Identification and characterization of long non-coding RNAs in intestinal immune regulation of largemouth bass, Micropterus salmoides, under acute heat stress.

Long noncoding RNAs (lncRNAs), which are RNA molecules that do not code for proteins and have a length exceeding 200 base pairs, have been found to play a crucial role in regulating intestinal immunity. The high mortality of various fish species induced by high temperatures is known to be associated with enteritis. Our investigation demonstrated that acute heat stress was responsible for inducing fish enteritis. However, the specific lncRNAs involved this process remains unknown. In this current study, we utilized intestinal sequencing data from the largemouth bass species Micropterus salmoides under acute heat stress, resulting in a total of 347,351,492 clean reads obtained from six cDNA libraries. A total of 3399 novel lncRNA transcripts originating from 2488 distinct lncRNA genes were successfully identified. Consistent with previous findings in other fish species, these lncRNAs demonstrated comparatively shorter transcript lengths when compared to protein-coding genes. Furthermore, a total of 216 novel lncRNA exhibited differential expression (DE) in the intestine of largemouth bass, meeting the criteria of absolute log2 fold change exceeding 2 and a p-value below 0.05. Additionally, these DE-lncRNAs were found to regulate 210 neighboring genes in a cis-regulatory manner. An examination of GO/KEGG enrichment revealed a notable enrichment of immune regulation (p < 0.05) among these cis-genes, with lncRNA MSTRG.8573.1 playing a significant role in regulating the jak-stat signaling pathway during this process. This study presents a comprehensive inventory of novel DE-lncRNA implicated in the development of enteritis in largemouth bass under acute heat stress. These findings offer valuable insights for future investigations on the regulation of lncRNAs to mitigate heat stress-induced fish enteritis.

Alleviating effects of essential oil from Artemisia vulgaris on enteritis in zebrafish via modulating oxidative stress and inflammatory response.

Artemisia vulgaris (A. vulgaris) is a traditional Chinese medicine widely distributed in China and contains many bioactive compounds with pharmacological effects. However, the anti-inflammatory effects and mechanism of essential oil from A. vulgaris on enteritis in fish are still unclear. In this study, in order to elucidate the underlying mechanism of essential oil from A. vulgaris on zebrafish enteritis, zebrafish were used for establishing animal models to observe the histopathological changes of intestines, determine the activities of immune-related enzymes and oxidative stress indicators, and the mRNA expression of genes in MyD88/TRAF6/NF-KB signaling pathways. The results showed that different doses of A. vulgaris essential oil could effectively alleviate zebrafish enteritis in a dose- and time-dependent manner by improving the intestinal histopathological damage, decreasing the intestinal oxidative stress, repairing the intestinal immune ability, changing the expression levels of IL-1ß, IL-10 and genes in MyD88/TRAF6/NF-κB pathway. In addition, co-treatment with oxazolone and MyD88 inhibitor could alleviate the morphological damage, the induction of oxidative stress, and the levels of immune-related enzymes and the mRNA expression of genes in MyD88/TRAF6/NF-κB signaling pathway. Moreover, essential oil from A. vulgaris had more significantly therapeutic effects on enteritis of male zebrafish than that of female zebrafish. This result will clarify the therapeutic effect and anti-inflammatory mechanism of essential oil from A. vulgaris on zebrafish enteritis, and provide a theoretical basis for further research on the rationality of A. vulgaris to replace feed antibiotics.

Gastrointestinal manifestations in PTEN hamartoma tumor syndrome.

The PTEN hamartoma tumor syndrome (PHTS) is a heterogeneous set of multisystem disorders caused by germline pathogenic variants in the PTEN tumor suppressor gene. Manifestations include developmental anomalies and proliferative lesions. Evidence of involvement of the GI tract has accrued over time, leading to the incorporation of GI manifestations (multiple hamartomas, glycogenic acanthosis and colorectal cancer) into the diagnostic criteria. Polyps of the upper and lower GI tract are found in most adult patients and in a significant fraction of children. Polyps tend to be of mixed histology, with a predominance of hamartomas and ganglioneuromas. PHTS patients are also at increased risk of colorectal cancer, and surveillance by colonoscopy is advised starting at the age of 35-40 years. A number of additional manifestations, including eosinophilic gastrointestinal disorders, have been observed in few or single cases, and their association with PHTS has yet to be determined.

Determining the protective effects of Ma-Mu-Ran Antidiarrheal Capsules against acute DSS-induced enteritis using 16S rRNA gene sequencing and fecal metabolomics.

Ma-Mu-Ran Antidiarrheal Capsules (MMRAC) is traditional Chinese medicine that has been used to treat diarrhea caused by acute enteritis (AE) and bacillary dysentery in Xinjiang (China) for many years. However, the potential therapeutic mechanism of MMRAC for AE and its regulatory mechanism on host metabolism is unclear. This study used fecal metabolomics profiling with GC/MS and 16S rRNA gene sequencing analysis to explore the potential regulatory mechanisms of MMRAC on a dextran sulfate sodium salt (DSS)-induced mouse model of AE. Fecal metabolomics-based analyses were performed to detect the differentially expressed metabolites and metabolic pathways. The 16S rRNA gene sequencing analysis was used to assess the altered gut microbes at the genus level and for functional prediction. Moreover, Pearson correlation analysis was used to integrate differentially expressed metabolites and altered bacterial genera. The results revealed that six intestinal bacteria and seven metabolites mediated metabolic disorders (i.e., metabolism of amino acid, carbohydrate, cofactors and vitamins, and lipid) in AE mice. Besides, ten altered microbes mediated the differential expression of eight metabolites and regulated these metabolisms after MMRAC administration. Overall, these findings demonstrate that AE is associated with metabolic disorders and microbial dysbiosis. Further, we present that MMRAC exerts protective effects against AE by improving host metabolism through the intestinal flora.

Transcriptome analysis of the potential molecular mechanism of enteritis in intestine and hepatopancreas of Litopenaeus vannamei.

The prawn, Litopenaeus vannamei (L. vannamei), is the most widely farmed species in the world but the incidence of enteritis in L. vannamei has increased in recent years. However, the pathogenesis of enteritis remains unclear. In this study, high-throughput sequencing was used to analyze the hepatopancreatic and intestinal transcriptome of healthy and enteritis-affected individuals from the same pond. In total, 1209 and 1608 differently-expressed genes (DEGs) were detected in the hepatopancreatic and intestinal transcriptomes, respectively. Significantly changed genes were enriched in the intestinal immune network for IgA Production, Lysosomes, Sphingolipid Metabolism and the Peroxisome Signaling Pathway. Expression of the integrin α4ß7 gene was significantly increased in the intestine of L. vannamei with enteritis, while expression of 38 DEGs associated with the lysosome was significantly down-regulated. Furthermore, the expression of sphingolipid metabolism-related enzymes and superoxide dismutase (SOD) genes was also significantly decreased, indicating that abnormal autoimmune function, weak intestinal resistance to external pathogenic microbial invasion, and self-healing ability were important factors associated with enteritis in L. vannamei. In addition, the expression of trypsin and pancreatic lipase was decreased in the hepatopancreas of L. vannamei with enteritis. This study provided new insights into the possible molecular pathogenesis of enteritis in L. vannamei.

2021 year in review: Spotlight on eosinophils.

This review highlights recent advances in the understanding of eosinophils and eosinophilic diseases, particularly eosinophilic gastrointestinal diseases during the last year. The increasing incidence of diseases marked by eosinophilia has been documented and highlighted the need to understand eosinophil biology and eosinophilic contributions to disease. Significant insight into the nature of eosinophilic diseases has been achieved using next-generation sequencing technologies, proteomic analysis, and machine learning to analyze tissue biopsies. These technologies have elucidated mechanistic underpinnings of eosinophilic inflammation, delineated patient endotypes, and identified patient responses to therapeutic intervention. Importantly, recent clinical studies using mAbs that interfere with type 2 cytokine signaling or deplete eosinophils point to multiple and complex roles of eosinophils in tissues. Several studies identified distinct activation features of eosinophils in different tissues and disease states. The confluence of these studies supports a new paradigm of tissue-resident eosinophils that have pro- and anti-inflammatory immunomodulatory roles in allergic disease. Improved understanding of unique eosinophil activation states is now poised to identify novel therapeutic targets for eosinophilic diseases.

Sinomenine Hydrochloride Ameliorates Fish Foodborne Enteritis via α7nAchR-Mediated Anti-Inflammatory Effect Whilst Altering Microbiota Composition.

Foodborne intestinal inflammation is a major health and welfare issue in aquaculture. To prevent enteritis, various additives have been incorporated into the fish diet. Considering anti-inflammatory immune regulation, an effective natural compound could potentially treat or prevent intestinal inflammation. Our previous study has revealed galantamine's effect on soybean induced enteritis (SBMIE) and has highlighted the possible role of the cholinergic anti-inflammatory pathway in the fish gut. To further activate the intestinal cholinergic related anti-inflammatory function, α7nAchR signaling was considered. In this study, sinomenine, a typical agonist of α7nAChR in mammals, was tested to treat fish foodborne enteritis via its potential anti-inflammation effect using the zebrafish foodborne enteritis model. After sinomenine's dietary inclusion, results suggested that there was an alleviation of intestinal inflammation at a pathological level. This outcome was demonstrated through the improved morphology of intestinal villi. At a molecular level, SN suppressed inflammatory cytokines' expression (especially for tnf-α) and upregulated anti-inflammation-related functions (indicated by expression of il-10, il-22, and foxp3a). To systematically understand sinomenine's intestinal effect on SBMIE, transcriptomic analysis was done on the SBMIE adult fish model. DEGs (sinomenine vs soybean meal groups) were enriched in GO terms related to the negative regulation of lymphocyte/leukocyte activation and alpha-beta T cell proliferation, as well as the regulation of lymphocyte migration. The KEGG pathways for glycolysis and insulin signaling indicated metabolic adjustments of α7nAchR mediated anti-inflammatory effect. To demonstrate the immune cells' response, in the SBMIE larva model, inflammatory gatherings of neutrophils, macrophages, and lymphocytes caused by soybean meal could be relieved significantly with the inclusion of sinomenine. This was consistent within the sinomenine group as CD4+ or Foxp3+ lymphocytes were found with a higher proportion at the base of mucosal folds, which may suggest the Treg population. Echoing, the sinomenine group's 16s sequencing result, there were fewer enteritis-related TM7, Sphingomonas and Shigella, but more Cetobacterium, which were related to glucose metabolism. Our findings indicate that sinomenine hydrochloride could be important in the prevention of fish foodborne enteritis at both immune and microbiota levels.

Multiomics analysis of soybean meal induced marine fish enteritis in juvenile pearl gentian grouper, Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂.

As an important protein source, soybean products can cause intestinal inflammation and injury in many animals including human beings, particularly infants and juvenile individuals. Research in this field has been performed for terrestrial animals and fish, but still lacks integrity and systematicness. In this study, the main biological processes in the intestinal tract of marine fish juvenile pearl gentian grouper in the state of soybean meal-induced enteritis (SBMIE) were analyzed. A total of 720 groupers with an approximate initial weight of 12.5 g were randomly divided into three groups: the fish meal (FM) control group, the 20% SBM group (SBM20), and the SBM40 group (n = 4). Three iso-nitrogenous and iso-lipidic diets were prepared and fed to fish for 10 weeks. Each barrel contained a water volume of about 1 m3 in and was exposed to natural light and temperature. Results indicated that the growth and physiology of groupers fed with SBM were significantly negatively affected, with the gene expressions of intestinal structural protein abnormal. 16SrDNA high-throughput sequencing showed that the intestinal microflora played an important role in the pathogenesis of pearl gentian grouper SBMIE, which may activate a variety of pathogen pattern recognition receptors, such as toll-like receptors (TLRs), RIG-I-like receptors, and nod-like receptors. Transcriptome analysis revealed that changes of the SBMIE signaling pathway in pearl gentian groupers were conservative to some extent than that of terrestrial animals and freshwater fish. Moreover, the TLRs-nuclear factor kappa-B signaling pathway becomes activated, which played an important role in SBMIE. Meanwhile, the signal pathways related to nutrient absorption and metabolism were generally inhibited. Metabolomics analysis showed that isoflavones and saponins accounted for a large proportion in the potential biomarkers of pearl gentian grouper SBMIE, and most of the biomarkers had significantly positive or negative correlations with each other; 56 metabolites were exchanged between intestinal tissues and contents, which may play an important role in the development of enteritis, including unsaturated fatty acids, organic acids, amino acids, vitamins, small peptides, and nucleotides, etc. These results provide a basic theoretical reference for solving the intestinal issues of fish SBMIE and research of inflammatory bowel disease in mammals.

No effect of deleted in malignant brain tumors 1 deficiency on chemotherapy induced murine intestinal mucositis.

Mucositis is a serious adverse effect of chemotherapeutic treatment. During intestinal mucositis, the mucosal barrier is compromised, increasing the risk of severe infections. Mucositis necessitates dose reduction or pauses in treatment, which affect the outcome of the treatment. Deleted in malignant brain tumors 1 (DMBT1) is a secreted scavenger protein with effects on innate immunity and epithelial regeneration. We have previously shown that jejunal DMBT1 expression is increased in piglets during chemotherapeutic treatment. We hypothesized that DMBT1 ameliorates doxorubicin-induced mucositis. Individually-caged Dmbt1+/+ (WT) and Dmbt1-/- (KO) female mouse littermates received intraperitoneal injections of either doxorubicin or saline. They were euthanized after three (D3) or seven days (D7). Weight loss was monitored every day, and serum citrulline levels were measured at termination. Intestinal tissue was analyzed for the expression of DMBT1 and proinflammatory cytokines (IL-1ß, IL-6, and TNF). Specimens from the small intestines and colon were scored for inflammation and epithelial and mucosal architecture changes. We detected no effect of DMBT1 on weight loss, serum citrulline levels, expression of proinflammatory cytokines, or histologic damage. We detected a significant increase in crypt depth in WT mice compared to that in KO mice on D3. In conclusion, DMBT1 does not affect doxorubicin-induced mucositis in mice.

Preventive Anti-inflammatory Diet to Reduce Gastrointestinal Inflammation in Familial Adenomatous Polyposis Patients: A Prospective Pilot Study.

Familial adenomatous polyposis (FAP) is an autosomal-dominant hereditary condition associated with germline mutations in the adenomatous polyposis coli gene. Patient management involves prophylactic surgery and intensive life-long endoscopic surveillance. Diet is a major concern for patients with FAP, who are generally free of symptoms before surgery but tend to have issues related to bowel function postoperatively. We hypothesized that a low-inflammatory diet based on the principles and recipes of the Mediterranean diet would reduce markers of local and systemic inflammation. Twenty-eight patients with FAP over 18 years of age who underwent rectum-sparing prophylactic colectomy and were included in our surveillance program participated in a pilot dietary intervention study. Blood and stool samples at baseline (T0), at the end of the dietary intervention (T1, three months), and at the end of the study (T2, six months after T0) were collected. Gastrointestinal inflammation markers including fecal calprotectin, cyclooxygenase-2, and 15-hydroxyprostaglandin dehydrogenase were evaluated. Serum calprotectin, insulin, insulin-like growth factor-1, C-reactive protein, and glycated hemoglobin were also assessed. Significant changes in serum calprotectin, insulin, and insulin-like growth factor-1 levels occurred over time. Borderline significant changes were observed in the neutrophil-lymphocyte ratio. These changes were noticeable immediately at the end of the 3-month active dietary intervention (T1). A significant increase in 15-hydroxyprostaglandin dehydrogenase expression in the normal crypts of matched samples was also observed between T0 and T2. This pilot study supports the hypothesis that a low-inflammatory diet can modulate gastrointestinal markers of inflammation in individuals with FAP. PREVENTION RELEVANCE: Cancer is known to be related to inflammatory conditions. This study suggests that anti-inflammatory dietary intervention may potentially prevent adenomas and cancer in FAP patients by reducing systemic and tissue inflammatory indices.

Transversal gene expression panel to evaluate intestinal health in broiler chickens in different challenging conditions.

There is a high interest on gut health in poultry with special focus on consequences of the intestinal diseases, such as coccidiosis and C. perfringens-induced necrotic enteritis (NE). We developed a custom gene expression panel, which could provide a snapshot of gene expression variation under challenging conditions. Ileum gene expression studies were performed through high throughput reverse transcription quantitative real-time polymerase chain reaction. A deep review on the bibliography was done and genes related to intestinal health were selected for barrier function, immune response, oxidation, digestive hormones, nutrient transport, and metabolism. The panel was firstly tested by using a nutritional/Clostridium perfringens model of intestinal barrier failure (induced using commercial reused litter and wheat-based diets without exogenous supplementation of enzymes) and the consistency of results was evaluated by another experiment under a coccidiosis challenge (orally gavaged with a commercial coccidiosis vaccine, 90× vaccine dose). Growth traits and intestinal morphological analysis were performed to check the gut barrier failure occurrence. Results of ileum gene expression showed a higher expression in genes involved in barrier function and nutrient transport in chickens raised in healthy conditions, while genes involved in immune response presented higher expression in C.perfringens-challenged birds. On the other hand, the Eimeria challenge also altered the expression of genes related to barrier function and metabolism, and increased the expression of genes related to immune response and oxidative stress. The panel developed in the current study gives us an overview of genes and pathways involved in broiler response to pathogen challenge. It also allows us to deep into the study of differences in gene expression pattern and magnitude of responses under either a coccidial vaccine or a NE.

Mitigation of Gliadin-Induced Inflammation and Cellular Damage by Curcumin in Human Intestinal Cell Lines.

Wheat is a major diet from many years; apart from its nutritious value, the wheat protein gliadin is responsible for many inflammatory diseases like celiac disease (CD), and non-celiac gluten sensitivity (NCGS). In this study, the gliadin-induced inflammation and associated cellular damage along with the protective role of curcumin was evaluated using human intestinal cell lines (HCT-116 and HT-29) as a model. Cells were cultured and exposed to 160 µg/ml of gliadin, 100 µM H2O2, and 10 µM curcumin (3 h pretreatment) followed by the assessment of inflammation. Spectrophotometric methods, real-time-PCR, ELISA, Western blotting, and confocal microscopy techniques were used to assess inflammatory markers such as advanced oxidation protein products (AOPPs) level, activity of myeloperoxidase (MPO) and NADPH oxidase (NOX), cytokines, and cell damage markers. The results show that gliadin increases the AOPPs level and the activity of MPO and NOX expression. It enhances inflammation by increasing expression of pro-inflammatory cytokines, altered expression of anti-inflammatory, and regulatory cytokines. It exacerbates the cellular damage by increasing MMP-2 and 9 and decreasing integrin α and ß expression. Gliadin promotes disease pathogenesis by inducing the inflammation and cellular damage which further alter the cellular homeostasis. The pretreatment of curcumin counteracts the adverse effect of gliadin and protect the cells via diminishing the inflammation and help the cell to regain the cellular morphology suggesting phytochemical-based remedial interventions against wheat allergies.

Polysaccharides extracted from Rheum tanguticum ameliorate radiation-induced enteritis via activation of Nrf2/HO-1.

Radiation-induced enteritis is a major side effect in cancer patients undergoing abdominopelvic radiotherapy. The Nrf2/HO-1 pathway is a critical endogenous antioxidant stress pathway, but its precise role in radiation-induced enteritis remains to be clarified. Polysaccharides extracted from Rheum tanguticum (RTP) can protect the intestinal cells from radiation-induced damage, but the underlying mechanism is unknown. SD rats and IEC-6 cells were exposed to 12 or 10 Gy X-ray radiation. Rat survival, and histopathological and immunohistochemical profiles were analyzed at different time points. Indicators of oxidative stress and inflammatory response were also assessed. Cell viability, apoptosis and Nrf2/HO-1 expression were evaluated at multiple time points. Significant changes were observed in the physiological and biochemical indexes of rats after radiation, accompanied by significant oxidative stress response. The mRNA and protein expression of Nrf2 peaked at 12 h after irradiation, and HO-1 expression peaked at 48 h after irradiation. RTP administration reduced radiation-induced intestinal damage, upregulated Nrf2/HO-1, improved physiological indexes, significantly decreased apoptosis and inflammatory factors, and upregulated HO-1, particularly at 48 h after irradiation. In conclusion, Nrf2 is activated in the early stage of radiation-induced intestinal injury and plays a protective role. RTP significantly ameliorates radiation-induced intestinal injury via the regulation of Nrf2 and its downstream protein HO-1.

Low lethal doses of Streptococcus iniae caused enteritis in Siberian sturgeon (Acipenser baerii).

In aquaculture, the incidence of enteritis due to Streptococcus iniae infection in Siberian sturgeon (Acipenser baerii) has increased in recent years. The pathogenesis of S. iniae is largely unknown due to the paucity of experimental studies on fish intestinal inflammation. In this study, S. iniae infection of A. baerii juveniles was induced by anal intubation of 0.15 mL at a low lethal dose (2 × 107 CFU/mL). Intestinal pathology and gene expression studies were conducted within 10 days of the experiment. Histopathological examination showed severe intestinal lesions, inflammatory cell infiltration, intestinal submucosa edema, epithelial cell shedding and necrosis. Predominant symptoms of exudative inflammation, metamorphic inflammation and proliferative inflammation on days 1-3, 4-6, and 7-10 post infection were shown, respectively. Ultrastructural observations also revealed fractured microvilli and shedding on days 4-6. Intestinal villi gradually repaired during the subsequent 7-10 days post infection. Expression of the pro-inflammatory cytokines, tumor necrosis factor and interleukin 1ß were up-regulated on days 1-3 followed by a significant decrease on day 5, ultimately reaching control levels on day 10 post infection. A similar pattern was shown in mucus cells, involving mucin secretion and expression of the mucin encoding gene, Mucin-2. These results showed the cellular response to S. iniae infection associated with inflammatory genes expression in the Siberian sturgeon.

Ionizing radiation induces BH4 deficiency by downregulating GTP-cyclohydrolase 1, a novel target for preventing and treating radiation enteritis.

Radiation enteritis (RE) is a common side effect after radiotherapy for abdominal cancer. RE pathogenesis is complicated, with no drugs available for prevention or treatments. Intestinal ischemia is a key factor in the occurrence and development of enteritis. The effect of ionizing radiation (IR) on intestinal ischemia is unknown. Deficiency of tetrahydrobiopterin (BH4) produced by GTP-cyclohydrolase 1 (Gch1) is important in ischemic diseases. This study focused on the relationship of Gch1/BH4 between intestinal ischemia in radiation enteritis. BH4 levels were analyzed by high-performance liquid chromatography in humans and rats after radiotherapy. Intestinal blood perfusion was measured by laser doppler flow imaging. Vascular ring tests determined the diastolic functions of rat mesenteric arteries. Gene, protein, and immunohistochemical staining experiments and inhibitor interventions were used to investigate Gch1 and endothelial NOS (eNOS) in rat mesenteric arteries and endothelial cells. The results showed that IR decreased BH4 levels in patients and rats after radiotherapy and decreased intestinal blood perfusion in rats. The degree of change in intestinal ischemia was consistent with intestinal villus injury. Gch1 mRNA and protein levels and nitric oxide (NO) production significantly decreased, while eNOS uncoupling in arterial and vascular endothelial cells strongly increased. BH4 supplementation improved eNOS uncoupling and NO levels in vascular endothelia after IR. The results of this study showed that downregulation of Gch1 in intestinal blood vessels after IR is an important target in RE. BH4 supplementation may prevent intestinal ischemia and improve vascular endothelial function after IR. These findings have clinical significance for the prevention and treatment of RE.

Dietary lipids fuel GPX4-restricted enteritis resembling Crohn's disease.

The increased incidence of inflammatory bowel disease (IBD) has become a global phenomenon that could be related to adoption of a Western life-style. Westernization of dietary habits is partly characterized by enrichment with the ω-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA), which entails risk for developing IBD. Glutathione peroxidase 4 (GPX4) protects against lipid peroxidation (LPO) and cell death termed ferroptosis. We report that small intestinal epithelial cells (IECs) in Crohn's disease (CD) exhibit impaired GPX4 activity and signs of LPO. PUFAs and specifically AA trigger a cytokine response of IECs which is restricted by GPX4. While GPX4 does not control AA metabolism, cytokine production is governed by similar mechanisms as ferroptosis. A PUFA-enriched Western diet triggers focal granuloma-like neutrophilic enteritis in mice that lack one allele of Gpx4 in IECs. Our study identifies dietary PUFAs as a trigger of GPX4-restricted mucosal inflammation phenocopying aspects of human CD.

MicroRNA gga-miR-200a-3p modulates immune response via MAPK signaling pathway in chicken afflicted with necrotic enteritis.

MicroRNAs (miRNAs) are small non-coding RNAs that contribute to host immune response as post-transcriptional regulation. The current study investigated the biological role of the chicken (Gallus gallus) microRNA-200a-3p (gga-miR-200a-3p), using 2 necrotic enteritis (NE) afflicted genetically disparate chicken lines, 6.3 and 7.2, as well as the mechanisms underlying the fundamental signaling pathways in chicken. The expression of gga-miR-200a-3p in the intestinal mucosal layer of NE-induced chickens, was found to be upregulated during NE infection in the disease-susceptible chicken line 7.2. To validate the target genes, we performed an overexpression analysis of gga-miR-200a-3p using chemically synthesized oligonucleotides identical to gga-miR-200a-3p, reporter gene analysis including luciferase reporter assay, and a dual fluorescence reporter assay in cultured HD11 chicken macrophage cell lines. Gga-miR-200a-3p was observed to be a direct transcriptional repressor of ZAK, MAP2K4, and TGFß2 that are involved in mitogen-activated protein kinase (MAPK) pathway by targeting the 3'-UTR of their transcripts. Besides, gga-miR-200a-3p may indirectly affect the expression of protein kinases including p38 and ERK1/2 at both transcriptional and translational levels, suggesting that this miRNA may function as an important regulator of the MAPK signaling pathway. Proinflammatory cytokines consisting of IL-1ß, IFN-γ, IL-12p40, IL-17A, and LITAF belonging to Th1 and Th17-type cytokines, were upregulated upon gga-miR-200a-3p overexpression. These findings have enhanced our knowledge of the immune function of gga-miR-200a-3p mediating the chicken immune response via regulation of the MAPK signaling pathway and indicate that this miRNA may serve as an important biomarker of diseases in domestic animals.

MicroRNA gga-miR-10a-mediated transcriptional regulation of the immune genes in necrotic enteritis afflicted chickens.

miRNAs are involved in both adaptive and innate immune systems of host animals; and play important roles in many immune-related pathways. The systemic biological roles of gga-miR-10a-5p chicken microRNA on immune response were investigated in two necrotic enteritis (NE) induced chicken lines, Marek's disease (MD) resistant (line 6.3) and susceptible (line 7.2). We determined the expression patterns of gga-miR-10a in the intestinal mucosal layer of chickens upon NE induction, and identified the target genes (MyD88, and SKP1) related to the host immune response to pathogens. We found that gga-miR-10a expression in the intestinal mucosal layer of MD-resistant chicken line 6.3 gga-miR-10a was significantly down-regulated (p < 0.01) during NE. Overexpression analysis of gga-miR-10a and reporter gene analysis using a wild- or mutant-type MyD88 3' untranslated region (3' UTR)-luciferase construct in chicken macrophage cell line HD11 and chicken fibroblast cell line OU2 showed that gga-miR-10a acted as a direct translational repressor of MyD88 by targeting the 3' UTR of this gene. Furthermore, miR-10a indirectly negatively influenced the expression of signaling molecules related to the MyD88-dependent pathway, including TRAF6, TAK1, and NF-κB1 at both transcriptional and translational levels. Downstream of the MyD88-dependent pathway, several proinflammatory cytokines such as IL-1ß, IFN-γ, IL-12p40, TNFSF15, and LITAF were down-regulated by overexpression of gga-miR-10a. These results suggest that gga-miR-10a is an important regulator of the Toll-like receptor signaling pathway. The findings of this study improve our understanding of the biological functions of miR-10a and the mechanisms underlying the TLR signaling pathway upon NE afflicted chickens, as well improving the overall understanding of the immune system function in domestic animals.

LGP2 plays a critical role in MDA5-mediated antiviral activity against duck enteritis virus.

Duck viral enteritis (DEV) is a DNA virus that leads to heavy economic losses in the commercial duck industry. As a key cytoplasmic sensor, melanoma differentiation-associated gene 5 (MDA5) can recognize viral RNA and enhance the antiviral immune response. Retinoic acid-inducible gene-I (RIG-I) and MDA5 both belong to the RIG-I-like receptors family, and RIG-I is known to be involved in the anti-DEV signaling pathway. However, the role of MDA5 in DEV infection remains unclear. In this study, we used overexpression and knockdown methods to determine if MDA5 affected DEV infection in ducks. We confirmed that DEV infection was significantly suppressed in MDA5-overexpressing DEF cells, while knockdown of MDA5 by siRNA markedly enhanced DEV growth. We demonstrated that overexpression of duck MDA5 significantly upregulated expression of interferon (IFN)-stimulated genes, including myxovirus resistance protein (Mx), IFN-induced oligodenylate synthetase-like (OASL), IFN-induced transmembrane protein 1 (IFITM1) and IFN-ß. In addition, the transcriptional level of MDA5 was upregulated both in vivo and in vitro upon DEV infection. We also showed that there was an association between MDA5 and laboratory of genetics and physiology 2 (LGP2) in antiviral signaling. LGP2 functioned as a concentration-dependent switch between MDA5-specific enhancement and interference. Overall, these findings indicated that MDA5 restricted DEV replication and LGP2 plays a critical role in MDA5-mediated antiviral activity against DEV.

Phosphoinositide 3-Kinase P110δ-Signaling Is Critical for Microbiota-Activated IL-10 Production by B Cells that Regulate Intestinal Inflammation.

The phosphoinositide 3-kinase catalytic subunit p110δ (PI3Kδ) gene maps to a human inflammatory bowel diseases (IBD) susceptibility locus, and genetic deletion of PI3Kδ signaling causes spontaneous colitis in mice. However, little is known regarding the role of PI3Kδ on IL-10-producing B cells that help regulate mucosal inflammation in IBD. We investigated the role of PI3Kδ signaling in B cell production of IL-10, following stimulation by resident bacteria and B cell regulatory function against colitis. In vitro, B cells from PI3KδD910A/D910A mice or wild-type B cells treated with PI3K specific inhibitors secreted significantly less IL-10 with greater IL-12p40 following bacterial stimulation. These B cells failed to suppress inflammatory cytokines by co-cultured microbiota-activated macrophages or CD4+ T cells. In vivo, co-transferred wild-type B cells ameliorated T cell-mediated colitis, while PI3KδD910A/D910A B cells did not confer protection from mucosal inflammation. These results indicate that PI3Kδ-signaling mediates regulatory B cell immune differentiation when stimulated with resident microbiota or their components, and is critical for induction and regulatory function of IL-10-producing B cells in intestinal homeostasis and inflammation.