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Haem oxygenase 1 (HO-1), an inducible enzyme responsible for the breakdown of haem, is primarily considered an antioxidant, and has long been overlooked by immunologists. However, research over the past two decades in particular has demonstrated that HO-1 also exhibits numerous anti-inflammatory properties. These emerging immunomodulatory functions have made HO-1 an appealing target for treatment of diseases characterized by high levels of chronic inflammation. In this Review, we present an introduction to HO-1 for immunologists, including an overview of its roles in iron metabolism and antioxidant defence, and the factors which regulate its expression. We discuss the impact of HO-1 induction in specific immune cell populations and provide new insights into the immunomodulation that accompanies haem catabolism, including its relationship to immunometabolism. Furthermore, we highlight the therapeutic potential of HO-1 induction to treat chronic inflammatory and autoimmune diseases, and the issues faced when trying to translate such therapies to the clinic. Finally, we examine a number of alternative, safer strategies that are under investigation to harness the therapeutic potential of HO-1, including the use of phytochemicals, novel HO-1 inducers and carbon monoxide-based therapies.This Review provides an overview of haem oxygenase 1 (HO-1) for immunologists, including its roles in iron metabolism and antioxidant defence, and the impact of HO-1 induction in specific immune cell populations. The authors highlight the therapeutic potential of HO-1 induction for treatment of chronic inflammatory and autoimmune diseases.

Fecal protease profiling represents a promising frontier in the non-invasive diagnosis of gastrointestinal disorders, particularly inflammatory bowel diseases (IBD), such as Crohn’s disease and ulcerative colitis, and irritable bowel syndrome (IBS). These conditions share overlapping symptoms but differ significantly in etiology and pathology, making accurate differentiation essential for appropriate management. This pilot study investigated protease activity in stool samples using a custom panel of fluorogenic peptide substrates across varying pH conditions to uncover disease-specific enzymatic signatures. Samples from IBD patients revealed broad protease activation involving both serine and cysteine classes, while the small IBS cohort showed a tendency toward a pattern enriched in furin-like serine proteases pattern dominated by furin-like serine proteases, especially at alkaline pH. Notably, one substrate, Ac-RSVL-AMC, showed higher activity in UC than in CD at acidic pH and moderate discriminatory ability in this pilot cohort. Inhibition assays confirmed the enzymatic contribution of furin-like proteases, and follow-up analysis in remission-phase IBD patients indicated persistent dysregulation, suggesting potential biomarker utility beyond active inflammation. The observed substrate-specific activity profiles highlight the importance of sequence context in proteolytic cleavage and underscore the complexity of protease involvement in gastrointestinal pathology. These findings support fecal protease profiling as a promising, rapid and low-cost approach that could contribute to distinguishing IBD from IBS and differentiating IBD subtypes, providing a foundation for future minimally invasive diagnostic strategies that require validation in larger cohorts.

Epithelial cell death in intestinal inflammatory disease Two groups identify the regulation of death-receptor-induced necroptosis as an epithelial intrinsic mechanism that is important for the maintenance of immune homeostasis and the prevention of intestinal inflammation in mice. Welz et al . describe an unexpected physiological function for FADD (Fas-associated protein with death domain), an adaptor protein required for death-receptor-induced apoptosis. Mice with intestinal epithelial specific knockout of FADD develop severe colon inflammation due to increased death of FADD-deficient colonic epithelial cells. Günther et al . report a novel and unexpected function of caspase-8 in maintaining immune homeostasis in the gut. Caspase-8 expression by gut epithelial cells is shown to protect mice from TNF-mediated Paneth cell death and intestinal inflammation. Increased expression of the protein RIP3 was associated with the TNF-induced pathology, and elevated RIP3 expression was also found in intestinal Paneth cells of patients with Crohn's disease. Intestinal immune homeostasis depends on a tightly regulated cross talk between commensal bacteria, mucosal immune cells and intestinal epithelial cells (IECs) 1 , 2 , 3 , 4 . Epithelial barrier disruption is considered to be a potential cause of inflammatory bowel disease; however, the mechanisms regulating intestinal epithelial integrity are poorly understood 1 , 5 . Here we show that mice with IEC-specific knockout of FADD (FADD IEC-KO ), an adaptor protein required for death-receptor-induced apoptosis 6 , spontaneously developed epithelial cell necrosis, loss of Paneth cells, enteritis and severe erosive colitis. Genetic deficiency in RIP3, a critical regulator of programmed necrosis 7 , 8 , 9 , prevented the development of spontaneous pathology in both the small intestine and colon of FADD IEC-KO mice, demonstrating that intestinal inflammation is triggered by RIP3-dependent death of FADD-deficient IECs. Epithelial-specific inhibition of CYLD, a deubiquitinase that regulates cellular necrosis 10 , prevented colitis development in FADD IEC-KO but not in NEMO IEC-KO mice 11 , showing that different mechanisms mediated death of colonic epithelial cells in these two models. In FADD IEC-KO mice, TNF deficiency ameliorated colon inflammation, whereas MYD88 deficiency and also elimination of the microbiota prevented colon inflammation, indicating that bacteria-mediated Toll-like-receptor signalling drives colitis by inducing the expression of TNF and other cytokines. However, neither CYLD, TNF or MYD88 deficiency nor elimination of the microbiota could prevent Paneth cell loss and enteritis in FADD IEC-KO mice, showing that different mechanisms drive RIP3-dependent necrosis of FADD-deficient IECs in the small and large bowel. Therefore, by inhibiting RIP3-mediated IEC necrosis, FADD preserves epithelial barrier integrity and antibacterial defence, maintains homeostasis and prevents chronic intestinal inflammation. Collectively, these results show that mechanisms preventing RIP3-mediated epithelial cell death are critical for the maintenance of intestinal homeostasis and indicate that programmed necrosis of IECs might be implicated in the pathogenesis of inflammatory bowel disease, in which Paneth cell and barrier defects are thought to contribute to intestinal inflammation.

Objective Gut microbiota metabolises bile acids (BA). As dysbiosis has been reported in inflammatory bowel diseases (IBD), we aim to investigate the impact of IBD-associated dysbiosis on BA metabolism and its influence on the epithelial cell inflammation response. Design Faecal and serum BA rates, expressed as a proportion of total BA, were assessed by high-performance liquid chromatography tandem mass spectrometry in colonic IBD patients (42) and healthy subjects (29). The faecal microbiota composition was assessed by quantitative real-time PCR. Using BA profiles and microbiota composition, cluster formation between groups was generated by ranking models. The faecal BA profiles in germ-free and conventional mice were compared. Direct enzymatic activities of BA biotransformation were measured in faeces. The impact of BA on the inflammatory response was investigated in vitro using Caco-2 cells stimulated by IL-1β. Results IBD-associated dysbiosis was characterised by a decrease in the ratio between Faecalibacterium prausntizii and Escherichia coli. Faecal-conjugated BA rates were significantly higher in active IBD, whereas, secondary BA rates were significantly lower. Interestingly, active IBD patients exhibited higher levels of faecal 3-OH-sulphated BA. The deconjugation, transformation and desulphation activities of the microbiota were impaired in IBD patients. In vitro, secondary BA exerted anti-inflammatory effects, but sulphation of secondary BAs abolished their anti-inflammatory properties. Conclusions Impaired microbiota enzymatic activity observed in IBD-associated dysbiosis leads to modifications in the luminal BA pool composition. Altered BA transformation in the gut lumen can erase the anti-inflammatory effects of some BA species on gut epithelial cells and could participate in the chronic inflammation loop of IBD.

Abstract Background Concentrations of the neutrophil protein myeloperoxidase are elevated in the feces of individuals with endoscopically active inflammatory bowel disease (IBD). Its enzyme activity could give an immediate readout of endoscopic inflammation. We investigated whether fecal myeloperoxidase activity (fMPOa) is associated with IBD endoscopic inflammation. We also investigated whether myeloperoxidase promotes oxidative stress in IBD. Methods Myeloperoxidase enzyme activity was measured using an enzyme-linked immunosorbent assay (ELISA fMPOa), a novel CM-sepharose extraction assay (CM-S fMPOa), or by quantifying urinary glutathione sulfonamide (GSA) by tandem mass spectrometry. GSA is a specific biomarker of myeloperoxidase activity. IBD activity was assessed using the ulcerative colitis endoscopic index of severity or the simple endoscopic score for Crohn’s disease (SES-CD). Spearman’s correlation and receiver operating characteristic curves evaluated biomarker utility. Results IBD patients (n = 172) were recruited prospectively (ulcerative colitis, n = 72; Crohn’s disease, n = 100). fMPO was mostly active. Its enzyme activity, measured either as ELISA fMPOa or CM-S fMPOa, correlated with endoscopic inflammation in both ulcerative colitis and Crohn’s disease. Urinary GSA is also correlated with endoscopic disease inflammation. Correlations of urinary GSA with disease measures and other biomarkers were stronger in ulcerative colitis than in Crohn’s disease. Conclusions Myeloperoxidase is active in IBD and its enzyme activity is a reliable marker of IBD endoscopic inflammation. Our results with the CM-S fMPOa assay demonstrate the potential for an immediate and accurate measure of fMPO enzyme activity as a robust, low-cost test for IBD activity. Myeloperoxidase may contribute to tissue damage in IBD. Lay Summary The enzyme activity of myeloperoxidase in feces signaled flares of endoscopic inflammation in patients with inflammatory bowel disease. Urinary glutathione sulfonamide indicated that myeloperoxidase is active during inflammation. Fecal myeloperoxidase activity could be measured immediately using an inexpensive test. Graphical Abstract Graphical Abstract

ObjectiveTo investigate, among children with inflammatory bowel disease (IBD) and elevated liver enzymes, what threshold of gamma-glutamyltransferase (GGT) best distinguishes those with and without primary sclerosing cholangitis (PSC).MethodDelayed-type diagnostic study with a paired design. Children with IBD were regularly screened with GGT (index test). Confirmation of PSC was based on magnetic resonance cholangiopancreatography (MRCP) and/or liver histology (preferred reference standard). Children at low risk of PSC continued regular GGT testing for latent PSC to become visible (alternative reference test). The primary outcome was the negative predictive value (NPV) using three predefined test thresholds, respectively, 1, 2 and 5× the upper limit of normal (ULN). The secondary outcome was the GGT threshold based on receiver operating characteristic analysis.Results132 of 469 children (28.1%) had elevated GGT levels at their first colonoscopy or during follow-up. Eventually, 34 children (7.2%) were diagnosed with PSC. Median GGT (IQR) for children with and without PSC was 227 (127–345) and 77 (59–138) U/L, respectively. Of the predefined GGT thresholds, 2× ULN (ie, 100 U/L) had the best test characteristics, including an NPV of 98% and a negative likelihood ratio of 0.04 (95% CI 0.01 to 0.31). The area under the curve was 0.83 (95% CI: 0.75 to 0.90) and the optimal GGT threshold was 103.5 U/L.ConclusionIn children with IBD who have GGT elevations less than 2× ULN, the likelihood of PSC is extremely low. In such cases, MRCP and liver biopsy can be omitted. Regular GGT monitoring is advised, as PSC may develop over time.

The class III PI3-kinase (PIK3C3) is an enzyme responsible for the generation of phosphatidylinositol 3-phosphate (PI3P), a critical component of vesicular membrane. Here, we report that PIK3C3 deficiency in zebrafish results in intestinal injury and inflammation. In pik3c3 mutants, gut tube forms but fails to be maintained. Gene expression analysis reveals that barrier-function-related inflammatory bowel disease (IBD) susceptibility genes ( e-cadherin , hnf4a , ttc7a ) are suppressed, while inflammatory response genes are stimulated in the mutants. Histological analysis shows neutrophil infiltration into mutant intestinal epithelium and the clearance of gut microbiota. Yet, gut microorganisms appear dispensable as mutants cultured under germ-free condition have similar intestinal defects. Mechanistically, we show that PIK3C3 deficiency suppresses the formation of PI3P and disrupts the polarized distribution of cell-junction proteins in intestinal epithelial cells. These results not only reveal a role of PIK3C3 in gut homeostasis, but also provide a zebrafish IBD model. The functions of the class III PI3-kinase (PIK3C3) in gut homeostasis and innate immunity are poorly understood. Here the authors show that PIK3C3-deficient zebrafishes develop intestinal injury and inflammation due to mislocalization of cell junction proteins.

Inflammatory bowel disease (IBD), which include Crohn’s disease (CD) and ulcerative colitis (UC), exhibits a complex multifactorial pathogenesis involving genetic susceptibility, imbalance of gut microbiota, mucosal immune disorder and environmental factors. Recent studies reported associations between ubiquitination and deubiquitination and the occurrence and development of inflammatory bowel disease. Ubiquitination modification, one of the most important types of post-translational modifications, is a multi-step enzymatic process involved in the regulation of various physiological processes of cells, including cell cycle progression, cell differentiation, apoptosis, and innate and adaptive immune responses. Alterations in ubiquitination and deubiquitination can lead to various diseases, including IBD. Here, we review the role of E3 ubiquitin ligases and deubiquitinases (DUBs) and their mediated ubiquitination and deubiquitination modifications in the pathogenesis of IBD. We highlight the importance of this type of posttranslational modification in the development of inflammation, and provide guidance for the future development of targeted therapeutics in IBD.

Dysregulation of the proteolytic balance is often associated with diseases. Serine proteases and matrix metalloproteases are involved in a multitude of biological processes and notably in the inflammatory response. Within the framework of digestive inflammation, several studies have stressed the role of serine proteases and matrix metalloproteases (MMPs) as key actors in its pathogenesis and pointed to the unbalance between these proteases and their respective inhibitors. Substantial efforts have been made in developing new inhibitors, some of which have reached clinical trial phases, notwithstanding that unwanted side effects remain a major issue. However, studies on the proteolytic imbalance and inhibitors conception are directed toward host serine/MMPs proteases revealing a hitherto overlooked factor, the potential contribution of their bacterial counterpart. In this review, we highlight the role of proteolytic imbalance in human digestive inflammation focusing on serine proteases and MMPs and their respective inhibitors considering both host and bacterial origin.

Genetic alterations that reduce the function of the immunoregulatory cytokine IL-10 contribute to colitis in mouse and man. Myeloid cells such as macrophages (MΦs) and dendritic cells (DCs) play an essential role in determining the relative abundance of IL-10 versus inflammatory cytokines in the gut. As such, using small molecules to boost IL-10 production by DCs–MΦs represents a promising approach to increase levels of this cytokine specifically in gut tissues. Toward this end, we screened a library of well-annotated kinase inhibitors for compounds that enhance production of IL-10 by murine bone-marrow–derived DCs stimulated with the yeast cell wall preparation zymosan. This approach identified a number of kinase inhibitors that robustly up-regulate IL-10 production including the Food and Drug Administration (FDA)-approved drugs dasatinib, bosutinib, and saracatinib that target ABL, SRC-family, and numerous other kinases. Correlating the kinase selectivity profiles of the active compounds with their effect on IL-10 production suggests that inhibition of salt-inducible kinases (SIKs) mediates the observed IL-10 increase. This was confirmed using the SIK-targeting inhibitor HG-9-91-01 and a series of structural analogs. The stimulatory effect of SIK inhibition on IL-10 is also associated with decreased production of the proinflammatory cytokines IL-1β, IL-6, IL-12, and TNF-α, and these coordinated effects are observed in human DCs–MΦs and anti-inflammatory CD11c ⁺ CX ₃CR1 ʰⁱ cells isolated from murine gut tissue. Collectively, these studies demonstrate that SIK inhibition promotes an anti-inflammatory phenotype in activated myeloid cells marked by robust IL-10 production and establish these effects as a previously unidentified activity associated with several FDA-approved multikinase inhibitors.