Cordyceps militaris Immunomodulatory Protein Promotes the Phagocytic Ability of Macrophages through the TLR4-NF-κB Pathway.

Enhancing the phagocytosis of immune cells with medicines provides benefits to the physiological balance by removing foreign pathogens and apoptotic cells. The fungal immunomodulatory protein (FIP) possessing various immunopotentiation functions may be a good candidate for such drugs. However, the effect and mechanism of FIP on the phagocytic activity is limitedly investigated. Therefore, the present study determined effects of Cordyceps militaris immunomodulatory protein (CMIMP), a novel FIP reported to induce cytokines secretion, on the phagocytosis using three different types of models, including microsphere, Escherichia Coli and Candida albicans. CMIMP not only significantly improved the phagocytic ability (p < 0.05), but also enhanced the bactericidal activity (p < 0.05). Meanwhile, the cell size, especially the cytoplasm size, was markedly increased by CMIMP (p < 0.01), accompanied by an increase in the F-actin expression (p < 0.001). Further experiments displayed that CMIMP-induced phagocytosis, cell size and F-actin expression were alleviated by the specific inhibitor of TLR4 (p < 0.05). Similar results were observed in the treatment with the inhibitor of the NF-κB pathway (p < 0.05). In conclusion, it could be speculated that CMIMP promoted the phagocytic ability of macrophages through increasing F-actin expression and cell size in a TLR4-NF-κB pathway dependent way.

Evaluation of pharmacotherapy recommendations in guidelines for inflammatory bowel disease.

WHAT IS KNOWN AND OBJECTIVE: The aim of this study was to systematically assess drug therapy in the guidelines for inflammatory bowel disease and to provide recommendations for the development of such guidelines. STUDY DESIGN: A systematic search was conducted in databases and on websites to identify guidelines for the treatment of inflammatory bowel disease. Qualified guidelines were assessed through the Appraisal of Guidelines for Research and Evaluation (AGREE II). Evidence from the guidelines was extracted from the guidelines themselves. The Oxford Centre for Evidence-based Medicine (OCEBM) evidence grading system was used to regrade and assess this evidence. RESULTS: A total of 11 guidelines for the medical treatment of inflammatory bowel disease (Crohn's disease and ulcerative colitis) (2015-2019) were finally included, and after scoring using the AGREE II tool, the median scores in each domain were as follows: Ⅰ. scope and purpose (median score=88.9%, range: 76.4%-91.7%), Ⅱ. stakeholder involvement (median =38.9%, range: 18.1%-61.1%), Ⅲ. rigour of development (median =69.3%, range: 39.6%-77.6%), Ⅳ. clarity and presentation (median =97.2%, range: 91.7%-100%), Ⅴ. applicability (median =45.8%, range: 24%-68.8%) and Ⅵ. editorial independence (median =94.0%, range: 0-100%). Most of the guidelines scored over 60%, which is worthy of clinical recommendation, but different guidelines suggest that there is a great difference in drug therapy, mainly due to various populations, diverse focuses of attention, distinct efficacy of drugs between Crohn's disease and ulcerative colitis, and the preference of guiding developers for select evidence. WHAT IS NEW AND CONCLUSION: The quality of medical treatment guidelines for inflammatory bowel disease varies considerably. Over the past 5 years, medical treatment has been heterogeneous among different guidelines. Consideration of factors leading to heterogeneity of recommendations for drug treatment, especially preferences for evidence selection, will help upgrade the guidelines.

Appraisal of the diagnostic procedures of acute pancreatitis in the guidelines.

OBJECTIVES: The purpose of this study was to comprehensively assess the heterogeneity of procedures in the diagnostic guidelines for acute pancreatitis and to identify gaps limiting knowledge in diagnosing this disease. METHODS: A systematic search of a number of databases was performed to determine the guidelines for the diagnosis of acute pancreatitis in patients with severe pancreatitis. The guidelines for the diagnosis of severe pancreatitis were evaluated by AGREE II. The Measurement Scale of Rate of Agreement (MSRA) was used to assess the guidelines (2015-2020) and extract evidence supporting these recommendations for analysis. RESULTS: Seven diagnostic guidelines for acute pancreatitis were included. Only the 2019 WSES Guidelines for the Management of Severe Acute Pancreatitis and the Japanese Guidelines for the Management of Acute Pancreatitis: Japanese Guidelines 2015 had a total score of more than 60%, which is worthy of clinical recommendation. The average scores of the Scope and Purpose domain and the Clarity and Expression domain were the highest at 71.62% and 75.59%, respectively, while the average score of the Applicability area was the lowest at 16.67%. The included guidelines were further analyzed to determine the heterogeneity of the diagnosis of acute pancreatitis. The main reasons for the heterogeneity were the citation of low-quality evidence, the presence of far too many indicators for the classification of acute pancreatitis, unclear depictions of the standard, and poorly comprehensive recommendations for the diagnosis of the aetiology in the primary diagnosis of acute pancreatitis, the severity classification, the aetiological diagnosis, and the diagnosis of comorbidities. CONCLUSIONS: The quality of different diagnostic guidelines for severe pancreatitis is uneven. The recommendations are largely based on low-quality evidence, and the guidelines still have much room for improvement to reach a high level of quality. The diagnostic procedures for acute pancreatitis vary widely in different guidelines. There are large differences between them, and resolving the abovementioned reasons would be a very wise choice for guideline developers to revise and upgrade the guidelines in the future.

Acute exposure to deoxynivalenol inhibits porcine enteroid activity via suppression of the Wnt/ß-catenin pathway.

The intake of food containing deoxynivalenol frequently causes damage to the intestine, the renewal of which is driven by intestinal stem cells (ISCs). Nevertheless, the toxicity of deoxynivalenol on ISCs and its underlying mechanisms remain to be elucidated. As pigs are the most sensitive animals to deoxynivalenol, we used piglets for investigation in this study. Here, we show that intestinal epithelial cell activity, B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi1) protein level, and Wnt/ß-catenin pathway activity were suppressed with acute expose to deoxynivalenol. We further established a novel system for porcine crypt isolation and ex vivo cultivation. Crypts and crypt cells expanded and budded with typical enteroid morphologies under this system. Our results show that both acute in vivo and in vitro administration of deoxynivalenol significantly decreased enteroid activity. Simultaneously, protein levels of ß-catenin and leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5) in enteroids were reduced by deoxynivalenol exposure. In conclusion, we established a reliable culture system for porcine enteroids and demonstrated for the first time that the activity of ISCs and the Wnt/ß-catenin pathway is sensitively suppressed by acute deoxynivalenol exposure.

CDX2 Stimulates the Proliferation of Porcine Intestinal Epithelial Cells by Activating the mTORC1 and Wnt/ß-Catenin Signaling Pathways.

Caudal type homeobox 2 (CDX2) is expressed in intestinal epithelial cells and plays a role in gut development and homeostasis by regulating cell proliferation. However, whether CDX2 cooperates with the mammalian target of rapamycin complex 1 (mTORC1) and Wnt/ß-catenin signaling pathways to stimulate cell proliferation remains unknown. The objective of this study was to investigate the effect of CDX2 on the proliferation of porcine jejunum epithelial cells (IPEC-J2) and the correlation between CDX2, the mTORC1 and Wnt/ß-catenin signaling pathways. CDX2 overexpression and knockdown cell culture models were established to explore the regulation of CDX2 on both pathways. Pathway-specific antagonists were used to verify the effects. The results showed that CDX2 overexpression increased IPEC-J2 cell proliferation and activated both the mTORC1 and Wnt/ß-catenin pathways, and that CDX2 knockdown decreased cell proliferation and inhibited both pathways. Furthermore, the mTORC1 and Wnt/ß-catenin pathway-specific antagonist rapamycin and XAV939 (3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)]-4H -thiopyrano[4,3-d]pyrimidin-4-one) both suppressed the proliferation of IPEC-J2 cells overexpressing CDX2, and that the combination of rapamycin and XAV939 had an additive effect. Regardless of whether the cells were treated with rapamycin or XAV939 alone or in combination, both mTORC1 and Wnt/ß-catenin pathways were down-regulated, accompanied by a decrease in CDX2 expression. Taken together, our data indicate that CDX2 stimulates porcine intestinal epithelial cell proliferation by activating the mTORC1 and Wnt/ß-catenin signaling pathways.

miR-142-3p acts as an essential modulator of neutrophil development in zebrafish.

Neutrophils play critical roles in vertebrate innate immune responses. As an emerging regulator in normal myelopoiesis, the precise roles of microRNA in the development of neutrophils have yet to be clarified. Using zinc-finger nucleases, we have successfully generated heritable mutations in miR-142a and miR-142b and showed that hematopoietic-specific miR-142-3p is completely deleted in miR-142 double mutant zebrafish. The lack of miR-142-3p resulted in aberrant reduction and hypermaturation of neutrophils in definitive myelopoiesis, as well as impaired inflammatory migration of neutrophils in the fetal stage. Furthermore, the adult myelopoiesis in the miR-142-3p-deficient zebrafish was also affected, producing irregular hypermature neutrophils with increased cell size and a decreased nucleocytoplasmic ratio. Additionally, miR-142-3p-deficient zebrafish are expected to develop a chronic failure of myelopoiesis with age. Transcriptome analysis showed an aberrant activation of the interferon γ (IFN-γ) signaling pathway in myelomonocytes after miR-142-3p deletion. We found that the reduced number and hypermaturation of neutrophils caused by loss of miR-142-3p was mainly mediated by the abnormally activated IFN-γ signaling, especially the upregulation of stat1a and irf1b. Taken together, we uncovered a novel role of miR-142-3p in maintaining normal neutrophil development and maturation.

Setdb2 controls convergence and extension movements during zebrafish gastrulation by transcriptional regulation of dvr1.

As the primary driving forces of gastrulation, convergence and extension (C&E) movements lead to a medio-lateral narrowing and an anterior-posterior elongation of the embryonic body axis. Histone methylation as a post-translational modification plays a critical role in early embryonic development, but its functions in C&E movements remain largely unknown. Here, we show that the setdb2-dvr1 transcriptional cascade plays a critical role in C&E movements during zebrafish gastrulation. Knockdown of Setdb2, a SET domain-containing protein possessing a potential histone H3K9 methyltransferase activity, induced abnormal C&E movements, resulting in anterior-posterior shortening and medio-lateral expansion of the embryonic axis, as well as abnormal notochord cell polarity. Furthermore, we found that Setdb2 functions through fine-tuning the expression of dvr1, a ligand of the TGF-ß superfamily, to an appropriate level to ensure proper C&E movements in a non-cell-autonomous manner. In addition, both overexpression and knockdown of Dvr1 at the one-cell stage resulted in defects at epiboly and C&E. These data demonstrate that Setdb2 is a novel regulator for C&E movements and acts by modulating the expression level of dvr1, suggesting that Dvr1 acts as a direct and essential mediator for C&E cell movements.

Pten regulates homeostasis and inflammation-induced migration of myelocytes in zebrafish.

BACKGROUND: Loss of the tumor suppressor phosphatase and tensin homolog (PTEN) is frequently observed in hematopoietic malignancies. Although PTEN has been implicated in maintaining the quiescence of hematopoietic stem cells (HSCs), its role in hematopoiesis during ontogeny remains largely unexplored. METHODS: The expression of hematopoietic marker genes was analyzed via whole mount in situ hybridization assay in ptena and ptenb double mutant zebrafish. The embryonic myelopoiesis was characterized by living imaging and whole mount in situ immunofluorescence with confocal microscopy, as well as cell-specific chemical staining for neutrophils and macrophages. Analyses of the involved signaling pathway were carried out by inhibitor treatment and mRNA injection. RESULTS: Pten-deficient zebrafish embryos exhibited a strikingly increased number of myeloid cells, which were further characterized as being immune deficient. In accordance with this finding, the inhibition of phosphoinositide 3-kinase (PI3K) or the mechanistic target of rapamycin (mTOR) corrected the expansive myelopoiesis in the pten-deficient embryos. Further mechanistic studies revealed that the expression of cebpa, a critical transcription factor in myeloid precursor cells, was downregulated in the pten-deficient myeloid cells, whereas the injection of cebpa mRNA markedly ameliorated the dysmyelopoiesis induced by the loss of pten. CONCLUSIONS: Our data provide in vivo evidence that definitive myelopoiesis in zebrafish is critically regulated by pten via the elevation of cebpa expression.

Cannabinoid receptor 2 suppresses leukocyte inflammatory migration by modulating the JNK/c-Jun/Alox5 pathway.

BACKGROUND: The role of cannabinoid receptor type 2 (Cnr2) in regulating immune function had been widely investigated, but the mechanism is not fully understood. RESULTS: Cnr2 activation down-regulates 5-lipoxygenase (Alox5) expression by suppressing the JNK/c-Jun activation. CONCLUSION: The Cnr2-JNK-Alox5 axis modulates leukocyte inflammatory migration. SIGNIFICANCE: Linking two important regulators in leukocyte inflammatory migration and providing a potential therapeutic strategy for treating human inflammation-associated diseases. Inflammatory migration of immune cells is involved in many human diseases. Identification of molecular pathways and modulators controlling inflammatory migration could lead to therapeutic strategies for treating human inflammation-associated diseases. The role of cannabinoid receptor type 2 (Cnr2) in regulating immune function had been widely investigated, but the mechanism is not fully understood. Through a chemical genetic screen using a zebrafish model for leukocyte migration, we found that both an agonist of the Cnr2 and inhibitor of the 5-lipoxygenase (Alox5, encoded by alox5) inhibit leukocyte migration in response to acute injury. These agents have a similar effect on migration of human myeloid cells. Consistent with these results, we found that inactivation of Cnr2 by zinc finger nuclease-mediated mutagenesis enhances leukocyte migration, while inactivation of Alox5 blocks leukocyte migration. Further investigation indicates that there is a signaling link between Cnr2 and Alox5 and that alox5 is a target of c-Jun. Cnr2 activation down-regulates alox5 expression by suppressing the JNK/c-Jun activation. These studies demonstrate that Cnr2, JNK, and Alox5 constitute a pathway regulating leukocyte migration. The cooperative effect between the Cnr2 agonist and Alox5 inhibitor also provides a potential therapeutic strategy for treating human inflammation-associated diseases.

Experimental validation and complexity of miRNA-mRNA target interaction during zebrafish primitive erythropoiesis.

MicroRNAs (miRNAs) are endogenous small non-protein coding RNAs that play important regulatory roles in animals and plants by binding to target transcripts for cleavage or translational repression. Despite increasing number of genes has been predicted to be miRNA targets by bioinformatics analysis and luciferase-based reporter assay in vitro (RA-In Vitro), few of them have been experimentally validated in physiological context. Using transient reporter assay in vivo (TRA-In Vivo), we identified hydroxymethylbilane synthase b (hmbsb) and Krüppel-like transcription factor d (klfd) as potential target gene for miR-451 and miR-144, respectively. Although hmbsb, miR-451, klfd and miR-144 are all co-expressed in the developing erythroid progenitors during zebrafish erythropoiesis, only klfd can be validated as a bona fide physiological target of miR-144 using a transgene-based physiological reporter assay in vivo (PRA-In Vivo). Thus, failure to verify hmbsb as miR-451 target in physiological context raises a crucial question as to how to determine bona fide target of miRNA. The results address the importance of using multiple approaches combined with Western blot analysis to validate the physiological target of a given miRNA.

Mir-144 selectively regulates embryonic alpha-hemoglobin synthesis during primitive erythropoiesis.

Precise transcriptional control of developmental stage-specific expression and switching of alpha- and beta-globin genes is significantly important to understand the general principles controlling gene expression and the pathogenesis of thalassemia. Although transcription factors regulating beta-globin genes have been identified, little is known about the microRNAs and trans-acting mechanism controlling alpha-globin genes transcription. Here, we show that an erythroid lineage-specific microRNA gene, miR-144, expressed at specific developmental stages during zebrafish embryogenesis, negatively regulates the embryonic alpha-globin, but not embryonic beta-globin, gene expression, through physiologically targeting klfd, an erythroid-specific Krüppel-like transcription factor. Klfd selectively binds to the CACCC boxes in the promoters of both alpha-globin and miR-144 genes to activate their transcriptions, thus forming a negative feedback circuitry to fine-tune the expression of embryonic alpha-globin gene. The selective effect of the miR-144-Klfd pathway on globin gene regulation may thereby constitute a novel therapeutic target for improving the clinical outcome of patients with thalassemia.