Yingwei Chen

The dynamic assembly and regulation of the IκB kinase (IKK) complex in the NF-κB pathway are central to the pathogenesis and progression of inflammatory bowel disease (IBD). We recently reported that the transcription factor hematopoietically expressed homeobox (HHEX) promotes colitis-associated colorectal cancer, but the potential role of HHEX in intestinal inflammation remains uncharacterized. Here, we found that HHEX is upregulated in inflamed colons in a colitis mouse model and in clinical IBD samples. HHEX overexpression increased inflammatory cytokine expression, and HHEX loss largely abrogated the inflammatory response in vitro and intestinal inflammation in vivo. Mechanistically, IKKα phosphorylates HHEX at S213 to stabilize HHEX in response to TNF-α by inhibiting the interaction of HHEX with the E3 ubiquitin ligase MID2 and subsequent K48-linked ubiquitination and protein degradation. Importantly, HHEX interacted with and stabilized the IKKα/IKKβ complex via its N-terminal domain, thereby activating the NF-κB pathway and establishing a positive feedback loop that exacerbates intestinal inflammation. Our study reveals a transcription-independent function of HHEX in promoting IKK complex assembly and colitis, identifying HHEX as an IBD susceptibility gene and a potential target for IBD treatment. HHEX as a positive regulator of the IKK complex that activates the NF-κB pathway. Inflammatory stimuli can stabilize HHEX by IKKα-dependent phosphorylation.

Background The lack of response to TNF-α (tumor necrosis factor-alpha) monoclonal antibodies (mAbs) in autoimmune disease poses a considerable clinical challenge. Trigger receptor expressed on myeloid cells 1 (TREM1) has been proposed as a predictor of anti-TNF-α mAb responsiveness in inflammatory bowel disease (IBD), although this remains controversial. We investigated the role of TREM1 in nonresponders to infliximab (IFX) and elucidated the underlying mechanisms using experimental colitis models and macrophages. Conclusions TREM1 diminishes the efficacy of IFX in IBD by promoting caspase-8-dependent macrophage pyroptosis through the p-SYK/ITCH/TAK1 axis. Therefore, targeting TREM1 and macrophage pyroptosis may provide new therapeutic strategies for IBD nonresponders to anti-TNF-α mAbs.

Purpose: Ulcerative colitis (UC) is a chronic inflammatory bowel disease with a relapsing-and-remitting cycle. Mucosal healing is a critical predictor of long-term outcomes; however, the discordance between mucosal healing at different depths and clinical activity complicates UC management. Patients and Methods: Cell death-related genesets (CDRGs) were analyzed in UC patients, with grouping using unsupervised hierarchical clustering. Concordance between grouping methods was assessed using Cohen’s Kappa and Sankey plots. The predictive value was quantified using the area under the curve. Subgroup analysis explored the additive effects and intrinsic associations between the CDRG-based grouping and other grouping methods. Differentially expressed genes (DEGs) were identified using Wilcoxon test. Robustness was validated in 3 independent cohorts. Single-cell analysis was used to assess cell type-specific impacts. Cytokine expression in colon organoids was measured using RT-qPCR. Results: CDRGs’ activities effectively predicted the clinical, histological, and endoscopic grouping. CDRG-based grouping emerged as an independent predictor, resolving inconsistence between histological and clinical grouping and improving concordance across clinical, histological, and endoscopic grouping. Integrating endoscopic or histological grouping with CDRG-based grouping improved the predictive value for clinical activity. CDRGs were intrinsically associated with DEGs, impacted non-immune cells, and reshaped immune cell infiltration through intercellular interactions. Epithelial cells were the main target of active CDRGs, exhibiting upregulated MIF, MDK, and LGALS9 in UC. In necroptotic colon organoids, MIF and MDK were significantly upregulated, whereas LGALS9 remained unchanged. Conclusion: CDRG-based grouping served as an independent predictor to improve the concordance between clinical, histological, and endoscopic grouping in UC, and was of potency to precisely identify UC patients achieving long-term treatment target, namely mucosal healing. Active CDRGs intrinsically associated with genetic and immunological alterations in UC, highlighting their potential as novel biomarkers and therapeutic targets.

IκB kinase (IKK) complex is central regulators of the NF-κB pathway, and dysregulation of IKK phosphorylation leads to hyperactivation of proinflammatory response in various chronic inflammatory diseases, including inflammatory bowel disease (IBD). However, the dynamic modulation of IKK phosphorylation and dephosphorylation in intestinal inflammation remains uncharacterized. Here, we found that autophagy/beclin-1 regulator 1 (AMBRA1) was highly expressed in inflamed colons in a colitis mouse model and in clinical IBD samples. Importantly, AMBRA1 deletion significantly decreased proinflammatory cytokine expression and enhanced the therapeutic effect of infliximab on intestinal inflammation. Mechanistically, the N-term F1 domain of AMBRA1 was required for AMBRA1 to competitively interact with protein phosphatase 4 regulatory subunit 1 (PP4R1) and catalytic protein phosphatase 4 (PP4c) to suppress their interactions with IKK, promote the dissociation of the PP4R1/PP4c complex, and antagonize the dephosphorylation activity of this complex towards the IKK complex. In response to TNF-α stimulation, IKKα phosphorylates AMBRA1 at S1043 to stabilize AMBRA1 expression by impairing its binding to Cullin4A (CUL4A) to decrease its CUL4A-mediated K48-linked ubiquitination. Overall, our study identifies an autophagy-independent function of AMBRA1 as a positive modulator of IKK phosphorylation to promote intestinal inflammation, thus providing a new targeted therapeutic strategy for patients with refractory IBD.

Dietary fructose is widely used in beverages, processed foods, and Western diets as food additives, and is closely related to the increased prevalence of multiple diseases, including inflammatory bowel disease (IBD). However, the detailed mechanism by which high fructose disrupts intestinal homeostasis remains elusive. This study showed that high fructose corn syrup (HFCS) administration exacerbated intestinal inflammation and deteriorated barrier integrity. Several in vivo experimental models were utilized to verify the importance of gut microbiota and immune cells in HFCS-mediated dextran sulfate sodium (DSS)-induced colitis. In addition, untargeted metabolomics analysis revealed the imbalance between primary bile acids (PBAs) and secondary bile acids (SBAs) in feces. Hence, high fructose was speculated to modulate gut microbiota community and reduced the relative abundance of Clostridium and Clostridium scindens at genus and species level respectively, followed by a decrease in SBAs, especially isoalloLCA, thereby affecting Th17/Treg cells equilibrium and promoting intestinal inflammation. These findings provide novel insights into the crosstalk between gut flora, bile acids, and mucosal immunity, and highlight potential strategies for precise treatment of IBD.

Metabolic-associated fatty liver disease (MAFLD) and alcoholic hepatitis (AH) are among the most prevalent liver diseases worldwide, and their coexistence is common in clinical practice. However, currently established models of MAFLD-AH coexistence do not fully replicate their pathological characteristics and require sophisticated experimental techniques. Therefore, we aimed to develop an easily replicable model that mimics obesity-induced MAFLD-AH in patients. Our goal was to establish a murine model that replicates MAFLD and AH coexistence, resulting in significant liver injury and inflammation. To this end, we administered a single ethanol gavage dose to ob/ob mice on a chow diet. The administration of a single dose of ethanol led to elevated serum transaminase levels, increased liver steatosis, and apoptosis in ob/ob mice. Furthermore, ethanol binge caused a significant increase in oxidative stress in ob/ob mice, as measured via 4-hydroxynonenal. Importantly, the single dose of ethanol also markedly exacerbated liver neutrophil infiltration and upregulated the hepatic mRNA expression of several chemokines and neutrophil-related proteins, including CXCL1, CXCL2, and LCN2. Whole-liver transcriptomic analysis revealed that ethanol-induced changes in gene expression profile shared similar features with AH and MAFLD. In ob/ob mice, a single dose of ethanol binge caused significant liver injury and neutrophil infiltration. This easy-to-replicate murine model successfully mimics the pathological and clinical features of patients with coexisting MAFLD and AH and closely resembles the transcriptional regulation seen in human disease.

Purpose Inflammatory bowel disease (IBD) is a multifactorial chronic disease of the gastrointestinal tract. Dietary intervention in the treatment of IBD has gradually attracted more attention. In this study, amino acid-balanced diets (AABD) based on grains were developed and their influences on the regulation of IBD were investigated. Methods Dextran sodium sulfate (DSS)-induced acute colitis mice model was employed to evaluate the effects of AABD. Pathological symptoms, intestinal inflammation, gut barrier proteins and gut microbiota were determined after AABD intake. Results It was shown that AABD alleviated the symptoms of colitis by reducing the histological scores of mice colon, suppressing the expression of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and upregulating the expression of tight junction proteins. Analysis of gut microbiota revealed that AABD altered the structure of gut microbiota by decreasing the abundance and richness of harmful bacteria induced by DSS (Escherichia-Shigella, Parasutterella, etc.) and increasing that of beneficial bacteria (Akkermansia, etc.). Correlation analysis indicated that the alterations of pro-inflammatory factors were related with the change of microbiota, suggesting that the inhibitory effects of AABD on inflammation might be due to its regulation gut microbiota. Conclusion The AABD could efficiently mitigate colitis, and this study indicated that AABD could be applied as a promising dietary regulation strategy of IBD.

Background and Aims: There is evidence for a disturbed necroptosis function in many inflammatory diseases, but its role in inflammatory bowel diseases [IBD] and the underlying mechanisms are unclear. Here, we studied the functional significance and molecular mechanisms of ABIN3, a ubiquitin-binding protein, in regulating the ubiquitination and activation of necroptosis in IBD. Methods: The expression of necroptosis hallmarks and ABIN3 were assessed in inflamed samples of IBD patients, dextran sodium sulphate [DSS]-induced colitis models, and azoxymethane [AOM]/DSS models in mice. ABIN3 was overexpressed and silenced to explore its function in regulating necroptosis, inflammation, and intestinal barrier function. Immuoprecipitiation [IP] and co-IP assays were performed to investigate the cross-talk between ABIN3 and deubiquitinating enzyme A20, and the mechanisms of coordinating ubiquitination modification to regulate necroptosis. Results: Excessive necroptosis is an important contributory factor towards the uncontrolled inflammation and intestinal barrier defects in IBD and experimental colitis. Blocking necroptosis by Nec-1s or GSK’872 significantly prevented cell death and alleviated DSS-induced colitis in vivo, whereas in the AOM/DSS model, necroptosis inhibitors aggravated the severity of colitis-associated colon carcinogenesis [CAC]. Mechanistically, ABIN3 is rapidly recruited to the TNF-RSC complex, which interacts and coordinates with deubiquitinating enzyme A20 to control the K63 deubiquitination modification and subsequent activation of the critical necroptosis kinase, RIPK3, to suppress necroptosis. Conclusions: ABIN3 regulates inflammatory response and intestinal barrier function by interacting with A20 and coordinating the K63 deubiquitination modification of necroptosis in IBD.