Mechanistic study of fructus aurantii (Quzhou origin) in regulating ileal reg3g in the treatment for NASH.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a critical stage in the progression of non-alcoholic fatty liver disease (NAFLD), characterized by obvious inflammation and fibrosis. Because of its high incidence rate and serious consequences, NASH is becoming a global health problem. The influence of endotoxin translocation on NASH is receiving attention. As a traditional Chinese herb that effectively improves hepatic inflammation, Fructus Aurantii (Quzhou origin, FAQ) is widely used in the clinical treatment of NASH. However, the intervention mechanism of FAQ on reg3g and related endotoxin translocation remains unclear. AIM: To study the mechanism of the impact by which ileal regenerating family member 3 gamma (reg3g) deficiency and subsequent endotoxin translocation impact the progression of NASH; To elucidate the efficacy and mechanism of FAQ in the treatment of NASH. METHODS: Clinical serum, ileal tissue, and dynamic NASH model-related analyses collectively confirmed that reg3g is a pivotal gene associated with NASH. Reg3g-/- mice were used to assess the impact of reg3g on liver injury, inflammation, and fibrosis, as well as the underlying mechanism involved. In vitro studies elucidated the regulatory effects of FAQ on reg3g, intestinal barrier function, and intestinal permeability. Subsequently, the efficacy of FAQ was investigated in NASH mouse models. Pathological examinations combined with Western blotting (WB), immunohistochemistry (IHC), and multiplex immunohistochemical (mIHC) analyses were used to evaluate the effects of FAQ on mucosal repair and barrier function. Transepithelial electrical resistance (TEER), fluorescein isothiocyanate-dextran 4 (FD-4) experiments, coupled with enzyme linked immunosorbent assay (ELISA) and chromogenic LAL endotoxin assay were used to confirm intestinal permeability and endotoxin translocation. The results of WB and mIHC reflected the levels of endotoxin recruitment and M1 macrophage polarization in the liver. Parameters such as body weight, transaminases, and cholesterol were utilized to assess the metabolic effects of FAQ. RESULTS: Decreased expression of reg3g was associated with the progression of NASH. Ileal deficiency in reg3g resulted in damage to the intestinal barrier and permeability, leading to the recruitment of endotoxins via the 'gut-liver' axis to the liver, causing the polarization of M1 macrophages, release of inflammatory factors, excessive inflammation, and activation of hepatic stellate cells (HSCs), leading to fibrosis. FAQ significantly upregulated ileal reg3g expression and the expression of intestinal barrier-related proteins tight junction protein 1 (ZO-1) and occludin (OLCN) in mice (p < 0.05), thereby improving intestinal barrier function and permeability. Reduced intestinal permeability led to decreases in endotoxins entering the bloodstream and accumulating in the liver (p < 0.05). The expression of CD68 suggested reduced polarization of M1 macrophages. Expression levels of actin alpha 2, smooth muscle actin (α-SMA) and extracellular matrix (ECM)-related proteins also decreased, indicating improved liver fibrosis. CONCLUSION: FAQ ameliorates NASH by upregulating the expression of reg3g. The upregulation of reg3g contributes to the repair of the intestinal barrier and permeability, reducing the recruitment of endotoxins and subsequent polarization of M1 macrophages, excessive inflammation, and fibrosis.

Reg2 treatment is protective but the induced Reg2 autoantibody is destructive to the islets in NOD mice.

Regenerating family protein 2 (Reg2) is a trophic factor which stimulates ß-cell replication and resists islet destruction. However, Reg2 also serves as an islet autoantigen, which makes it complicated to judge the effectiveness in treating diabetes. How Reg2 treatment behaves in non-obese diabetic (NOD) mice is to be investigated. NOD mice were treated with recombinant Reg2 protein, Complete Freund's adjuvant (CFA) + PBS and CFA+Reg2 vaccinations, CFA+PBS- and CFA+Reg2-immunized antisera, and single chain variable fragment (scFv)-Reg2 and mIgG2a-Reg2 antibodies. Glycemic level, bodyweight, serum Reg2 antibody titer, glucose tolerance, and insulin secretion were determined. Islet morphological characteristics, insulitis, cell apoptosis, islet cell components, and T cell infiltration were analyzed by histological examinations. The autoantigenicity of constructed Reg2C and Reg2X fragments was determined in healthy BALB/c mice, and the bioactivity in stimulating cell proliferation and survival was assessed in insulinoma MIN6 cells. Reg2 administration alleviated diabetes in NOD mice with improved glucose tolerance and insulin secretion but elevated serum Reg2 autoantibodies. Histomorphometry showed reduced inflammatory area, TUNEL signal and CD8 + T cell infiltration, and increased ß-cell proportion in support of the islet-protective effect of Reg2 treatment. CFA+PBS and CFA+Reg2 immunizations prevented diabetic onset and alleviated insulitis while injections of the antisera offered mild protections. Antibody treatments accelerated diabetic onset without increasing the overall incidence. Reg2C fragment depletes antigenicity, but reserves protective activity in streptozotocin (STZ)-treated MIN6 cells. In conclusion, Reg2 treatment alleviates type 1 diabetes (T1D) by preserving islet ß-cells, but induces Reg2 autoantibody production which poses a potential risk of accelerating diabetic progression.

Transcription factors BZR1 and PAP1 cooperate to promote anthocyanin biosynthesis in Arabidopsis shoots.

Anthocyanins play critical roles in protecting plant tissues against diverse stresses. The complicated regulatory networks induced by various environmental factors modulate the homeostatic level of anthocyanins. Here, we show that anthocyanin accumulation is induced by brassinosteroids (BRs) in Arabidopsis (Arabidopsis thaliana) shoots and shed light on the underlying regulatory mechanism. We observed that anthocyanin levels are altered considerably in BR-related mutants, and BRs induce anthocyanin accumulation by upregulating the expression of anthocyanin biosynthetic genes. Our genetic analysis indicated that BRASSINAZOLE RESISTANT 1 (BZR1) and PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) are essential for BR-induced anthocyanin accumulation. The BR-responsive transcription factor BZR1 directly binds to the PAP1 promoter, regulating its expression. In addition, we found that intense anthocyanin accumulation caused by the pap1-D-dominant mutation is significantly reduced in BR mutants, implying that BR activity is required for PAP1 function after PAP1 transcription. Moreover, we demonstrated that BZR1 physically interacts with PAP1 to cooperatively regulate the expression of PAP1-target genes, such as TRANSPARENT TESTA 8, DIHYDROFLAVONOL 4-REDUCTASE, and LEUKOANTHOCYANIDIN DIOXYGENASE. Our findings indicate that BZR1 functions as an integral component of the PAP1-containing transcription factor complex, contributing to increased anthocyanin biosynthesis. Notably, we also show that functional interaction of BZR1 with PAP1 is required for anthocyanin accumulation induced by low nitrogen stress. Taken together, our results demonstrate that BR-regulated BZR1 promotes anthocyanin biosynthesis through cooperative interaction with PAP1 of the MBW complex.

Increased expression of REG3A promotes tumorigenic behavior in triple negative breast cancer cells.

BACKGROUND: Identifying new targets in triple negative breast cancer (TNBC) remains critical. REG3A (regenerating islet-derived protein 3 A), a calcium-dependent lectin protein, was thoroughly investigated for its expression and functions in breast cancer. METHODS: Bioinformatics and local tissue analyses were employed to identify REG3A expression in breast cancer. Genetic techniques were employed to modify REG3A expression, and the resulting effects on the behaviors of breast cancer cells were examined. Subcutaneous xenograft models were established to investigate the involvement of REG3A in the in vivo growth of breast cancer cells. RESULTS: Analysis of the TCGA database uncovered increased REG3A levels in human breast cancer tissues. Additionally, REG3A mRNA and protein levels were elevated in TNBC tissues of locally treated patients, contrasting with low expression in adjacent normal tissues. In primary human TNBC cells REG3A shRNA notably hindered cell proliferation, migration, and invasion while triggering caspase-mediated apoptosis. Similarly, employing CRISPR-sgRNA for REG3A knockout showed significant anti-TNBC cell activity. Conversely, REG3A overexpression bolstered cell proliferation and migration. REG3A proved crucial for activating the Akt-mTOR cascade, as evidenced by decreased Akt-S6K1 phosphorylation upon REG3A silencing or knockout, which was reversed by REG3A overexpression. A constitutively active mutant S473D Akt1 (caAkt1) restored Akt-mTOR activation and counteracted the proliferation inhibition and apoptosis induced by REG3A knockdown in breast cancer cells. Crucially, REG3A played a key role in maintaining mTOR complex integrity. Bioinformatics identified zinc finger protein 680 (ZNF680) as a potential REG3A transcription factor. Knocking down or knocking out ZNF680 reduced REG3A expression, while its overexpression increased it in primary breast cancer cells. Additionally, enhanced binding between ZNF680 protein and the REG3A promoter was observed in breast cancer tissues and cells. In vivo, REG3A shRNA significantly inhibited primary TNBC cell xenograft growth. In REG3A-silenced xenograft tissues, reduced REG3A levels, Akt-mTOR inhibition, and activated apoptosis were evident. CONCLUSION: ZNF680-caused REG3A overexpression drives tumorigenesis in breast cancer possibly by stimulating Akt-mTOR activation, emerging as a promising and innovative cancer target.

Development and validation of an ELISA to measure regenerating island-derived protein 3E in canine blood.

BACKGROUND: Regenerating island-derived proteins (REG) are upregulated in people with sepsis, pancreatitis, and gastrointestinal diseases. One member of the REG family, namely REG3E, was recently identified in pancreatic tissue and plasma of dogs, with high expression in pancreatitis and sepsis. OBJECTIVES: We aimed to develop and validate an ELISA to measure REG3E concentrations in canine blood. METHODS: An indirect sandwich ELISA was developed using recombinant canine REG3E protein and polyclonal anti-canine REG3E antibodies raised in guinea pigs and rabbits. Antibody specificity was assessed using western blot and mass spectrometric analysis of protein purified from canine plasma. Assay validation included evaluation of dilutional linearity, parallelism, spiking recovery, repeatability and reproducibility, stability, interferences, and comparison of serum and heparinized plasma. RESULTS: Antibodies bound specifically to REG3E with no evidence of cross-reactivity with other proteins. The limit of detection of the ELISA was 15 ng/mL, and the lower limit of quantification was 30 ng/mL. The assay demonstrated good to excellent linearity, dilutional and mixing parallelism, and recovery, with mean observed-to-expected ratios of 104%, 107%, 102%, and 92%, respectively, and no evidence of a hook effect. Coefficients of variation were ≤8.5% for repeatability and ≤14.3% for reproducibility at three different levels. Measurements of REG3E in plasma were not significantly influenced by different storage conditions, freeze-thawing cycles, hemolysis, lipemia, or icterus. There was no significant difference between REG3E concentrations in heparinized plasma and serum samples. CONCLUSIONS: The canine REG3E ELISA has acceptable precision, accuracy, linearity, and reproducibility for the measurement of REG3E in canine plasma and serum.

Single-cell transcriptome reveals the heterogeneity of malignant ductal cells and the prognostic value of REG4 and SPINK1 in primary pancreatic ductal adenocarcinoma.

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related deaths, with very limited therapeutic options available. This study aims to comprehensively depict the heterogeneity and identify prognostic targets for PDAC with single-cell RNA sequencing (scRNA-seq) analysis. Methods: ScRNA-seq analysis was performed on 16 primary PDAC and three adjacent lesions. A series of analytical methods were applied for analysis in cell clustering, gene profiling, lineage trajectory analysis and cell-to-cell interactions. In vitro experiments including colony formation, wound healing and sphere formation assay were performed to assess the role of makers. Results: A total of 32,480 cells were clustered into six major populations, among which the ductal cell cluster expressing high copy number variants (CNVs) was defined as malignant cells. Malignant cells were further subtyped into five subgroups which exhibited specific features in immunologic and metabolic activities. Pseudotime trajectory analysis indicated that components of various oncogenic pathways were differentially expressed along tumor progression. Furthermore, intensive substantial crosstalk between ductal cells and stromal cells was identified. Finally, genes (REG4 and SPINK1) screened out of differentially expressed genes (DEGs) were upregulated in PDAC cell lines. Silencing either of them significantly impaired proliferation, invasion, migration and stemness of PDAC cells. Conclusions: Our findings offer a valuable resource for deciphering the heterogeneity of malignant ductal cells in PDAC. REG4 and SPINK1 are expected to be promising targets for PDAC therapy.

Commensal bacterial outer membrane protein A induces interleukin-22 production.

Interleukin (IL)-22 promotes host-microbiota homeostasis. We sought to identify microbiota metabolite(s) that drive intestinal IL-22 production. We observed that exposing Peyer's patch cells (PPCs), ex vivo, to fecal supernatants (FSs) recapitulates fermentable fiber- and microbiota-dependent IL-22 production, and cellular sources thereof, thus supporting the use of this model. An interrogation of FSs generated from mice fed the fermentable fiber inulin (FS-Inu) revealed that its IL-22-inducing activity is mediated by heat-labile protein. Fractionation of FS-Inu by ion-exchange chromatography, and subsequent proteomic analysis of IL-22-inducing fractions, indicates that outer membrane protein A (OmpA) might be a microbial driver of IL-22 expression. Concomitantly, recombinant OmpA from Parabacteroides goldsteinii, which is enriched by an inulin diet, induces IL-22 production and expression of the IL-22-dependent genes REG3γ and -ß, in PPCs and mice. Thus, OmpA is one bacterial inducer of IL-22 expression, potentially linking diet, mucosal immune homeostasis, and gut health.

Reg4 deficiency aggravates pancreatitis by increasing mitochondrial cell death and fibrosis.

Regenerating gene family member 4 (Reg4) has been implicated in acute pancreatitis, but its precise functions and involved mechanisms have remained unclear. Herein, we sought to investigate the contribution of Reg4 to the pathogenesis of pancreatitis and evaluate its therapeutic effects in experimental pancreatitis. In acute pancreatitis, Reg4 deletion increases inflammatory infiltrates and mitochondrial cell death and decreases autophagy recovery, which are rescued by the administration of recombinant Reg4 (rReg4) protein. In chronic pancreatitis, Reg4 deficiency aggravates inflammation and fibrosis and inhibits compensatory cell proliferation. Moreover, C-X-C motif ligand 12 (CXCL12)/C-X-C motif receptor 4 (CXCR4) axis is sustained and activated in Reg4-deficient pancreas. The detrimental effects of Reg4 deletion are attenuated by the administration of the approved CXCR4 antagonist plerixafor (AMD3100). Mechanistically, Reg4 mediates its function in pancreatitis potentially via binding its receptor exostosin-like glycosyltransferase 3 (Extl3). In conclusion, our findings suggest that Reg4 exerts a therapeutic effect during pancreatitis by limiting inflammation and fibrosis and improving cellular regeneration.

Identification of biomarkers and potential therapeutic targets for pancreatic cancer by proteomic analysis in two prospective cohorts.

Pancreatic cancer (PC) is the deadliest malignancy due to late diagnosis. Aberrant alterations in the blood proteome might serve as biomarkers to facilitate early detection of PC. We designed a nested case-control study of incident PC based on a prospective cohort of 38,295 elderly Chinese participants with ∼5.7 years' follow-up. Forty matched case-control pairs passed the quality controls for the proximity extension assay of 1,463 serum proteins. With a lenient threshold of p < 0.005, we discovered regenerating family member 1A (REG1A), REG1B, tumor necrosis factor (TNF), and phospholipase A2 group IB (PLA2G1B) in association with incident PC, among which the two REG1 proteins were replicated using the UK Biobank Pharma Proteomics Project, with effect sizes increasing steadily as diagnosis time approaches the baseline. Mendelian randomization analysis further supported the potential causal effects of REG1 proteins on PC. Taken together, circulating REG1A and REG1B are promising biomarkers and potential therapeutic targets for the early detection and prevention of PC.

The imbalance of pulmonary Th17/Treg cells in BALB/c suckling mice infected with respiratory syncytial virus-mediated intestinal immune damage and gut microbiota changes.

The immune response induced by respiratory syncytial virus (RSV) infection is closely related to changes in the composition and function of gastrointestinal microorganisms. However, the specific mechanism remains unknown and the pulmonary-intestinal axis deserves further study. In this study, the mRNA levels of ROR-γt and Foxp3 in the lung and intestine increased first and then decreased. IL-17 and IL-22 reached the maximum on the third day after infection in the lung, and on the second day after infection in the small intestine and colon, respectively. RegⅢγ in intestinal tissue reached the maximum on the third day after RSV infection. Moreover, the genus enriched in the RSV group was Aggregatibacter, and Proteus was reduced. RSV infection not only causes Th17/Treg cell imbalance in the lungs of mice but also leads to the release of excessive IL-22 from the lungs through blood circulation which binds to IL-22 receptors on the intestinal surface, inducing RegⅢγ overexpression, impaired intestinal Th17/Treg development, and altered gut microbiota composition. Our research reveals a significant link between the pulmonary and intestinal axis after RSV infection. IMPORTANCE: RSV is the most common pathogen causing acute lower respiratory tract infections in infants and young children, but the complex interactions between the immune system and gut microbiota induced by RSV infection still requires further research. In this study, it was suggested that RSV infection in 7-day-old BALB/c suckling mice caused lung inflammation and disruption of Th17/Treg cells development, and altered the composition of gut microbiota through IL-22 induced overexpression of RegⅢγ, leading to intestinal immune injury and disruption of gut microbiota. This research reveals that IL-22 may be the link between the lung and gut. This study may provide a new insight into the intestinal symptoms caused by RSV and other respiratory viruses and the connection between the lung and gut axis, as well as new therapeutic ideas for the treatment of RSV-infected children.

IL-22-dependent responses and their role during Citrobacter rodentium infection.

The mouse pathogen Citrobacter rodentium is utilized as a model organism for studying infections caused by the human pathogens enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) and to elucidate mechanisms of mucosal immunity. In response to C. rodentium infection, innate lymphoid cells and T cells secrete interleukin (IL)-22, a cytokine that promotes mucosal barrier function. IL-22 plays a pivotal role in enabling mice to survive and recover from C. rodentium infection, although the exact mechanisms involved remain incompletely understood. Here, we investigated whether particular components of the host response downstream of IL-22 contribute to the cytokine's protective effects during C. rodentium infection. In line with previous research, mice lacking the IL-22 gene (Il22-/- mice) were highly susceptible to C. rodentium infection. To elucidate the role of specific antimicrobial proteins modulated by IL-22, we infected the following knockout mice: S100A9-/- (calprotectin), Lcn2-/- (lipocalin-2), Reg3b-/- (Reg3ß), Reg3g-/- (Reg3γ), and C3-/- (C3). All knockout mice tested displayed a considerable level of resistance to C. rodentium infection, and none phenocopied the lethality observed in Il22-/- mice. By investigating another arm of the IL-22 response, we observed that C. rodentium-infected Il22-/- mice exhibited an overall decrease in gene expression related to intestinal barrier integrity as well as significantly elevated colonic inflammation, gut permeability, and pathogen levels in the spleen. Taken together, these results indicate that host resistance to lethal C. rodentium infection may depend on multiple antimicrobial responses acting in concert, or that other IL-22-regulated processes, such as tissue repair and maintenance of epithelial integrity, play crucial roles in host defense to attaching and effacing pathogens.

Amphiregulin, ST2, and REG3α biomarker risk algorithms as predictors of nonrelapse mortality in patients with acute GVHD.

ABSTRACT: Graft-versus-host disease (GVHD) is a major cause of nonrelapse mortality (NRM) after allogeneic hematopoietic cell transplantation. Algorithms containing either the gastrointestinal (GI) GVHD biomarker amphiregulin (AREG) or a combination of 2 GI GVHD biomarkers (suppressor of tumorigenicity-2 [ST2] + regenerating family member 3 alpha [REG3α]) when measured at GVHD diagnosis are validated predictors of NRM risk but have never been assessed in the same patients using identical statistical methods. We measured the serum concentrations of ST2, REG3α, and AREG by enzyme-linked immunosorbent assay at the time of GVHD diagnosis in 715 patients divided by the date of transplantation into training (2004-2015) and validation (2015-2017) cohorts. The training cohort (n = 341) was used to develop algorithms for predicting the probability of 12-month NRM that contained all possible combinations of 1 to 3 biomarkers and a threshold corresponding to the concordance probability was used to stratify patients for the risk of NRM. Algorithms were compared with each other based on several metrics, including the area under the receiver operating characteristics curve, proportion of patients correctly classified, sensitivity, and specificity using only the validation cohort (n = 374). All algorithms were strong discriminators of 12-month NRM, whether or not patients were systemically treated (n = 321). An algorithm containing only ST2 + REG3α had the highest area under the receiver operating characteristics curve (0.757), correctly classified the most patients (75%), and more accurately risk-stratified those who developed Minnesota standard-risk GVHD and for patients who received posttransplant cyclophosphamide-based prophylaxis. An algorithm containing only AREG more accurately risk-stratified patients with Minnesota high-risk GVHD. Combining ST2, REG3α, and AREG into a single algorithm did not improve performance.

Loss of NLRP6 expression increases the severity of intestinal injury after syngeneic hematopoietic stem cell transplantation.

NLRP6 plays a crucial role in maintaining intestinal homeostasis by regulating the interaction between the intestinal mucosa and the microbiota. However, the impact of NLRP6 deficiency on intestinal damage following hematopoietic stem cell transplantation (HSCT) remains poorly understood. In this study, we established a syngeneic HSCT mouse model using C57BL/6 mice as donors and NLRP6-/- or C57BL/6 mice as recipients. Our findings revealed that NLRP6 deficiency had minimal influence on peripheral blood cell counts and splenic immune cell proportions in transplanted mice. However, it exacerbated pathological changes in the small intestine on day 14 post-transplantation, accompanied by increased proportions of macrophages, dendritic cells, and neutrophils. Furthermore, the NLRP6 deficiency resulted in elevated expression of MPO and CD11b, while reducing the levels mature caspase-1 and mature IL-1ß in the intestine. Moreover, the NLRP6 deficiency disturbed the expression of apoptosis-related molecules and decreased the tight junction protein occludin. Notably, recipient mice with NLRP6 deficiency exhibited lower mRNA expression levels of antimicrobial genes, such as Reg3γ and Pla2g2a. The short-term increase in inflammatory cell infiltration caused by NLRP6 deficiency was associated with intestinal damage, increased apoptosis, reduced expression of antimicrobial peptides, and impaired intestinal repair. Taken together, our findings demonstrate that the loss of NLRP6 exacerbates post-transplantation intestinal damage in recipient mice.

The role of morphine- and fentanyl-induced impairment of intestinal epithelial antibacterial activity in dysbiosis and its impact on the microbiota-gut-brain axis.

Recent evidence suggests that chronic exposure to opioid analgesics such as morphine disrupts the intestinal epithelial layer and causes intestinal dysbiosis. Depleting gut bacteria can preclude the development of tolerance to opioid-induced antinociception, suggesting an important role of the gut-brain axis in mediating opioid effects. The mechanism underlying opioid-induced dysbiosis, however, remains unclear. Host-produced antimicrobial peptides (AMPs) are critical for the integrity of the intestinal epithelial barrier as they prevent the pathogenesis of the enteric microbiota. Here, we report that chronic morphine or fentanyl exposure reduces the antimicrobial activity in the ileum, resulting in changes in the composition of bacteria. Fecal samples from morphine-treated mice had increased levels of Akkermansia muciniphila with a shift in the abundance ratio of Firmicutes and Bacteroidetes. Fecal microbial transplant (FMT) from morphine-naïve mice or oral supplementation with butyrate restored (a) the antimicrobial activity, (b) the expression of the antimicrobial peptide, Reg3γ, (c) prevented the increase in intestinal permeability and (d) prevented the development of antinociceptive tolerance in morphine-dependent mice. Improved epithelial barrier function with FMT or butyrate prevented the enrichment of the mucin-degrading A. muciniphila in morphine-dependent mice. These data implicate impairment of the antimicrobial activity of the intestinal epithelium as a mechanism by which opioids disrupt the microbiota-gut-brain axis.

Dissecting the respective roles of microbiota and host genetics in the susceptibility of Card9-/- mice to colitis.

BACKGROUND: The etiology of inflammatory bowel disease (IBD) is unclear but involves both genetics and environmental factors, including the gut microbiota. Indeed, exacerbated activation of the gastrointestinal immune system toward the gut microbiota occurs in genetically susceptible hosts and under the influence of the environment. For instance, a majority of IBD susceptibility loci lie within genes involved in immune responses, such as caspase recruitment domain member 9 (Card9). However, the relative impacts of genotype versus microbiota on colitis susceptibility in the context of CARD9 deficiency remain unknown. RESULTS: Card9 gene directly contributes to recovery from dextran sodium sulfate (DSS)-induced colitis by inducing the colonic expression of the cytokine IL-22 and the antimicrobial peptides Reg3ß and Reg3γ independently of the microbiota. On the other hand, Card9 is required for regulating the microbiota capacity to produce AhR ligands, which leads to the production of IL-22 in the colon, promoting recovery after colitis. In addition, cross-fostering experiments showed that 5 weeks after weaning, the microbiota transmitted from the nursing mother before weaning had a stronger impact on the tryptophan metabolism of the pups than the pups' own genotype. CONCLUSIONS: These results show the role of CARD9 and its effector IL-22 in mediating recovery from DSS-induced colitis in both microbiota-independent and microbiota-dependent manners. Card9 genotype modulates the microbiota metabolic capacity to produce AhR ligands, but this effect can be overridden by the implantation of a WT or "healthy" microbiota before weaning. It highlights the importance of the weaning reaction occurring between the immune system and microbiota for host metabolism and immune functions throughout life. A better understanding of the impact of genetics on microbiota metabolism is key to developing efficient therapeutic strategies for patients suffering from complex inflammatory disorders. Video Abstract.

Quantifying redox transcription factor dynamics as a tool to investigate redox signalling.

A critical feature of the cellular antioxidant response is the induction of gene expression by redox-sensitive transcription factors. In many cells, activating these transcription factors is a dynamic process involving multiple redox steps, but it is unclear how these dynamics should be measured. Here, we show how the dynamic profile of the Schizosaccharomyces pombe Pap1 transcription factor is quantifiable by three parameters: signal amplitude, signal time and signal duration. In response to increasing hydrogen peroxide concentrations, the Pap1 amplitude decreased while the signal time and duration showed saturable increases. In co-response plots, these parameters showed a complex, non-linear relationship to the mRNA levels of four Pap1-regulated genes. We also demonstrate that hydrogen peroxide and tert-butyl hydroperoxide trigger quantifiably distinct Pap1 activation profiles and transcriptional responses. Based on these findings, we propose that different oxidants and oxidant concentrations modulate the Pap1 dynamic profile, leading to specific transcriptional responses. We further show how the effect of combination and pre-exposure stresses on Pap1 activation dynamics can be quantified using this approach. This method is therefore a valuable addition to the redox signalling toolbox that may illuminate the role of dynamics in determining appropriate responses to oxidative stress.

Antibacterial peptide Reg4 ameliorates Pseudomonas aeruginosa-induced pulmonary inflammation and fibrosis.

Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative facultative anaerobe that has become an important cause of severe infections in humans, particularly in patients with cystic fibrosis. The development of efficacious methods or mendicants against P. aeruginosa is still needed. We previously reported that regenerating islet-derived family member 4 (Reg4) has bactericidal activity against Salmonella Typhimurium, a Gram-negative flagellated bacterium. We herein explore whether Reg4 has bactericidal activity against P. aeruginosa. In the P. aeruginosa PAO1-chronic infection model, Reg4 significantly inhibits the colonization of PAO1 in the lung and subsequently ameliorates pulmonary inflammation and fibrosis. Reg4 recombinant protein suppresses the growth motility and biofilm formation capability of PAO1 in vitro. Mechanistically, Reg4 not only exerts bactericidal action via direct binding to the P. aeruginosa cell wall but also enhances the phagocytosis of alveolar macrophages in the host. Taken together, our study demonstrates that Reg4 may provide protection against P. aeruginosa-induced pulmonary inflammation and fibrosis via its antibacterial activity.IMPORTANCEChronic lung infection with Pseudomonas aeruginosa is a leading cause of morbidity and mortality in patients with cystic fibrosis. Due to the antibiotic resistance of Pseudomonas aeruginosa, antimicrobial peptides appear to be a potential alternative to combat its infection. In this study, we report an antimicrobial peptide, regenerating islet-derived 4 (Reg4), that showed killing activity against clinical strains of Pseudomonas aeruginosa PAO1 and ameliorated PAO1-induced pulmonary inflammation and fibrosis. Experimental data also showed Reg4 directly bound to the bacterial cell membrane and enhanced the phagocytosis of host alveolar macrophages. Our presented study will be a helpful resource in searching for novel antimicrobial peptides that could have the potential to replace conventional antibiotics.

The deletion of ppr2 interferes iron sensing and leads to oxidative stress response in Schizosaccharomyces pombe.

Pentatricopeptide repeat proteins are involved in mitochondrial both transcriptional and posttranscriptional regulation. Schizosaccharomyces pombe Ppr2 is a general mitochondrial translation factor that plays a critical role in the synthesis of all mitochondrial DNA-encoded oxidative phosphorylation subunits, which are essential for mitochondrial respiration. Our previous analysis showed that ppr2 deletion resulted in increased expression of iron uptake genes and caused ferroptosis-like cell death in S. pombe. In the present work, we showed that deletion of ppr2 reduced viability on glycerol- and galactose-containing media.Php4 is a transcription repressor that regulates iron homeostasis in fission yeast. We found that in the ppr2 deletion strain, Php4 was constitutively active and accumulated in the nucleus in the stationary phase. We also found that deletion of ppr2 decreased the ferroptosis-related protein Gpx1 in the mitochondria. Overexpression of Gpx1 improves the viability of Δppr2 cells. We showed that the deletion of ppr2 increased the production of ROS, downregulated heme synthesis and iron-sulfur cluster proteins, and induced stress proteins. Finally, we observed the nuclear accumulation of Pap1-GFP and Sty1-GFP, suggesting that Sty1 and Pap1 in response to cellular stress in the ppr2 deletion strain. These results suggest thatppr2 deletion may cause mitochondrial dysfunction, which is likely to lead to iron-sensing defect and iron starvation response, resulting in perturbation of iron homeostasis and increased hydroxyl radical production. The increased hydroxyl radical production triggers cellular responses in theppr2 deletion strain.

A Validated Risk Stratification That Incorporates MAGIC Biomarkers Predicts Long-Term Outcomes in Pediatric Patients with Acute GVHD.

Acute graft versus host disease (GVHD) is a common and serious complication of allogeneic hematopoietic cell transplantation (HCT) in children but overall clinical grade at onset only modestly predicts response to treatment and survival outcomes. Two tools to assess risk at initiation of treatment were recently developed. The Minnesota risk system stratifies children for risk of nonrelapse mortality (NRM) according to the pattern of GVHD target organ severity. The Mount Sinai Acute GVHD International Consortium (MAGIC) algorithm of 2 serum biomarkers (ST2 and REG3α) predicts NRM in adult patients but has not been validated in a pediatric population. We aimed to develop and validate a system that stratifies children at the onset of GVHD for risk of 6-month NRM. We determined the MAGIC algorithm probabilities (MAPs) and Minnesota risk for a multicenter cohort of 315 pediatric patients who developed GVHD requiring treatment with systemic corticosteroids. MAPs created 3 risk groups with distinct outcomes at the start of treatment and were more accurate than Minnesota risk stratification for prediction of NRM (area under the receiver operating curve (AUC), .79 versus .62, P = .001). A novel model that combined Minnesota risk and biomarker scores created from a training cohort was more accurate than either biomarkers or clinical systems in a validation cohort (AUC .87) and stratified patients into 2 groups with highly different 6-month NRM (5% versus 38%, P < .001). In summary, we validated the MAP as a prognostic biomarker in pediatric patients with GVHD, and a novel risk stratification that combines Minnesota risk and biomarker risk performed best. Biomarker-based risk stratification can be used in clinical trials to develop more tailored approaches for children who require treatment for GVHD.