CrebH protects against liver injury associated with colonic inflammation via modulation of exosomal miRNA

CrebH protects against liver injury associated with colonic inflammation via modulation of exosomal miRNA | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CrebH protects against liver injury associated with colonic inflammation via modulation of exosomal miRNA Sang-Hee Lee, Sung-Je Moon, Seung Hee Woo, Gwangsook Ahn, Won Kon Kim, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2636684/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Jun, 2023 Read the published version in Cell & Bioscience → Version 1 posted 9 You are reading this latest preprint version Abstract Background Hepatic liver disease, including primary sclerosing cholangitis (PSC), is a serious extraintestinal manifestations of colonic inflammation. Cyclic adenosine monophosphate (cAMP)-responsive element-binding protein H (CrebH) is a transcription factor expressed mostly in the liver and small intestine. However, CrebH’s roles in the gut–liver axis remain unknown. Methods Inflammatory bowel disease (IBD) and PSC disease models were established in wild-type and CrebH -/- mice treated with dextran sulfate sodium, dinitrobenzene sulfonic acid, and diethoxycarbonyl dihydrocollidine diet, respectively. RNA sequencing were conducted to investigate differential gene expression. Exosomes were isolated from plasma and culture media. miRNA expression profiling was performed using the NanoString nCounter Mouse miRNA Panel. Effects of miR-29a-3p on adhesion molecule expression were investigated in bEnd.3 brain endothelial cells. Results CrebH -/- mice exhibited accelerated liver injury without substantial differences in the gut after administration of dextran sulfate sodium (DSS), and had similar features to PSC, including enlarged bile ducts, enhanced inflammation, and aberrant MAdCAM-1 expression. Furthermore, RNA-sequencing analysis showed that differentially expressed genes in the liver of CrebH -/- mice after DSS overlapped significantly with genes changed in PSC-liver. Analysis of plasma exosome miRNA isolated from WT and CrebH -/- mice indicates that CrebH can contribute to the exosomal miRNA profile. We also identified miR-29a-3p as an effective mediator for MAdCAM-1 expression. Administration of plasma exosome from CrebH -/- mice led to prominent inflammatory signals in the liver of WT mice with inflammatory bowel disease (IBD). Conclusions CrebH deficiency led to increased susceptibility to IBD-induced liver diseases via enhanced expression of adhesion molecules and concomitant infiltration of T lymphocytes. Exosomes can contribute to the progression of IBD-induced liver injury in CrebH -/- mice. These study provide novel insights into the role of CrebH in IBD-induced liver injury. CrebH exosomes inflammatory bowel disease liver damage primary sclerosing cholangitis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Inflammatory bowel diseases (IBDs), including Crohn’s disease (CD) and ulcerative colitis (UC), are associated with the destruction of gut structure and function, resulting in chronic intestinal inflammation [ 1 ]. Genetic and environmental factors are implicated in the immunopathologic process of IBD, leading to chronic inflammation in the gut [ 2 ]. Pathogenesis of IBD is closely related to an aberrant local immune response to intestinal microflora and uncontrolled endogenous regulator mechanisms [ 3 , 4 ]. In addition, IBDs are considered systemic diseases because their symptoms occur in the gastrointestinal tract and cause problems outside the gut, commonly called extraintestinal manifestations (EIMs) [ 5 ]. However, the pathogenic factors leading to EIMs are not fully understood. The liver is a critical site for antigen exposure and the response to invading pathogens during IBD. Liver-associated EIMs include primary sclerosing cholangitis (PSC), hepatitis, hepatic cirrhosis, fatty liver, and cholelithiasis, resulting in a high incidence of biliary cancer and colorectal cancer [ 6 ]. Among them, PSC is a severe IBD manifestation characterized by increased inflammation of the intrahepatic and extrahepatic bile ducts [ 7 – 9 ]. The prevalence of PSC patients suffering from concurrent IBD is approximately 70%, whereas the rate of PSC occurrence among IBD patients is only 1–5% [ 10 ]. Although the mechanistic links between IBD and PSC remain largely unknown, a widely accepted hypothesis is translocation of microbiota and their products from the gut to the liver, triggering an aberrant cholangiocytic response [ 11 ]. Another hypothesis is that the proportion of T-lymphocytes expressing α4β7 integrin increases in the liver of humans and animals with PSC [ 12 , 13 ]. In the gut, lymphocyte infiltration and activation are essential to protect the intestine from invading pathogens and play key roles in IBD pathogenesis [ 14 ]. Association of adhesion molecules with their ligand leads to tissue-specific trafficking of lymphocytes. Mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1), a gut-specific adhesion molecule, is a key player in lymphocyte trafficking with the vascular cell-adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1) [ 15 , 16 ]. In the liver, MAdCAM-1 is commonly detected in the hepatic sinusoids and has an important role in recruiting lymphocytes [ 13 ]. Although vascular adhesion protein 1 (VAP1) may regulate MAdCAM-1 expression in the liver [ 17 ], the regulation of MAdCAM-1 expression in the liver of PSC patients remains unclear. Cyclic adenosine monophosphate (cAMP)-responsive element-binding protein H (CrebH, known as CREB3L3) is a transcription factor related to a member of the CREB/ATF family [ 18 ]. CrebH was initially known as a liver-specific transcription factor owing to its marked expression in the liver; its roles in hepatic glucose and lipid metabolism are commonly studied [ 19 ]. The expression and role of CrebH in the small intestine have been reported recently [ 20 ]. Therefore, we hypothesized that CrebH plays an important role in the gut–liver axis. Methods Animal studies All mice were maintained at a constant temperature (20–22 ℃) under scheduled light: dark conditions (12:12 h); this animal study was approved by the guidelines of the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology (KRIBB-AEC-21129). We selected 10-week-old C57BL/6J and CrebH −/− male mice to be administered with 2.5% (w/v) dextran sulfate sodium (DSS, molecular weight = 36–50 kDa; MP Biomedicals, CA, USA) supplemented in drinking water to generate UC-mimetic animal models. For the CD model, anesthetized male mice were intrarectally injected with 3 mg dinitrobenzene (DNBS, St. Louis, MO, USA) in 100 µL 50% ethanol (EtOH). For the PSC animal model, 8-week-old mice were fed a 3.5-dieythioxycarbonyl-1,4-dihydrocollidine (DDC, Sigma-Aldrich, St. Louis, MO, USA) diet (standard rodent diet supplemented with 0.1% [w/w] DDC) for 7 days. WT male mice were twice injected with exosome (200 µg protein concentration/mice) isolated from WT and CrebH −/− male mice treated with DSS for 7 days and then administered with 2.5% DSS for 7 days. Cell Culture And Establishment Of Stable Cells HepG2 and bEnd.3 cell lines purchased from ATCC were cultured in Dulbecco’s modified Eagle’s medium (DMEM; HyClone, Logan, UT, USA) containing 10% fetal bovine serum (FBS, HyClone), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, MA, USA) at 37°C and 5% CO 2 . HepG2 cell lines constantly expressing the CrebH gene were generated by transfection with pcDNA3-Flag- CrebH [ 21 ] using the Lipofectamine LTX plus reagent system (Invitrogen, CA, USA) and selected by adding G418 solution (Sigma-Aldrich). Cell Transfection And Experiments bEnd.3 cells were seeded in a 6 cm dish and cultured at 37 ℃ in a 5% CO 2 incubator overnight. The cells were replaced with fresh complete media and transfected with miR-29a-3p mimic (Bioneer, Korea) and negative control (Bioneer) at 250 pM (final concentration) using RNAiMAX reagents (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s protocol. After additional incubation for 24 h, the cells were treated with 50 ng/mL TNFα or vehicle and harvested after further 24 h incubation. The miR-29a-3p mimic sequence was 5′-ACUGAUUUCUUUUGGUGUUCAG-3′. Plasma Analysis Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations were measured automatically using a chemical analyzer (Hitachi, Tokyo, Japan). Alkaline phosphatase (ALP) levels were measured using an ALP assay kit (Abcam, MA, USA) according to the manufacturer’s protocol [ 22 ]. Histological Analysis The colon, ileum, and liver tissues were cut at 5 µm thickness and stained separately with hematoxylin and eosin (H&E), similar to a previous study [ 23 , 24 ]. The severity of colitis was blindly assessed by a pathologist and scored using the histological scoring method [ 23 ]. For immunohistochemistry, the slides were incubated with a boiled citrate solution for 15 min, blocked for 1 h, and further incubated with primary antibodies against MAdCAM-1 (MyBioSource, CA, USA), cleaved caspase 3 (Cell Signaling, MA, USA), F4/80 (Abcam), and CD3 (Abcam) at 4 ℃ overnight. Then, slides were washed with PBS and stained with secondary antibodies and a Vectastain Elite ABC kit (Vector Labs, Burlington, ON, Canada) according to the manufacturer’s protocol. The positive cells were detected using diaminobenzidine (DAB, Vector Labs) and mounted with the antifade agent (Sigma-Aldrich). Tunel staining was performed according to the manufacturer’s manual. Fluorescence In Situ Hybridization (Fish) Analysis Sense and antisense probes were generated from a partial mouse CrebH gene encoded into pGEM-T vector (Promega, WI, USA) by using PCR with T7 or SP6 primers. The tissue expression of CrebH mRNA was investigated using a FISH kit (Thermo Fisher) according to the manufacturer’s protocol. Myeloperoxidase (Mpo) Activity MPO activity in the livers of WT and CrebH −/− mice was measured using an MPO activity colorimetric assay kit (Biovision Inc., CA, USA), according to the manufacturer’s protocol [ 25 ]. Rna Isolation And Quantitative Real Time-pcr Total RNA was prepared using TRIzol reagent (Thermo Fisher) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized using 1 µg template RNA and an iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Relative gene expression was determined using AccuPower 2 × Greenstar qPCR Master Mix (Bioneer) and a StepOnePlus™ Real-Time PCR device (Applied Biosystems, CA, USA). Each gene was normalized to 18s rRNA. The primers used are listed in Additional file 1 (Table S1 ). A thermal cycler was set for 40 cycles at an annealing temperature of 60 ℃. Fluorescence-activated Cell Sorting (Facs) Analysis Liver cells were prepared by passing through a 70 µm Falcon™ Cell strainer (Life sciences, MA, USA) and centrifugation of the supernatant into 40% Percoll (GE Healthcare, UK). The isolated cells were labeled with fluorophore-conjugated antibodies against PE-CD45 (BioLegend, CA, USA), FITC-CD3ε (BD Pharmingen, CA, USA), APC-CD8a (BD Pharmingen), and PerCP/Cy5.5-CD4 (BioLegend) for 30 min at 4 ℃. Labeled cells were assayed using a Gallio™ Flow Cytometer (Beckman Coulter, FL, USA). Data were analyzed using the FlowJo software (TreeStar, CA, USA). Western Blotting Western blotting The samples were homogenized in RIPA buffer (Sigma-Aldrich) supplemented with a protease inhibitor (Roche Applied Science, Germany). The proteins were separated by electrophoresis on a 10–12% sodium dodecyl sulfate-polyacrylamide gel, transferred to PVDF membranes, and blocked in TBST buffer with 5% skim milk. The membranes were incubated with primary antibodies against MAdCAM-1, α-tubulin (Cell Signaling), AKT1 (Cell Signaling), AKT2 (Cell Signaling), and TNF-R1 (Cell Signaling). Exosome Isolation Plasma exosome isolation was performed using the ExoQuick exosome precipitation kit (SBI System Biosciences, Mountain View, CA, USA) according to the manufacturer’s protocol [ 26 , 27 ]. HepG2 cell lines constantly expressing CrebH were seeded in a 10 cm culture dish overnight and changed with fresh DMEM containing 10% EV-depleted FBS (SBI System Biosciences) for 48 h. The culture medium was centrifuged at 1,000 × g for 5 min at 4 ℃ to eliminate suspended cells, filtered through a 0.22 µm syringe filter, and transferred to a Macrosep 100 KD filter system (PALL Laboratory, MA, USA) to enrich the particles with 30–90 nm molecular size. The exosomes contained in the enriched particles were isolated using ExoQuick-TC exosome precipitation solution (SBI System Biosciences). The exosome pellet was resuspended in 100 µL filtered-PBS and stored at − 80 ℃. Exosome Characterization And Treatment The exosome size was determined using a dynamic light scattering system (Otsuka ELS-Z, Japan). Exosomes isolated from the plasma of WT and CrebH −/− mice were quantified using an ExoELISA-ULTRA assay kit (SBI System Biosciences) according to the manufacturer’s guidelines [ 28 ]. Exosomal markers were estimated by immunoblotting using antibodies against TSG101 (Abcam) and CD9 (Abcam). Exosomes were labeled using an ExoGlow-Membrane EV labeling kit (SBI System Biosciences) following the manufacturer’s instructions. Labeled exosomes were cocultured with bEnd.3 cell lines for the indicated time. Images were obtained using fluorescence microscopy (Olympus, Tokyo, Japan). Exosomal proteins were quantified using Bradford protein assay and bEnd.3 cells were treated with exosomes (30 µg/mL protein concentration contained in exosome) for 24 h. Exosomal Mirna Profiling Exosomal total RNA was profiled using a NanoString nCounter Mouse miRNA Panel (NanoString Technologies, WA, USA) according to the manufacturer’s instructions [ 29 ]. Each RNA sample (50 ng) was added to the miRNA-tag ligation reaction. Ligated miRNA was diluted (1:5), added to hybridization, and subjected to 3 h of automated processing per cartridge. The acquired data were normalized by a set of six positive and negative control probes included in the system and processed using nSolver software (version 4.0, NanoString Technologies). Relative miRNA was expressed as fold-change. Transcriptome Analysis As described above, total RNA was isolated from liver tissues of WT-DSS and CrebH −/− -DSS or control and DDC-treated mice. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. RNA-sequencing (RNA-seq) libraries were constructed and sequenced on an Illumina X Ten. The number of reads mapped was counted using featureCounts (v1.5.0-p3); then, the fragments per kilobase of transcript per million mapped reads of each gene was calculated based on the length of the gene. Differential expression of each group was performed using the DESeq2 R package (v1.20.0). The resulting P values were adjusted using Benjamini and Hochberg’s approach [ 30 ], and genes with an adjusted P-value < 0.05 were assigned as differentially expressed. Gene ontology (GO) analysis using the WEB-based Gene SeT AnaLysis Toolkit ( https://www.webgestalt.org ) was applied to analyze the molecular functions of overlapped genes with significant differences. Heatmaps were generated by the TreeView3 program ( https://bitbucket.org/TreeView3Dev/treeview3 ) Statistical analysis Data were analyzed using the GraphPad Prism software (version 8.0; San Diego, CA, USA) and expressed as mean ± standard error of the mean (SEM). Differences between the two groups were analyzed using the Student’s t -test. Survival differences between groups were analyzed using the log-rank (Mantel–Cox) test. Statistical significance was set at P -value < 0.05. Uncropped blots are shown in the Original Western blots of Figures. Results CrebH deficiency does not affect the development of IBD pathogenesis CrebH is highly detected in both the liver and small intestine [ 19 , 20 ]. To confirm this observation, mRNA expression of CrebH was evaluated by FISH in ileum and colon tissues. Similar to the findings of another study [ 20 ], high-intensity CrebH-positive cell response was observed in the ileum (Fig. 1 A), while its expression was very rare in the colon. Firstly, the ablation effects of CrebH in DSS-induced colitis were determined. DSS administration to WT and CrebH −/− mice led to death and the development of severe pathogenesis in colon tissues, as evidenced by body weight loss, reduced hematocrit percentage and colon length, and severe inflammation with no significant differences between WT and CrebH −/− mice (Fig. 1 B–F). Consistently, pro-inflammatory cytokine expression did not differ between the groups (Fig. 1 G), suggesting that rare expression of CrebH in the colon is not enough to alter the pathogenesis of UC. Similar to the UC model, in CD animal models, CrebH did not affect IBD progression, as evidenced by the lack of significant differences (Additional file 2: Fig. S1 A–C). Ablation Of Crebh Deteriorates A Liver Injury Of Mice With Ibd IBD leads to concomitant disease development outside the gut [ 31 ]. Therefore, we estimated the liver pathology to confirm the role of CrebH in IBD-associated liver damage. The liver of CrebH −/− mice after administering DSS showed a pale color compared with those of WT mice (Fig. 2 A). Levels of ALT and AST, two liver damage markers, were increased in the plasma of CrebH −/− mice compared with that in the plasma of WT mice (Fig. 2 B, C). Excitingly, liver histology of CrebH −/− mice displayed enlarged bile ducts (Fig. 2 D). In contrast to the gut data, the expression of pro-inflammatory cytokines, such as IL-1β , IL-6 , and TNFα , was significantly increased in the livers of CrebH −/− mice compared with WT mice (Fig. 2 E). Furthermore, MPO activity was more enhanced in the livers of CrebH −/− mice than in those of WT mice (Fig. 2 F). These data show that CrebH −/− mice have high inflammatory conditions in their livers. Furthermore, apoptotic cells significantly increased in the livers of CrebH −/− mice (Fig. 2 G). Consistent with DSS-liver, in the CD mouse model, CrebH −/− mice showed severe pathogenesis compared with WT mice (Additional file 3: Fig. S2 A–D). Loss of CrebH leads to enhanced CD8 + T lymphocyte infiltration via up-regulation of adhesion molecules in the liver In IBD-associated liver disease, T lymphocytes infiltrate the liver and play a critical role in hepatic duct inflammation [ 16 ]. FACS analysis showed that CD8 + T lymphocytes were significantly upregulated in the liver of CrebH −/− mice after DSS treatment, whereas CD4 + T lymphocyte regulation was not significantly altered (Fig. 3 A). MAdCAM-1 and VAP1 , aberrantly expressed in the liver of PSC patients and animal models with IBD, were significantly increased in the livers of CrebH −/− mice compared with WT mice (Fig. 3 B–D). Histological data show that MAdCAM-1 was primarily distributed in the endothelium of vessels or sinusoids after DSS treatment (Fig. 3 E). CrebH is mostly expressed in the small intestine, and we investigated the MAdCAM-1 mRNA expression in ileum and colon tissues of mice with CD to determine the CrebH dependency on MAdCAM-1 expression. Consistent with CrebH expression levels, differential expressions of adhesion molecules containing MAdCAM-1 were more prominent in the small intestine than the large intestine (Fig. 3 F), explaining the CrebH contribution to local expression of MAdCAM-1 and the lack of difference in pathogenesis in the large intestines of both groups. Different gene expressions in the liver of CrebH −/− mice are closely related with PSC-liver As described, we observed that the livers of CrebH −/− mice after DSS have similar histological and pathogenic characteristics as PSC. Interestingly, CrebH −/− mice was more susceptible to the pathogenesis of PSC caused by DDC treatments than were WT mice. Therefore, we performed differential gene expression analysis to estimate the similarity of CrebH −/− mice liver with PSC-liver. Gene expression analyzed by RNA-seq in the liver of CrebH −/− -DSS vs. WT-DSS groups was compared with the dataset of DDC-treated vs. control groups. The volcano plot of differentially expressed genes (DEGs) shows significant genes, including 3,906 upregulated and 3,088 downregulated genes in DDC vs. control groups and 246 upregulated and 128 downregulated genes in CrebH −/− -DSS vs. WT-DSS (Fig. 4 A). The top 20 genes upregulated in CrebH −/− -DSS vs. WT-DSS are listed in the heatmap, and several genes were validated by qRT-PCR (Fig. 4 B). Excitingly, 41.5% upregulated and 41.4% downregulated genes in CrebH −/− -DSS vs. WT-DSS overlapped with genes altered in DDC vs. control, respectively (Fig. 4 C). On analyzing the gene ontology terms, the three most enriched in molecular function (MF) terms of the upregulated genes among overlapped genes were “extracellular matrix structural constituents conferring compression resistance,” “extracellular matrix binding,” and “collagen-binding,” which are closely related to tissue fibrosis (Fig. 4 D). In MF terms, the 10 genes associated with extracellular matrix binding, including Adamts15 , Anxa2 , Bgn , Ctss , Dcn , Itgb3 , Lgals1 , Nid1 , Sparc , and Tgfbi , were observed in the heatmap (Fig. 4 E, left ), and several genes validated by qRT-PCR showed a similar pattern with RNA-seq analysis (Fig. 4 E, right ). These data suggest that liver injury in CrebH −/− after administration of DSS is similar to the pathogenic characteristics of PSC. Exosomes play an important role in regulating adhesion molecules during liver injury of CrebH −/− mice To identify systemic mediators affected by CrebH ablation, we performed multiplex plasma analysis. No significant differences between CrebH − /− and WT mice were detected in the plasma (Additional file 5: Table S2 ). Exosomes play an important role in cell-to-cell communication in systemic and local systems [ 32 ]. Therefore, we hypothesized that exosomes might be potential mediators contributing to the aberrant pathogenesis in CrebH −/− -DSS mice. First, we characterized exosomes isolated from the plasma of each mice group to estimate their successful isolation (Fig. 5 A–D). bEnd.3 cells, an endothelial cell line, were stimulated with exosomes isolated from the plasma of WT-water exosome (WC-exo), WT-DSS exosome (WD-exo), CrebH −/− -water exosome (KC-exo), and CrebH −/− -DSS exosome (KD-exo). KC-exo treatment led to significantly higher MAdCAM-1 expression than WC-exo treatment (Fig. 5 E). Furthermore, KD-exo additionally elevated the effects of KC-exo (Fig. 5 E). VAP1 expression was upregulated only in KD-exo-treated cells (Fig. 5 F). To further investigate the liver-specific effects of CrebH, HepG2 cell lines constantly overexpressing CrebH were generated (Additional file 6: Fig. S4 ). Isolated exosomes from the medium of the stable cells were characterized (Fig. 5 G). Consistent with another study [ 33 ], TNFα treatment stimulated MAdCAM-1 expression, and co-treatment with pcDNA-exo significantly enhanced its expression in bEnd.3 cells (Fig. 5 H). Interestingly, CrebH-exo eliminated pcDNA-exo-induced MAdCAM-1 expression (Fig. 5 H), suggesting that CrebH can modulate MAdCAM-1 expression in an exosome dependent manner. Immunoblotting data for MAdCAM-1 further supported this result (Fig. 5 I). Profiling Of Exosomal Mirna And Identification Of Mir-29a-3p As An Effector Mirna To identify exosomal mediators affecting liver pathogenesis during IBD, we performed miRNA profiling of exosomes using NanoString analysis. The Venn diagram revealed that altered miRNA between WC-exo and WD-exo shared 17 miRNAs compared with WD-exo and KD-exo (Fig. 6 A). The heatmap shows aberrant expression of various miRNAs from each group (Fig. 6 B and Additional file 7–9: Table S3 –5). Excitingly, most altered miRNA was increased in the plasma of CrebH −/− mice compared with that of WT after administration of DSS, suggesting that CrebH can regulate exosomal miRNA contents (Fig. 6 B). To identify hepatocyte-specific mediators, we performed miRNA profiling in the exosomes derived from HepG2 cells constantly overexpressing pcDNA3 or CrebH and found miR-29a as a potential target miRNA. The sequence of mouse miR-29a was the same as that of humans and rats. miR-29a-3p was found in exosomes from HepG2 cells overexpressing CrebH protein with upregulated expression (Fig. 6 C), and its levels were validated by qRT-PCR (Fig. 6 D). In contrast, miR-29a-3p was downregulated in the plasma of CrebH −/− mice compared with that in WT mice (Fig. 6 E). Previously, Deng et al. reported that miR-29a-3p regulates several TNFα-induced adhesion molecules, including VCAM1 , ICAM1 , and E-selectin , by targeting TNF receptor-1 (TNF-R1) in various endothelial cell lines [ 34 ]. Therefore, we generated a miR-29a-3p mimic to investigate its effect on MAdCAM-1 expression. Transfection of the mimic into bEnd.3 cells led to an approximately 700-fold increase in the miR-29a-3p level compared with the negative control (Fig. 6 F). We found that the mimic inhibited MAdCAM-1 and VAP1 expression in bEnd.3 cells (Fig. 6 G, H). Recent studies have suggested that MAdCAM-1 expression is regulated by TNF-α and/or AKT signaling pathways [ 33 ]. We found a target sequence in the AKT2 3′-untranslated region (UTR; DIANA tools; http://diana.imis.athena-innovation.gr ; Fig. 6 I). Our western blot data showed that AKT2 levels were slightly higher in the liver of CrebH −/− mice than in that of WT mice under healthy conditions (Fig. 6 I). However, this pattern was not observed after DSS treatment. Interestingly, TNF-R1 levels were dramatically increased in the livers of CrebH −/− mice, and this induction was more strongly enhanced by DSS administration (Fig. 6 I), suggesting that aberrant expression of adhesion molecules in the liver of CrebH −/− mice might have occurred owing to increased TNF-R1. Plasma exosomes isolated from CrebH −/− mice with IBD aggravate DSS-induced liver injury in WT mice To investigate the exosome effects against liver damages, plasma exosomes from WT and CrebH −/− mice administered with DSS for 7 days were isolated and then injected into WT mice followed by DSS (Fig. 7 A). Plasma AST levels were significantly increased in mice injected with CrebH −/− -exo compared with PBS controls (Fig. 7 B). Although ALT levels were not significant, they increased in mice treated with CrebH −/− -exo compared with both controls (Fig. 7 B). This suggests that the exosomes can contribute to the development of liver injury in WT mice. Adhesion molecules increased in the liver of CrebH −/− mice were also up-regulated by CrebH −/− -exo without VAP1 (Fig. 7 C), resulting in enhanced hepatic infiltration of immune cells evidenced by increased F4/80 positive cells and expression of F4/80 and CD3 markers (Fig. 7 D). Levels of proinflammatory cytokines were significantly higher in liver treated with CrebH −/− -exo compared with PBS and WT-exo controls (Fig. 7 E). Interestingly, the expressions of IL-1β and TNFα showed a downward tendency in liver treated with WT-exo compared with PBS controls, providing a protective role of exosome against hepatic inflammation during IBD (Fig. 7 E). Finally, we confirmed the DEGs observed in NGS dataset. The Acot3 and Mcam genes increased in CrebH −/− mice compared with WT mice, and were significantly higher in mice treated with CrebH −/− -exo than in the only-PBS group; no significantly different expression was observed between mice with CrebH −/− -exo and those with WT-exo (Fig. 7 F). Excitingly, Anxa2, Ctss, Dcn, and Lgals1 among DEGs overlapped with PSC-liver were significantly increased by treatment of CrebH −/− -exo compared with both control groups (Fig. 7 G). As these genes are involved in extracellular matrix binding, our results might suggest that exosomes from CrebH −/− mice are strongly associated with IBD induced-fibrosis. Discussion Liver homeostasis is closely related to the gut environment because approximately 70% of blood derived from the gut reaches the liver [ 35 ]. This gut–liver axis has been implicated in various liver diseases such as PSC [ 36 ]. Membrane-bound transcriptional factor CrebH is primarily expressed in the liver and regulates genes related to triglyceride metabolism and fatty acid oxidation [ 19 ]. Recently, CrebH expression has also been reported in the small intestine [ 20 ]. However, the potential roles of CrebH in the gut–liver axis remain completely unknown. This study employed a genetic mouse model to determine the effects of CrebH ablation on IBD in the gut and liver. To the best of our knowledge, this study is the first to show the role of CrebH in the gut–liver axis. PSC, a well-known clinically IBD-connected chronic liver disease, is evidence of an interaction between the gut and the liver [ 6 ]. Clinically, most PSC patients have concurrent IBD, while IBD patients have only 1–5% PSC, suggesting that liver might have protective systems against IBD-induced PSC progression. PSC-liver is characterized by inflammation and onion skin-type fibrotic lesions around bile ducts, resulting in bile duct strictures and accumulation of bile acids into the liver [ 37 ]. Lymphocyte infiltration is a common characterization important for disease progression in IBD and IBD-related liver disorders [ 15 , 16 ]. Interestingly, CrebH −/− mice exhibited enlarged bile ducts, enhanced inflammation, and DEGs highly overlapped with PCS-liver. Furthermore, MAdCAM-1, demonstrated as a gut-specific adhesion molecule [ 12 ] and known as a marker for PSC liver [ 13 ], significantly increased in the liver of CrebH −/− mice. Therefore, our study suggests that CrebH might be involved in the progression of IBD-related liver diseases such as PSC. Reciprocal interactions between the gut and liver during IBD is established through the portal vein, which carries gut-origin products to the liver. The systemic effectors involved in liver injury progression during IBD contain various cytokines [ 38 ] and chemokines [ 39 ] together with bacteria and their products [ 40 ]. However, we did not find any differences in the plasma in our experimental conditions, suggesting another potential factors regulating liver injury. Exosomes play a critical role in cell-to-cell communication and protect their contents, including protein, RNA, DNA, and metabolites in the blood system [ 32 ]. Therefore, we hypothesized that exosomes might play an important role in the accelerated pathogenesis of the liver in CrebH −/− mice. Fortunately, exosomes isolated from the plasma of CrebH −/− mice can effectively stimulate MAdCAM-1 and VAP1 expression in bEnd.3 cells. Conversely, exosomes from the culture medium of HepG2 cells expressing exogenous CrebH inhibited TNFα-induced MAdCAM-1 expression. Furthermore, exosomes isolated from CrebH −/− mice stimulated infiltration and activation of immune cells in the liver of WT mice with IBD, while exosomes isolated from WT mice did not. These data suggest that exosomes have a protective role in IBD-induced hepatic inflammation and CrehH can regulate the contents of exosomes. Notably, many miRNAs of exosomes isolated in plasma from CrebH −/− mice were significantly increased compared with those of WT mice in both healthy and diseased conditions, suggesting that CrebH might regulate exosomal miRNA by unknown mechanisms. Mechanistically, exosomal miRNAs mediate post-transcriptional gene silencing by binding to the 3′-UTR or open reading frame region of the target gene [ 41 ]. Recently, Deng et al. demonstrated that miR-29a-3p could inhibit the expression of ICAM1 , VCAM1 , and E-selectin induced by TNFα stimulation in vitro and in vivo [ 34 ]. They also suggested that miR-29a-3p specifically suppresses TNF-R1 expression by targeting the 3′-UTR of TNFRSF1A [ 34 ]. Furthermore, MAdCAM-1 was induced by the TNFα signaling pathway [ 33 ]. Therefore, miR-29a-3p was selected as a potential miRNA for testing. We found that miR-29a-3p was increased in exosomes from HepG2 cells overexpressing CrebH compared with those from control cells expressing pcDNA3 and was reduced in the exosomes isolated from plasma of CrebH −/− mice; however, the difference was not statistically significant. The miR-29a-3p mimic inhibited MAdCAM-1 and VAP1 expression in bEnd.3 cells. Furthermore, we found a dramatic upregulation of TNF-R1 in the livers of CrebH −/− mice. These data suggest that miR-29a-3p might be a critical factor in regulating MAdCAM-1 expression along with another adhesion molecule such as ICAM1 , VCAM1 , or E-selectin. According to previous studies, miRNAs are preferentially sorted into exosomes by four potential modes, including the neural sphingomyelinase 2-associated pathway [ 42 ], heterogeneous nuclear ribonucleoprotein-associated pathway [ 43 ], 3′-end of the miRNA sequence-associated pathway, and miRNA-induced silencing complex-related pathway [ 44 ]. Thus, CrebH might be involved in these miRNA sorting systems; however, further studies are necessary to confirm this. Conclusion This study demonstrates that liver of CrebH −/− mice after DSS treatment show characteristic phenotypes resembling the PSC-liver, as evidenced by increased biliary inflammation, enlarged bile ducts, upregulation of adhesion molecules such as MAdCAM-1, and high similarity of altered genes compared with PSC-liver, suggesting that CrebH −/− mice might be a potential animal model for investigating the initial pathogenesis of liver during the progression of PSC-IBD. This study also demonstrates that exosomes play a pivotal role in protecting the subsequent pathogenesis of IBD and IBD-related liver inflammation (Fig. 8 ). Exosomal miRNAs are potential effector molecules, and CrebH can affect some exosomal miRNAs. Based on recent clinical studies on monoclonal antibodies against MAdCAM-1 or α4β7 [ 45 , 46 ], miR-29a-3p could be an effective therapeutic strategy for IBD treatment. Therefore, this study provides new insight for future human studies. Abbreviations ALP alkaline phosphatase ALT alanine aminotransferase AST aspartate aminotransferase CD Crohn’s disease cAMP cyclic adenosine monophosphate cDNA complementary DNA CrebH cyclic adenosine monophosphate-responsive element-binding protein H DDC 3.5-dieythioxycarbonyl-1,4-dihydrocollidine DEGs differentially expressed genes DNBS dinitrobenzene DSS dextran sulfate sodium EIMs extraintestinal manifestations FISH fluorescence in situ hybridization IBD inflammatory bowel disease ICAM1 intercellular adhesion protein 1 MAdCAM-1 mucosal vascular addressin cell adhesion molecule 1 MF molecular function MPO myeloperoxidase PSC primary sclerosing cholangitis TNF-R1” TNF receptor-1 UC ulcerative colitis VAP1 vascular adhesion protein 1 VCAM1 vascular cell-adhesion molecule 1. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The RNA-seq raw data has been deposited in the Korean Nucleotide Archive (KoNA, https://kobic.re.kr/kona) under accession numbers PRJKA220165 and PRJKA220166. The datasets used and/or analysed during the current study are available from the corresponding authors on reasonable request. Competing interest The authors declare no competing interests. Funding This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP; 2019R1A2C1086436 and 2022R1A2C1006815) and a grant from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM5392312 and KGS1042322). Author contributions S.-H.L. & S.-J.M. performed most animal experiments and analyzed the data. S.H.W. performed in vitro experiments and formal analysis. H.J.H. and C.-H.L. conceived of the study and designed experimental strategies. H.J.H., C.-H.L., and W.K.K. contributed to funding acquisition. 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Soler D, Chapman T, Yang LL, Wyant T, Egan R, Fedyk ER. The binding specificity and selective antagonism of vedolizumab, an anti-alpha4beta7 integrin therapeutic antibody in development for inflammatory bowel diseases. J Pharmacol Exp Ther. 2009;330:864–75. Vermeire S, Sandborn WJ, Danese S, Hebuterne X, Salzberg BA, Klopocka M, et al. Anti-MAdCAM antibody (PF-00547659) for ulcerative colitis (TURANDOT): a phase 2, randomised, double-blind, placebo-controlled trial. Lancet. 2017;390:135–44. Supplementary Files Additionalfile1.docx Additional file 1 (Word, txt): Table S1. Primer sequences used in this study. Additionalfile2.pptx Additional file 2 (ppt): Figure S1. Ablation effects of CrebH on the development of DNBS-induced IBD pathogenesis. A, Survival of WT (n=10) and KO (n=9) mice response to DNBS. B, Colon length of WT (n=3) and CrebH -/- (n=5 ~ 6) mice treated with a vehicle or DNBS for 3 days. C, Representative images of the colon and ileum. Bar represents 200 μm. Additionalfile3.pptx Additional file 3 (ppt): Figure S2. Ablation effects of CrebH on DNBS-induced liver injury. A, Growth images of WT and CrebH -/- mice and their liver. WT and CrebH -/- mice were intrarectally administrated with 3 mg of DNBS for 3 d. B-C, Plasma ALT and AST levels of WT and CrebH -/- mice. *P < 0.05, or ***P < 0.001. D, Liver histology of WT and CrebH -/- mice. Bar represents 200 μm. Additionalfile5.docx Additional file 5 (Word, txt): Table S2. Plasma parameters analyzed by Multiplex cytokine bead assay. Data are expressed as mean ± SEM. Additionalfile6.pptx Additional file 6 (ppt): Figure S4.CrebH protein expression was determined by western blotting using antibody against CrebH. HepG2 cells were transiently transfected with plasmid expressing pcDNA3, CrebH-full form, and CrebH-active form (N-terminal region) and selected by incubation with G418. Additionalfile7.docx Additional file 7 (Word, txt): Table S3: Differently regulated miRNA lists (WC-exo vs. WD-exo). Additionalfile8.docx Additional file 8 (Word, txt): Table S4: Differently regulated miRNA lists (WD-exo vs. KD-exo). Additionalfile9.docx Additional file 9 (Word, txt): Table S5: Differently regulated miRNA lists (WC-exo vs. KC-exo). Cite Share Download PDF Status: Published Journal Publication published 27 Jun, 2023 Read the published version in Cell & Bioscience → Version 1 posted Editorial decision: Major revision 13 Apr, 2023 Reviewer # 2 agreed at journal 17 Mar, 2023 Reviewers agreed at journal 17 Mar, 2023 Reviewers invited by journal 16 Mar, 2023 Reviewer # 1 agreed at journal 16 Mar, 2023 Editor assigned by journal 03 Mar, 2023 Submission checks completed at journal 02 Mar, 2023 Editor invited by journal 02 Mar, 2023 First submitted to journal 01 Mar, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2636684","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":184277625,"identity":"356f7172-eb25-4b04-a08d-300c84533b8c","order_by":0,"name":"Sang-Hee Lee","email":"","orcid":"","institution":"Korea Research Institute of Bioscience and Biotechnology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sang-Hee","middleName":"","lastName":"Lee","suffix":""},{"id":184277626,"identity":"e20e69de-902b-42c1-86f9-7f687ab9b0f1","order_by":1,"name":"Sung-Je Moon","email":"","orcid":"","institution":"Korea Research Institute of Bioscience and Biotechnology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sung-Je","middleName":"","lastName":"Moon","suffix":""},{"id":184277627,"identity":"6adce89c-ec31-434f-a743-becb56548f75","order_by":2,"name":"Seung Hee Woo","email":"","orcid":"","institution":"Korea Research Institute of Bioscience and Biotechnology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Seung","middleName":"Hee","lastName":"Woo","suffix":""},{"id":184277628,"identity":"46ec0f2a-1ad6-41a6-986c-7e7a57ef8d22","order_by":3,"name":"Gwangsook Ahn","email":"","orcid":"","institution":"Daejeon University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gwangsook","middleName":"","lastName":"Ahn","suffix":""},{"id":184277629,"identity":"d1165023-46a8-49fb-81dc-1db92f995634","order_by":4,"name":"Won Kon Kim","email":"","orcid":"","institution":"Korea Research Institute of Bioscience and Biotechnology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Won","middleName":"Kon","lastName":"Kim","suffix":""},{"id":184277630,"identity":"06ca26c8-8863-403e-9c0f-3035e04e3364","order_by":5,"name":"Chul-Ho Lee","email":"","orcid":"","institution":"Korea Research Institute of Bioscience and Biotechnology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chul-Ho","middleName":"","lastName":"Lee","suffix":""},{"id":184277631,"identity":"6cc7be1b-ed03-46c6-b5ad-8a50568e4d55","order_by":6,"name":"Jung Hwang Hwang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYNCCChs5CQbGNmYwh4coLWfSjEnUwth2OHEGAwMbcVr425sffvzZdjh95uzDbY8LGO7JMfCcfYBXi8SZY8bSPOfSc2fzJbYbz2AoNmbgbTfAq8VAIgfonjLr3Hk8jG3SPAwJiQ38bPgdBtLC+IONOV0OqqWeKC0MPG3OCdJQLQkMvG34tUD8cibNcGYPI9AvBgmGbTzH8GsBh9iPCht5iTPszx4XVCTI8/Ok4deC7k5g7JCkYRSMglEwCkYBVgAAgqU4AJsM5d8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-5779-6666","institution":"Korea Research Institute of Bioscience and Biotechnology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jung","middleName":"Hwang","lastName":"Hwang","suffix":""}],"badges":[],"createdAt":"2023-02-28 05:43:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2636684/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2636684/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13578-023-01065-9","type":"published","date":"2023-06-27T21:26:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":34588881,"identity":"b30bd96d-6f4a-42af-abc8-ad101c6ad40d","added_by":"auto","created_at":"2023-03-21 14:28:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3777468,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of CrebH mRNA and effects of CrebH ablation on the progression of IBD pathogenesis. A,\u003c/strong\u003e Expression of CrebH mRNA was determined in the colon and ileum by FISH (left, anti-sense probe; right, sense probe). The scale bar represents 50 μm. \u003cstrong\u003eB,\u003c/strong\u003e Survival differences of WT and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice after 2.5% DSS were monitored daily. WT (n = 11) and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (n = 10). \u003cstrong\u003eC,\u003c/strong\u003e Bodyweight loss was estimated in the mouse groups; WT-con (n = 4, open square), \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e-con (n = 4, blue square), WT-DSS, (n = 4, open circle), and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e-DSS (n = 4, red circle). \u003cstrong\u003eD-E,\u003c/strong\u003e Hematocrit and colon length of WT (n = 4) and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (n = 4) mice. \u003cstrong\u003eF,\u003c/strong\u003e Liver histopathology of WT (n = 8) and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (n = 7) mice were estimated by hematoxylin and eosin (H\u0026amp;E) staining (left) and scoring (right). The scale bar represents 200 μm. \u003cstrong\u003eG\u003c/strong\u003e The levels of \u003cem\u003eIL-1β\u003c/em\u003e, \u003cem\u003eIL-6\u003c/em\u003e, and \u003cem\u003eTNFα\u003c/em\u003e were measured by qRT-PCR in the colon tissues from WT (blue) and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (red) mice treated with 2.5% DSS for 7 days. Statistical analysis was performed using two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. Error bars represent the mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2636684/v1/4609b33010768516a8dce0bb.png"},{"id":34588878,"identity":"fad3ecd6-ca77-4c09-bec0-4b8a0cc75cda","added_by":"auto","created_at":"2023-03-21 14:28:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3750016,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCrebH deficiency accelerates IBD-associated liver damage.\u003c/strong\u003e \u003cstrong\u003eA,\u003c/strong\u003e Representative liver aspect of WT and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice treated with 2.5% DSS for 7 days. \u003cstrong\u003eB-C,\u003c/strong\u003e ALT and AST levels were measured in the plasma of WT (n = 7) and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (n = 8) mice. \u003cem\u003e***P \u003c/em\u003e\u0026lt; 0.001. \u003cstrong\u003eD,\u003c/strong\u003e Representative liver histology of WT and CrebH\u003csup\u003e-/-\u003c/sup\u003e mice were stained with H\u0026amp;E. The bar represents 200 μm. \u003cstrong\u003eE,\u003c/strong\u003e The levels of \u003cem\u003eIL-1β\u003c/em\u003e, \u003cem\u003eIL-6\u003c/em\u003e, and \u003cem\u003eTNFα\u003c/em\u003e in the liver from WT (n = 7) and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (n = 7) mice were analyzed by qRT-PCR. *\u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.01 or \u003cem\u003e***P \u003c/em\u003e\u0026lt; 0.001. \u003cstrong\u003eF,\u003c/strong\u003e Liver MPO activity was measured using a commercially available assay kit. *\u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.01. \u003cstrong\u003eG,\u003c/strong\u003e Liver images of tunel (left images) and cleaved caspase 3 (right graph) in WT and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice. The scale bar represents 200 μm. \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05, *\u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.01. Positive cells per field were counted using the ImageJ software. Statistical analysis was performed using a two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. Error bars represent the mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2636684/v1/5449b4647e2ae093851c85e6.png"},{"id":34588879,"identity":"4d0f2164-fdc2-454a-8221-8de5c4a0f0ea","added_by":"auto","created_at":"2023-03-21 14:28:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3425957,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCrebH deficiency leads to increased T lymphocyte infiltration via enhanced adhesion molecules in the liver.\u003c/strong\u003e \u003cstrong\u003eA,\u003c/strong\u003e Cd3\u003csup\u003e+\u003c/sup\u003eCd4\u003csup\u003e+\u003c/sup\u003e and Cd3\u003csup\u003e+\u003c/sup\u003eCd8\u003csup\u003e+\u003c/sup\u003e double-positive cells were determined by flow cytometric analysis of cell population in the liver of WT (n = 6) and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (n = 6) mice after DSS treatment. \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05. \u003cstrong\u003eB-C,\u003c/strong\u003e mRNA levels of \u003cem\u003eMAdCAM-1\u003c/em\u003e and \u003cem\u003eVAP1\u003c/em\u003e in the liver from WT and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice were measured by qRT-PCR. *\u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.01, or \u003cem\u003e***P \u003c/em\u003e\u0026lt; 0.001. \u003cstrong\u003eD,\u003c/strong\u003e Protein levels of MAdCAM-1 in the liver from WT and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice were evaluated by immunoblotting using specific antibodies against each protein, and their optical intensities were normalized by b-actin. \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05. \u003cstrong\u003eE,\u003c/strong\u003e Representative immunohistochemistry images on MAdCAM-1 in the liver from WT and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice. The scale bar represents 200 μm. \u003cstrong\u003eF,\u003c/strong\u003e Gene expression of \u003cem\u003eMAdCAM-1\u003c/em\u003e, \u003cem\u003eVAP1\u003c/em\u003e, and \u003cem\u003eICAM1\u003c/em\u003e was estimated in the colon (left) and ileum (right) of WT (n = 5) and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (n = 6) mice treated with DNBS for 2 days. \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05, *\u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.01. Statistical analysis was performed using a two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. Error bars represent the mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2636684/v1/9ce407c2417d7c8f3e198537.png"},{"id":34590679,"identity":"5682d8a7-1217-4d10-bf22-0a30b9bd88bf","added_by":"auto","created_at":"2023-03-21 14:36:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2642212,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene expressions in the liver of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCrebH\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/- \u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003emice overlap with those expressed differentially in mice with PSC. A,\u003c/strong\u003e Volcano plot of genes significantly altered between DDC (n = 3) vs. control groups (n = 3) and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e-DSS (n = 3) vs. WT-DSS (n = 3). \u003cstrong\u003eB,\u003c/strong\u003e Heatmap (left, RNA-seq data) showing top 20 genes significantly upregulated in the liver of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003emice compared with WT mice; several genes were validated by qRT-PCR. WT (n = 5) and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003e(n = 5) mice. \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05 or \u003cem\u003e**P \u003c/em\u003e\u0026lt; 0.01. Acot3, acyl-CoA thioesterase 3; Cyp4a10, cytochrome P450 4A10; Cyp4a14, cytochrome P450 omega-hydroxylase 4a14; Fabp4, fatty acid-binding protein 4; Mcam, melanoma cell adhesion molecule. \u003cstrong\u003eC,\u003c/strong\u003e Comparison of transcriptome analysis of DEGs between \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e-DSS vs. WT-DSS and DDC vs. control groups. Venn diagram of overlapping differentially upregulated (upper) and downregulated (lower) gene expression. \u003cstrong\u003eD,\u003c/strong\u003e Top 10 enriched in “molecular function” of the RNA-seq dataset. GO enrichment of DEGs was analyzed using the WEB-based Gene SeT AnaLysis Toolkit (\u003ca href=\"http://www.webgestalt.org\"\u003ewww.webgestalt.org\u003c/a\u003e). \u003cstrong\u003eE,\u003c/strong\u003e Heatmap of genes enriched in extracellular matrix binding category; several genes were validated by qRT-PCR. \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e**P \u003c/em\u003e\u0026lt; 0.01, \u003cem\u003e***P \u003c/em\u003e\u0026lt; 0.001. Adamts15, ADAM metallopeptidase with thrombospondin type 1 motif 15; Anxa2, annexin A2; Ctss, cathepsin S; Dcn, decorin; Itgb3, integrin subunit beta 3; Lgals1, galectin 1. Statistical analysis was performed using a two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. Error bars represent the mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2636684/v1/01ef29c226ff34c3051dfafb.png"},{"id":34591959,"identity":"79637fb8-b783-408b-8718-62c3b8b54502","added_by":"auto","created_at":"2023-03-21 14:44:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2979185,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExosomes isolated from plasma of both mouse groups or culture media of cells expressing exogenous CrebH stimulate the expression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMAdCAM-1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVAP1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in endothelial cell lines.\u003c/strong\u003e \u003cstrong\u003eA,\u003c/strong\u003e Immunoblot data for TSG101 and CD9 of exosome lysates. \u003cstrong\u003eB,\u003c/strong\u003e Exosome numbers isolated from WT (blue) and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (red) mice were estimated using a commercially available exosome quantification kit in 100 mL plasma. \u003cstrong\u003eC,\u003c/strong\u003e Exosomal protein levels were measured by the Bradford protein quantification method. Water (n = 3) and DSS (n = 5). \u003cstrong\u003eD,\u003c/strong\u003e Exosome uptake was investigated in bEnd.3 cells. \u003cstrong\u003eE-F,\u003c/strong\u003e Expressions of \u003cem\u003eMAdCAM-1\u003c/em\u003e and \u003cem\u003eVAP1 \u003c/em\u003eresponse to exosome isolated from WT and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003emice. WT-control exosome (WC-exo), WT-DSS exosome (WD-exo), \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e-control exosome (KC-exo), or \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e-DSS exosome (KD-exo) groups. \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05 or \u003cem\u003e**P \u003c/em\u003e\u0026lt; 0.01. \u003cstrong\u003eG,\u003c/strong\u003e Size distribution of exosomes isolated from the culture medium was determined by a dynamic light scattering system. \u003cstrong\u003eH, \u003c/strong\u003eTNFa-induced \u003cem\u003eMAdCAM-1\u003c/em\u003e expression was estimated by qRT-PCR after treatment of pcDNA–exo or CrebH-exo. \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05 or \u003cem\u003e***P \u003c/em\u003e\u0026lt; 0.001. \u003cstrong\u003eI,\u003c/strong\u003e Protein levels of MAdCAM-1. Alpha-tubulin was used as a loading control. Statistical analysis was performed using a two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. Error bars represent the mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2636684/v1/de37adb1cf1b81d2e7b72258.png"},{"id":34593636,"identity":"d2fe02a3-941c-4a21-844d-b7801f795774","added_by":"auto","created_at":"2023-03-21 15:00:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2782564,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExosomal miRNA from plasma of WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCrebH\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/- \u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003emice\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand the effect of miR-29a-3p on expression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMAdCAM-1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVAP1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003eA,\u003c/strong\u003e Venn diagram of exosomal miRNA altered between WT-control vs. WT-DSS or WT-DSS vs. \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e-DSS. \u003cstrong\u003eB,\u003c/strong\u003e Heatmaps showing hierarchical clustering of differentially expressed exosomal miRNA between DSS vs. control (left) and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e-DSS vs. WT-DSS (right). \u003cstrong\u003eC,\u003c/strong\u003e miR-29a-3p levels analyzed by miRNA profiling of stable HepG2 cells overexpressing pcDNA3 or CrebH. \u003cstrong\u003eD,\u003c/strong\u003e Exosomal miR-29a-3p concentration was validated by qRT-PCR in exosomes released from HepG2 cells overexpressing pcDNA3.1 or CrebH. \u003cstrong\u003eE,\u003c/strong\u003e miRNA profiling data for exosomal miR-29a levels of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e-DSS compared with WT-DSS. \u003cstrong\u003eF,\u003c/strong\u003e Effect of the mimic on miR-29a-3p expression in bEnd.3 cells. \u003cstrong\u003eG-H,\u003c/strong\u003e Gene expression of \u003cem\u003eMAdCAM-1\u003c/em\u003e and \u003cem\u003eVAP1\u003c/em\u003e response to miR-29a mimic. \u003cstrong\u003eI,\u003c/strong\u003e Target sequence on AKT 3’ UTR against miR-29a-3p (upper) and representative western blot data for AKT1, AKT2, and TNF-R1 (lower). \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05 or \u003cem\u003e**P \u003c/em\u003e\u0026lt; 0.01. Statistical analysis was performed using a two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. Representative data are from at least two independent experiments. Error bars represent the mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2636684/v1/ebc7898af48ac8f3e26c1540.png"},{"id":34591963,"identity":"39340ed2-4b4c-4168-9ac7-fc42351a1728","added_by":"auto","created_at":"2023-03-21 14:44:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4732203,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlasma exosomes isolated from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCrebH\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/- \u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003emice \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ewith IBD stimulate hepatic inflammation in the liver of WT mice treated with DSS. A, \u003c/strong\u003eSchematic representation of the experimental schedule. Plasma exosomes from WT and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003emice after administration of DSS for 7 days were isolated and then WT mice were treated with isolated exosomes (200 ug protein concentration) at 0 and 4 days of DSS schedule. PBS were used as a control for exosome treatment. \u003cstrong\u003eB,\u003c/strong\u003e AST and ALT levels. \u003cstrong\u003eC,\u003c/strong\u003e Gene expression of \u003cem\u003eMAdCAM-1, ICAM1, VCAM1, \u003c/em\u003eand\u003cem\u003e VAP\u003c/em\u003e in the livers of each group. \u003cstrong\u003eD,\u003c/strong\u003e Immunohistochemistry for F4/80 and CD3 (upper), confirmed by cell counting and mRNA expression (lower). The scale bars represent 200 mm (for F4/80) and 100 mm (for CD3), respectively. \u003cstrong\u003eE,\u003c/strong\u003e Gene expression of \u003cem\u003eIL-1b, IL-6, TNFa, \u003c/em\u003eand\u003cem\u003e IL-10\u003c/em\u003e in the livers of each group. \u003cstrong\u003eF,\u003c/strong\u003e Gene expression of \u003cem\u003eAcot3, Cyp4a10, Cyp4a14, Fabp4, \u003c/em\u003eand\u003cem\u003e Mcam\u003c/em\u003e in the livers of each group. \u003cstrong\u003eG,\u003c/strong\u003e Gene expression of \u003cem\u003eAdamts, Anxa2, Ctss, Dcn, Itgb3, \u003c/em\u003eand\u003cem\u003e Lgals1\u003c/em\u003e in the livers of each group. \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e**P \u003c/em\u003e\u0026lt; 0.01, *\u003cem\u003e**P \u003c/em\u003e\u0026lt; 0.001. Statistical analysis was performed using a two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. Error bars represent the mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-2636684/v1/f7a933fa52a9cfe5ca7ed4aa.png"},{"id":34588891,"identity":"0f01f26e-745c-453f-87a6-add474c9efda","added_by":"auto","created_at":"2023-03-21 14:28:32","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":4166475,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of the roles of CrebH on IBD-induced liver injury.\u003c/strong\u003eBacteria and their products, cytokines, growth factors, and other factors move from the gut to liver through the blood stream during IBD. These contribute to liver injury via stimulation of inflammation. In normal condition, exosomal miRNA blocks the expression of MAdCAM-1 via regulation of TNF-R1. However, absence of CrebH lead to aberrant exosomal miRNA, resulting in severe liver injury.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-2636684/v1/6418395a750a6fffcefd206a.png"},{"id":44732085,"identity":"687d2822-105a-48a6-a815-a5d1666167b9","added_by":"auto","created_at":"2023-10-16 21:52:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5393209,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2636684/v1/3903071e-b3be-4996-8364-b7bea0e5d3f2.pdf"},{"id":34588877,"identity":"fae541ba-845e-42d7-947c-9f39777e0f39","added_by":"auto","created_at":"2023-03-21 14:28:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14370,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1 (Word, txt):\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable S1. Primer sequences used in this study.\u003c/p\u003e","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-2636684/v1/7381627762d9e900203ce0de.docx"},{"id":34590688,"identity":"e90323d4-a8ce-48d6-9629-0e9668e4d0f3","added_by":"auto","created_at":"2023-03-21 14:36:32","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":466304,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2 (ppt):\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S1\u003c/strong\u003e.\u003cstrong\u003e Ablation effects of CrebH on the development of DNBS-induced IBD pathogenesis. A,\u003c/strong\u003e Survival of WT (n=10) and KO (n=9) mice response to DNBS. \u003cstrong\u003eB,\u003c/strong\u003e Colon length of WT (n=3) and CrebH\u003csup\u003e-/-\u003c/sup\u003e (n=5 ~ 6) mice treated with a vehicle or DNBS for 3 days. \u003cstrong\u003eC,\u003c/strong\u003e Representative images of the colon and ileum. Bar represents 200 μm.\u003c/p\u003e","description":"","filename":"Additionalfile2.pptx","url":"https://assets-eu.researchsquare.com/files/rs-2636684/v1/b1067402603b46c7725ecb63.pptx"},{"id":34592895,"identity":"f754f549-725a-4809-b8b2-27d6b40c2c22","added_by":"auto","created_at":"2023-03-21 14:52:32","extension":"pptx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":708347,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 3 (ppt):\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S2. Ablation effects of CrebH on DNBS-induced liver injury. A,\u003c/strong\u003e Growth images of WT and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice and their liver. WT and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice were intrarectally administrated with 3 mg of DNBS for 3 d. \u003cstrong\u003eB-C,\u003c/strong\u003e Plasma ALT and AST levels of WT and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice. \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05, or \u003cem\u003e***P \u003c/em\u003e\u0026lt; 0.001. \u003cstrong\u003eD,\u003c/strong\u003e Liver histology of WT and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice. Bar represents 200 μm.\u003c/p\u003e","description":"","filename":"Additionalfile3.pptx","url":"https://assets-eu.researchsquare.com/files/rs-2636684/v1/d9b2896de8881061ef1086f4.pptx"},{"id":34588884,"identity":"89ec31f7-ecc3-4490-ac80-2b26d581ba25","added_by":"auto","created_at":"2023-03-21 14:28:32","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":14053,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 5 (Word, txt):\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable S2. Plasma parameters analyzed by Multiplex cytokine bead assay. Data are expressed as mean ± SEM.\u003c/p\u003e","description":"","filename":"Additionalfile5.docx","url":"https://assets-eu.researchsquare.com/files/rs-2636684/v1/9ba469357fd55795ee66a251.docx"},{"id":34588893,"identity":"1aa537e9-76b3-4796-9294-ab6f4a45e4b7","added_by":"auto","created_at":"2023-03-21 14:28:32","extension":"pptx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":159214,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 6 (ppt):\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S4.\u003c/strong\u003eCrebH protein expression was determined by western blotting using antibody against CrebH. HepG2 cells were transiently transfected with plasmid expressing pcDNA3, CrebH-full form, and CrebH-active form (N-terminal region) and selected by incubation with G418.\u003c/p\u003e","description":"","filename":"Additionalfile6.pptx","url":"https://assets-eu.researchsquare.com/files/rs-2636684/v1/0653c411062b7738e9b565e4.pptx"},{"id":34590680,"identity":"bcba565b-76e3-4dc1-8ada-91525e291409","added_by":"auto","created_at":"2023-03-21 14:36:32","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":15898,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 7 (Word, txt):\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable S3: Differently regulated miRNA lists (WC-exo vs. WD-exo).\u003c/p\u003e","description":"","filename":"Additionalfile7.docx","url":"https://assets-eu.researchsquare.com/files/rs-2636684/v1/a4d676aefdc5b39ccc0ce4d7.docx"},{"id":34591958,"identity":"254c362a-bd13-45cd-9dd1-d2a398397bda","added_by":"auto","created_at":"2023-03-21 14:44:32","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":14425,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 8 (Word, txt):\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable S4: Differently regulated miRNA lists (WD-exo vs. KD-exo).\u003c/p\u003e","description":"","filename":"Additionalfile8.docx","url":"https://assets-eu.researchsquare.com/files/rs-2636684/v1/fa8e8d13e624eb811bd16ba9.docx"},{"id":34590683,"identity":"90668aa2-ed24-4cbd-a456-eb1b73370fc1","added_by":"auto","created_at":"2023-03-21 14:36:32","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":13679,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 9 (Word, txt):\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable S5: Differently regulated miRNA lists (WC-exo vs. KC-exo).\u003c/p\u003e","description":"","filename":"Additionalfile9.docx","url":"https://assets-eu.researchsquare.com/files/rs-2636684/v1/a5e0afd2064e3dc2c29c21e8.docx"}],"financialInterests":"","formattedTitle":"CrebH protects against liver injury associated with colonic inflammation via modulation of exosomal miRNA","fulltext":[{"header":"Background","content":"\u003cp\u003eInflammatory bowel diseases (IBDs), including Crohn\u0026rsquo;s disease (CD) and ulcerative colitis (UC), are associated with the destruction of gut structure and function, resulting in chronic intestinal inflammation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Genetic and environmental factors are implicated in the immunopathologic process of IBD, leading to chronic inflammation in the gut [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Pathogenesis of IBD is closely related to an aberrant local immune response to intestinal microflora and uncontrolled endogenous regulator mechanisms [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addition, IBDs are considered systemic diseases because their symptoms occur in the gastrointestinal tract and cause problems outside the gut, commonly called extraintestinal manifestations (EIMs) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, the pathogenic factors leading to EIMs are not fully understood.\u003c/p\u003e \u003cp\u003eThe liver is a critical site for antigen exposure and the response to invading pathogens during IBD. Liver-associated EIMs include primary sclerosing cholangitis (PSC), hepatitis, hepatic cirrhosis, fatty liver, and cholelithiasis, resulting in a high incidence of biliary cancer and colorectal cancer [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Among them, PSC is a severe IBD manifestation characterized by increased inflammation of the intrahepatic and extrahepatic bile ducts [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The prevalence of PSC patients suffering from concurrent IBD is approximately 70%, whereas the rate of PSC occurrence among IBD patients is only 1\u0026ndash;5% [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Although the mechanistic links between IBD and PSC remain largely unknown, a widely accepted hypothesis is translocation of microbiota and their products from the gut to the liver, triggering an aberrant cholangiocytic response [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Another hypothesis is that the proportion of T-lymphocytes expressing α4β7 integrin increases in the liver of humans and animals with PSC [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In the gut, lymphocyte infiltration and activation are essential to protect the intestine from invading pathogens and play key roles in IBD pathogenesis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Association of adhesion molecules with their ligand leads to tissue-specific trafficking of lymphocytes. Mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1), a gut-specific adhesion molecule, is a key player in lymphocyte trafficking with the vascular cell-adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the liver, MAdCAM-1 is commonly detected in the hepatic sinusoids and has an important role in recruiting lymphocytes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Although vascular adhesion protein 1 (VAP1) may regulate MAdCAM-1 expression in the liver [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], the regulation of MAdCAM-1 expression in the liver of PSC patients remains unclear.\u003c/p\u003e \u003cp\u003eCyclic adenosine monophosphate (cAMP)-responsive element-binding protein H (CrebH, known as CREB3L3) is a transcription factor related to a member of the CREB/ATF family [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. CrebH was initially known as a liver-specific transcription factor owing to its marked expression in the liver; its roles in hepatic glucose and lipid metabolism are commonly studied [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The expression and role of CrebH in the small intestine have been reported recently [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Therefore, we hypothesized that CrebH plays an important role in the gut\u0026ndash;liver axis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal studies\u003c/h2\u003e \u003cp\u003e All mice were maintained at a constant temperature (20\u0026ndash;22 ℃) under scheduled light: dark conditions (12:12 h); this animal study was approved by the guidelines of the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology (KRIBB-AEC-21129). We selected 10-week-old C57BL/6J and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e male mice to be administered with 2.5% (w/v) dextran sulfate sodium (DSS, molecular weight\u0026thinsp;=\u0026thinsp;36\u0026ndash;50 kDa; MP Biomedicals, CA, USA) supplemented in drinking water to generate UC-mimetic animal models. For the CD model, anesthetized male mice were intrarectally injected with 3 mg dinitrobenzene (DNBS, St. Louis, MO, USA) in 100 \u0026micro;L 50% ethanol (EtOH). For the PSC animal model, 8-week-old mice were fed a 3.5-dieythioxycarbonyl-1,4-dihydrocollidine (DDC, Sigma-Aldrich, St. Louis, MO, USA) diet (standard rodent diet supplemented with 0.1% [w/w] DDC) for 7 days. WT male mice were twice injected with exosome (200 \u0026micro;g protein concentration/mice) isolated from WT and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e male mice treated with DSS for 7 days and then administered with 2.5% DSS for 7 days.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell Culture And Establishment Of Stable Cells\u003c/h3\u003e\n\u003cp\u003eHepG2 and bEnd.3 cell lines purchased from ATCC were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM; HyClone, Logan, UT, USA) containing 10% fetal bovine serum (FBS, HyClone), 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin (Gibco, MA, USA) at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. HepG2 cell lines constantly expressing the \u003cem\u003eCrebH\u003c/em\u003e gene were generated by transfection with pcDNA3-Flag-\u003cem\u003eCrebH\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] using the Lipofectamine LTX plus reagent system (Invitrogen, CA, USA) and selected by adding G418 solution (Sigma-Aldrich).\u003c/p\u003e\n\u003ch3\u003eCell Transfection And Experiments\u003c/h3\u003e\n\u003cp\u003ebEnd.3 cells were seeded in a 6 cm dish and cultured at 37 ℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator overnight. The cells were replaced with fresh complete media and transfected with miR-29a-3p mimic (Bioneer, Korea) and negative control (Bioneer) at 250 pM (final concentration) using RNAiMAX reagents (Thermo Fisher Scientific, MA, USA) according to the manufacturer\u0026rsquo;s protocol. After additional incubation for 24 h, the cells were treated with 50 ng/mL TNFα or vehicle and harvested after further 24 h incubation. The miR-29a-3p mimic sequence was 5\u0026prime;-ACUGAUUUCUUUUGGUGUUCAG-3\u0026prime;.\u003c/p\u003e\n\u003ch3\u003ePlasma Analysis\u003c/h3\u003e\n\u003cp\u003ePlasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations were measured automatically using a chemical analyzer (Hitachi, Tokyo, Japan). Alkaline phosphatase (ALP) levels were measured using an ALP assay kit (Abcam, MA, USA) according to the manufacturer\u0026rsquo;s protocol [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eHistological Analysis\u003c/h3\u003e\n\u003cp\u003eThe colon, ileum, and liver tissues were cut at 5 \u0026micro;m thickness and stained separately with hematoxylin and eosin (H\u0026amp;E), similar to a previous study [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The severity of colitis was blindly assessed by a pathologist and scored using the histological scoring method [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For immunohistochemistry, the slides were incubated with a boiled citrate solution for 15 min, blocked for 1 h, and further incubated with primary antibodies against MAdCAM-1 (MyBioSource, CA, USA), cleaved caspase 3 (Cell Signaling, MA, USA), F4/80 (Abcam), and CD3 (Abcam) at 4 ℃ overnight. Then, slides were washed with PBS and stained with secondary antibodies and a Vectastain Elite ABC kit (Vector Labs, Burlington, ON, Canada) according to the manufacturer\u0026rsquo;s protocol. The positive cells were detected using diaminobenzidine (DAB, Vector Labs) and mounted with the antifade agent (Sigma-Aldrich). Tunel staining was performed according to the manufacturer\u0026rsquo;s manual.\u003c/p\u003e\n\u003ch3\u003eFluorescence In Situ Hybridization (Fish) Analysis\u003c/h3\u003e\n\u003cp\u003eSense and antisense probes were generated from a partial mouse \u003cem\u003eCrebH\u003c/em\u003e gene encoded into pGEM-T vector (Promega, WI, USA) by using PCR with T7 or SP6 primers. The tissue expression of \u003cem\u003eCrebH\u003c/em\u003e mRNA was investigated using a FISH kit (Thermo Fisher) according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003ch3\u003eMyeloperoxidase (Mpo) Activity\u003c/h3\u003e\n\u003cp\u003eMPO activity in the livers of WT and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice was measured using an MPO activity colorimetric assay kit (Biovision Inc., CA, USA), according to the manufacturer\u0026rsquo;s protocol [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eRna Isolation And Quantitative Real Time-pcr\u003c/h3\u003e\n\u003cp\u003eTotal RNA was prepared using TRIzol reagent (Thermo Fisher) according to the manufacturer\u0026rsquo;s protocol. Complementary DNA (cDNA) was synthesized using 1 \u0026micro;g template RNA and an iScript\u0026trade; cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Relative gene expression was determined using AccuPower 2 \u003cem\u003e\u0026times;\u003c/em\u003e Greenstar qPCR Master Mix (Bioneer) and a StepOnePlus\u0026trade; Real-Time PCR device (Applied Biosystems, CA, USA). Each gene was normalized to 18s rRNA. The primers used are listed in Additional file 1 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). A thermal cycler was set for 40 cycles at an annealing temperature of 60 ℃.\u003c/p\u003e\n\u003ch3\u003eFluorescence-activated Cell Sorting (Facs) Analysis\u003c/h3\u003e\n\u003cp\u003eLiver cells were prepared by passing through a 70 \u0026micro;m Falcon\u0026trade; Cell strainer (Life sciences, MA, USA) and centrifugation of the supernatant into 40% Percoll (GE Healthcare, UK). The isolated cells were labeled with fluorophore-conjugated antibodies against PE-CD45 (BioLegend, CA, USA), FITC-CD3ε (BD Pharmingen, CA, USA), APC-CD8a (BD Pharmingen), and PerCP/Cy5.5-CD4 (BioLegend) for 30 min at 4 ℃. Labeled cells were assayed using a Gallio\u0026trade; Flow Cytometer (Beckman Coulter, FL, USA). Data were analyzed using the FlowJo software (TreeStar, CA, USA).\u003c/p\u003e\n\u003ch3\u003eWestern Blotting\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blotting\u003c/div\u003e \u003cp\u003eThe samples were homogenized in RIPA buffer (Sigma-Aldrich) supplemented with a protease inhibitor (Roche Applied Science, Germany). The proteins were separated by electrophoresis on a 10\u0026ndash;12% sodium dodecyl sulfate-polyacrylamide gel, transferred to PVDF membranes, and blocked in TBST buffer with 5% skim milk. The membranes were incubated with primary antibodies against MAdCAM-1, α-tubulin (Cell Signaling), AKT1 (Cell Signaling), AKT2 (Cell Signaling), and TNF-R1 (Cell Signaling).\u003c/p\u003e\n\u003ch3\u003eExosome Isolation\u003c/h3\u003e\n\u003cp\u003ePlasma exosome isolation was performed using the ExoQuick exosome precipitation kit (SBI System Biosciences, Mountain View, CA, USA) according to the manufacturer\u0026rsquo;s protocol [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. HepG2 cell lines constantly expressing CrebH were seeded in a 10 cm culture dish overnight and changed with fresh DMEM containing 10% EV-depleted FBS (SBI System Biosciences) for 48 h. The culture medium was centrifuged at 1,000 \u003cem\u003e\u0026times; g\u003c/em\u003e for 5 min at 4 ℃ to eliminate suspended cells, filtered through a 0.22 \u0026micro;m syringe filter, and transferred to a Macrosep 100 KD filter system (PALL Laboratory, MA, USA) to enrich the particles with 30\u0026ndash;90 nm molecular size. The exosomes contained in the enriched particles were isolated using ExoQuick-TC exosome precipitation solution (SBI System Biosciences). The exosome pellet was resuspended in 100 \u0026micro;L filtered-PBS and stored at \u0026minus;\u0026thinsp;80 ℃.\u003c/p\u003e\n\u003ch3\u003eExosome Characterization And Treatment\u003c/h3\u003e\n\u003cp\u003eThe exosome size was determined using a dynamic light scattering system (Otsuka ELS-Z, Japan). Exosomes isolated from the plasma of WT and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were quantified using an ExoELISA-ULTRA assay kit (SBI System Biosciences) according to the manufacturer\u0026rsquo;s guidelines [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Exosomal markers were estimated by immunoblotting using antibodies against TSG101 (Abcam) and CD9 (Abcam). Exosomes were labeled using an ExoGlow-Membrane EV labeling kit (SBI System Biosciences) following the manufacturer\u0026rsquo;s instructions. Labeled exosomes were cocultured with bEnd.3 cell lines for the indicated time. Images were obtained using fluorescence microscopy (Olympus, Tokyo, Japan). Exosomal proteins were quantified using Bradford protein assay and bEnd.3 cells were treated with exosomes (30 \u0026micro;g/mL protein concentration contained in exosome) for 24 h.\u003c/p\u003e\n\u003ch3\u003eExosomal Mirna Profiling\u003c/h3\u003e\n\u003cp\u003eExosomal total RNA was profiled using a NanoString nCounter Mouse miRNA Panel (NanoString Technologies, WA, USA) according to the manufacturer\u0026rsquo;s instructions [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Each RNA sample (50 ng) was added to the miRNA-tag ligation reaction. Ligated miRNA was diluted (1:5), added to hybridization, and subjected to 3 h of automated processing per cartridge. The acquired data were normalized by a set of six positive and negative control probes included in the system and processed using nSolver software (version 4.0, NanoString Technologies). Relative miRNA was expressed as fold-change.\u003c/p\u003e\n\u003ch3\u003eTranscriptome Analysis\u003c/h3\u003e\n\u003cp\u003eAs described above, total RNA was isolated from liver tissues of WT-DSS and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-DSS or control and DDC-treated mice. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. RNA-sequencing (RNA-seq) libraries were constructed and sequenced on an Illumina X Ten. The number of reads mapped was counted using featureCounts (v1.5.0-p3); then, the fragments per kilobase of transcript per million mapped reads of each gene was calculated based on the length of the gene. Differential expression of each group was performed using the DESeq2 R package (v1.20.0). The resulting P values were adjusted using Benjamini and Hochberg\u0026rsquo;s approach [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], and genes with an adjusted P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were assigned as differentially expressed. Gene ontology (GO) analysis using the WEB-based Gene SeT AnaLysis Toolkit (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.webgestalt.org\u003c/span\u003e\u003cspan address=\"https://www.webgestalt.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was applied to analyze the molecular functions of overlapped genes with significant differences. Heatmaps were generated by the TreeView3 program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bitbucket.org/TreeView3Dev/treeview3\u003c/span\u003e\u003cspan address=\"https://bitbucket.org/TreeView3Dev/treeview3\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using the GraphPad Prism software (version 8.0; San Diego, CA, USA) and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Differences between the two groups were analyzed using the Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. Survival differences between groups were analyzed using the log-rank (Mantel\u0026ndash;Cox) test. Statistical significance was set at \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Uncropped blots are shown in the Original Western blots of Figures.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCrebH deficiency does not affect the development of IBD pathogenesis\u003c/h2\u003e \u003cp\u003eCrebH is highly detected in both the liver and small intestine [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. To confirm this observation, mRNA expression of \u003cem\u003eCrebH\u003c/em\u003e was evaluated by FISH in ileum and colon tissues. Similar to the findings of another study [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], high-intensity CrebH-positive cell response was observed in the ileum (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), while its expression was very rare in the colon. Firstly, the ablation effects of CrebH in DSS-induced colitis were determined. DSS administration to WT and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice led to death and the development of severe pathogenesis in colon tissues, as evidenced by body weight loss, reduced hematocrit percentage and colon length, and severe inflammation with no significant differences between WT and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u0026ndash;F). Consistently, pro-inflammatory cytokine expression did not differ between the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), suggesting that rare expression of \u003cem\u003eCrebH\u003c/em\u003e in the colon is not enough to alter the pathogenesis of UC. Similar to the UC model, in CD animal models, CrebH did not affect IBD progression, as evidenced by the lack of significant differences (Additional file 2: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u0026ndash;C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAblation Of Crebh Deteriorates A Liver Injury Of Mice With Ibd\u003c/h3\u003e\n\u003cp\u003eIBD leads to concomitant disease development outside the gut [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, we estimated the liver pathology to confirm the role of CrebH in IBD-associated liver damage. The liver of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice after administering DSS showed a pale color compared with those of WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Levels of ALT and AST, two liver damage markers, were increased in the plasma of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared with that in the plasma of WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). Excitingly, liver histology of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice displayed enlarged bile ducts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In contrast to the gut data, the expression of pro-inflammatory cytokines, such as \u003cem\u003eIL-1β\u003c/em\u003e, \u003cem\u003eIL-6\u003c/em\u003e, and \u003cem\u003eTNFα\u003c/em\u003e, was significantly increased in the livers of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared with WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Furthermore, MPO activity was more enhanced in the livers of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice than in those of WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These data show that \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice have high inflammatory conditions in their livers. Furthermore, apoptotic cells significantly increased in the livers of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Consistent with DSS-liver, in the CD mouse model, \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice showed severe pathogenesis compared with WT mice (Additional file 3: Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA\u0026ndash;D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLoss of CrebH leads to enhanced CD8\u003c/b\u003e \u003csup\u003e \u003cb\u003e+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eT lymphocyte infiltration via up-regulation of adhesion molecules in the liver\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn IBD-associated liver disease, T lymphocytes infiltrate the liver and play a critical role in hepatic duct inflammation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. FACS analysis showed that CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes were significantly upregulated in the liver of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice after DSS treatment, whereas CD4\u003csup\u003e+\u003c/sup\u003e T lymphocyte regulation was not significantly altered (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). MAdCAM-1 and \u003cem\u003eVAP1\u003c/em\u003e, aberrantly expressed in the liver of PSC patients and animal models with IBD, were significantly increased in the livers of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared with WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026ndash;D). Histological data show that MAdCAM-1 was primarily distributed in the endothelium of vessels or sinusoids after DSS treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). CrebH is mostly expressed in the small intestine, and we investigated the MAdCAM-1 mRNA expression in ileum and colon tissues of mice with CD to determine the CrebH dependency on MAdCAM-1 expression. Consistent with \u003cem\u003eCrebH\u003c/em\u003e expression levels, differential expressions of adhesion molecules containing MAdCAM-1 were more prominent in the small intestine than the large intestine (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), explaining the CrebH contribution to local expression of \u003cem\u003eMAdCAM-1\u003c/em\u003e and the lack of difference in pathogenesis in the large intestines of both groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDifferent gene expressions in the liver of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eCrebH\u003c/span\u003e \u003csup\u003e \u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e \u003c/sup\u003e \u003cb\u003emice are closely related with PSC-liver\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs described, we observed that the livers of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice after DSS have similar histological and pathogenic characteristics as PSC. Interestingly, \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice was more susceptible to the pathogenesis of PSC caused by DDC treatments than were WT mice. Therefore, we performed differential gene expression analysis to estimate the similarity of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice liver with PSC-liver. Gene expression analyzed by RNA-seq in the liver of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-DSS vs. WT-DSS groups was compared with the dataset of DDC-treated vs. control groups. The volcano plot of differentially expressed genes (DEGs) shows significant genes, including 3,906 upregulated and 3,088 downregulated genes in DDC vs. control groups and 246 upregulated and 128 downregulated genes in \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-DSS vs. WT-DSS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The top 20 genes upregulated in \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-DSS vs. WT-DSS are listed in the heatmap, and several genes were validated by qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Excitingly, 41.5% upregulated and 41.4% downregulated genes in \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-DSS vs. WT-DSS overlapped with genes altered in DDC vs. control, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). On analyzing the gene ontology terms, the three most enriched in molecular function (MF) terms of the upregulated genes among overlapped genes were \u0026ldquo;extracellular matrix structural constituents conferring compression resistance,\u0026rdquo; \u0026ldquo;extracellular matrix binding,\u0026rdquo; and \u0026ldquo;collagen-binding,\u0026rdquo; which are closely related to tissue fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In MF terms, the 10 genes associated with extracellular matrix binding, including \u003cem\u003eAdamts15\u003c/em\u003e, \u003cem\u003eAnxa2\u003c/em\u003e, \u003cem\u003eBgn\u003c/em\u003e, \u003cem\u003eCtss\u003c/em\u003e, \u003cem\u003eDcn\u003c/em\u003e, \u003cem\u003eItgb3\u003c/em\u003e, \u003cem\u003eLgals1\u003c/em\u003e, \u003cem\u003eNid1\u003c/em\u003e, \u003cem\u003eSparc\u003c/em\u003e, and \u003cem\u003eTgfbi\u003c/em\u003e, were observed in the heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, \u003cem\u003eleft\u003c/em\u003e), and several genes validated by qRT-PCR showed a similar pattern with RNA-seq analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, \u003cem\u003eright\u003c/em\u003e). These data suggest that liver injury in \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e after administration of DSS is similar to the pathogenic characteristics of PSC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExosomes play an important role in regulating adhesion molecules during liver injury of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eCrebH\u003c/span\u003e \u003csup\u003e \u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e \u003c/sup\u003e \u003cb\u003emice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo identify systemic mediators affected by CrebH ablation, we performed multiplex plasma analysis. No significant differences between \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e/\u0026minus;\u003c/sup\u003e and WT mice were detected in the plasma (Additional file 5: Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Exosomes play an important role in cell-to-cell communication in systemic and local systems [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, we hypothesized that exosomes might be potential mediators contributing to the aberrant pathogenesis in \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-DSS mice. First, we characterized exosomes isolated from the plasma of each mice group to estimate their successful isolation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;D). bEnd.3 cells, an endothelial cell line, were stimulated with exosomes isolated from the plasma of WT-water exosome (WC-exo), WT-DSS exosome (WD-exo), \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-water exosome (KC-exo), and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-DSS exosome (KD-exo). KC-exo treatment led to significantly higher \u003cem\u003eMAdCAM-1\u003c/em\u003e expression than WC-exo treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Furthermore, KD-exo additionally elevated the effects of KC-exo (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). VAP1 expression was upregulated only in KD-exo-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). To further investigate the liver-specific effects of CrebH, HepG2 cell lines constantly overexpressing \u003cem\u003eCrebH\u003c/em\u003e were generated (Additional file 6: Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Isolated exosomes from the medium of the stable cells were characterized (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Consistent with another study [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], TNFα treatment stimulated \u003cem\u003eMAdCAM-1\u003c/em\u003e expression, and co-treatment with pcDNA-exo significantly enhanced its expression in bEnd.3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Interestingly, CrebH-exo eliminated pcDNA-exo-induced \u003cem\u003eMAdCAM-1\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH), suggesting that \u003cem\u003eCrebH\u003c/em\u003e can modulate \u003cem\u003eMAdCAM-1\u003c/em\u003e expression in an exosome dependent manner. Immunoblotting data for MAdCAM-1 further supported this result (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eProfiling Of Exosomal Mirna And Identification Of Mir-29a-3p As An Effector Mirna\u003c/h3\u003e\n\u003cp\u003eTo identify exosomal mediators affecting liver pathogenesis during IBD, we performed miRNA profiling of exosomes using NanoString analysis. The Venn diagram revealed that altered miRNA between WC-exo and WD-exo shared 17 miRNAs compared with WD-exo and KD-exo (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The heatmap shows aberrant expression of various miRNAs from each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and Additional file 7\u0026ndash;9: Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u0026ndash;5). Excitingly, most altered miRNA was increased in the plasma of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared with that of WT after administration of DSS, suggesting that CrebH can regulate exosomal miRNA contents (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). To identify hepatocyte-specific mediators, we performed miRNA profiling in the exosomes derived from HepG2 cells constantly overexpressing pcDNA3 or CrebH and found miR-29a as a potential target miRNA. The sequence of mouse miR-29a was the same as that of humans and rats. miR-29a-3p was found in exosomes from HepG2 cells overexpressing CrebH protein with upregulated expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), and its levels were validated by qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). In contrast, miR-29a-3p was downregulated in the plasma of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared with that in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Previously, Deng et al. reported that miR-29a-3p regulates several TNFα-induced adhesion molecules, including \u003cem\u003eVCAM1\u003c/em\u003e, \u003cem\u003eICAM1\u003c/em\u003e, and \u003cem\u003eE-selectin\u003c/em\u003e, by targeting TNF receptor-1 (TNF-R1) in various endothelial cell lines [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, we generated a miR-29a-3p mimic to investigate its effect on \u003cem\u003eMAdCAM-1\u003c/em\u003e expression. Transfection of the mimic into bEnd.3 cells led to an approximately 700-fold increase in the miR-29a-3p level compared with the negative control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). We found that the mimic inhibited \u003cem\u003eMAdCAM-1\u003c/em\u003e and \u003cem\u003eVAP1\u003c/em\u003e expression in bEnd.3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, H). Recent studies have suggested that \u003cem\u003eMAdCAM-1\u003c/em\u003e expression is regulated by TNF-α and/or AKT signaling pathways [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. We found a target sequence in the AKT2 3\u0026prime;-untranslated region (UTR; DIANA tools; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://diana.imis.athena-innovation.gr\u003c/span\u003e\u003cspan address=\"http://diana.imis.athena-innovation.gr\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Our western blot data showed that AKT2 levels were slightly higher in the liver of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice than in that of WT mice under healthy conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). However, this pattern was not observed after DSS treatment. Interestingly, TNF-R1 levels were dramatically increased in the livers of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, and this induction was more strongly enhanced by DSS administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI), suggesting that aberrant expression of adhesion molecules in the liver of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice might have occurred owing to increased TNF-R1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePlasma exosomes isolated from\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eCrebH\u003c/span\u003e \u003csup\u003e \u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e \u003c/sup\u003e \u003cb\u003emice with IBD aggravate DSS-induced liver injury in WT mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the exosome effects against liver damages, plasma exosomes from WT and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice administered with DSS for 7 days were isolated and then injected into WT mice followed by DSS (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Plasma AST levels were significantly increased in mice injected with \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-exo compared with PBS controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Although ALT levels were not significant, they increased in mice treated with \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-exo compared with both controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). This suggests that the exosomes can contribute to the development of liver injury in WT mice. Adhesion molecules increased in the liver of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were also up-regulated by \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-exo without \u003cem\u003eVAP1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), resulting in enhanced hepatic infiltration of immune cells evidenced by increased F4/80 positive cells and expression of \u003cem\u003eF4/80\u003c/em\u003e and \u003cem\u003eCD3\u003c/em\u003e markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Levels of proinflammatory cytokines were significantly higher in liver treated with \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-exo compared with PBS and WT-exo controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Interestingly, the expressions of \u003cem\u003eIL-1β\u003c/em\u003e and \u003cem\u003eTNFα\u003c/em\u003e showed a downward tendency in liver treated with WT-exo compared with PBS controls, providing a protective role of exosome against hepatic inflammation during IBD (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Finally, we confirmed the DEGs observed in NGS dataset. The Acot3 and Mcam genes increased in \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared with WT mice, and were significantly higher in mice treated with \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-exo than in the only-PBS group; no significantly different expression was observed between mice with \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-exo and those with WT-exo (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). Excitingly, Anxa2, Ctss, Dcn, and Lgals1 among DEGs overlapped with PSC-liver were significantly increased by treatment of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-exo compared with both control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). As these genes are involved in extracellular matrix binding, our results might suggest that exosomes from \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice are strongly associated with IBD induced-fibrosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eLiver homeostasis is closely related to the gut environment because approximately 70% of blood derived from the gut reaches the liver [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This gut\u0026ndash;liver axis has been implicated in various liver diseases such as PSC [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Membrane-bound transcriptional factor CrebH is primarily expressed in the liver and regulates genes related to triglyceride metabolism and fatty acid oxidation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Recently, CrebH expression has also been reported in the small intestine [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, the potential roles of CrebH in the gut\u0026ndash;liver axis remain completely unknown. This study employed a genetic mouse model to determine the effects of \u003cem\u003eCrebH\u003c/em\u003e ablation on IBD in the gut and liver. To the best of our knowledge, this study is the first to show the role of CrebH in the gut\u0026ndash;liver axis.\u003c/p\u003e \u003cp\u003ePSC, a well-known clinically IBD-connected chronic liver disease, is evidence of an interaction between the gut and the liver [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Clinically, most PSC patients have concurrent IBD, while IBD patients have only 1\u0026ndash;5% PSC, suggesting that liver might have protective systems against IBD-induced PSC progression. PSC-liver is characterized by inflammation and onion skin-type fibrotic lesions around bile ducts, resulting in bile duct strictures and accumulation of bile acids into the liver [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Lymphocyte infiltration is a common characterization important for disease progression in IBD and IBD-related liver disorders [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Interestingly, \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice exhibited enlarged bile ducts, enhanced inflammation, and DEGs highly overlapped with PCS-liver. Furthermore, MAdCAM-1, demonstrated as a gut-specific adhesion molecule [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and known as a marker for PSC liver [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], significantly increased in the liver of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. Therefore, our study suggests that CrebH might be involved in the progression of IBD-related liver diseases such as PSC.\u003c/p\u003e \u003cp\u003eReciprocal interactions between the gut and liver during IBD is established through the portal vein, which carries gut-origin products to the liver. The systemic effectors involved in liver injury progression during IBD contain various cytokines [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and chemokines [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] together with bacteria and their products [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, we did not find any differences in the plasma in our experimental conditions, suggesting another potential factors regulating liver injury. Exosomes play a critical role in cell-to-cell communication and protect their contents, including protein, RNA, DNA, and metabolites in the blood system [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, we hypothesized that exosomes might play an important role in the accelerated pathogenesis of the liver in \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. Fortunately, exosomes isolated from the plasma of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice can effectively stimulate \u003cem\u003eMAdCAM-1\u003c/em\u003e and \u003cem\u003eVAP1\u003c/em\u003e expression in bEnd.3 cells. Conversely, exosomes from the culture medium of HepG2 cells expressing exogenous CrebH inhibited TNFα-induced \u003cem\u003eMAdCAM-1\u003c/em\u003e expression. Furthermore, exosomes isolated from \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice stimulated infiltration and activation of immune cells in the liver of WT mice with IBD, while exosomes isolated from WT mice did not. These data suggest that exosomes have a protective role in IBD-induced hepatic inflammation and CrehH can regulate the contents of exosomes.\u003c/p\u003e \u003cp\u003eNotably, many miRNAs of exosomes isolated in plasma from \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were significantly increased compared with those of WT mice in both healthy and diseased conditions, suggesting that CrebH might regulate exosomal miRNA by unknown mechanisms. Mechanistically, exosomal miRNAs mediate post-transcriptional gene silencing by binding to the 3\u0026prime;-UTR or open reading frame region of the target gene [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Recently, Deng et al. demonstrated that miR-29a-3p could inhibit the expression of \u003cem\u003eICAM1\u003c/em\u003e, \u003cem\u003eVCAM1\u003c/em\u003e, and E-selectin induced by TNFα stimulation \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. They also suggested that miR-29a-3p specifically suppresses TNF-R1 expression by targeting the 3\u0026prime;-UTR of \u003cem\u003eTNFRSF1A\u003c/em\u003e [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Furthermore, MAdCAM-1 was induced by the TNFα signaling pathway [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Therefore, miR-29a-3p was selected as a potential miRNA for testing. We found that miR-29a-3p was increased in exosomes from HepG2 cells overexpressing CrebH compared with those from control cells expressing pcDNA3 and was reduced in the exosomes isolated from plasma of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice; however, the difference was not statistically significant. The miR-29a-3p mimic inhibited \u003cem\u003eMAdCAM-1\u003c/em\u003e and \u003cem\u003eVAP1\u003c/em\u003e expression in bEnd.3 cells. Furthermore, we found a dramatic upregulation of TNF-R1 in the livers of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. These data suggest that miR-29a-3p might be a critical factor in regulating MAdCAM-1 expression along with another adhesion molecule such as \u003cem\u003eICAM1\u003c/em\u003e, \u003cem\u003eVCAM1\u003c/em\u003e, or E-selectin. According to previous studies, miRNAs are preferentially sorted into exosomes by four potential modes, including the neural sphingomyelinase 2-associated pathway [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], heterogeneous nuclear ribonucleoprotein-associated pathway [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], 3\u0026prime;-end of the miRNA sequence-associated pathway, and miRNA-induced silencing complex-related pathway [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Thus, CrebH might be involved in these miRNA sorting systems; however, further studies are necessary to confirm this.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that liver of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice after DSS treatment show characteristic phenotypes resembling the PSC-liver, as evidenced by increased biliary inflammation, enlarged bile ducts, upregulation of adhesion molecules such as MAdCAM-1, and high similarity of altered genes compared with PSC-liver, suggesting that \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice might be a potential animal model for investigating the initial pathogenesis of liver during the progression of PSC-IBD. This study also demonstrates that exosomes play a pivotal role in protecting the subsequent pathogenesis of IBD and IBD-related liver inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Exosomal miRNAs are potential effector molecules, and CrebH can affect some exosomal miRNAs. Based on recent clinical studies on monoclonal antibodies against MAdCAM-1 or α4β7 [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], miR-29a-3p could be an effective therapeutic strategy for IBD treatment. Therefore, this study provides new insight for future human studies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eALP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ealkaline phosphatase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eALT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ealanine aminotransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003easpartate aminotransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCrohn\u0026rsquo;s disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ecAMP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecyclic adenosine monophosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ecDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecomplementary DNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCrebH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecyclic adenosine monophosphate-responsive element-binding protein H\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDDC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e3.5-dieythioxycarbonyl-1,4-dihydrocollidine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDEGs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edifferentially expressed genes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDNBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edinitrobenzene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDSS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edextran sulfate sodium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEIMs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eextraintestinal manifestations\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFISH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIBD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einflammatory bowel disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eICAM1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eintercellular adhesion protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMAdCAM-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emucosal vascular addressin cell adhesion molecule 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emolecular function\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMPO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emyeloperoxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eprimary sclerosing cholangitis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNF-R1\u0026rdquo; TNF receptor-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eulcerative colitis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVAP1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evascular adhesion protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVCAM1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evascular cell-adhesion molecule 1.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA-seq raw data has been deposited in the Korean Nucleotide Archive (KoNA, https://kobic.re.kr/kona) under accession numbers PRJKA220165 and PRJKA220166. The datasets used and/or analysed during the current study are available from the corresponding authors on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP; 2019R1A2C1086436 and 2022R1A2C1006815) and a grant from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM5392312 and KGS1042322).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.-H.L. \u0026amp; S.-J.M. performed most animal experiments and analyzed the data. S.H.W. performed in vitro experiments and formal analysis. H.J.H. and C.-H.L. conceived of the study and designed experimental strategies. H.J.H., C.-H.L., and W.K.K. contributed to funding acquisition. H.J.H., S.-H.L., S.-J.M., and C.-H.L. wrote original draft. W.K.K. and G.A. reviewed and edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to thank Dong-Hee Choi, Young-Keun Choi, In-Bok Lee, Jung-Hyun Choi, and Yun-Jeong Seo for animal care and technical support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003ePodolsky DK. Inflammatory bowel disease. N Engl J Med. 2002;347:417\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLoddo I, Romano C. Inflammatory Bowel Disease: Genetics, Epigenetics, and Pathogenesis. 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Lancet. 2017;390:135\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-and-bioscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbio","sideBox":"Learn more about [Cell \u0026 Bioscience](http://cellandbioscience.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cbio/default.aspx","title":"Cell \u0026 Bioscience","twitterHandle":"@OACellBiology","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"CrebH, exosomes, inflammatory bowel disease, liver damage, primary sclerosing cholangitis","lastPublishedDoi":"10.21203/rs.3.rs-2636684/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2636684/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHepatic liver disease, including primary sclerosing cholangitis (PSC), is a serious extraintestinal manifestations of colonic inflammation. Cyclic adenosine monophosphate (cAMP)-responsive element-binding protein H (CrebH) is a transcription factor expressed mostly in the liver and small intestine. However, CrebH’s roles in the gut–liver axis remain unknown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInflammatory bowel disease (IBD) and PSC disease models were established in wild-type and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice treated with dextran sulfate sodium, dinitrobenzene sulfonic acid, and diethoxycarbonyl dihydrocollidine diet, respectively. RNA sequencing were conducted to investigate differential gene expression. Exosomes were isolated from plasma and culture media. miRNA expression profiling was performed using the NanoString nCounter Mouse miRNA Panel. Effects of miR-29a-3p on adhesion molecule expression were investigated in bEnd.3 brain endothelial cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice exhibited accelerated liver injury without substantial differences in the gut after administration of dextran sulfate sodium (DSS), and had similar features to PSC, including enlarged bile ducts, enhanced inflammation, and aberrant MAdCAM-1 expression. Furthermore, RNA-sequencing analysis showed that differentially expressed genes in the liver of \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice after DSS overlapped significantly with genes changed in PSC-liver. Analysis of plasma exosome miRNA isolated from WT and \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice indicates that CrebH can contribute to the exosomal miRNA profile. We also identified miR-29a-3p as an effective mediator for MAdCAM-1 expression. Administration of plasma exosome from \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice led to prominent inflammatory signals in the liver of WT mice with inflammatory bowel disease (IBD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCrebH\u003c/em\u003e deficiency led to increased susceptibility to IBD-induced liver diseases via enhanced expression of adhesion molecules and concomitant infiltration of T lymphocytes. Exosomes can contribute to the progression of IBD-induced liver injury in \u003cem\u003eCrebH\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice. These study provide novel insights into the role of CrebH in IBD-induced liver injury.\u003c/p\u003e","manuscriptTitle":"CrebH protects against liver injury associated with colonic inflammation via modulation of exosomal miRNA","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-21 14:28:27","doi":"10.21203/rs.3.rs-2636684/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-04-14T00:49:35+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2023-03-18T00:00:00+00:00","index":2,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2023-03-17T09:12:39+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-03-17T00:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2023-03-17T00:00:00+00:00","index":1,"fulltext":""},{"type":"editorAssigned","content":"","date":"2023-03-03T13:05:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-03-02T23:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2023-03-02T23:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell \u0026 Bioscience","date":"2023-03-01T19:29:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-and-bioscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbio","sideBox":"Learn more about [Cell \u0026 Bioscience](http://cellandbioscience.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cbio/default.aspx","title":"Cell \u0026 Bioscience","twitterHandle":"@OACellBiology","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"42bef90d-60f0-4dc3-ae37-8e6e406a2669","owner":[],"postedDate":"March 21st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T21:36:22+00:00","versionOfRecord":{"articleIdentity":"rs-2636684","link":"https://doi.org/10.1186/s13578-023-01065-9","journal":{"identity":"cell-and-bioscience","isVorOnly":false,"title":"Cell \u0026 Bioscience"},"publishedOn":"2023-06-27 21:26:38","publishedOnDateReadable":"June 27th, 2023"},"versionCreatedAt":"2023-03-21 14:28:27","video":"","vorDoi":"10.1186/s13578-023-01065-9","vorDoiUrl":"https://doi.org/10.1186/s13578-023-01065-9","workflowStages":[]},"version":"v1","identity":"rs-2636684","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2636684","identity":"rs-2636684","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}