mRNA Levels Of IL-17A Research Articles

Human urine-derived stem cells: potential therapy for psoriasis-like dermatitis in mice

Over the past decade, significant advancements in stem cell research led by mesenchymal stem cells (MSCs) have facilitated their practical application in clinical settings, including inflammatory skin diseases. Urine-derived stem cells (USCs) are obtained from healthy human urine in a noninvasive approach with properties similar to mesenchymal stem cells (MSCs). However, the therapeutic potential of USCs for inflammatory skin diseases has not yet been fully explored. Herein, we report the therapeutic effects of USCs-derived culture supernatants on mice with psoriasis-like dermatitis using our originally established human USCs model. We examined the isolation of USCs from human urine using a simple centrifugation process. Cell markers related to MSCs-like cell were positive for CD29, CD44, CD73, CD90, and negative for HLA-DR, CD34, and CD45 by FACS analysis. Differentiation assays revealed that the cells possessed the capability to differentiate into adipocytes, chondrocytes, and osteocytes. USCs-conditioned medium (CM) treatment significantly suppressed the severity of dermatitis in imiquimod (IMQ)-treated psoriasis mice model. Histopathological examination revealed that USCs-CM treatment attenuated epidermal thickness and the numbers of infiltrating inflammatory cells, including neutrophils, T-cells, and macrophages in dermatitis-affected areas in IMQ-treated psoriasis mice. Furthermore, USCs-CM treatment decreased mRNA levels of IL-17A, IL-17F, and IL-23p19 was reduced in dermatitis area. In summary, our findings revealed new potential strategies for utilizing USCs and USCs-CM as therapeutic agents for inflammatory skin diseases, including psoriasis.

Ex-vivo Cell-based Assay for Assessment of Response to TNF Inhibitors in Patients with Rheumatic Diseases.

TNF inhibitors (TNFi) comprise 5 products whose structure and signalling differ. An individual patient with a rheumatic disease may respond to one TNFi but not to another. In addition, 30-40% of the patients respond inadequately to TNFi. The different TNFi downstream signalling may lead to their clinical efficacy. Several reports showed that TNFi exhibited differential effects on Th17 cells. We analyzed the different TNFi effects on IL-17A expression in the peripheral blood mononuclear cells (PBMCs) of patients with rheumatic diseases in order to evaluate the predictive capability of responses in an ex-vivo setting. PBMCs were co-cultured with the different TNFi or medium (control), and IL-17A mRNA levels were analyzed by qPCR. IL-17A expression levels in response to 4 TNFi (except certolizumab pegol) were compared with control. IL-17A expression in the assay was correlated to the clinical responses. Assay sensitivity and specificity for distinguishing responders from non-responders was calculated by receiver-operating characteristic (ROC) analysis. The results of a retrospective cohort of patients with rheumatic diseases (n = 82) correlated with their therapeutic responses to the different TNFi with 89.5% accuracy. The assay predicted the responses of a prospective cohort (n = 54) to specific TNFi with 79% accuracy. This functional assay could assist in predicting the odds for response to TNFi therapy, indicating whether a given patient is likely to respond to a specific TNFi.

Quercetin through miR-147–5p/Clip3 axis reducing Th17 cell differentiation to alleviate periodontitis

BackgroundQuercetin (QU) plays an important role in treating periodontitis; however, the mechanism through which microRNAs regulate Th17 cell differentiation has not been determined. MethodsQU was administered intragastrically to periodontitis rats once a day for one month. The morphology of alveolar bone was observed by micro-CT, gingival tissue structure was observed by HE staining, IL-6, TNF-α, IL-17A, RORγt, FOXP3 and IL-10 were detected by immunohistochemical staining, and Th17 and Treg cells in the peripheral blood were detected by flow cytometry. CD4+T cells were induced to differentiate into Th17 cells in vitro. Cell viability was determined by CCK8, and IL-17A and RORγt were detected by qPCR. Th17 cells were detected by flow cytometry, microRNA sequencing and bioinformatics analysis were used to screen key microRNAs, the phenotypic changes of Th17 cells were observed after overexpressed microRNAs via mimics. TargetScan database, in situ hybridization, and dual-luciferase reporter experiment were used to predict and prove target genes of microRNAs. The phenotype of Th17 cells was observed after overexpression of microRNA and target gene. ResultsCompared with periodontitis group, the distance from cementoenamel junction(CEJ) to alveolar bone(AB) was decreased, the structure of gingival papilla was improved, IL-6, TNF-α, IL-17, and RORγt were downregulated, FOXP3 and IL-10 were upregulated, the proportion of Th17 decreased and Treg increased in peripheral blood after QU treatment. Compared with Th17 cell group, mRNA levels of IL-17A and RORγt were decreased, and proportion of Th17 cells was significantly lower in the coculture group. MiR-147–5p was low in control group, upregulated in Th17 cell group, and downregulated after QU intervention, it's eight bases were inversely related to 3′UTR of Clip3, miR-147–5p with Clip3 were co-located in cells of periodontal tissue. Compared with those in Th17-mimicsNC + QU cells, the mRNA levels of RORγt and IL-17A upregulated, and proportion of Th17 cells increased in Th17-miR-147–5p + QU cells. The miR-147–5p mimics inhibited the luciferase activity of the WT Clip3 3′UTR but had no effect on the Mut Clip3 3′UTR. Clip3 was significantly downregulated after the overexpression of miR-147–5p. Mimics transfected with miR-147–5p reversed the decrease in the proportion of Th17 cells induced by QU, while the overexpression of Clip3 antagonized the effect of miR-147–5p and further reduced the proportion of Th17 cells. Moreover, the overexpression of miR-147–5p reversed the decreases in the mRNA levels of IL-17 and RORγt induced by QU treatment, while pcDNA3.1 Clip3 treatment further decreased the mRNA levels of IL-17 and RORγt. ConclusionQU reducing inflammatory response and promoting alveolar bone injury and repair, which closely relative to inhibit the differentiation of CD4+T cells into Th17 cells by downregulating miR-147–5p to promote the activation of Clip3.

Associations of Inflammation, Oxidative Stress and Prognosis with IL-37 Expression in Sepsis Rats with Lung Injury

This study aimed to investigate the role of interleukin-37 (IL-37) expression in lung tissues of sepsis-induced acute lung injury (ALI) rats and its impact on ALI, along with the underlying mechanisms. Sprague-Dawley (SD) rats were categorized into three groups: Control, sepsis-induced ALI (via cecal ligation and puncture, CLP), and sepsis-induced ALI with antibiotics (CLP+An). ALI models were established, and lung tissue injuries were assessed through hematoxylineosin staining. mRNA levels of IL-1α, IL-1β, IL-37, and tumor necrosis factor-α (TNF-α) were measured via RT-PCR, while IL-37 protein levels in lung tissues were determined using Western blotting. Additionally, bronchoalveolar lavage fluid (BALF) and blood samples were collected to assess inflammatory factors through ELISA. In the CLP group, there was an increase in pro-inflammatory factors (IL-1α, IL-1β, and TNF-α) in lung tissues and serum. However, in the CLP+An group, these factors decreased, IL-37 expression increased, and oxidative stress levels decreased. IL-37 demonstrated an inhibitory effect on the release of pro-inflammatory factors (IL-1α, IL-1β, and TNF-α) in sepsis rats, leading to a reduction in lung tissue inflammation. Furthermore, IL-37 exhibited a protective role by reducing oxidative stress in sepsis-induced lung tissues. These findings highlight IL-37 as a potential therapeutic target for mitigating ALI in sepsis.

Propionic acid ameliorates cognitive function through immunomodulatory effects on Th17 cells in perioperative neurocognitive disorders

BackgroundElderly patients undergoing surgery are prone to cognitive decline known as perioperative neurocognitive disorders (PND). Several studies have shown that the microglial activation and the decrease of short-chain fatty acids (SCFAs) in gut induced by surgery may be related to the pathogenesis of PND. The purpose of this study was to determine whether microglia and short-chain fatty acids were involved in cognitive dysfunction in aged rats. MethodsMale wild-type Wistar rats aged 11–12 months were randomly divided into control group (Ctrl: Veh group), propionic acid group (Ctrl: PA group), exploratory laparotomy group (LP: Veh group) and propionic acid + exploratory laparotomy group (LP: PA group) according to whether exploratory laparotomy (LP) or PA pretreatment for 21 days was performed. The motor ability of the rats was evaluated by open field test on postoperative day 3 (POD3), and then the cognitive function was evaluated by Y-maze test and fear conditioning test. The expression of IL-1β, IL-6, RORγt and IL-17A mRNA in hippocampus was detected by RT-qPCR, the expression of IL-17A and IL-17RA in hippocampus was detected by Western blot, and the activation of microglia was detected by immunofluorescence. ResultsThe PND rat model was successfully established by laparotomy. Compared with Ctrl: Veh group, the body weight of LP: Veh group decreased, the percentage of spontaneous alternations in Y maze decreased (P < 0.001), and the percentage of freezing time in contextual fear test decreased (P < 0.001). Surgery triggers neuroinflammation, manifested as the elevated levels of the inflammatory cytokines IL-1β (P < 0.001) and IL-6 (P < 0.001), the increased expression of the transcription factor RORγt (P = 0.0181, POD1; P = 0.0073, POD5)and major inflammatory cytokines IL-17A (P = 0.0215, POD1; P = 0.0071, POD5), and the increased average fluorescence intensity of Iba1 (P < 0.001, POD1; P < 0.001, POD5). After PA preconditioning, the recovery of rats in LP: PA group was faster than that in LP: Veh group as the body weight lost on POD1 (P = 0.0148) was close to the baseline level on POD5 (P = 0.1846), and they performed better in behavioral tests. The levels of IL-1β (P < 0.001) and IL-6 (P = 0.0035) inflammatory factors in hippocampus decreased on POD1 and the average fluorescence intensity of Iba1 decreased (P = 0.0024, POD1; P < 0.001, POD5), representing the neuroinflammation was significantly improved. Besides, the levels of RORγt mRNA (P = 0.0231, POD1; P = 0.0251, POD5) and IL-17A mRNA (P = 0.0208, POD1; P = 0.0071, POD5) in hippocampus as well as the expression of IL-17A (P = 0.0057, POD1; P < 0.001, POD5) and IL-17RA (P = 0.0388) decreased. ConclusionPA pretreatment results in reduced postoperative neuroinflammation and improved cognitive function, potentially attributed to the regulatory effects of PA on Th17-mediated immune responses.

Rho-kinase inhibitor alleviates CD4+T cell mediated corneal graft rejection by modulating its STAT3 and STAT5 activation

Penetrating keratoplasty remains the most common treatment to restore vision for corneal diseases. Immune rejection after corneal transplantation is one of the major causes of graft failure. In recent years, Rho-associated protein kinase (ROCK) inhibitors have been found to be associated with the activation of the STATs pathway and are widely studied in autoimmune diseases. Therefore, it may be possible that the ROCK inhibitors also participate in the local and systemic immune regulation in corneal transplantation through activation of the STATs pathway and affect the CD4+ T cell differentiation. This study aimed to explore the role of ROCK-STATs pathway in the occurrence of immune rejection in corneal transplantation by applying Y27632, a ROCK inhibitor, to the recipient mice and peripheral CD4+ T cells. We found that Y27632 significantly up-regulated the phosphorylation level of STAT5 in both spleen and lymph nodes, down-regulated the phosphorylation level of STAT3 in the CD4+ T cells in the spleen. It also increased the proportion of CD4+CD25+Foxp3+Helios+ Tregs while decreased CD4+IL17A+ -Th17 cells. Moreover, Y27632 also reduced the proportion of dendritic cells in both spleen and lymph nodes, as well as the expression level of CD86 on their surfaces in the spleen, while the proportion of macrophages was not affected. The expression levels of ROCK1, ROCK2, CD11c and IL-17A mRNA were also found to be low in the graft tissue while the expression of Helios was upregulated. Rho-kinase inhibitor can modulate the balance of Tregs/Th17 by regulating the phosphorylation levels of both STAT3 and STAT5, thereby inhibiting the occurrence of immune rejection in allogeneic corneal transplantation.

HMGB1 regulates Th17 cell differentiation and function in patients with psoriasis.

Psoriasis is an immune-mediated chronic inflammatory skin disease, in which T helper 17 (Th17) cells and its effective cytokine interleukin (IL)-17A play a pivotal pathogenic role. High mobility group box 1 (HMGB1) is an important proinflammatory cytokine, which has been confirmed to be highly expressed in the peripheral circulation and epidermis tissues of psoriasis patients. The regulatory effect of HMGB1 on IL-17A expression and function has been reported in some inflammatory and autoimmune diseases by theHMGB1-Toll-like receptor 4 (TLR4)-interleukin (IL)-23-IL-17A pathway. While, in the pathological environment of psoriasis, whether HMGB1 can exert the regulatory effect on IL-17A is not clear. We aimed to evaluate the role of HMGB1-TLR4-IL-23-IL-17A pathway in the pathogenesis of psoriasis and explore the possible regulatory mechanism of HMGB1 on Th17 cell differentiation. Serum levels of HMGB1, TLR4, IL-23, and IL-17A were quantified in 50 patients with moderate-to-severe plaque psoriasis and 30 healthy controls. Peripheral blood mononuclear cells were acquired from 10 severe psoriasis patients and administrated by different concentrations of recombinant-HMGB1 (rHMGB1) to detect the Th17 cell percentage, mRNA and protein levels of TLR4, IL-23, IL-17A and retinoid-related orphan receptor γt (RORγt). The serum levels of HMGB1, TLR4, IL-23, and IL-17A in psoriasis patients were significantly higher than healthy controls, especially in severe patients, and positively correlated with the severity index. There were also positive correlations between every two detected indicators of HMGB1, TLR4, IL-23, and IL-17A. In vitro study, rHMGB1 can promote the elevated expression of Th17 cell percentage as well as TLR4, IL-23, IL-17A, and RORγt in a dose-dependent manner. HMGB1 can contribute to the pathogenesis of psoriasis by regulating Th17 cell differentiation through HMGB1-TLR4-IL-23-RORγt pathway, then promotesIL-17A production and aggravates inflammation process. Targeting HMGB1 may be a possible potential candidate for the immunotherapy of psoriasis.

Unbalanced Aryl Hydrocarbon Receptor Expression in Peripheral and Lesional T Cell Subsets of Atopic Dermatitis.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, which is involved in the pathogenesis of a variety of skin diseases such as atopic dermatitis (AD). In this study, we aimed to study the AhR-expressing cells in T helper 17 (Th17), T helper 22 (Th22), regulatory T cells (Treg) and B cells in peripheral blood and in AD skin lesions. Twenty AD patients defined according to the Chinese criteria of atopic dermatitis and eighteen healthy subjects were included in our study. The AhR-expressing Th17, Th22, Treg and total B cells in peripheral blood were measured by flow cytometry. The AhR+ Th17 cells and AhR+ Th22 cells in AD skin lesions were measured by immunofluorescence. The mRNA of AhR, interleukin (IL)-22, IL-17A, IL-10, Foxp3, RORγT and TGF-β in peripheral blood mononuclear cells (PBMCs) was measured by real-time quantitative polymerase chain reaction. The expression of AhR in peripheral CD4+ T cells, Th22 cells, Treg cells and total B cells was significantly increased in AD. AhR+IL-17A+ and AhR+IL-22+ lymphocytes were also increased in AD skin lesions. The mRNA levels of AhR, IL-22 and IL-17A in PBMCs in AD patients were significantly higher. AhR mRNA levels in PBMCs positively correlated with peripheral basophil count, peripheral eosinophil count and mRNA levels of IL-22. AhR was highly expressed in subpopulations of CD4+ T cells in peripheral blood and skin lesions of AD, suggesting that AhR might contribute to the pathogenesis of AD.

Empagliflozin suppresses the differentiation/maturation of human epicardial preadipocytes and improves paracrine secretome profile

Abstract Aims Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce the epicardial adipose tissue (EAT) in human, which could enhance the cardioprotective effect in heart failure and atrial fibrillation. However, the precise mechanism of EAT reduction remains unclear. We investigated the direct effect of SGLT2 inhibitor, empagliflozin, on human primary epicardial adipocyte and preadipocyte. Methods and Results In Arm1, human EAT biopsies obtained during surgery and isolated preadipocyte/adipocytes were used for evaluating SGLT2 expression. In Arm2 and Arm3, we investigated the direct effect of empagliflozin on the differentiation/maturation and secretome profile of human preadipocyte/adipocyte. We validated the effect of empagliflozin on paracrine effect of EAT using co-culture with human iPS-derived atrial cardiomyocytes and empagliflozin-treated human epicardial adipocyte in Arm4. The validity of findings obtained from Arm1-4 were verified in view of clinical perspective in Arm5 and Arm6. SGLT2 was mainly expressed in the human pre-adipocyte in EAT. The expression levels of SGLT2 significantly diminished when the pre-adipocyte terminally differentiated. The formation of lipid droplets and fatty acid binding protein 4 (FABP4) expression in adipocyte were attenuated by empagliflozin treatment without affecting cell proliferation. mRNA levels of IL1α, IL1β, IL6, TGFβ and MCP1 in empagliflozin-treated adipocyte were significantly decreased. Of these, secreted protein levels of IL6 and MCP1 were significantly downregulated in supernatant of empagliflozin-treated cells. Co-culture of human iPS-derived atrial cardiomyocytes and adipocyte pre-treated with or without empagliflozin revealed that empagliflozin had striking suppressive effects on myocardial oxidative stress. Only IL6 mRNA expression in human EAT showed the clinically relevant association in terms of the difference of AF prevalence, anti-diabetic drug and N-terminal pro-brain natriuretic peptide. Conclusions We demonstrated that empagliflozin suppresses human epicardial pre-adipocyte differentiation/maturation, possibly leading to the reduction of EAT volume, and improves paracrine secretome profile of EAT. IL6 might be a key adipokine regulated by SGLT2 inhibitors.

Topical anti-TNF-a ssDNA aptamer decreased the imiquimod induced psoriatic inflammation in BALB/c mice

BackgroundTumor Necrosis Factor-α (TNF-α) is a pro-inflammatory factor that plays a pivotal role in psoriasis. Due to limitations of monoclonal antibody-based therapies, it is needed to discover new anti-TNF-α factors instead of usual anti-TNF-α monoclonal antibodies. Compared to antibodies, single-stranded DNA or RNA molecules named aptamers, have advantages such as time-saving, less risk for immunogenicity and cost-effectiveness. Therefore, the aim of the present study was to assess the therapeutic effects of T1-T4 dimer anti-TNF-ɑ ssDNA aptamer topical treatment in the imiquimod (IMQ)-induced psoriasis animal model. Methods5% IMQ cream was prescribed on the right ear of BALB/c to induce psoriasis model. The hydrogel-containing anti-TNF-ɑ aptamer or treatment control aptamer (anti- Interleukin (IL)17A) was topically prescribed to the mice's ears 10 min before IMQ cream treatment. The psoriasis area severity index (PASI) score was used to evaluate psoriasis intensity. Histopathology analysis was done for mice ears sections. Mass, size, and cell number of mice spleens were measured. The IL-17 level was determined in culture supernatants of axillary lymph node cells using ELISA. The mRNA expression levels of IL-17A, IL-1β, STAT3, and S100a9, were evaluated in mice treated ear with quantitative Real Time-PCR. ResultsThe anti-TNF-ɑ ssDNA aptamer lower doses had significant decrease in IMQ-induced PASI score (p < 0.05). In addition, in these groups, the IL-17A, STAT3, and S100a9 mRNA levels were significantly lower than the IMQ group (p < 0.05). ConclusionAccording to our findings, this aptamer seems to be a prospective candidate for treating psoriatic inflammation especially in lower concentrations.

MAP kinase inhibitor PD98059 regulates Th1, Th9, Th17, and natural T regulatory cells in an experimental autoimmune encephalomyelitis mouse model of multiple sclerosis

Experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis (MS), provides significant insights into the mechanisms that initiate and drive autoimmunity. MS is a chronic autoimmune disease of the central nervous system, characterized by inflammatory infiltration associated with demyelination. T lymphocyte cells play a crucial role in MS, whereas natural T regulatory (nTreg) cells prevent autoimmune inflammation by suppressing lymphocyte activity. This study sought to investigate the role of PD98059, a selective MAP kinase inhibitor, in Th1, Th9, Th17, and nTreg cells using the SJL/J mouse model of EAE. Following EAE development, the mice were intraperitoneally administered PD98059 (5 mg/kg for two weeks) daily. We evaluated the effects of PD98059 on Th1 (IFN-γ and T-bet), Th9 (IL-9 and IRF4), Th17 (IL-17A and RORγT), and nTreg (FoxP3 and Helios) cells in the spleen using flow cytometry. Moreover, we explored the effects of PD98059 on the IFN-γ, T-bet, IL-9, IRF4, IL-17A, RORγT, FoxP3, and Helios mRNA and protein levels in brain tissues using qRT-PCR and Western blot analyses. PD98059 treatment significantly decreased the proportion of CD4+IFN-γ+, CD4+T-bet+, CD4+IL-9+, CD4+IRF4+, CD4+IL-17A+, CD4+RORγT+, CD4+IL-17A+, and CD4+RORγT+ cells while increasing that of CD4+FoxP3+ and CD4+Helios+ cells. In addition, PD98059 administration decreased the mRNA and protein levels of IFN-γ, T-bet, IL-9, IRF4, IL-17A, and RORγT but increased those of FoxP3 and Helios in the brain tissue of EAE mice. Our findings suggest that PD98059 corrects immune dysfunction in EAE mice, which is concurrent with the modulation of multiple signaling pathways.

Dietary Fiber Inulin Improves Murine Imiquimod-Induced Psoriasis-like Dermatitis.

Psoriasis is a chronic skin disease with interleukin (IL)-17-dominated inflammation and hyperproliferation of epidermis. Dietary fiber is fermented by the gut microbiome into short-chain fatty acids (SCFAs) that manifest anti-inflammatory effects. We examined if feeding with an inulin-enriched high-fiber diet (HFD) might improve topical imiquimod-induced psoriasis-like dermatitis in mice. HFD reduced thickening and total severity scores of imiquimod-induced dermatitis and reduced epidermal thickness, inflammatory infiltrates, including Ly6G+ neutrophils, and epidermal Ki67+ proliferating cells. HFD reduced mRNA levels of IL-17A, IL-17F, IL-22, IL-1β, tumor necrosis factor (TNF)-α, CXCL1, CXCL2, and keratin 16 and increased those of transforming growth factor (TGF)-β1 and cyclin-dependent kinase inhibitor 1A in imiquimod-induced dermatitis. In 16S rRNA sequencing of the gut microbiome, imiquimod increased relative abundance of phylum Firmicutes, while HFD increased that of phylum Bacteroidota and genus Bacteroides. HFD increased serum and fecal concentrations of SCFA propionate. Oral propionate reduced inflammatory infiltrates and epidermal Ki67+ cells and reduced mRNA levels of IL-17A, IL-17F, IL-17C, IL-22, IL-1β, IL-6, TNF-α, CXCL1, CCL20 and increased those of TGF-β1and IL-10 in imiquimod-indued dermatitis. Dietary inulin supplementation improves imiquimod-induced psoriasis-like dermatitis partially via propionate, and may be a promising adjunctive therapy for psoriasis.

Gentiopicroside-Loaded Chitosan Nanoparticles Inhibit TNF-α-Induced Proliferation and Inflammatory Response in HaCaT Keratinocytes and Ameliorate Imiquimod-Induced Dermatitis Lesions in Mice.

In this study, we aimed to report the biological characteristics of the first successful synthesis of gentiopicroside-loaded chitosan nanoparticles and to evaluate the therapeutic effects and preliminary mechanisms of gentiopicrin-loaded chitosan on psoriasis-like cell and mouse models. Gentiopicroside-loaded chitosan nanoparticles (CHI-GEN) were prepared, and their biological characteristics were evaluated. HaCaT keratinocytes were stimulated with TNF-α to establish a psoriatic keratinocyte model. MTT assay and flow cytometry were used to measure cell viability and apoptosis, respectively. mRNA levels of K17, VEGF A, and IL-6 and IL-23A were detected using qRT-PCR. These tests were used to preliminarily assess the effects of CHI-GEN on keratinocyte proliferation and inflammation. Imiquimod was used to construct a psoriasis-like mice model. The severity of psoriasis was scored based on the psoriasis area severity index (PASI), H&E staining was used to observe the histological changes and the level of inflammation and cell proliferation of skin lesions was evaluated by measuring the mRNA levels of K17, IL-23A, and IL-17A using qRT-PCR. The average particle size of CHI-GEN nanoparticles was approximately 100 nm, and the zeta potential was 2.69 ± 0.87 mV. The cumulative release was 67.2% in solutions of pH 5.5 at 24 h. GEN reduced TNF-α-induced excessive proliferation of HaCaT keratinocytes and downregulated mRNA levels of K17, VEGF A, and inflammatory cytokines IL-6 and IL-23A, which was more obvious in the CHI-GEN treatment group. Additionally, CHI-GEN significantly improved the severity of skin lesions in psoriasis-like mice and downregulated the mRNA expressions of IL-6, IL-23A, and IL-17A in mice skin lesions. In conclusion, we successfully prepared gentiopicrin-chitosan nanoparticles. Our results show that these nanoparticles have anti-psoriasis activity, inhibits keratinocyte proliferation and improves symptoms in psoriasis model mice and can be used to develop an effective strategy for the treatment of psoriasis.

The Gliadin Hydrolysis Capacity of B. longum, L. acidophilus, and L. plantarum and Their Protective Effects on Caco-2 Cells against Gliadin-Induced Inflammatory Responses.

Non-celiac wheat sensitivity (NCWS) is a poorly understood gluten-related disorder (GRD) and its prominent symptoms can be ameliorated by gluten avoidance. This study aimed to determine the effectiveness of a probiotic mixture in hydrolyzing gliadin peptides (toxic components of gluten) and suppressing gliadin-induced inflammatory responses in Caco-2 cells. Wheat dough was fermented with a probiotic mix for 0, 2, 4, and 6 h. The effect of the probiotic mix on gliadin degradation was monitored by SDS-PAGE. The expression levels of IL-6, IL-17A, INF-γ, IL-10, and TGF-β were evaluated using ELISA and qRT-PCR methods. According to our findings, fermenting wheat dough with a mix of B. longum, L. acidophilus, and L. plantarum for 6 h was effective in gliadin degradation. This process also reduced levels of IL-6 (p = 0.004), IL-17A (p = 0.004), and IFN-γ (p = 0.01) mRNA, as well as decreased IL-6 (p = 0.006) and IFN-γ (p = 0.0009) protein secretion. 4 h fermentation led to a significant decrease in IL-17A (p = 0.001) and IFN-γ (p = 0.003) mRNA, as well as reduced levels of IL-6 (p = 0.002) and IFN-γ (p < 0.0001) protein secretion. This process was also observed to increase the expression levels of IL-10 (p < 0.0001) and TGF-β (p < 0.0001) mRNA. 4 h fermentation of wheat flour with the proposed probiotic mix might be a good strategy to develop an affordable gluten-free wheat dough for NCWS and probably other GRD patients.

CXCR3 antagonist NBI-74330 mitigates joint inflammation in Collagen-Induced arthritis model in DBA/1J mice.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by uncontrolled synovial proliferation, pannus formation, cartilage injury, and bone destruction. We used the CXCR3-specific antagonist NBI-74330 to block T-cell-mediated signaling in a DBA/1J mouse model of collagen-induced arthritis (CIA). After CIA induction, DBA/1J mice were treated with NBI-74330 (100mg/kg) daily from day 21 until day 34 and evaluated for arthritic score and histopathological changes. Furthermore, using flow cytometry, we investigated the effects of NBI-74330 on Th1 (IFN-γ, TNF-α, T-bet, STAT4, Notch-3, and RANKL), Th17 (IL-21, IL-17A, STAT3, and RORγt), and Th22 (IL-22) cells in splenic CD4+ and CXCR3+T-cells. We also used RT-PCR to assess the effect of mRNA levels of IFN-γ, TNF-α, T-bet, RANKL, IL-17A, RORγt, and IL-22 in knee tissues. The IFN-γ, TNF-α, and IL-17A serum protein levels were measured using ELISA. Compared to vehicle-treated CIA mice, the severity of arthritic scores and histological severity of inflammation decreased significantly in NBI-74330-treated CIA mice. Moreover, compared to vehicle-treated CIA mice, the percentages of CD4+IFN-γ+, CD4+TNF-α+, CD4+T-bet+, CD4+STAT4+, CD4+Notch-3+, CXCR3+IFN-γ+, CXCR3+TNF-α+, CXCR3+T-bet+, CXCR3+STAT4+, CXCR3+Notch-3+, CD4+RANKL+, CD4+IL-21+, CD4+IL-17A+, CD4+STAT3+, CD4+RORγt+, and CD4+IL-22+ cells decreased in NBI-74330-treated CIA mice. Furthermore, NBI-74330-treatment downregulated IFN-γ, TNF-α, T-bet, RANKL, STAT3, IL-17A, RORγt, and IL-22 mRNA levels. Serum IFN-γ, TNF-α, and IL-17A levels were significantly lower in NBI-74330-treated CIA mice than in vehicle-treated CIA mice. This study demonstrates the antiarthritic effects of NBI-74330 in CIA mice. Therefore, these data suggest that NBI-74330 could be considered a potential RA treatment.

Pathogenic NKT cells attenuate urogenital chlamydial clearance and enhance infertility.

Urogenital chlamydial infections continue to increase with over 127 million people affected annually, causing significant economic and public health pressures. While the role of traditional MHCI and II peptide presentation is well defined in chlamydial infections, the role of lipid antigens in immunity remains unclear. Natural killer (NK) T cells are important effector cells that recognize and respond to lipid antigens during infections. Chlamydial infection of antigen-presenting cells facilitates presentation of lipid on the MHCI-like protein, CD1d, which stimulates NKT cells to respond. During urogenital chlamydial infection, wild-type (WT) female mice had significantly greater chlamydial burden than CD1d-/- (NKT-deficient) mice, and had significantly greater incidence and severity of immunopathology in both primary and secondary infections. WT mice had similar vaginal lymphocytic infiltrate, but 59% more oviduct occlusion compared to CD1d-/- mice. Transcriptional array analysis of oviducts day 6 post-infection revealed WT mice had elevated levels of Ifnγ (6-fold), Tnfα (38-fold), Il6 (2.5-fold), Il1β (3-fold) and Il17a (6-fold) mRNA compared to CD1d-/- mice. In infected females, oviduct tissues had an elevated infiltration of CD4+ -invariant NKT (iNKT) cells, however, iNKT-deficient Jα18-/- mice had no significant differences in hydrosalpinx severity or incidence compared to WT controls. Lipid mass spectrometry of surface-cleaved CD1d in infected macrophages revealed an enhancement of presented lipids and cellular sequestration of sphingomyelin. Taken together, these data suggest an immunopathogenic role for non-invariant NKT cells in urogenital chlamydial infections, facilitated by lipid presentation via CD1d via infected antigen-presenting cells.

Oroxylin A inhibited autoimmune hepatitis-induced liver injury and shifted Treg/Th17 balance to Treg differentiation.

Autoimmune hepatitis (AIH) is a kind of autoimmune disease mediated by T cells, and its incidence is gradually increasing in the world. Oroxylin A (OA) is one of the major bioactive flavonoids that has been reported to inhibit inflammatory. Here, an AIH model of mouse was induced by Concanavalin A (Con A). It found that serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were decreased in mice with the treatment of OA. Hematoxylin-eosin staining showed that the liver injury was attenuated by OA, and TUNEL staining indicated that the cells apoptosis of liver was weakened in mice with OA treatment. ELISA analysis of cytokines and chemokines suggested that OA reduced the expression of IL-6, IL-17A, chemokine ligand 2 (CCL2), C-X-C motif chemokine ligand 1 (CXCL1) and CXCL10, but promoted the expression of IL-10 and TGF-β in mice. The mRNA levels of Il-17a in liver and spleen tissues were also significantly decreased, on the contrary, the mRNA levels of Il-10 in liver and spleen tissues were increased. The proportion of Treg/Th17 detected by flow cytometry revealed that OA promoted the differentiation of Treg and inhibited the differentiation of Th17 both in the liver and spleen. The results of this study demonstrated the inhibitory effects of OA on AIH-induced liver injury and the inflammatory response of AIH, and revealed that OA affected the balance of Treg/Th17 and shifted the balance toward Treg differentiation. It provided new potential drugs for the prevention of AIH.

The correlation of Th22 and regulatory T cells with Helicobacter pylori infection in patients with chronic gastritis.

Helicobacter pyloriis planted in the human stomach and is the most common cause of chronic gastritis, which produced specific local and systemic humoral immunity, while the associations of these immune responses and H. pylori in the development of chronic gastritis remain unclear. This study analyzed histology, the number of Th22 and regulatory T (Treg) cells, and the levels of inflammation- and gastritis-related indicators between 22 H. pylori-infected and 24 non-H. pylori-infected chronic gastritis patients by hematoxylin-eosinstaining, enzyme-linked immunosorbent assay, quantitative reverse transcription PCR, and flow cytometry analysis. This study found that the pathological damage degree of gastric mucosa in H. pylori infection patients was more serious. In the H. pylori-infected patient serum, the gastrin, G-17, interleukins(IL)-22, transforming growth factor(TGF)-β, tumor necrosis factor(TNF)-α, IL-4, and IL-17A levels were notably raised, while the interferon(IFN)-γ level was inhibited, and in gastric mucosa, and except IFN-γ, the IL-22, forkhead box P3 (Foxp3), TNF-α, IL-4, and IL-17A mRNA levels were raised too. The receiver operating characteristiccurve analysis indicates serum IL-22, TGF-β, TNF-α, IL-4, and IL-17A are suitable for differential diagnosis of H. pylori infection. In addition, in the peripheral blood, the percentages of the IL-22+ CD4+ and Foxp3+ CD4+ T cells were raised with H. pylori infection. The positive correlation between IL-22 and Foxp3 mRNA levels and the degree of H. pylori colonization and gastric mucositis by Pearson'scorrelation analysis. Treg and Th22 cells were positively associated with the degree of H. pylori infection and the severity of gastritis. In summary, this study provides an experimental basis for the study of the eradication of H. pylori and the biological mechanism of chronic gastritis.

IL-22 inhibits bleomycin-induced pulmonary fibrosis in association with inhibition of IL-17A in mice

BackgroundInterstitial lung disease, a common extra-articular complication of connective tissue disease, is characterized by progressive and irreversible pulmonary inflammation and fibrosis, which causes significant mortality. IL-22 shows a potential in regulating chronic inflammation and possibly plays an anti-fibrotic role by protecting epithelial cells. However, the detailed effects and underlying mechanisms are still unclear. In this study, we explored the impact of IL-22 on pulmonary fibrosis both in vivo and in vitro.MethodsTo induce pulmonary fibrosis, wild-type mice and IL-22 knockout mice were intratracheally injected with bleomycin followed by treatments with recombinant IL-22 or IL-17A neutralizing antibody. We investigated the role of IL-22 on bleomycin-induced pulmonary fibrosis and the mechanism in the possible interaction between IL-22 and IL-17A. Fibrosis-related genes were detected using RT-qPCR, western blot, and immunofluorescence. Inflammatory and fibrotic changes were assessed based on histological features. We also used A549 human alveolar epithelial cells, NIH/3T3 mouse fibroblast cells, and primary mouse lung fibroblasts to study the impact of IL-22 on fibrosis in vitro.ResultsIL-22 knockout mice showed aggravated pulmonary fibrosis compared with wild-type mice, and injection of recombinant IL-22 decreased the severe fibrotic manifestations in IL-22 knockout mice. In cell culture assays, IL-22 decreased protein levels of Collagen I in A549 cells, NIH/3T3 cells, and primary mouse lung fibroblasts. IL-22 also reduced the protein level of Collagen I in NIH/3T3 cells which were co-cultured with T cells. Mechanistically, IL-22 reduced the Th17 cell proportion and IL-17A mRNA level in lung tissues, and treatment with an IL-17A neutralizing antibody alleviated the severe pulmonary fibrosis in IL-22 knockout mice. The IL-17A neutralizing antibody also reduced Collagen I expression in NIH/3T3 cells in vitro. Knockdown of IL-17A with siRNAs or administration of IL-22 in NIH/3T3 cells and MLFs decreased expression of Collagen I, an effect blocked by concurrent use of recombinant IL-17A.ConclusionsIL-22 mediated an anti-fibrogenesis effect in the bleomycin-induced pulmonary fibrosis model and this effect was associated with inhibition of IL-17A.

Gain-of-function of TRPM4 predisposes mice to psoriasiform dermatitis.

Transient receptor potential melastatin 4 (TRPM4) is a Ca2+-activated, monovalent cation channel that is expressed in a wide range of cells. We previously reported two gain-of-function (GoF) mutations of TRPM4 as the cause of progressive symmetric erythrokeratodermia (PSEK), which shares similar clinical and histopathological features with psoriasis. Using CRISPR/Cas9 technology, we generated TRPM4I1029M mice that have the equivalent mutation to one of the two genetic mutations found in human PSEK (equivalent to human TRPM4I1033M). Using this mutant mice, we examined the effects of TRPM4 GoF at the cellular and phenotypic levels to elucidate the pathological mechanisms underlying PSEK. In the absence of experimental stimulation, TRPM4I1029M mice did not show a phenotype. When treated with imiquimod (IMQ), however, TRPM4I1029M mice were predisposed to more severe psoriasiform dermatitis (PsD) than wild-type (WT), which was characterized by greater accumulation of CCR6-expressing γδ T cells and higher mRNA levels of Il17a. In TRPM4I1029M mice, dendritic cells showed enhanced migration and keratinocytes exhibited increased proliferation. Moreover, a TRPM4 inhibitor, glibenclamide, ameliorated PsD in WT and TRPM4I1029M mice. Our results indicate elevated TRPM4 activities boosted susceptibility to cutaneous stimuli, likely through elevation of membrane potential and alteration of downstream cellular signaling, resulting in enhanced inflammation. Our results further suggest a possible therapeutic application of TRPM4 inhibitors in psoriasis.