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.

Reg3γ: current understanding and future therapeutic opportunities in metabolic disease.

Regenerating family member gamma, Reg3γ (the mouse homolog of human REG3A), belonging to the antimicrobial peptides (AMPs), functions as a part of the host immune system to maintain spatial segregation between the gut bacteria and the host in the intestine via bactericidal activity. There is emerging evidence that gut manipulations such as bariatric surgery, dietary supplementation or drug treatment to produce metabolic benefits alter the gut microbiome. In addition to changes in a wide range of gut hormones, these gut manipulations also induce the expression of Reg3γ in the intestine. Studies over the past decades have revealed that Reg3γ not only plays a role in the gut lumen but can also contribute to host physiology through interaction with the gut microbiota. Herein, we discuss the current knowledge regarding the biology of Reg3γ, its role in various metabolic functions, and new opportunities for therapeutic strategies to treat metabolic disorders.

Role and mechanism of REG2 depletion in insulin secretion augmented by glutathione peroxidase-1 overproduction.

We previously reported a depletion of murine regenerating islet-derived protein 2 (REG2) in pancreatic islets of glutathione peroxidase-1 (Gpx1) overexpressing (OE) mice. The present study was to explore if and how the REG2 depletion contributed to an augmented glucose stimulated insulin secretion (GSIS) in OE islets. After we verified a consistent depletion (90%, p < 0.05) of REG2 mRNA, transcript, and protein in OE islets compared with wild-type (WT) controls, we treated cultured and perifused OE islets (70 islets/sample) with REG2 (1 µg/ml or ml · min) and observed 30-40% (p < 0.05) inhibitions of GSIS by REG2. Subsequently, we obtained evidences of co-immunoprecipitation, cell surface ligand binding, and co-immunofluorescence for a ligand-receptor binding between REG2 and transmembrane, L-type voltage-dependent Ca2+ channel (CaV1.2) in beta TC3 cells. Mutating the C-type lectin binding domain of REG2 or deglycosylating CaV1.2 removed the inhibition of REG2 on GSIS and(or) the putative binding between the two proteins. Treating cultured OE and perifused WT islets with REG2 (1 µg/ml or ml · min) decreased (p < 0.05) Ca2+ influx triggered by glucose or KCl. An intraperitoneal (ip) injection of REG2 (2 µg/g) to OE mice (6-month old, n = 10) decreased their plasma insulin concentration (46%, p < 0.05) and elevated their plasma glucose concentration (25%, p < 0.05) over a 60 min period after glucose challenge (ip, 1 g/kg). In conclusion, our study identifies REG2 as a novel regulator of Ca2+ influx and insulin secretion, and reveals a new cascade of GPX1/REG2/CaV1.2 to explain how REG2 depletion in OE islets could decrease its binding to CaV1.2, resulting in uninhibited Ca2+ influx and augmented GSIS. These findings create new links to bridge redox biology, tissue regeneration, and insulin secretion.

The effects of REG4 expression on chemoresistance of ovarian cancer.

Although ovarian cancer usually responds well to platinum- and taxane-based first-line chemotherapy, most patients develop recurrence and chemoresistance. Regenerating gene 4 (REG4) is a secretory protein involved in cell differentiation and proliferation. We found higher REG4 expression in ovarian cancer than in normal tissues (p < .05). Regenerating gene 4 expression was negatively associated with overall, progression-free or post-progression survival rates of patients with ovarian cancer receiving platinum or paclitaxel treatment (p < .05) according to a Kaplan-Meier plotter. Regenerating gene 4 overexpression resulted in either cisplatin or paclitaxel resistance, and apoptosis resistance in CAOV3 ovarian cancer cells (p < .05). REG4-transfected ovarian cancer cells showed stronger migration and invasion treated with cisplatin or paclitaxel (p < .05). Additionally, cisplatin or paclitaxel exposure led to the overexpression of phosphorylated phosphoinositide 3-kinase (p-PI3K), p-Akt, phosphorylated mammalian target of rapamycin (p-mTOR), glutathione S-transferase-π, survivin, and B-cell lymphoma 2 in REG4 transfectants compared with control cells (p < .05). These findings suggested that REG4 expression was up-regulated in ovarian cancer, and associated with poor survival and chemotherapy resistance. REG4 promoted the occurrence, development, and chemotherapy resistance of ovarian cancer by regulating cell proliferation, apoptosis, migration, and invasion, and PI3K/Akt/m-TOR signalling pathways. IMPACT STATEMENTWhat is already known on this subject? REG4 mRNA expression is up-regulated in many digestive cancers. High REG4 expression was associated with an adverse prognosis, high tumour and nodal stages, poor differentiation, and hepatic and peritoneal metastases of digestive cancers. REG4 expression conferred cancer cells with increased resistance to chemoradiotherapy, especially 5-FU-based treatment, by activating the MAPK/Erk/Bim signalling pathway.What do the results of this study add? REG4 was highly expressed in ovarian cancer. The expression of p-PI3K, p-AKT, p-mTOR, GST-π, survivin, and Bcl-2 was increased in REG4-overexpressing cells. High REG4 expression was significantly associated with inferior OS, PFS, and PPS rates in patients with ovarian cancer receiving platinum chemotherapy. REG4 mediated cisplatin and paclitaxel resistance in CAOV3 ovarian cancer cells. The percentage of apoptotic cells was markedly lower in REG4-transfected compared to mock-transfected cells after cisplatin or paclitaxel treatment.What are the implications of these findings for clinical practice and/or further research? This study aimed to evaluate the prognostic significance of REG4 expression in ovarian cancer treated with platinum and paclitaxel, to explore REG4 chemoresistance mechanisms to platinum and paclitaxel, and to provide a scientific experimental basis for the clinical treatment and outcome evaluation of ovarian cancer. In order to provide comprehensive clinical treatment of ovarian cancer, it is helpful to improve our understanding of multi-drug resistance and identify new cancer diagnostic biomarkers.

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.

Nociceptor-derived Reg3γ prevents endotoxic death by targeting kynurenine pathway in microglia.

Nociceptors can fine-tune local or systemic immunity, but the mechanisms of nociceptive modulation in endotoxic death remain largely unknown. Here, we identified C-type lectin Reg3γ as a nociceptor-enriched hormone that protects the host from endotoxic death. During endotoxemia, nociceptor-derived Reg3γ penetrates the brain and suppresses the expression of microglial indoleamine dioxygenase 1, a critical enzyme of the kynurenine pathway, via the Extl3-Bcl10 axis. Endotoxin-administered nociceptor-null mice and nociceptor-specific Reg3γ-deficient mice exhibit a high mortality rate accompanied by decreased brain HK1 phosphorylation and ATP production despite normal peripheral inflammation. Such metabolic arrest is only observed in the brain, and aberrant production of brain quinolinic acid, a neurotoxic metabolite of the kynurenine pathway, causes HK1 suppression. Strikingly, the central administration of Reg3γ protects mice from endotoxic death by enhancing brain ATP production. By identifying nociceptor-derived Reg3γ as a microglia-targeted hormone, this study provides insights into the understanding of tolerance to endotoxic death.

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 gamma (Reg3g) ameliorates tacrolimus-induced pancreatic ß-cell dysfunction in mice by restoring mitochondrial function.

BACKGROUND AND PURPOSE: Tacrolimus a first-line medication used after transplantation can induce ß-cell dysfunction, causing new-onset diabetes mellitus (NODM). Regenerating islet-derived protein 3 gamma (Reg3g), a member of the pancreatic regenerative gene family, has been reported to improve type 1 diabetes by promoting ß-cell regeneration. We aim to investigate the role of Reg3g in reversing tacrolimus-induced ß-cell dysfunction and NODM in mice. EXPERIMENTAL APPROACH: Circulating REG3A (the human homologue of mouse Reg3g) in heart transplantation patients treated with tacrolimus was detected. The glucose-stimulated insulin secretion and mitochondrial functions, including mitochondria membrane potential (MMP), mitochondria calcium, ATP production, oxygen consumption rate and mitochondrial morphology were investigated in ß-cells. Additionally, effects of Reg3g on tacrolimus-induced NODM in mice were analysed. KEY RESULTS: Circulating REG3A level in heart transplantation patients with NODM significantly decreased compared with those without diabetes. Tacrolimus down-regulated Reg3g via inhibiting STAT3-mediated transcription activation. Moreover, Reg3g restored glucose-stimulated insulin secretion suppressed by tacrolimus in ß-cells by improving mitochondrial functions, including increased MMP, mitochondria calcium uptake, ATP production, oxygen consumption rate and contributing to an intact mitochondrial morphology. Mechanistically, Reg3g increased accumulation of pSTAT3(Ser727) in mitochondria by activating ERK1/2-STAT3 signalling pathway, leading to restoration of tacrolimus-induced mitochondrial impairment. Reg3g overexpression also effectively mitigated tacrolimus-induced NODM in mice. CONCLUSION AND IMPLICATIONS: Reg3g can significantly ameliorate tacrolimus-induced ß-cell dysfunction by restoring mitochondrial function in a pSTAT3(Ser727)-dependent manner. Our observations identify a novel Reg3g-mediated mechanism that is involved in tacrolimus-induced NODM and establish the novel role of Reg3g in reversing tacrolimus-induced ß-cell dysfunction.

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.

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.

Tumour-Derived Reg3A Educates Dendritic Cells to Promote Pancreatic Cancer Progression.

As a pancreatic inflammatory marker, regenerating islet-derived protein 3A (Reg3A) plays a key role in inflammation-associated pancreatic carcinogenesis by promoting cell proliferation, inhibiting apoptosis, and regulating cancer cell migration and invasion. This study aimed to reveal a novel immuno-regulatory mechanism by which Reg3A modulates tumour-promoting responses during pancreatic cancer (PC) progression. In an in vitro Transwell system that allowed the direct co-culture of human peripheral blood-derived dendritic cells (DCs) and Reg3A-overexpressing/ silenced human PC cells, PC cell-derived Reg3A was found to downregulate CD80, CD83 and CD86 expression on educated DCs, increase DC endocytic function, inhibit DC-induced T lymphocyte proliferation, reduce IL-12p70 production, and enhance IL-23 production by DCs. The positive effect of tumour-derived Reg3A-educated human DCs on PC progression was demonstrated in vivo by intraperitoneally transferring them into PC-implanted severe combined immunodeficiency (SCID) mice reconstituted with human T cells. A Reg3A-JAK2/STAT3 positive feedback loop was identified in DCs educated with Reg3A. In conclusion, as a tumour-derived factor, Reg3A acted to block the differentiation and maturation of the most important antigen-presenting cells, DCs, causing them to limit their potential anti-tumour responses, thus facilitating PC escape and progression.

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.

The microbiota coordinates diurnal rhythms in innate immunity with the circadian clock.

Environmental light cycles entrain circadian feeding behaviors in animals that produce rhythms in exposure to foodborne bacteria. Here, we show that the intestinal microbiota generates diurnal rhythms in innate immunity that synchronize with feeding rhythms to anticipate microbial exposure. Rhythmic expression of antimicrobial proteins was driven by daily rhythms in epithelial attachment by segmented filamentous bacteria (SFB), members of the mouse intestinal microbiota. Rhythmic SFB attachment was driven by the circadian clock through control of feeding rhythms. Mechanistically, rhythmic SFB attachment activated an immunological circuit involving group 3 innate lymphoid cells. This circuit triggered oscillations in epithelial STAT3 expression and activation that produced rhythmic antimicrobial protein expression and caused resistance to Salmonella Typhimurium infection to vary across the day-night cycle. Thus, host feeding rhythms synchronize with the microbiota to promote rhythms in intestinal innate immunity that anticipate exogenous microbial exposure.

REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway.

Persistent acinar to ductal metaplasia (ADM) is a recently recognized precursor of pancreatic ductal adenocarcinoma (PDAC). Here we show that the ADM area of human pancreas tissue adjacent to PDAC expresses significantly higher levels of regenerating protein 3A (REG3A). Exogenous REG3A and its mouse homolog REG3B induce ADM in the 3D culture of primary human and murine acinar cells, respectively. Both Reg3b transgenic mice and REG3B-treated mice with caerulein-induced pancreatitis develop and sustain ADM. Two out of five Reg3b transgenic mice with caerulein-induced pancreatitis show progression from ADM to pancreatic intraepithelial neoplasia (PanIN). Both in vitro and in vivo ADM models demonstrate activation of the RAS-RAF-MEK-ERK signaling pathway. Exostosin-like glycosyltransferase 3 (EXTL3) functions as the receptor for REG3B and mediates the activation of downstream signaling proteins. Our data indicates that REG3A/REG3B promotes persistent ADM through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway. Targeting REG3A/REG3B, its receptor EXTL3, or other downstream molecules could interrupt the ADM process and prevent early PDAC carcinogenesis.

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.

Reg4 regulates pancreatic regeneration following pancreatitis via modulating the Notch signaling.

Pancreatic regeneration after acute pancreatitis is critical in the normal restoration of pancreatic exocrine function, the inhibition of which can cause severe complications including pancreatic exocrine insufficiency. However, the regulators of pancreatic regeneration and the underlying mechanisms remain uncovered. Here, using the inducible Tet-on system, we found that regenerating family member 4 (Reg4) knockdown significantly impaired pancreatic regeneration after pancreatitis. Both acinar-to-ductal metaplasia and the resolution of pancreatitis during regeneration were affected by Reg4 knockdown. Further investigations confirmed that Reg4 exerted its function through regulating Notch activation both in vitro and in vivo. Our study revealed Reg4 as a new regulator and potential therapeutic target for pancreatic regeneration.

Myocardial Accumulations of Reg3A, Reg3γ and Oncostatin M Are Associated with the Formation of Granulomata in Patients with Cardiac Sarcoidosis.

Cardiac sarcoidosis (CS) is a poorly understood disease and is characterized by the focal accumulation of immune cells, thus leading to the formation of granulomata (GL). To identify the developmental principles of fatal GL, fluorescence microscopy and Western blot analysis of CS and control patients is presented here. CS is visualized macroscopically by positron emission tomography (PET)/ computed tomography (CT). A battery of antibodies is used to determine structural, cell cycle and inflammatory markers. GL consist of CD68+, CD163+ and CD206+ macrophages surrounded by T-cells within fibrotic areas. Cell cycle markers such as phospho-histone H3, phospho-Aurora and Ki67 were moderately present; however, the phosphorylated ERM (ezrin, radixin and moesin) and Erk1/2 proteins, strong expression of the myosin motor protein and the macrophage transcription factor PU.1 indicate highly active GL. Mild apoptosis is consistent with PI3 kinase and Akt activation. Massive amounts of the IL-1R antagonist reflect a mild activation of stress and inflammatory pathways in GL. High levels of oncostatin M and the Reg3A and Reg3γ chemokines are in accordance with macrophage accumulation in areas of remodeling cardiomyocytes. We conclude that the formation of GL occurs mainly through chemoattraction and less by proliferation of macrophages. Furthermore, activation of the oncostatin/Reg3 axis might help at first to wall-off substances but might initiate the chronic development of heart failure.

Helicobacter pylori-Induced Rev-erbα Fosters Gastric Bacteria Colonization by Impairing Host Innate and Adaptive Defense.

BACKGROUND & AIMS: Rev-erbα represents a powerful transcriptional repressor involved in immunity. However, the regulation, function, and clinical relevance of Rev-erbα in Helicobacter pylori infection are presently unknown. METHODS: Rev-erbα was examined in gastric samples from H pylori-infected patients and mice. Gastric epithelial cells (GECs) were isolated and infected with H pylori for Rev-erbα regulation assays. Gastric tissues from Rev-erbα-/- and wild-type (littermate control) mice or these mice adoptively transferred with CD4+ T cells from IFN-γ-/- and wild-type mice, bone marrow chimera mice and mice with in vivo pharmacological activation or inhibition of Rev-erbα were examined for bacteria colonization. GECs, CD45+CD11c-Ly6G-CD11b+CD68- myeloid cells and CD4+ T cells were isolated, stimulated and/or cultured for Rev-erbα function assays. RESULTS: Rev-erbα was increased in gastric mucosa of H pylori-infected patients and mice. H pylori induced GECs to express Rev-erbα via the phosphorylated cagA that activated ERK signaling pathway to mediate NF-κB directly binding to Rev-erbα promoter, which resulted in increased bacteria colonization within gastric mucosa. Mechanistically, Rev-erbα in GECs not only directly suppressed Reg3b and ß-defensin-1 expression, which resulted in impaired bactericidal effects against H pylori of these antibacterial proteins in vitro and in vivo; but also directly inhibited chemokine CCL21 expression, which led to decreased gastric influx of CD45+CD11c-Ly6G-CD11b+CD68- myeloid cells by CCL21-CCR7-dependent migration and, as a direct consequence, reduced bacterial clearing capacity of H pylori-specific Th1 cell response. CONCLUSIONS: Overall, this study identifies a model involving Rev-erbα, which collectively ensures gastric bacterial persistence by suppressing host gene expression required for local innate and adaptive defense against H pylori.