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Group 3 innate lymphoid cells (ILC3) are the major subset of gut-resident ILC with essential roles in infections and tissue repair, but how they adapt to the gut environment to maintain tissue residency is unclear. We report that Tox2 is critical for gut ILC3 maintenance and function. Gut ILC3 highly expressed Tox2, and depletion of Tox2 markedly decreased ILC3 in gut but not at central sites, resulting in defective control of Citrobacter rodentium infection. Single-cell transcriptional profiling revealed decreased expression of Hexokinase-2 in Tox2-deficient gut ILC3. Consistent with the requirement for hexokinases in glycolysis, Tox2-/- ILC3 displayed decreased ability to utilize glycolysis for protein translation. Ectopic expression of Hexokinase-2 rescued Tox2-/- gut ILC3 defects. Hypoxia and interleukin (IL)-17A each induced Tox2 expression in ILC3, suggesting a mechanism by which ILC3 adjusts to fluctuating environments by programming glycolytic metabolism. Our results reveal the requirement for Tox2 to support the metabolic adaptation of ILC3 within the gastrointestinal tract.

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Objectives: Peripheral blood immune cell profiling of atopic dermatitis patients before and after treatment by single-cell RNA sequencing technique has not been reported. To study the immune Cell Profiling of Atopic Dermatitis Patients Before and After Treatment with Halometasone Cream Wet-Wrap Therapy. Methods: We used single cell sequencing to detect the proportion change and gene expression change of immune cells in 2 patients before and after treatment, and then used real-time PCR to confirm the mRNA level of differential genes. Results: In this study, scRNA-seq in two patients with severe AD before and after halometasone cream wet-wrap therapy showed that in the mild severity of AD after treatment, Th2 cells were significantly decreased (41.2% vs 13.4%), Th1 and Th17 cells were increased (23.3% vs 43.7%, 2.3% vs 4.8% respectively). The proportion of Th22 cells did not change much (1.3% vs 1.9%). Tregs were significantly increased also (1.5% vs 5.0%). In the regulatory T cells, the expression of IL-27, PD-1, CD103, CTLA-4, ZNF-66, IL-ß, CD7 gene was specifically increased after treatment, and CD39, P21, TOX2, CD151, CD79A, S100A12, TRAP1 gene was specifically decreased after treatment. In the TH2 cells, the expression of CD27, CD68, EZH1, RAD1, EGFR, CCR10, BCL11A, KLF4 gene was specifically increased after treatment and CCL26, CD180, IL-31, CCL22, LEF1, OX40 gene was specifically decreased after treatment. Conclusions: These genes may be new target for further study.

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Colon cancer was the second leading cause of cancer-related deaths in Japan in 2019. The effects of geniposide isolated from Gardenia jasminoides fructus (Rubiaceae) on the azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced growth of colon tumors and changes in interleukin (IL)-1 ß, monocyte chemoattractant protein (MCP)-1, IL-10, and programmed cell death-1 (PD-1) levels in the colon were investigated. The intraperitoneal administration of AOM (10 mg/kg) on days 0 and 27 induced colorectal carcinogenesis. Free access to 1% (w/v) DSS drinking water was given to mice on days 7-15, 32-33, and 35-38. Geniposide (30 and 100 mg/kg) was orally administered on days 1-16, discontinued for 11 days (days 16 to 26), and then administered again on days 27-41. Colonic levels of cytokines, chemokine, and PD-1 were measured using by enzyme-linked immunosorbent assay (ELISA). Increases in colorectal tumor numbers and areas were significantly inhibited by geniposide. In addition, geniposide (100 mg/kg) reduced colonic levels of IL-1 ß, MCP-1, PD-1 and IL-10 by 67.4, 57.2, 100%, and 100% respectively. Cyclooxygenase (COX)-2- and thymocyte selection high mobility group box proteins (TOX/TOX2)-positive cell numbers were significantly reduced by geniposide. Geniposide (30 and 100 mg/kg) decreased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) expressions in immunohistochemical analysis by 64.2 and 98.2%, respectively. Thus, the inhibitory effects of geniposide on colon tumor growth may be associated with reductions in the colonic levels of IL-1 ß, MCP-1, IL-10, and PD-1 via the down-regulated expression of COX-2 and TOX/TOX2 through the inhibition of Phospho-STAT3 expression (in vivo and in vitro).

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BACKGROUND: Extranodal natural killer/T-cell lymphoma (NKTL) is an aggressive type of non-Hodgkin lymphoma with dismal outcome. A better understanding of disease biology and key oncogenic process is necessary for the development of targeted therapy. Super-enhancers (SEs) have been shown to drive pivotal oncogenes in various malignancies. However, the landscape of SEs and SE-associated oncogenes remain elusive in NKTL. METHODS: We used Nano-ChIP-seq of the active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) to profile unique SEs NKTL primary tumor samples. Integrative analysis of RNA-seq and survival data further pinned down high value, novel SE oncogenes. We utilized shRNA knockdown, CRISPR-dCas9, luciferase reporter assay, ChIP-PCR to investigate the regulation of transcription factor (TF) on SE oncogenes. Multi-color immunofluorescence (mIF) staining was performed on an independent cohort of clinical samples. Various function experiments were performed to evaluate the effects of TOX2 on the malignancy of NKTL in vitro and in vivo. RESULTS: SE landscape was substantially different in NKTL samples in comparison with normal tonsils. Several SEs at key transcriptional factor (TF) genes, including TOX2, TBX21(T-bet), EOMES, RUNX2, and ID2, were identified. We confirmed that TOX2 was aberrantly overexpressed in NKTL relative to normal NK cells and high expression of TOX2 was associated with worse survival. Modulation of TOX2 expression by shRNA, CRISPR-dCas9 interference of SE function impacted on cell proliferation, survival and colony formation ability of NKTL cells. Mechanistically, we found that RUNX3 regulates TOX2 transcription by binding to the active elements of its SE. Silencing TOX2 also impaired tumor formation of NKTL cells in vivo. Metastasis-associated phosphatase PRL-3 has been identified and validated as a key downstream effector of TOX2-mediated oncogenesis. CONCLUSIONS: Our integrative SE profiling strategy revealed the landscape of SEs, novel targets and insights into molecular pathogenesis of NKTL. The RUNX3-TOX2-SE-TOX2-PRL-3 regulatory pathway may represent a hallmark of NKTL biology. Targeting TOX2 could be a valuable therapeutic intervene for NKTL patients and warrants further study in clinic.

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Colon cancer was the second leading cause of cancer-related death in 2019. We herein investigated the effects of acertannin containing Acer species on azoxymethane (AOM)/dextran sulfate sodium (DDS)-induced colon cancer growth and changes in the colonic levels of interleukin (IL)-1ß, monocyte chemoattractant protein (MCP)-1, IL-10, and programmed cell death-1 (PD-1). Colorectal carcinogenesis was induced by an intraperitoneal injection of AOM (10 mg/kg) on days 0 and 27. Mice were given 1% (w/v) DSS drinking water ad libitum on days 7-14, 32-33, and 35-38. Acertannin (30 and 100 mg/kg) was orally administered on days 1-16, discontinued for 11 days (days 16-26), and then administered again on days 27-41. The colonic levels of cytokines, a chemokine, and PD-1 were measured using the respective ELISA kits. The number and area of tumors in mice treated with acertannin (100 mg/kg) decreased by 53.9 and 63.1%, respectively. Furthermore, the colonic levels of IL-1ß, MCP-1, IL-10, and PD-1 showed reductions of 57.3, 62.9, 62.8, and 100%, respectively, while the numbers of cyclooxygenase-2 (COX-2)-, thymocyte selection-associated high mobility group box proteins (TOX)/TOX2-, PD-1-, and signal transducer and activator of transcription 3 (STAT3) phosphorylation-positive numbers decreased by 79.6, 77.9, 93.8, and 100%, respectively. In conclusion, the inhibitory effects of acertannin on AOM/DSS-induced colon tumor growth appear to be associated with reductions in the colonic levels of IL-1ß, MCP-1, IL-10, and PD-1 through the down-regulated expression of COX-2 and TOX/TOX2 in the tumor microenvironment.

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BACKGROUND: Colorectal cancer was the second leading cause of mortality in 2019 and the number of new colorectal cancer cases was the highest in 2018 and 2019 in Japan. PURPOSE: The present study investigated the inhibitory effects of 2(S)-2',5,6',7-tetrahydroxyflavanone and 2 (R), 3(R)-2',3,5,6'-7-pentahydroxyflavanone on the incidence and growth of tumors in azoxymethane (AOM) plus dextran sulfate sodium (DSS)-treated mice. METHODS: The intraperitoneal administration of AOM (10 mg/kg) on day 0 induced colorectal carcinogenesis. Mice were given free and unlimited access to drinking water containing 1.5% (w/v) DSS on days 5 - 8, 30 - 33, and 56 - 57. They were orally administered tetra- and penta-hydroxyflavanones (10 and 30 mg/kg) for 10, 11, and 14 days followed by discontinuation intervals of 20 and 15 days. Cytokine, chemokine, programmed cell death-1 (PD-1), cyclooxygenase (COX)-2, and thymocyte selection-associated high mobility group box protein (TOX)/TOX2 expression levels were measured using their respective ELISA kits and an immunohistochemical analysis. RESULTS: The number and area of tumors decreased by 60.6 and 72.9% in mice administered 10 mg/kg tetra- and pentahydroxyflavanones, respectively, with reductions of 95.0 and 87.0% in Ki-67-positive cells, 91.7 and 92.7% in COX-2-postive cells, and 83.1 and 93.8% in TOX/TOX2-positive cells, respectively, in the colon. On the other hand, two tera- and pentahydroxyflavanone had no effect on p53 (a tumor suppressor by cell cycle arrest and apoptosis)-positive cells. The administration of 10 mg/kg tetra- and pentahydroxyflavanones to AOM/DSS-treated mice also resulted in decreases of 59.5 and 42.5% in IL-10 levels and 58.1 and 93.9% in PD-1 levels, respectively, in the colon. CONCLUSION: The inhibitory effects of tetra- and pentahydroxyflavanones on the growth of colon tumors in AOM/DSS-treated mice appear to be associated with decreases in the colon levels of IL-10 and PD-1 through the down-regulated expression of COX-2 and CD8+ T-cell exhaustion by TOX/TOX2 in the tumor microenvironment.

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Increased hepatic glucose production (HGP) contributes to hyperglycemia in type 2 diabetes. Hormonal regulation of this process is primarily, but not exclusively, mediated by the AKT-FoxO1 pathway. Here, we show that cAMP and dexamethasone regulate the high-mobility group superfamily member TOX4 to mediate HGP, independent of the insulin receptor/FoxO1 pathway. TOX4 inhibition decreases glucose production in primary hepatocytes and liver and increases glucose tolerance. Combined genetic ablation of TOX4 and FoxO1 in liver has additive effects on glucose tolerance and gluconeogenesis. Moreover, TOX4 ablation fails to reverse the metabolic derangement brought by insulin receptor knockout. TOX4 expression is increased in livers of patients with steatosis and diabetes and in diet-induced obese and db/db mice. In the latter two murine models, knockdown Tox4 decreases glycemia and improves glucose tolerance. We conclude that TOX4 is an insulin receptor-independent regulator of HGP and a candidate contributor to the pathophysiology of diabetes.

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Nowadays, although dispersants have been widely applied for emergency response to oil spills, they are potentially hazardous to the marine ecosystem. Therefore, it is necessary to evaluate dispersants' toxicity in a practical and integrated way before their large-scale application. Here, we compared the acute toxicity of five chemical dispersants (concentrate RS-I, conventional RS-I, HLD-501, Fuken-2, and Weipu) to three species (a microalgae Platymonas helgolandica, a mollusk Ruditapes philippinarum, and a luminescent bacterium Acinetobacter sp. Tox2) which represent different trophic levels. Our results showed that (1) conventional RS-I was slightly toxic to all the three test organisms; (2) concentrate RS-I and Weipu were slightly toxic to R. philippinarum, but were not toxic to the other two test species; (3) Fuken-2 and HLD-501 exhibited no acute toxicity to the three test organisms. Our results could provide information on toxicity data derived from multiple test organisms for the use of these five dispersants in the future.

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Sulfonamides (SAs) are conventional veterinary antibiotics that pose ecological risks in the aquatic environment. This study aims to evaluate the environmental concerns of SAs in the Three Gorges Reservoir Area (TGRA) and their toxicogenetic implications. Here, we employed various in vitro and in vivo bioassays to determine the combine toxicogenetic effects of SAs, which were further confirmed through applying Combination Index (CI) and Independent Action (IA) models. Among the investigated SAs, sulfamethoxazole (SMX) appeared as the individual chemical with relatively high environmental effects and elevated in vitro and in vivo toxicity. Importantly, exposure to the binary mixtures of SAs induced higher developmental toxicity and significantly perturbed the detoxification pathway in zebrafish, compared to that of individual compound exposure. Moreover, the CI and IA models indicated greater synergistic effects of SAs binary mixtures as SMX-SMR, SMX-ST, and SPY-ST on the Acinetobacter sp. Tox2 at Fa = 0.5. Contrarily, IA model predicted the additive, antagonistic and synergistic effects of SAs mixtures on the transcriptional responses of detoxification pathways in zebrafish, implying the different mode of actions (MoAs) for SAs to induce mixture toxicity in vivo. Thus, the nature of toxicological interactions of SAs should be considered while performing their ecological risk assessment.

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T follicular helper (Tfh) cells provide essential help to B cells in germinal center (GC) reactions. Bcl6 is the obligatory lineage transcription factor in Tfh cells. Here, we examined the molecular pathways that induce Bcl6 gene expression and underscore Bcl6-dependent function during Tfh cell commitment. Integration of genome-wide Bcl6 occupancy in Tfh cells and differential gene expression analyses suggested an important role for the transcription factor Tox2 in Tfh cell differentiation. Ectopic expression of Tox2 was sufficient to drive Bcl6 expression and Tfh development. In genome-wide ChIP-seq analyses, Tox2-bound loci associated with Tfh cell differentiation and function, including Bcl6. Tox2 binding was associated with increased chromatin accessibility at these sites, as measured by ATAC-seq. Tox2-/- mice exhibited defective Tfh differentiation, and inhibition of both Tox2 and the related transcription factor Tox abolished Tfh differentiation. Thus, a Tox2-Bcl6 axis establishes a transcriptional feed-forward loop that promotes the Tfh program.

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T cells expressing chimeric antigen receptors (CAR T cells) have shown impressive therapeutic efficacy against leukemias and lymphomas. However, they have not been as effective against solid tumors because they become hyporesponsive ("exhausted" or "dysfunctional") within the tumor microenvironment, with decreased cytokine production and increased expression of several inhibitory surface receptors. Here we define a transcriptional network that mediates CD8+ T cell exhaustion. We show that the high-mobility group (HMG)-box transcription factors TOX and TOX2, as well as members of the NR4A family of nuclear receptors, are targets of the calcium/calcineurin-regulated transcription factor NFAT, even in the absence of its partner AP-1 (FOS-JUN). Using a previously established CAR T cell model, we show that TOX and TOX2 are highly induced in CD8+ CAR+ PD-1high TIM3high ("exhausted") tumor-infiltrating lymphocytes (CAR TILs), and CAR TILs deficient in both TOX and TOX2 (Tox DKO) are more effective than wild-type (WT), TOX-deficient, or TOX2-deficient CAR TILs in suppressing tumor growth and prolonging survival of tumor-bearing mice. Like NR4A-deficient CAR TILs, Tox DKO CAR TILs show increased cytokine expression, decreased expression of inhibitory receptors, and increased accessibility of regions enriched for motifs that bind activation-associated nuclear factor κB (NFκB) and basic region-leucine zipper (bZIP) transcription factors. These data indicate that Tox and Nr4a transcription factors are critical for the transcriptional program of CD8+ T cell exhaustion downstream of NFAT. We provide evidence for positive regulation of NR4A by TOX and of TOX by NR4A, and suggest that disruption of TOX and NR4A expression or activity could be promising strategies for cancer immunotherapy.

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BACKGROUND: Major depressive disorder (MDD) is the second largest cause of global disease burden. It has an estimated heritability of 37%, but published genome-wide association studies have so far identified few risk loci. Haplotype-block-based regional heritability mapping (HRHM) estimates the localized genetic variance explained by common variants within haplotype blocks, integrating the effects of multiple variants, and may be more powerful for identifying MDD-associated genomic regions. METHODS: We applied HRHM to Generation Scotland: The Scottish Family Health Study, a large family- and population-based Scottish cohort (N = 19,896). Single-single nucleotide polymorphism (SNP) and haplotype-based association tests were used to localize the association signal within the regions identified by HRHM. Functional prediction was used to investigate the effect of MDD-associated SNPs within the regions. RESULTS: A haplotype block across a 24-kb region within the TOX2 gene reached genome-wide significance in HRHM. Single-SNP- and haplotype-based association tests demonstrated that five of nine genotyped SNPs and two haplotypes within this block were significantly associated with MDD. The expression of TOX2 and a brain-specific long noncoding RNA RP1-269M15.3 in frontal cortex and nucleus accumbens basal ganglia, respectively, were significantly regulated by MDD-associated SNPs within this region. Both the regional heritability and single-SNP associations within this block were replicated in the UK-Ireland group of the most recent release of the Psychiatric Genomics Consortium (PGC), the PGC2-MDD (Major Depression Dataset). The SNP association was also replicated in a depressive symptom sample that shares some individuals with the PGC2-MDD. CONCLUSIONS: This study highlights the value of HRHM for MDD and provides an important target within TOX2 for further functional studies.

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