The Transcription Factor Ets1 Suppresses T Follicular Helper Type 2 Cell Differentiation to Halt the Onset of Systemic Lupus Erythematosus.

Single-nucleotide polymorphisms in ETS1 are associated with systemic lupus erythematosus (SLE). Ets1-/- mice develop SLE-like symptoms, suggesting that dysregulation of this transcription factor is important to the onset or progression of SLE. We used conditional deletion approaches to examine the impact of Ets1 expression in different immune cell types. Ets1 deletion on CD4+ T cells, but not B cells or dendritic cells, resulted in the SLE autoimmunity, and this was associated with the spontaneous expansion of T follicular helper type 2 (Tfh2) cells. Ets1-/- Tfh2 cells exhibited increased expression of GATA-3 and interleukin-4 (IL-4), which induced IgE isotype switching in B cells. Neutralization of IL-4 reduced Tfh2 cell frequencies and ameliorated disease parameters. Mechanistically, Ets1 suppressed signature Tfh and Th2 cell genes, including Cxcr5, Bcl6, and Il4ra, thus curbing the terminal Tfh2 cell differentiation process. Tfh2 cell frequencies in SLE patients correlated with disease parameters, providing evidence for the relevance of these findings to human disease.

Emodin modulates gut microbial community and triggers intestinal immunity.

BACKGROUND: The gut microbiota (GM) plays an important role in human health and is being investigated as a possible target for new therapies. Although there are many studies showing that emodin can improve host health, emodin-GM studies are scarce. Here, the effects of emodin on the GM were investigated in vitro and in vivo. RESULTS: In vitro single bacteria cultivation showed that emodin stimulated the growth of beneficial bacteria Akkermansia, Clostridium, Roseburia, and Ruminococcus but inhibited major gut enterotypes (Bacteroides and Prevotella). Microbial community analysis from a synthetic gut microbiome model through co-culture indicated the consistent GM change by emodin. Interestingly, emodin stimulated Clostridium and Ruminococcus (which are related to Roseburia and Faecalibacterium) in a mice experiment and induced anti-inflammatory immune cells, which may correlate with its impact on specific gut bacteria. CONCLUSION: Emodin (i) showed similar GM changes in monoculture, co-culture, and in an in vivo mice experiment and (ii) simulated regulatory T-cell immune responses in vivo. This suggest that emodin may be used to modulate the GM and improve health. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Locus-Specific Reversible DNA Methylation Regulates Transient IL-10 Expression in Th1 Cells.

IL-10 is a pleiotropic cytokine with multifaceted functions in establishing immune homeostasis. Although expressed by Th1 and Th2 cells, conventional Th1 cells produce marginal levels of IL-10 compared with their Th2 counterparts. In this study, we investigated the epigenetic mechanisms of Il-10 gene expression in Th1 cells. Bioinformatics EMBOSS CpG plot analysis and bisulfite pyrosequencing revealed three CpG DNA methylation sites in the Il-10 gene locus. Progressive DNA methylation at all of the CpG regions of interest (ROIs) established a repressive program of Il-10 gene expression in Th1 cells. Interestingly, Th1 cells treated with IL-12 and IL-27 cytokines, thereby mimicking a chronic inflammatory condition in vivo, displayed a significant increase in IL-10 production that was accompanied by selective DNA demethylation at ROI 3 located in intron 3. IL-10-producing T cells isolated from lymphocytic choriomeningitis virus-infected mice also showed enhanced DNA demethylation at ROI 3. Binding of STAT1 and STAT3 to demethylated ROI 3 enhanced IL-10 expression in an IL-12/IL-27-dependent manner. Accordingly, CD4+ T cells isolated from STAT1- or STAT3-knockout mice were significantly defective in IL-10 production. Our data suggest that, although stably maintained DNA methylation at the promoter may repress IL-10 expression in Th1 cells, locus-specific reversible DNA demethylation may serve as a threshold platform to control transient Il-10 gene expression.

Correction to: Cinnamon extract induces tumor cell death through inhibition of NFκB and AP1.

Following publication of the original article [1], the authors have re-evaluated the authorship for this article. The updated author group is.

NFAT1 Regulates Systemic Autoimmunity through the Modulation of a Dendritic Cell Property.

The transcription factor NFAT1 plays a pivotal role in the homeostasis of T lymphocytes. However, its functional importance in non-CD4+ T cells, especially in systemic immune disorders, is largely unknown. In this study, we report that NFAT1 regulates dendritic cell (DC) tolerance and suppresses systemic autoimmunity using the experimental autoimmune myasthenia gravis (EAMG) as a model. Myasthenia gravis and EAMG are T cell-dependent, Ab-mediated autoimmune disorders in which the acetylcholine receptor is the major autoantigen. NFAT1-knockout mice showed higher susceptibility to EAMG development with enhanced Th1/Th17 cell responses. NFAT1 deficiency led to a phenotypic alteration of DCs that show hyperactivation of NF-κB-mediated signaling pathways and enhanced binding of NF-κB (p50) to the promoters of IL-6 and IL-12. As a result, NFAT1-knockout DCs produced much higher levels of proinflammatory cytokines such as IL-1ß, IL-6, IL-12, and TNF-α, which preferentially induce Th1/Th17 cell differentiation. Our data suggest that NFAT1 may limit the hyperactivation of the NF-κB-mediated proinflammatory response in DCs and suppress autoimmunity by serving as a key regulator of DC tolerance.

Hypoxia-inducible factor-2α is an essential catabolic regulator of inflammatory rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder that manifests as chronic inflammation and joint tissue destruction. However, the etiology and pathogenesis of RA have not been fully elucidated. Here, we explored the role of the hypoxia-inducible factors (HIFs), HIF-1α (encoded by HIF1A) and HIF-2α (encoded by EPAS1). HIF-2α was markedly up-regulated in the intimal lining of RA synovium, whereas HIF-1α was detected in a few cells in the sublining and deep layer of RA synovium. Overexpression of HIF-2α in joint tissues caused an RA-like phenotype, whereas HIF-1α did not affect joint architecture. Moreover, a HIF-2α deficiency in mice blunted the development of experimental RA. HIF-2α was expressed mainly in fibroblast-like synoviocytes (FLS) of RA synovium and regulated their proliferation, expression of RANKL (receptor activator of nuclear factor-κB ligand) and various catabolic factors, and osteoclastogenic potential. Moreover, HIF-2α-dependent up-regulation of interleukin (IL)-6 in FLS stimulated differentiation of TH17 cells-crucial effectors of RA pathogenesis. Additionally, in the absence of IL-6 (Il6-/- mice), overexpression of HIF-2α in joint tissues did not cause an RA phenotype. Thus, our results collectively suggest that HIF-2α plays a pivotal role in the pathogenesis of RA by regulating FLS functions, independent of HIF-1α.

6-Methoxyflavone inhibits NFAT translocation into the nucleus and suppresses T cell activation.

NFAT plays a crucial role in the immune system by regulating the transcription of inducible genes during immune responses. In T cells, NFAT proteins govern various cellular events related to T cell development, activation, tolerance induction, and differentiation. We previously reported the NFAT1-dependent enhancer activity of conserved noncoding sequence (CNS)-9, a distal cis-acting element, in the regulation of IL-10 transcription in T cells. In this study, we developed a T cell-based reporter system to identify compounds that modulate the regulatory activity of CNS-9. Among the identified candidates, 6-methoxyflavone (6-MF) significantly inhibited the enhancer activity of CNS-9, thereby reducing IL-10 expression in T cells without affecting cell viability. 6-MF also downregulated the transcription of NFAT1 target genes such as IL-4, IL-13, and IFN-γ. Treatment of 6-MF inhibited the translocation of NFAT1 into the nucleus, which consequently interrupted NFAT1 binding to the target loci, without affecting the expression or dephosphorylation of NFAT1. Treatment of 6-MF to CD4(+) T cells or B cells isolated from mice with atopic dermatitis significantly reduced disease-associated cytokine production, as well as the levels of IgE. In addition, oral administration of 6-MF to atopic dermatitis mice ameliorated disease symptoms by reducing serum IgE levels and infiltrating lymphocytes. Conclusively, our results suggest that 6-MF can be a potential candidate for the development of an effective immunomodulator via the suppression of NFAT-mediated T cell activation.

Interaction of Ets-1 with HDAC1 represses IL-10 expression in Th1 cells.

IL-10 is a multifunctional cytokine that plays a crucial role in immunity and tolerance. IL-10 is produced by diverse immune cell types, including B cells and subsets of T cells. Although Th1 produce IL-10, their expression levels are much lower than Th2 cells under conventional stimulation conditions. The potential role of E26 transformation-specific 1 (Ets-1) transcription factor as a negative regulator for Il10 gene expression in CD4(+) T cells has been implicated previously. In this study, we investigated the underlying mechanism of Ets-1-mediated Il10 gene repression in Th1 cells. Compared with wild type Th1 cells, Ets-1 knockout Th1 cells expressed a significantly higher level of IL-10, which is comparable with that of wild type Th2 cells. Upregulation of IL-10 expression in Ets-1 knockout Th1 cells was accompanied by enhanced chromatin accessibility and increased recruitment of histone H3 acetylation at the Il10 regulatory regions. Reciprocally, Ets-1 deficiency significantly decreased histone deacetylase 1 (HDAC1) enrichment at the Il10 regulatory regions. Treatment with trichostatin A, an inhibitor of HDAC family, significantly increased Il10 gene expression by increasing histone H3 acetylation recruitment. We further demonstrated a physical interaction between Ets-1 and HDAC1. Coexpression of Ets-1 with HDAC1 synergistically repressed IL-10 transcription activity. In summary, our data suggest that an interaction of Ets-1 with HDAC1 represses the Il10 gene expression in Th1 cells.

Dexamethasone-induced muscle atrophy and bone loss in six genetically diverse collaborative cross founder strains demonstrates phenotypic variability by Rg3 treatment.

Background: Osteosarcopenia is a common condition characterized by the loss of both bone and muscle mass, which can lead to an increased risk of fractures and disability in older adults. The study aimed to elucidate the response of various mouse strains to treatment with Rg3, one of the leading ginsenosides, on musculoskeletal traits and immune function, and their correlation. Methods: Six Collaborative Cross (CC) founder strains induced muscle atrophy and bone loss with dexamethasone (15 mg/kg) treatment for 1 month, and half of the mice for each strain were orally administered Rg3 (20 mg/kg). Different responses were observed depending on genetic background and Rg3 treatment. Results: Rg3 significantly increased grip strength, running performance, and expression of muscle and bone health-related genes in a two-way analysis of variance considering the genetic backgrounds and Rg3 treatment. Significant improvements in grip strength, running performance, bone area, and muscle mass, and the increased gene expression were observed in specific strains of PWK/PhJ. For traits related to muscle, bone, and immune functions, significant correlations between traits were confirmed following Rg3 administration compared with control mice. The phenotyping analysis was compiled into a public web resource called Rg3-OsteoSarco. Conclusion: This highlights the complex interplay between genetic determinants, pathogenesis of muscle atrophy and bone loss, and phytochemical bioactivity and the need to move away from single inbred mouse models to improve their translatability to genetically diverse humans. Rg3-OsteoSarco highlights the use of CC founder strains as a valuable tool in the field of personalized nutrition.

Stat6 and c-Jun mediate Th2 cell-specific IL-24 gene expression.

TCR signaling regulates multiple aspects of T cell function by controlling expression of various cytokine genes. IL-24 is a multifunctional cytokine belonging to the IL-10 family. It displays anticancer effects in diverse cancer cells and regulates immunopathology of psoriasis and rheumatoid arthritis. IL-24 also plays an important role in B cell differentiation. Mouse IL-24 gene is selectively expressed in activated Th2 cells upon TCR stimulation. However, the molecular mechanisms by which TCR stimulation induces IL-24 gene expression are still unclear. In this study, to elucidate the mechanism of Th2 cell-specific expression of IL-24, we identified a proximal promoter region (-157/+95 bp) that plays critical role in activating the IL-24 gene in Th2 cells. This region has a Th2 cell-specific open chromatin structure along with permissive histone modifications. In vivo binding of Stat6 and AP-1 (c-Jun) to the IL-24 promoter locus in Th2 cells synergistically transactivated the IL-24 promoter. Stat6 and c-Jun proteins were found to physically cooperate with each other and upregulated IL-24 gene transcription. Knockdown of either Stat6 or c-Jun suppressed endogenous IL-24 gene expression in Th2 cells. In summary, TCR stimulation induces IL-24 expression in Th2 cells by the coordinate action of Stat6 and c-Jun transcription factors at the transcriptional level.

Generation of regulatory dendritic cells and CD4+Foxp3+ T cells by probiotics administration suppresses immune disorders.

The beneficial effects of probiotics have been described in many diseases, but the mechanism by which they modulate the immune system is poorly understood. In this study, we identified a mixture of probiotics that up-regulates CD4(+)Foxp3(+) regulatory T cells (Tregs). Administration of the probiotics mixture induced both T-cell and B-cell hyporesponsiveness and down-regulated T helper (Th) 1, Th2, and Th17 cytokines without apoptosis induction. It also induced generation of CD4(+)Foxp3(+) Tregs from the CD4(+)CD25(-) population and increased the suppressor activity of naturally occurring CD4(+)CD25(+) Tregs. Conversion of T cells into Foxp3(+) Tregs is directly mediated by regulatory dendritic cells (rDCs) that express high levels of IL-10, TGF-beta, COX-2, and indoleamine 2,3-dioxygenase. Administration of probiotics had therapeutical effects in experimental inflammatory bowel disease, atopic dermatitis, and rheumatoid arthritis. The therapeutical effect of the probiotics is associated with enrichment of CD4(+)Foxp3(+) Tregs in the inflamed regions. Collectively, the administration of probiotics that enhance the generation of rDCs and Tregs represents an applicable treatment of inflammatory immune disorders.

Exploring probiotic effector molecules and their mode of action in gut-immune interactions.

Probiotics, live microorganisms that confer health benefits when consumed in adequate amounts, have gained significant attention for their potential therapeutic applications. The beneficial effects of probiotics are believed to stem from their ability to enhance intestinal barrier function, inhibit pathogens, increase beneficial gut microbes, and modulate immune responses. However, clinical studies investigating the effectiveness of probiotics have yielded conflicting results, potentially due to the wide variety of probiotic species and strains used, the challenges in controlling the desired number of live microorganisms, and the complex interactions between bioactive substances within probiotics. Bacterial cell wall components, known as effector molecules, play a crucial role in mediating the interaction between probiotics and host receptors, leading to the activation of signaling pathways that contribute to the health-promoting effects. Previous reviews have extensively covered different probiotic effector molecules, highlighting their impact on immune homeostasis. Understanding how each probiotic component modulates immune activity at the molecular level may enable the prediction of immunological outcomes in future clinical studies. In this review, we present a comprehensive overview of the structural and immunological features of probiotic effector molecules, focusing primarily on Lactobacillus and Bifidobacterium. We also discuss current gaps and limitations in the field and propose directions for future research to enhance our understanding of probiotic-mediated immunomodulation.

Gut Microbial Metabolites on Host Immune Responses in Health and Disease.

Intestinal microorganisms interact with various immune cells and are involved in gut homeostasis and immune regulation. Although many studies have discussed the roles of the microorganisms themselves, interest in the effector function of their metabolites is increasing. The metabolic processes of these molecules provide important clues to the existence and function of gut microbes. The interrelationship between metabolites and T lymphocytes in particular plays a significant role in adaptive immune functions. Our current review focuses on 3 groups of metabolites: short-chain fatty acids, bile acids metabolites, and polyamines. We collated the findings of several studies on the transformation and production of these metabolites by gut microbes and explained their immunological roles. Specifically, we summarized the reports on changes in mucosal immune homeostasis represented by the Tregs and Th17 cells balance. The relationship between specific metabolites and diseases was also analyzed through latest studies. Thus, this review highlights microbial metabolites as the hidden treasure having potential diagnostic markers and therapeutic targets through a comprehensive understanding of the gut-immune interaction.

Chlorella vulgaris Modulates Gut Microbiota and Induces Regulatory T Cells to Alleviate Colitis in Mice.

Chlorella vulgaris (C. vulgaris) is unicellular green algae consumed worldwide as a functional food. The immune stimulatory function of C. vulgaris is known; however, no study has elucidated its immune regulatory potential and associated microbiome modulation. In the current study, we aimed to validate the immune regulatory role of C. vulgaris mediated through two mechanisms. Initially, we assessed its ability to promote the expansion of the regulatory T cell (Treg) population. Subsequently, we investigated its impact on gut microbiota composition and associated metabolites. The supplementation of C. vulgaris altered the gut microbiota composition, accompanied by increased short-chain fatty acid (SCFAs) production in mice at homeostasis. We later used C. vulgaris in the treatment of a DSS-induced colitis model. C. vulgaris intervention alleviated the pathological symptom of colitis in mice, with a corresponding increase in Treg levels. As C. vulgaris is a safe and widely used food supplement, it can be a feasible strategy to instigate cross-talk between the host immune system and the intestinal flora for the effective management of inflammatory bowel disease (IBD).

IRF4 regulates IL-10 gene expression in CD4(+) T cells through differential nuclear translocation.

CD4(+) T cells play critical roles in the generation of protective immunity against a variety of pathogens. The main two types of effector CD4(+) T cells, Th1 and Th2 are characterized by their ability to produce signature cytokines. Among them, IL-10 is a multi-functional cytokine that plays a crucial role in maintaining the balance between immunity and tolerance. Although IL-10 is produced by both differentiated primary Th1 and Th2 cells, Th2 cells produce much higher levels of IL-10 upon stimulation. However, little information is available on the molecular mechanisms of IL-10 gene regulation at the transcriptional level. Interferon regulatory factor IRF4 is a member of the IRF family of transcription factors and plays critical roles in the development of CD4(+) T cells into Th2 cells. In this present study, we elucidate the underlying mechanism of IRF4 mediated IL-10 gene transcription in primary CD4(+) T cells. Th2 specific binding of IRF4 to the IRF4 responsive elements in IL-10 locus potentiated IL-10 expression in Th2 cells. Knockdown of IRF4 by siRNA decreased IL-10 expression level in Th2 cells. Nuclear translocation of IRF4 was much higher in Th2 cells upon stimulation, which contribute to maintain IL-10(high) phenotype of Th2 cells. Collectively, our results suggest that stimulation driven quantitative differences of IRF4 in the nucleus and its binding to IL-10 regulatory elements are crucial mechanisms to induce IL-10(high) gene expression in Th2 cells.

Obtusifolin, an Anthraquinone Extracted from Senna obtusifolia (L.) H.S.Irwin & Barneby, Reduces Inflammation in a Mouse Osteoarthritis Model.

Osteoarthritis (OA) is an age-related degenerative disease that causes cartilage dysfunction and inflammation. Obtusifolin, an anthraquinone extracted from Senna obtusifolia (L.) H.S.Irwin & Barneby seeds, has anti-inflammatory functions; it could be used as a drug component to relieve OA symptoms. In this study, we investigated the effects of obtusifolin on OA inflammation. In vitro, interleukin (IL)-1ß (1 ng/mL)-treated mouse chondrocytes were co-treated with obtusifolin at different concentrations. The expression of matrix metalloproteinase (Mmp) 3, Mmp13, cyclooxygenase 2 (Cox2), and signaling proteins was measured by polymerase chain reaction and Western blotting; collagenase activity and the PGE2 level were also determined. In vivo, OA-induced C57BL/6 mice were administered obtusifolin, and their cartilage was stained with Safranin O to observe damage. Obtusifolin inhibited Mmp3, Mmp13, and Cox2 expression to levels similar to or more than those after treatment with celecoxib. Additionally, obtusifolin decreased collagenase activity and the PGE2 level. Furthermore, obtusifolin regulated OA via the NF-κB signaling pathway. In surgically induced OA mouse models, the cartilage destruction decreased when obtusifolin was administered orally. Taken together, our results show that obtusifolin effectively reduces cartilage damage via the regulation of MMPs and Cox2 expression. Hence, we suggest that obtusifolin could be a component of another OA symptom reliever.

Schisandrol A Suppresses Catabolic Factor Expression by Blocking NF-κB Signaling in Osteoarthritis.

Schisandrol A possesses pharmacological properties and is used to treat various diseases; however, its effects on osteoarthritis (OA) progression remain unclear. Here, we investigated Schisandrol A as a potential therapeutic agent for OA. In vitro, Schisandrol A effects were confirmed based on the levels of expression of catabolic factors (MMPs, ADAMTS5, and Cox2) induced by IL-1ß or Schisandrol A treatment in chondrocytes. In vivo, experimental OA in mice was induced using a destabilized medial meniscus (DMM) surgical model or oral gavage of Schisandrol A in a dose-dependent manner, and demonstrated using histological analysis. In vitro and in vivo analyses demonstrated that Schisandrol A inhibition attenuated osteoarthritic cartilage destruction via the regulation of Mmp3, Mmp13, Adamts5, and Cox2 expression. In the NF-κB signaling pathway, Schisandrol A suppressed the degradation of IκB and the phosphorylation of p65 induced by IL-1ß. Overall, and Schisandrol A reduced the expression of catabolic factors by blocking NF-κB signaling and prevented cartilage destruction. Therefore, Schisandrol A attenuated OA progression, and can be used to develop novel OA drug therapies.

Cinnamon extract induces tumor cell death through inhibition of NFkappaB and AP1.

BACKGROUND: Cinnamomum cassia bark is the outer skin of an evergreen tall tree belonging to the family Lauraceae containing several active components such as essential oils (cinnamic aldehyde and cinnamyl aldehyde), tannin, mucus and carbohydrate. They have various biological functions including anti-oxidant, anti-microbial, anti-inflammation, anti-diabetic and anti-tumor activity. Previously, we have reported that anti-cancer effect of cinnamon extracts is associated with modulation of angiogenesis and effector function of CD8+ T cells. In this study, we further identified that anti-tumor effect of cinnamon extracts is also link with enhanced pro-apoptotic activity by inhibiting the activities NFkappaB and AP1 in mouse melanoma model. METHODS: Water soluble cinnamon extract was obtained and quality of cinnamon extract was evaluated by HPLC (High Performance Liquid Chromatography) analysis. In this study, we tested anti-tumor activity and elucidated action mechanism of cinnamon extract using various types of tumor cell lines including lymphoma, melanoma, cervix cancer and colorectal cancer in vitro and in vivo mouse melanoma model. RESULTS: Cinnamon extract strongly inhibited tumor cell proliferation in vitro and induced active cell death of tumor cells by up-regulating pro-apoptotic molecules while inhibiting NFkappaB and AP1 activity and their target genes such as Bcl-2, BcL-xL and survivin. Oral administration of cinnamon extract in melanoma transplantation model significantly inhibited tumor growth with the same mechanism of action observed in vitro. CONCLUSION: Our study suggests that anti-tumor effect of cinnamon extracts is directly linked with enhanced pro-apoptotic activity and inhibition of NFkappaB and AP1 activities and their target genes in vitro and in vivo mouse melanoma model. Hence, further elucidation of active components of cinnamon extract could lead to development of potent anti-tumor agent or complementary and alternative medicine for the treatment of diverse cancers.