCAM-1, this study provides insight into the mechanism of action of HPS. In summary, HPS can inhibit TNFα-mediated vascular inflammatory responses and has potential as a new anti-atherosclerotic drug.

Senescence and odontoblastic differentiation of dental pulp cells.

Cellular senescence has been suggested to be involved in physiological changes of cytokine production. Previous studies showed that the concentration of tumor necrosis factor-α (TNF-α) is higher in the blood of aged people compared with that of young people. So far, the precise effects of TNF-α on theodontoblastic differentiation of pulp cells have been controversial. Therefore, we aimed to clarify how this cytokine affected pulp cells during aging. Human dental pulp cells (HDPCs) were cultured until reaching the plateau of their growth, and the cells were isolated at actively (young HDPCs; yHDPCs) or inactively (senescent HDPCs; sHDPCs) proliferating stages. sHDPCs expressed senescence-related molecules while yHDPCs did not. When these HDPCs were cultured in anodontoblast-inductive medium, both young and senescent cells showed mineralization, but mineralization in sHDPCs was lower compared with yHDPCs. However, the administration of TNF-α to this culture medium altered these responses: yHDPCs showed downregulated mineralization, while sHDPCs exhibited significantly increased mineralization. Furthermore, the expression of tumor necrosis factor receptor 1 (TNFR1), a receptor of TNF-α, was significantly upregulated in sHDPCs compared with yHDPCs. Downregulation of TNFR1 expression led to decreased mineralization of TNF-α-treated sHDPCs, whereas restored the reduction in TNF-α-treated yHDPCs. These results suggested that sHDPCs preserved the odontoblastic differentiation capacity and TNF-α promoted odontoblastic differentiation of HDPCs with theprogress of their population doublings through increased expression of TNFR1. Thus, TNF-α might exert a different effect on the odontoblastic differentiation of HDPCs depending on their proliferating activity. In addition, the calcification of pulp chamber with age may be related with increased reactivity of pulp cells to TNF-α.