Identification and immunoinfiltration analysis of key genes in ulcerative colitis using WGCNA.

Objective: Ulcerative colitis (UC) is a chronic non-specific inflammatory bowel disease characterized by an unclear pathogenesis. This study aims to screen out key genes related to UC pathogenesis. Methods: Bioinformatics analysis was conducted for screening key genes linked to UC pathogenesis, and the expression of the screened key genes was verified by establishing a UC mouse model. Results: Through bioinformatics analysis, five key genes were obtained. Subsequent infiltration analysis revealed seven significantly different immune cell types between the UC and general samples. Additionally, animal experiment results illustrated markedly decreased body weight, visible colonic shortening and damage, along with a significant increase in the DAI score of the DSS-induced mice in the UC group in comparison with the NC group. In addition, H&E staining results demonstrated histological changes including marked inflammatory cell infiltration, loss of crypts, and epithelial destruction in the colon mucosa epithelium. qRT-PCR analysis indicated a down-regulation of ABCG2 and an up-regulation of IL1RN, REG4, SERPINB5 and TRIM29 in the UC mouse model. Notably, this observed trend showed a significant dependence on the concentration of DSS, with the mouse model of UC induced by 7% DSS demonstrating a more severe disease state compared to that induced by 5% DSS. Conclusion: ABCG2, IL1RN, REG4, SERPINB5 and TRIM29 were screened out as key genes related to UC by bioinformatics analysis. The expression of ABCG2 was down-regulated, and that of IL1RN, REG4, SERPINB5 and TRIM29 were up-regulated in UC mice as revealed by animal experiments.

Structural modifications of INGAP-PP present in HTD4010 peptide potentiate its effect on rat islet gene expression and insulin secretion.

Type 2 diabetes (T2D) is characterized by peripheral insulin resistance and altered insulin secretion due to a progressive loss of ß-cell mass and function. Today, most antidiabetic agents are designed to resolve impaired insulin secretion and/or insulin resistance, and only GLP-1-based formulations contribute to stopping the decline in ß-cell mass. HTD4010, a peptide carrying two modifications of the amino acid sequence of INGAP-PP (N-terminus acetylation and substitution of Asn13 by Ala) showed greater plasma stability and could be a good candidate for proposal as a drug that could improve ß cell mass and function lost in T2D. In the present study, we showed that HTD4010 included in the culture media of normal rat islets at a dose 100 times lower than that used for INGAP-PP was able to modulate, in the same way as the original peptide, both insulin secretion in response to glucose and the expression of key genes related to insular function, insulin and leptin intracellular pathways, neogenesis, apoptosis, and inflammatory response. Our results confirm the positive effect of HTD4010 on ß-cell function and gene expression of factors involved in the maintenance of ß-cell mass. Although new assays in animal models of prediabetes and T2D must be performed to be conclusive, our results are very encouraging, and they suggest that the use of HTD4010 at a dose 100 times lower than that of INGAP-PP could minimize its side effects in a future clinical trial.

Transcriptional signature of islet neogenesis-associated protein peptide-treated rat pancreatic islets reveals induction of novel long non-coding RNAs.

Background: Diabetes mellitus is characterized by chronic hyperglycemia with loss of ß-cell function and mass. An attractive therapeutic approach to treat patients with diabetes in a non-invasive way is to harness the innate regenerative potential of the pancreas. The Islet Neogenesis-Associated Protein pentadecapeptide (INGAP-PP) has been shown to induce ß-cell regeneration and improve their function in rodents. To investigate its possible mechanism of action, we report here the global transcriptional effects induced by the short-term INGAP-PP in vitro treatment of adult rat pancreatic islets. Methods and findings: Rat pancreatic islets were cultured in vitro in the presence of INGAP-PP for 4 days, and RNA-seq was generated from triplicate treated and control islet samples. We performed a de novo rat gene annotation based on the alignment of RNA-seq reads. The list of INGAP-PP-regulated genes was integrated with epigenomic data. Using the new gene annotation generated in this work, we quantified RNA-seq data profiled in INS-1 cells treated with IL1ß, IL1ß+Calcipotriol (a vitamin D agonist) or vehicle, and single-cell RNA-seq data profiled in rat pancreatic islets. We found 1,669 differentially expressed genes by INGAP-PP treatment, including dozens of previously unannotated rat transcripts. Genes differentially expressed by the INGAP-PP treatment included a subset of upregulated transcripts that are associated with vitamin D receptor activation. Supported by epigenomic and single-cell RNA-seq data, we identified 9 previously unannotated long noncoding RNAs (lncRNAs) upregulated by INGAP-PP, some of which are also differentially regulated by IL1ß and vitamin D in ß-cells. These include Ri-lnc1, which is enriched in mature ß-cells. Conclusions: Our results reveal the transcriptional program that could explain the enhancement of INGAP-PP-mediated physiological effects on ß-cell mass and function. We identified novel lncRNAs that are induced by INGAP-PP in rat islets, some of which are selectively expressed in pancreatic ß-cells and downregulated by IL1ß treatment of INS-1 cells. Our results suggest a relevant function for Ri-lnc1 in ß-cells. These findings are expected to provide the basis for a deeper understanding of islet translational results from rodents to humans, with the ultimate goal of designing new therapies for people with diabetes.

Regenerating gene 4 promotes chemoresistance of colorectal cancer by affecting lipid droplet synthesis and assembly.

BACKGROUND: Regenerating gene 4 (REG4) has been proved to be carcinogenic in some cancers, but its manifestation and possible carcinogenic mechanisms in colorectal cancer (CRC) have not yet been elucidated. Our previous study found that the drug resistance of CRC cells may be closely linked to their fat metabolism. AIM: To explore the role of REG4 in CRC and its association with lipid droplet formation and chemoresistance. METHODS: We conducted a meta-analysis and bioinformatics and pathological analyses of REG4 expression in CRC. The effects of REG4 on the phenotypes and related protein expression were also investigated in CRC cells. We detected the impacts of REG4 on the chemoresistance and lipid droplet formation in CRC cells. Finally, we analyzed how REG4 regulated the transcription and proteasomal degradation of lipogenic enzymes in CRC cells. RESULTS: Compared to normal mucosa, REG4 mRNA expression was high in CRC (P < 0.05) but protein expression was low. An inverse correlation existed between lymph node and distant metastases, tumor-node-metastasis staging or short overall survival and REG4 mRNA overexpression (P < 0.05), but vice versa for REG4 protein expression. REG4-related genes included: Chemokine activity; taste receptors; protein-DNA and DNA packing complexes; nucleosomes and chromatin; generation of second messenger molecules; programmed cell death signals; epigenetic regulation and DNA methylation; transcription repression and activation by DNA binding; insulin signaling pathway; sugar metabolism and transfer; and neurotransmitter receptors (P < 0.05). REG4 exposure or overexpression promoted proliferation, antiapoptosis, migration, and invasion of DLD-1 cells in an autocrine or paracrine manner by activating the epidermal growth factor receptor-phosphoinositide 3-kinase-Akt-nuclear factor-κB pathway. REG4 was involved in chemoresistance not through de novo lipogenesis, but lipid droplet assembly. REG4 inhibited the transcription of acetyl-CoA carboxylase 1 (ACC1) and ATP-citrate lyase (ACLY) by disassociating the complex formation of anti-acetyl (AC)-acetyl-histone 3-AC-histone 4-inhibitor of growth protein-5-si histone deacetylase;-sterol-regulatory element binding protein 1 in their promoters and induced proteasomal degradation of ACC1 or ACLY. CONCLUSION: REG4 may be involved in chemoresistance through lipid droplet assembly. REG4 reduces expression of de novo lipid synthesis key enzymes by inhibiting transcription and promoting ubiquitination-mediated proteasomal degradation.

The bioinformatics analysis of the clinicopathological and prognostic significances of REG4 mRNA in gynecological cancers.

To clarify the clinicopathological importance of REG4 mRNA expression, we used GEO, TCGA, xiantao, UALCAN, and Kaplan-Meier plotter for a bioinformatics analysis in breast, cervical, endometrial and ovarian cancers. Compared to normal tissues, REG4 expression was found to be upregulated in breast, cervical, endometrial, and ovarian cancers (p < 0.05). Breast cancer had a higher level of REG4 methylation than normal tissues (p < 0.05), which was negatively correlated with its mRNA expression. REG4 expression was positively correlated with oestrogen and progesterone receptor expression, and aggressiveness of PAM50 classification of breast cancer patients (p < 0.05). Breast infiltrating lobular carcinomas expressed more REG4 than ductal carcinomas (p < 0.05). The REG4-related signal pathways mainly included peptidase, keratinisation, brush border and digestion and so forth in gynecological cancers. Our results indicated that REG4 overexpression was associated with gynecological carcinogenesis and their histogenesis, and may be used as a marker for aggressive behaviour and prognosis of breast or cervical cancer.IMPACT STATEMENTWhat is already known on this subject? REG4 encodes a secretory c-type lectin and plays an essential role in inflammation, carcinogenesis, apoptotic and radiochemotherapeutic resistance.What do the results of this study add? As a standalone predictor, REG4 expression was positively correlated with progression-free survival. Expression of REG4 mRNA was positively associated with the T stage and adenosquamous cell carcinoma of cervical cancer. The top signal pathways related to REG4 included smell and chemical stimulus, peptidase, intermediate filament, and keratinisation in breast cancer; ligand-receptor interaction, metabolism of hormone, xenobiotic and retinol, peptidase, brush border and digestion in cervical and ovarian cancers; bile secretion, intermediate filament, chemical carcinogenesis, brush border and keratinisation in endometrial cancer. REG4 mRNA expression was positively correlated with DC cell infiltration in breast cancer, positively with Th17 cells, TFH, cytotoxic cells and T cells in cervical and endometrial cancers, and negatively with DC cell infiltration, cytotoxic cells and T cells in ovarian cancer. The top hub genes mainly included small proline rich protein 2B in breast cancer; fibrinogens and apoproteins in cervical, endometrial and ovarian cancers.What are the implications of these finding for clinical practice and/or further research? Our study has showed that REG4 mRNA expression is a potential biomarker or therapeutic target for gynaecologic cancers.

Upregulation of REG IV gene in human intestinal epithelial cells by lipopolysaccharide via downregulation of microRNA-24.

The pathophysiology of inflammatory bowel diseases (IBD) reflects a balance between mucosal injury and reparative mechanisms. Some regenerating gene (Reg) family members (REG Iα, REG Iß and REG IV) are expressed in Crohn's disease (CD) and ulcerative colitis (UC) and involved as proliferative mucosal factors in IBD. We revealed that REG Iα and REG Iß were induced in cell culture system by IL-6/IL-22. Although REG IV was upregulated in IBD biopsy samples, the upregulation of REG IV was not at all induced in cell culture by autoimmune-related cytokines such as IL-6, IL-22 and TNFα. Here, we analysed REG IV expression in LS-174 T and HT-29 human intestinal epithelial cells by real-time RT-PCR and elisa. REG IV expression was induced by lipopolysaccharide (LPS). However, LPS did not activate REG IV promoter activity. As the LPS-induced upregulation of REG IV was considered to be regulated post-transcriptionally, we searched targeted microRNA (miR), which revealed that REG IV mRNA has a potential target sequence for miR-24. We measured the miR-24 level of LPS-treated cells and found that the level was significantly lower. The LPS-induced increase of REG IV mRNA was abolished by the introduction of miR-24 mimic but not by non-specific control RNA.

A de novo regulation design shows an effectiveness in altering plant secondary metabolism.

Introduction: Transcription factors (TFs) and cis-regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for De Novo regulation designs to engineer plants. Objectives: We selected two Arabidopsis TF types and two tobacco CRE types to design a De Novo regulation and evaluated its effectiveness in plant engineering. Methods: G-box and MYB recognition elements (MREs) were identified in four Nicotiana tabacum JAZs (NtJAZs) promoters. MRE-like and G-box like elements were identified in one nicotine pathway gene promoter. TF screening led to select Arabidopsis Production of Anthocyanin Pigment 1 (PAP1/MYB) and Transparent Testa 8 (TT8/bHLH). Two NtJAZ and two nicotine pathway gene promoters were cloned from commercial Narrow Leaf Madole (NL) and KY171 (KY) tobacco cultivars. Electrophoretic mobility shift assay (EMSA), cross-linked chromatin immunoprecipitation (ChIP), and dual-luciferase assays were performed to test the promoter binding and activation by PAP1 (P), TT8 (T), PAP1/TT8 together, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex. A DNA cassette was designed and then synthesized for stacking and expressing PAP1 and TT8 together. Three years of field trials were performed by following industrial and GMO protocols. Gene expression and metabolic profiling were completed to characterize plant secondary metabolism. Results: PAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activated NtJAZ promoters but did not bind to nicotine pathway gene promoters. The engineered red P + T plants significantly upregulated four NtJAZs but downregulated the tobacco alkaloid biosynthesis. Field trials showed significant reduction of five tobacco alkaloids and four carcinogenic tobacco specific nitrosamines in most or all cured leaves of engineered P + T and PAP1 genotypes. Conclusion: G-boxes, MREs, and two TF types are appropriate molecular tools for a De Novo regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.

Colonic interleukin-22 protects intestinal mucosal barrier and microbiota abundance in severe acute pancreatitis.

Intestinal barrier dysfunction plays a critical role in the pathophysiology of many diseases including severe acute pancreatitis (SAP). Interleukin-22 (IL-22) is a critical regulator of intestinal epithelial homeostasis. However, the mechanism, origin site, and characteristics of IL-22 in the intestinal barrier dysfunction remains elusive. Studies were conducted in patients with SAP and SAP mice model. SAP mice model was induced by intraductal infusion of 5% taurocholic acid. The level and source of IL-22 were analyzed by flow cytometry. The effect of IL-22 in SAP-associated intestinal injury were examined through knockout of IL-22 (IL-22-/- ) or administration of recombinant IL-22 (rIL-22). IL-22 increased in the early phase of SAP but declined more quickly than that of proinflammatory cytokines, such as IL-6 and TNF-α. CD177+ neutrophils contributed to IL-22 expression in SAP. IL-22 was activated in the colon rather than the small intestine during SAP. Deletion of IL-22 worse the severity of colonic injury, whereas administration of rIL-22 reduced colonic injury. Mechanistically, IL-22 ameliorates the intestinal barrier dysfunction in SAP through decreasing colonic mucosal permeability, upregulation of E-cadherin and ZO-1 expression, activation of pSTAT3/Reg3 pathway and restoration of fecal microbiota abundance. This study revealing that early decreased colonic IL-22 aggravates intestinal mucosal barrier dysfunction and microbiota dysbiosis in SAP. Colonic IL-22 is likely a promising treating target in the early phase of SAP management. Research in context Evidence before this study Intestinal barrier dysfunction plays a critical role in the pathophysiology of severe acute pancreatitis (SAP). Interleukin-22 (IL-22) is a critical regulator of intestinal epithelial homeostasis. However, the mechanism, origin site and characteristics of IL-22 in the intestinal barrier dysfunction remains elusive. Added value of this study Firstly, we determined the dynamic expression profile of IL-22 in SAP and found that IL-22 was mostly activated in the pancreas and colon and decreased earlier than proinflammatory cytokines. CD177+ neutrophils contributed to IL-22 expression in SAP. Furthermore, we found that IL-22 ameliorates intestinal barrier dysfunction in SAP through decreasing colonic mucosal permeability, upregulation of E-cadherin and ZO-1 expression, activation of pSTAT3/Reg3 pathway and restoration of fecal microbiota abundance. Implications of all the available evidence This study highlights the role of colonic injury and colonic IL-22 in SAP. IL-22 is likely a promising treating target in the early phase of SAP management.

Regenerating islet-derived protein 3-α is a prognostic biomarker for gastrointestinal chronic graft-versus-host disease.

Prognostic biomarkers used to identify likelihood of disease progression have not been identified for chronic graft-versus-host disease (cGVHD), the leading cause of late nonrelapse mortality (NRM) in survivors of allogeneic hematopoietic cell transplantation. Gastrointestinal cGVHD (GI-cGVHD) has been particularly challenging to classify. Here, we analyzed 3 proteomics markers (Regenerating islet-derived protein 3-α [Reg3α], C-X-C motif ligand 9 [CXCL9], and Stimulation-2 [ST2]) in 2 independent cohorts of patients with cGVHD totaling 289 patients. Plasma concentrations of Reg3α were significantly increased in patients with GI-cGVHD (P = .0012) compared with those without (P = .01), but plasma concentrations of CXCL9 and ST2 were not. Patients with high Reg3α (≥72 ng/mL) vs low Reg3α had higher NRM (23% vs 11%; P = .015). Because Reg3α has been identified as a lower GI tract marker in acute GVHD, we correlated Reg3α with lower acute-like GI-cGVHD vs classical fibrotic-like esophageal manifestations and found that Reg3α did not differ between the subtypes. No difference was observed between upper GI tract and lower GI tract subtypes. Patients with extremely high Reg3α (≥180 ng/mL) had higher GI scores but not higher scores for the lower GI tract. In a multivariable Cox regression model, patients with high Reg3α were 1.9 times more likely to die without relapse. Our findings demonstrate the utility of Reg3α as a prognostic marker for GI-cGVHD. These data warrant prospective biomarker validation studies.

Colonic healing requires Wnt produced by epithelium as well as Tagln+ and Acta2+ stromal cells.

Although Wnt signaling is clearly important for the intestinal epithelial homeostasis, the relevance of various sources of Wnt ligands themselves remains incompletely understood. Blocking the release of Wnt in distinct stromal cell types suggests obligatory functions of several stromal cell sources and yields different observations. The physiological contribution of epithelial Wnt to tissue homeostasis remains unclear. We show here that blocking epithelial Wnts affects colonic Reg4+ epithelial cell differentiation and impairs colonic epithelial regeneration after injury in mice. Single-cell RNA analysis of intestinal stroma showed that the majority of Wnt-producing cells were contained in transgelin (Tagln+) and smooth muscle actin α2 (Acta2+) expressing populations. We genetically attenuated Wnt production from these stromal cells using Tagln-Cre and Acta2-CreER drivers, and found that blockage of Wnt release from either epithelium or Tagln+ and Acta2+ stromal cells impaired colonic epithelial healing after chemical-induced injury. Aggregated blockage of Wnt release from both epithelium and Tagln+ or Acta2+ stromal cells drastically diminished epithelial repair, increasing morbidity and mortality. These results from two uncharacterized stromal populations suggested that colonic recovery from colitis-like injury depends on multiple Wnt-producing sources.

Reg4 Interacts with CD44 to Regulate Proliferation and Stemness of Colorectal and Pancreatic Cancer Cells.

Regenerating Gene 4 (Reg4) is highly upregulated in gastrointestinal (GI) malignancies including colorectal and pancreatic cancers. Numerous studies demonstrated an association between higher Reg4 expression and tumor aggressiveness, intrinsic resistance to apoptotic death, and poor outcomes from GI malignancies. However, the precise receptor and underlying signaling mechanism have remained unknown. Although we previously reported a Reg4-mediated induction of EGFR activity in colorectal cancer cells, a direct interaction between Reg4 and EGFR was not observed. This study is focused on identifying the cell surface binding partner of Reg4 and dissecting its role in colorectal cancer and pancreatic cancer growth and stem cell survival. In vitro models of human colorectal cancer and pancreatic cancer were used to evaluate the results. Results of this study find: (i) Reg4 interacts with CD44, a transmembrane protein expressed by a population of colorectal cancer and pancreatic cancer cells; (ii) Reg4 activates regulated intramembrane proteolysis of CD44 resulting in γ-secretase-mediated cleavage and release of the CD44 intracytoplasmic domain (CD44ICD) that functions as a transcriptional activator of D-type cyclins involved in the regulation of cancer cell proliferation and Klf4 and Sox2 expression involved in regulating pluripotency of cancer stem cells; and (iii) Reg4 significantly increases colorectal cancer and pancreatic cancer cell proliferation and their clonogenic potential in stem cell assays. IMPLICATIONS: These results suggest that pro-proliferative and pro-stemness effects of Reg4 are mediated through γ-secretase-mediated CD44/CD44ICD signaling, hence strategies to disrupt Reg4-CD44-γ-secretase-CD44ICD signaling axis may increase cancer cell susceptibility to chemo- and radiotherapeutics.

The regenerating protein 3A: a crucial molecular with dual roles in cancer.

INTRODUCTION: REG3A, a member of the third subclass of the Reg family, has been found in a variety of tissues but is not detected in immune cells. In the past decade, it has been determined that REG3A expression is regulated by injury, infection, inflammatory stimuli, and pro-cytokines via different signaling pathways, and it acts as a tissue-repair, bactericidal, and anti-inflammatory molecule in human diseases. Recently, the role of REG3A in cancer has received increasing attention. The present article aims to investigate the structure, expression, regulation, function of REG3A, and to highlight the potential role of REG3A in tumors. METHODS: A detailed literature search and data organization were conducted to find information about the role of REG3A in variety of physiological functions and tumors. RESULTS: Contradictory roles of REG3A have been reported in different tumor models. Some studies have demonstrated that high expression of REG3A in cancers can be oncogenic. Other studies have shown decreased REG3A expression in cancer cells as well as suppressed tumor growth. CONCLUSIONS: Taken together, better understanding of REG3A may lead to new insights that make it a potentially useful target for cancer therapy.

Reg3ß: A Potential Therapeutic Target for Tissue Injury and Inflammation-Associated Disorders.

Since regenerating islet-derived 3ß (Reg3ß) was first reported, various studies have been conducted to explore the involvement of Reg3ß in a gamut of maladies, such as diabetes, pancreatitis, pancreatic ductal adenocarcinoma, and extrapancreatic maladies such as inflammatory bowel disease, acute liver failure, and myocardial infarction. Surprisingly, there is currently no systematic review of Reg3ß. Therefore, we summarize the structural characteristics, transcriptional regulation, putative receptors, and signaling pathways of Reg3ß. The exact functional roles in various diseases, especially gastrointestinal and liver diseases, are also discussed. Reg3ß plays multiple roles in promoting proliferation, inducing differentiation, preventing apoptosis, and resisting bacteria. The present review may provide new directions for the diagnosis and treatment of gastrointestinal, liver, and pancreatic diseases.

Reg3A (regenerating family member 3 alpha) acts as a tumor suppressor by targeting DMBT1 (deleted in malignant brain tumors 1) in gastric cancer.

Regenerating family member 3 alpha (Reg3A) encodes a pancreatic secretory protein that may be involved in cell proliferation or differentiation. However, the function and downstream regulatory mechanism of Reg3A in gastric cancer (GC) remains elusive. This study aimed to clarify the function and mechanism of Reg3A regulating cell proliferation in GC. The expression levels of Reg3A were confirmed in GC patients and cells using qRT-PCR and western blotting. TCGA datasets and clinical samples were used to explore the correlation between Reg3A and clinicopathologic features in GC. Cell viability, colony formation, and xenograft tumorigenesis assays were performed to detect the function of Reg3A on cell proliferation. Besides, we predicted the correlated genes of Reg3A by analyzing TCGA datasets, and further investigated the downstream regulatory mechanism of Reg3A in GC. Our results demonstrated that Reg3A is down-regulated in vitro and vivo (P < 0.05). Reg3A expression are negatively correlated with TNM classification (P < 0.001), lymph node (P < 0.001) in GC. Reg3A significantly suppresses cell proliferation in GC (P < 0.05). Bioinformatic analysis and experimental results confirmed that Reg3A positively regulates the expression of deleted in malignant brain tumor 1 (DMBT1, P < 0.05). Besides, Reg3A and DMBT1 all prolong the overall survival (OS, P < 0.01), post-progression survival (PPS, P < 0.05), and first progression survival (FP, P < 0.01). The function of Reg3A inhibiting cell proliferation is abolished by DMBT1 siRNA in GC (P < 0.05). In conclusion, Reg3A may act as a novel tumor suppressor by promoting DMBT1 expression, which may be a potential therapeutic target in patients with GC.

Secretome screening reveals immunomodulating functions of IFNα-7, PAP and GDF-7 on regulatory T-cells.

Regulatory T cells (Tregs) are the key cells regulating peripheral autoreactive T lymphocytes. Tregs exert their function by suppressing effector T cells. Tregs have been shown to play essential roles in the control of a variety of physiological and pathological immune responses. However, Tregs are unstable and can lose the expression of FOXP3 and suppressive functions as a consequence of outer stimuli. Available literature suggests that secreted proteins regulate Treg functional states, such as differentiation, proliferation and suppressive function. Identification of secreted proteins that affect Treg cell function are highly interesting for both therapeutic and diagnostic purposes in either hyperactive or immunosuppressed populations. Here, we report a phenotypic screening of a human secretome library in human Treg cells utilising a high throughput flow cytometry technology. Screening a library of 575 secreted proteins allowed us to identify proteins stabilising or destabilising the Treg phenotype as suggested by changes in expression of Treg marker proteins FOXP3 and/or CTLA4. Four proteins including GDF-7, IL-10, PAP and IFNα-7 were identified as positive regulators that increased FOXP3 and/or CTLA4 expression. PAP is a phosphatase. A catalytic-dead version of the protein did not induce an increase in FOXP3 expression. Ten interferon proteins were identified as negative regulators that reduced the expression of both CTLA4 and FOXP3, without affecting cell viability. A transcriptomics analysis supported the differential effect on Tregs of IFNα-7 versus other IFNα proteins, indicating differences in JAK/STAT signaling. A conformational model experiment confirmed a tenfold reduction in IFNAR-mediated ISG transcription for IFNα-7 compared to IFNα-10. This further strengthened the theory of a shift in downstream messaging upon external stimulation. As a summary, we have identified four positive regulators of FOXP3 and/or CTLA4 expression. Further exploration of these Treg modulators and their method of action has the potential to aid the discovery of novel therapies for both autoimmune and infectious diseases as well as for cancer.

The Potential Role of REG Family Proteins in Inflammatory and Inflammation-Associated Diseases of the Gastrointestinal Tract.

Regenerating gene (REG) family proteins serve as multifunctional secretory molecules with trophic, antiapoptotic, anti-inflammatory, antimicrobial and probably immuno-regulatory effects. Since their discovery, accumulating evidence has clarified the potential roles of the REG family in the occurrence, progression and development of a wide range of inflammatory and inflammation-associated diseases of the gastrointestinal (GI) tract. However, significant gaps still exist due to the undefined nature of certain receptors, regulatory signaling pathways and possible interactions among distinct Reg members. In this narrative review, we first describe the structural features, distribution pattern and purported regulatory mechanisms of REG family proteins. Furthermore, we summarize the established and proposed roles of REG proteins in the pathogenesis of various inflammation-associated pathologies of the GI tract and the body as a whole, focusing particularly on carcinogenesis in the ulcerative colitis-colitic cancer sequence and gastric cancer. Finally, the clinical relevance of REG products in the context of diagnosis, treatment and prognostication are also discussed in detail. The current evidence suggests a need to better understanding the versatile roles of Reg family proteins in the pathogenesis of inflammatory-associated diseases, and their broadened future usage as therapeutic targets and prognostic biomarkers is anticipated.

Temporal overexpression of IL-22 and Reg3γ differentially impacts the severity of experimental autoimmune encephalomyelitis.

IL-22 is an alpha-helical cytokine which belongs to the IL-10 family of cytokines. IL-22 is produced by RORγt+ innate and adaptive lymphocytes, including ILC3, γδ T, iNKT, Th17 and Th22 cells and some granulocytes. IL-22 receptor is expressed primarily by non-haematopoietic cells. IL-22 is critical for barrier immunity at the mucosal surfaces in the steady state and during infection. Although IL-22 knockout mice were previously shown to develop experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), how temporal IL-22 manipulation in adult mice would affect EAE course has not been studied previously. In this study, we overexpressed IL-22 via hydrodynamic gene delivery or blocked it via neutralizing antibodies in C57BL/6 mice to explore the therapeutic impact of IL-22 modulation on the EAE course. IL-22 overexpression significantly decreased EAE scores and demyelination, and reduced infiltration of IFN-γ+IL-17A+Th17 cells into the central nervous system (CNS). The neutralization of IL-22 did not alter the EAE pathology significantly. We show that IL-22-mediated protection is independent of Reg3γ, an epithelial cell-derived antimicrobial peptide induced by IL-22. Thus, overexpression of Reg3γ significantly exacerbated EAE scores, demyelination and infiltration of IFN-γ+IL-17A+ and IL-17A+GM-CSF+Th17 cells to CNS. We also show that Reg3γ may inhibit IL-2-mediated STAT5 signalling and impair expansion of Treg cells in vivo and in vitro. Finally, Reg3γ overexpression dramatically impacted intestinal microbiota during EAE. Our results provide novel insight into the role of IL-22 and IL-22-induced antimicrobial peptide Reg3γ in the pathogenesis of CNS inflammation in a murine model of MS.

IL-22-dependent dysbiosis and mononuclear phagocyte depletion contribute to steroid-resistant gut graft-versus-host disease in mice.

Efforts to improve the prognosis of steroid-resistant gut acute graft-versus-host-disease (SR-Gut-aGVHD) have suffered from poor understanding of its pathogenesis. Here we show that the pathogenesis of SR-Gut-aGVHD is associated with reduction of IFN-γ+ Th/Tc1 cells and preferential expansion of IL-17-IL-22+ Th/Tc22 cells. The IL-22 from Th/Tc22 cells causes dysbiosis in a Reg3γ-dependent manner. Transplantation of IFN-γ-deficient donor CD8+ T cells in the absence of CD4+ T cells produces a phenocopy of SR-Gut-aGVHD. IFN-γ deficiency in donor CD8+ T cells also leads to a PD-1-dependent depletion of intestinal protective CX3CR1hi mononuclear phagocytes (MNP), which also augments expansion of Tc22 cells. Supporting the dual regulation, simultaneous dysbiosis induction and depletion of CX3CR1hi MNP results in full-blown Gut-aGVHD. Our results thus provide insights into SR-Gut-aGVHD pathogenesis and suggest the potential efficacy of IL-22 antagonists and IFN-γ agonists in SR-Gut-aGVHD therapy.

Oral administration of PEGylated TLR7 ligand ameliorates alcohol-associated liver disease via the induction of IL-22.

Effective therapies for alcohol-associated liver disease (ALD) are limited; therefore, the discovery of new therapeutic agents is greatly warranted. Toll-like receptor 7 (TLR7) is a pattern recognition receptor for single-stranded RNA, and its activation prevents liver fibrosis. We examined liver and intestinal damage in Tlr7-/- mice to determine the role of TLR7 in ALD pathogenesis. In an alcoholic hepatitis (AH) mouse model, hepatic steatosis, injury, and inflammation were induced by chronic binge ethanol feeding in mice, and Tlr7 deficiency exacerbated these effects. Because these results demonstrated that endogenous TLR7 signaling activation is protective in the AH mouse model, we hypothesized that TLR7 activation may be an effective therapeutic strategy for ALD. Therefore, we investigated the therapeutic effect of TLR7 agonistic agent, 1Z1, in the AH mouse model. Oral administration of 1Z1 was well tolerated and prevented intestinal barrier disruption and bacterial translocation, which thus suppressed ethanol-induced hepatic injury, steatosis, and inflammation. Furthermore, 1Z1 treatment up-regulated the expression of antimicrobial peptides, Reg3b and Reg3g, in the intestinal epithelium, which modulated the microbiome by decreasing and increasing the amount of Bacteroides and Lactobacillus, respectively. Additionally, 1Z1 up-regulated intestinal interleukin (IL)-22 expression. IL-22 deficiency abolished the protective effects of 1Z1 in ethanol-induced liver and intestinal damage, suggesting intestinal IL-22 as a crucial mediator for 1Z1-mediated protection in the AH mouse model. Collectively, our results indicate that TLR7 signaling exerts protective effects in the AH mouse model and that a TLR7 ligand, 1Z1, holds therapeutic potential for the treatment of AH.

Bacteria-derived long chain fatty acid exhibits anti-inflammatory properties in colitis.

OBJECTIVE: Data from clinical research suggest that certain probiotic bacterial strains have the potential to modulate colonic inflammation. Nonetheless, these data differ between studies due to the probiotic bacterial strains used and the poor knowledge of their mechanisms of action. DESIGN: By mass-spectrometry, we identified and quantified free long chain fatty acids (LCFAs) in probiotics and assessed the effect of one of them in mouse colitis. RESULTS: Among all the LCFAs quantified by mass spectrometry in Escherichia coli Nissle 1917 (EcN), a probiotic used for the treatment of multiple intestinal disorders, the concentration of 3-hydroxyoctadecaenoic acid (C18-3OH) was increased in EcN compared with other E. coli strains tested. Oral administration of C18-3OH decreased colitis induced by dextran sulfate sodium in mice. To determine whether other bacteria composing the microbiota are able to produce C18-3OH, we targeted the gut microbiota of mice with prebiotic fructooligosaccharides (FOS). The anti-inflammatory properties of FOS were associated with an increase in colonic C18-3OH concentration. Microbiota analyses revealed that the concentration of C18-3OH was correlated with an increase in the abundance in Allobaculum, Holdemanella and Parabacteroides. In culture, Holdemanella biformis produced high concentration of C18-3OH. Finally, using TR-FRET binding assay and gene expression analysis, we demonstrated that the C18-3OH is an agonist of peroxisome proliferator activated receptor gamma. CONCLUSION: The production of C18-3OH by bacteria could be one of the mechanisms implicated in the anti-inflammatory properties of probiotics. The production of LCFA-3OH by bacteria could be implicated in the microbiota/host interactions.