Infliximab modifies CD74-mediated lymphatic abnormalities and adipose tissue alterations in creeping fat of Crohn's disease.

BACKGROUND: Lymphatic abnormalities are essential for pathophysiologic changes of creeping fat (CrF) in Crohn's disease (CD). Anti-tumor necrosis factor (TNF) therapy has been proved to alleviate CrF lesions, however, whether it achieves these by remodeling lymphatics is unknown. METHODS: CD74 expression was detected in CrF and uninvolved mesentery of CD patients. Lymphatic functions in vitro were evaluated and lymphatic endothelium barrier were checked by transendothelial electrical resistance (TEER) and FITC-Dextran permeability. Protein level of tight junction and signaling pathways were detected by western blotting. RESULTS: CD74 was upregulated in LECs of CrF and positively correlated with TNF-α synthesis. This was suppressed by IFX administration. In vitro, TNF-α stimulated LECs to express CD74 through NF-κB signaling pathway, and this was rescued by IFX. CD74 downregulation suppressed the abilities of LECs in proliferation, migration and tube formation. Interaction of CD74-MIF impaired LECs' barrier via reducing tight junction proteins in an ERK1/2-dependent manner, which was reversed by CD74 downregulation. Consistently, the CD patients receiving IFX therapy displayed decreased lymphangiogenesis and improved mesenteric lymphatic endothelium barrier, companied with reduced adipocyte size and adipokine levels in CrF. CONCLUSIONS: Anti-TNF therapy could modify pathological changes in CrF by alleviating CD74-mediated lymphatic abnormalities.

ATF4 Deficiency Promotes Intestinal Inflammation in Mice by Reducing Uptake of Glutamine and Expression of Antimicrobial Peptides.

BACKGROUND & AIMS: Activating transcription factor 4 (ATF4) regulates genes involved in the inflammatory response, amino acid metabolism, autophagy, and endoplasmic reticulum stress. We investigated whether its activity is altered in patients with inflammatory bowel diseases (IBDs) and mice with enterocolitis. METHODS: We obtained biopsy samples during endoscopy from inflamed and/or uninflamed regions of the colon from 21 patients with active Crohn's disease (CD), 22 patients with active ulcerative colitis (UC), and 38 control individuals without IBD and of the ileum from 19 patients with active CD and 8 individuals without IBD in China. Mice with disruption of Atf4 specifically in intestinal epithelial cells (Atf4ΔIEC mice) and Atf4-floxed mice (controls) were given dextran sodium sulfate (DSS) to induce colitis. Some mice were given injections of recombinant defensin α1 (DEFA1) and supplementation of l-alanyl-glutamine or glutamine in drinking water. Human and mouse ileal and colon tissues were analyzed by quantitative real-time polymerase chain reaction, immunoblots, and immunohistochemistry. Serum and intestinal epithelial cell (IEC) amino acids were measured by high-performance liquid chromatography-tandem mass spectrometry. Levels of ATF4 were knocked down in IEC-18 cells with small interfering RNAs. Microbiomes were analyzed in ileal feces from mice by using 16S ribosomal DNA sequencing. RESULTS: Levels of ATF4 were significantly decreased in inflamed intestinal mucosa from patients with active CD or active UC compared with those from uninflamed regions or intestinal mucosa from control individuals. ATF4 was also decreased in colonic epithelia from mice with colitis vs mice without colitis. Atf4ΔIEC mice developed spontaneous enterocolitis and colitis of greater severity than control mice after administration of DSS. Atf4ΔIEC mice had decreased serum levels of glutamine and reduced levels of antimicrobial peptides, such as Defa1, Defa4, Defa5, Camp, and Lyz1, in ileal Paneth cells. Atf4ΔIEC mice had alterations in ileal microbiomes compared with control mice; these changes were reversed by administration of glutamine. Injections of DEFA1 reduced the severity of spontaneous enteritis and DSS-induced colitis in Atf4ΔIEC mice. We found that expression of solute carrier family 1 member 5 (SLC1A5), a glutamine transporter, was directly regulated by ATF4 in cell lines. Overexpression of SLC1A5 in IEC-18 or primary IEC cells increased glutamine uptake and expression of antimicrobial peptides. Knockdown of ATF4 in IEC-18 cells increased expression of inflammatory cytokines, whereas overexpression of SLC1A5 in the knockdown cells reduced cytokine expression. Levels of SLC1A5 were decreased in inflamed intestinal mucosa of patients with CD and UC and correlated with levels of ATF4. CONCLUSIONS: Levels of ATF4 are decreased in inflamed intestinal mucosa from patients with active CD or UC. In mice, ATF4 deficiency reduces glutamine uptake by intestinal epithelial cells and expression of antimicrobial peptides by decreasing transcription of Slc1a5. ATF4 might therefore be a target for the treatment of IBD.

Anti-TNF-α Monoclonal Antibody Therapy Improves Anemia through Downregulating Hepatocyte Hepcidin Expression in Inflammatory Bowel Disease.

Anemia is one of the most common complications in patients with inflammatory bowel disease (IBD). Hepcidin as a key regulator of iron metabolism is pivotal in mediating the occurrence of anemia of chronic disease. Herein, we analyzed the levels of hepcidin in sera from IBD patients by enzyme-linked immunosorbent assay and investigated its potential role in regulating the anemia in IBD. We observed that the levels of serum hepcidin were increased in active IBD patients compared with those in remitted IBD patients and healthy controls and that serum hepcidin was associated with disease activity, CRP, and ESR, respectively. Importantly, we found that the increased levels of serum hepcidin were positively correlated with the severity of anemia and the imbalance of iron metabolism in anemic UC and CD patients. Proinflammatory factors (e.g., IL-6, IL-17, and TNF-α) were positively correlated with the concentrations of serum hepcidin in IBD patients. Interestingly, hepcidin was found to be decreased in patients with Crohn's disease after successful therapy with anti-TNF-α mAb (i.e., infliximab), indicating the underlying association between TNF-α and hepcidin expression. To investigate the specific mechanisms involved, we cultured LO2 and HepG2 cell lines in vitro under stimulation with TNF-α and observed that the levels of hepcidin mRNA were markedly upregulated in caspase-3/8- and NF-κB-dependent manners. Therefore, our data suggest that TNF-α stimulates the expression of hepcidin in IBD patients, resulting in aggravated anemia and that blockage of TNF-α or the caspase-3/8 and NF-κB pathways could downregulate hepcidin expression. This study provides inspiration for the therapy and management of anemia in IBD.

CD177+ neutrophils suppress epithelial cell tumourigenesis in colitis-associated cancer and predict good prognosis in colorectal cancer.

Neutrophils are found to be infiltrated in tumour tissues of patients with colitis-associated cancer (CAC) and colorectal cancer (CRC), and CD177 is mainly expressed in neutrophils. In our study, expression of CD177 in tumour tissues from patients with CAC or CRC was analysed byquantitative real-time polymerase chain reaction, flow cytometry and immunohistochemistry. We recruited 378 patients with CRC, determined CD177 expression in tumours and examined its correlation with clinicopathological features. Moreover, CAC model was induced in wild-type and CD177-/- mice by azoxymethane/dextran sodium sulphate. CD177+ neutrophils were significantly increased in colon tumour tissues from patients with CRC or CAC compared with controls. Expression of CD177 mRNA and percentages of CD177+ neutrophils were also markedly increased in tumour tissues from CRC patients compared with controls. Patients with high density of CD177+ neutrophils had better overall survival and disease-free survival compared with controls. Multivariate analyses revealed that the density of CD177+ neutrophils was an independent factor in predicting overall survival and disease-free survival. Consistently, CD177 depletion aggravated azoxymethane/dextran sodium sulphate-induced CAC in mice. Expression of Ki67 and proliferating cell nuclear antigen was increased in tumour tissues from CD177-/- mice compared with wild-type counterparts. Moreover, CD177-/- neutrophils failed to migrate in response to fMLP[AU: Please expand fMLP, DN, TNM and HIF-1α.] stimulation compared with wild-type controls. Our data indicate that CD177+ neutrophils suppress epithelial cell tumourigenesis and act as an independent factor in predicting the prognosis in patients with CRC. CD177+ neutrophils may serve as a novel therapeutic target in the treatment and predict the prognosis of CAC and CRC.

Clinical significance of soluble immunoglobulins A and G and their coated bacteria in feces of patients with inflammatory bowel disease.

BACKGROUND: Immunoglobulin A (IgA) and IgG are major components in human intestinal mucosal surface and sera, and IgA- or IgG-coated bacteria play a vital role in the intestinal homeostasis. However, the correlation of IgA, IgG and their coated bacteria with the clinical characteristics of inflammatory bowel disease (IBD) has not been fully clarified. METHODS: The levels of soluble IgA and IgG in sera and feces were detected by ELISA, and the percentage of IgA- and IgG-coated bacteria in feces was analyzed by flow cytometry. Crohn's disease activity index (CDAI) and Simple Endoscopic Score for Crohn's disease (SES-CD) for Crohn's disease (CD) or Mayo score and ulcerative colitis endoscopic index of severity (UCEIS) for ulcerative colitis (UC), erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were used to evaluate the disease activity. RESULTS: 178 patients with CD, 75 patients with UC and 41 healthy donors were recruited in this study. We found that the concentrations of soluble IgA and IgG in feces of active IBD patients were significantly higher than those in healthy controls and that the levels of soluble IgA and IgG in feces from IBD patients were positively correlated with CRP, ESR, Mayo score, UCEIS, SES-CD, and CDAI, respectively. Moreover, we also observed that the percentage of IgA- and IgG-coated bacteria markedly increased in feces of IBD patients, especially in CD patients at the age of 17 to 40 years old, with terminal ileal lesions and perianal lesions, as well as from E2 UC patients, and was closely associated with disease activities. CONCLUSIONS: The levels of soluble IgA and IgG and the percentage of IgA- and IgG-coated bacteria strikingly increase in feces of IBD patients and correlate with disease activity.

Anti-TNF-α Therapy Suppresses Proinflammatory Activities of Mucosal Neutrophils in Inflammatory Bowel Disease.

Neutrophils have been found to play an important role in the pathogenesis of inflammatory bowel disease (IBD), and anti-TNF-α mAb (i.e., infliximab) therapy is demonstrated to be effective in the induction of clinical remission and mucosal healing in these patients. However, how anti-TNF-α mAb regulates the functions of neutrophils is still unknown. Herein, we found that anti-TNF-α therapy significantly downregulated infiltration of neutrophils in inflamed mucosa of IBD patients. Importantly, anti-TNF-α mAb could inhibit neutrophils to produce proinflammatory mediators, such as ROS, calprotectin, IL-8, IL-6, and TNF-α. These data indicate that TNF-α plays a critical role in the induction of mucosal inflammatory response, and that blockade of TNF-α modulates intestinal homeostasis through balancing immune responses of neutrophils.

Single-cell Expression Atlas Reveals Cell Heterogeneity in the Creeping Fat of Crohn's Disease.

BACKGROUND: Creeping fat (CrF) has been recognized to play a positive role in Crohn's disease (CD) progression, yet the cellular compositions within mesenteric adipose tissue (MAT) and their potential mechanism in CrF formation are poorly understood. METHODS: Analysis of 10X single-cell RNA sequencing was performed on 67 064 cells from 3 pairs of surgically resected samples of CrF and their uninvolved MAT. The results were validated in another cohort with 6 paired MAT samples by immunofluorescence. RESULTS: All samples manifested excellent consistency and repeatability in our study, and 10 cell types from the transcriptome atlas, including 20 clusters, were identified. In CrF, a specific vascular endothelial cell subpopulation highly expressing lipoprotein lipase was first identified, with a significantly increased proportion. This vascular endothelial cell subpopulation manifested robust peroxisome proliferator-activated receptor γ (PPARγ) transcription activity and an upregulated PPAR signaling pathway and was involved in lipid metabolism and the antibacterial response. A novel fibroblast subpopulation (FC3) with remarkable GREM1 and RFLNB expression was identified and validated to predominantly accumulate in the CrF. The FC3 was annotated as inflammation-associated fibroblasts, which are characterized by inflammatory responses and the regulation of Smad phosphorylation related to intestinal fibrosis. The trajectory of fibroblasts revealed their pro-inflammatory and profibrotic conversion tendency during CrF formation with corresponding gene dynamics. Additionally, we unprecedently dissected the different origins and functions of 6 macrophage subclusters within the myeloid compartment. CONCLUSIONS: Our results uncover the cellular heterogeneity in the MAT of CD and the role of these various cellular compositions in CrF development. This comprehensive understanding of CrF provides future directions for in-depth research on and potential targets for MAT-based treatment.

This is the first study that provides a comprehensive single-cell transcriptomic atlas in mesenteric adipose tissue (MAT) of Crohn's disease and elaborates the functional diversity and dynamic changes of the cellular components during creeping fat (CrF) formation.

ADSCs stimulated by VEGF-C alleviate intestinal inflammation via dual mechanisms of enhancing lymphatic drainage by a VEGF-C/VEGFR-3-dependent mechanism and inhibiting the NF-κB pathway by the secretome.

BACKGROUND: Adipose-derived stem cells (ADSCs) have provided promising applications for Crohn's disease (CD). However, the practical efficacy of ADSCs remains controversial, and their mechanism is still unclear. Based on the pathogenesis of dysregulated immune responses and abnormal lymphatic alterations in CD, vascular endothelial growth factor-C (VEGF-C) is thought to be a favourable growth factor to optimize ADSCs. We aimed to investigate the efficacy of VEGF-C-stimulated ADSCs and their dual mechanisms in both inhibiting inflammation "IN" and promoting inflammation "OUT" in the intestine. METHODS: Human stem cells isolated from adipose tissues were identified, pretreated with or without 100 ng/ml VEGF-C and analysed for the secretion of cell culture supernatants in vitro. Lymphatic endothelial cells (LECs) were treated with ADSCs-conditioned medium or co-cultured with ADSCs and VEGF-C stimulated ADSCs. Changes in LECs transmigration, and VEGF-C/VEGFR-3 mRNA levels were assessed by transwell chamber assay and qRT-PCR. ADSCs and VEGF-C-stimulated ADSCs were intraperitoneally injected into mice with TNBS-induced chronic colitis. ADSCs homing and lymphatic vessel density (LVD) were evaluated by immunofluorescence staining. Lymphatic drainage was assessed using Evans blue. Cytokines and growth factors expression was detected respectively by ELISA and qRT-PCR. The protein levels of VEGF-C/VEGFR-3-mediated downstream signals and the NF-κB pathway were assayed by western blot. Faecal microbiota was measured by 16S rRNA sequencing. RESULTS: ADSCs stimulated with VEGF-C released higher levels of growth factors (VEGF-C, TGF-ß1, and FGF-2) and lower expression of cytokines (IFN-γ and IL-6) in cell supernatants than ADSCs in vitro (all P < 0.05). Secretome released by VEGF-C stimulated ADSCs exhibited a stronger LEC migratory capability and led to elevated VEGF-C/VEGFR-3 expression, but these effects were markedly attenuated by VEGFR-3 inhibitor. VEGF-C-stimulated ADSCs homing to the inflamed colon and mesenteric lymph nodes (MLNs) can exert stronger efficacy in improving colitis symptoms, reducing inflammatory cell infiltration, and significantly enhancing lymphatic drainage. The mRNA levels and protein concentrations of anti-inflammatory cytokines and growth factors were markedly increased with decreased proinflammatory cytokines in the mice treated with VEGF-C-stimulated ADSCs. Systemic administration of VEGF-C-stimulated ADSCs upregulated the colonic VEGF-C/VEGFR-3 pathway and activated downstream AKT and ERK phosphorylation signalling, accompanied by decreased NF-κB p65 expression. A higher abundance of faecal p-Bacteroidetes and lower p-Firmicutes were detected in mice treated with VEGF-C-stimulated ADSCs (all P < 0.05). CONCLUSION: VEGF-C-stimulated ADSCs improve chronic intestinal inflammation by promoting lymphatic drainage and enhancing paracrine signalling via activation of VEGF-C/VEGFR-3-mediated signalling and inhibition of the NF-κB pathway. Our study may provide a new insight into optimizing ADSCs treatment and investigating potential mechanisms in CD.

Anti-TNF Therapy Induces CD4+ T-Cell Production of IL-22 and Promotes Epithelial Repairs in Patients With Crohn's Disease.

Background: Anti-tumor necrosis factor (TNF) therapy appears to be effective in the treatment of Crohn's disease (CD), a chronic inflammatory disease of the gastrointestinal tract. However, the mechanisms involved are not completely understood. Methods: Fifty-seven active CD patients were enrolled, and cytokine profiles in colonic biopsies of patients with active CD receiving anti-TNF monoclonal antibody (mAb) (infliximab [IFX]) treatment were determined using quantitative real-time polymerase chain reaction (qRT-PCR). Colonic biopsies of active CD patients and healthy donors were cultured with IFX in vitro, and cytokine profiles were measured by qRT-PCR. Peripheral blood (PB)-CD4+ T cells were stimulated with anti-CD3 and anti-CD28 mAbs in the presence of human immunoglobin (HIg), IFX, recombinant human TNF-α converting enzyme (rhTACE), and aryl hydrocarbon receptor (AhR) inhibitor (CH-223191), respectively, to determine interleukin (IL)-22 expression by CD4+ T cells. Caco2 cells were also utilized to study their potential role in modulating epithelial cell barrier repairs in vitro. Results: IFX therapy markedly upregulated IL-22 mRNA expression in the gut mucosa of CD patients. In vitro treatment with IFX greatly promoted CD CD4+ T cells to express IL-22, which was inhibited by rhTACE, indicating that reverse signaling through binding to membrane-bound TNF mediates anti-TNF-induced IL-22 expression of CD CD4+ T cells. However, blockade of AhR markedly inhibited anti-TNF-induced IL-22+CD4+ T (Th22) cell differentiation in CD patients. Moreover, treatment with IL-22 induced intestinal epithelial cell expression of tight junction proteins (eg, claudin1 and ZO-1) and facilitated transepithelial resistance, indicating that IL-22 protects intestinal mucosa from inflammation via maintenance of epithelial barrier integrity. Conclusions: Our results uncover a novel mechanism whereby anti-TNF therapy upregulates IL-22 production in CD patients through promoting Th22 cell differentiation and contributes to intestinal epithelial barrier repairs.