Ulcerative colitis (UC) is a chronic inflammatory bowel disease marked by immune cell infiltration, crypt erosion, and severe ulceration. In phase 3 studies with etrolizumab, the results of a transcriptional analysis of colonic biopsies revealed that etrolizumab-mediated integrin β7 blockade, but not adalimumab (a TNF-blocking antibody), reduced genes associated with integrin αEβ7+ intraepithelial lymphocytes (IELs). Both treatments significantly reduced stromal and myeloid cell-related genes linked to Mayo Clinic Score (MCS) remission status. A single-cell atlas from UC biopsies identified 36 distinct cell populations, including myeloid cells. This atlas enabled cell-specific signatures and cellular deconvolution of the phase 3 data, showing reductions in neutrophils, monocyte-derived macrophages, and inflammatory fibroblasts, along with increases in epithelial cells common to both treatments. Pseudo-time analyses identified four neutrophil subsets, transitioning from PADI4hi, OSMhi, and MX1hi to CXCR4hi populations. PADI4hi and OSMhi neutrophils exhibited high protease, cytokine (CXCL1, IL1B, OSM), and chemokine receptor (CXCR1, CXCR2) levels, while MX1hi expressed markers of IFN exposure. CXCR4hi neutrophils showed elevated CXCL2, TNF, and VEGFA levels. Notably, interactions between PADI4hi and OSMhi neutrophils and inflammatory fibroblasts, such as OSM and IL1B, were associated with MCS remission with both drugs. CXCR4hi neutrophils showed only minor changes unrelated to clinical outcomes. These findings suggest that neutrophils are highly heterogeneous, with abundant interactions in inflamed colonic tissue, potentially perpetuating chronic disease. Disrupting neutrophil interactions with myeloid and resident cells like inflammatory fibroblasts could reduce inflammation, possibly enhancing clinical remission rates.

BackgroundThe noncanonical NF-κB pathway, driven by NF-κB-inducing kinase (NIK), plays a central role in inflammatory bowel disease (IBD) pathogenesis by regulating immune responses across epithelial, myeloid, and lymphoid compartments. Despite advances in IBD therapies, novel therapeutic options targeting NIK remain underexplored.MethodsWe utilized cell-type-specific knockout models for intestinal epithelial cells, dendritic cells, as well as systemic NIK-deleted mice, to investigate the role of NIK in IBD pathogenesis. A small molecule NIK inhibitor (NIK-smi) was evaluated using pharmacokinetic/pharmacodynamic (PK/PD) modeling and its efficacy tested in preclinical colitis models, including Helicobacter hepaticus + anti-IL-10R, adoptive T cell transfer, and DSS-induced colitis. Biomarker analyses were performed using flow cytometry and multiplex cytokine detection.ResultsNIK deletion in epithelial cells disrupted M-cell differentiation, reducing CCL20-mediated recruitment of CCR6-expressing Th17 cells and ILC3s. In dendritic cells, NIK activity amplified CD40-mediated innate immune responses, while in T cells, NIK skewed differentiation toward pro-inflammatory Th1 cells at the expense of regulatory T cells. Systemic NIK deletion and pharmacologic inhibition with NIK-smi attenuated inflammation in preclinical colitis models, reducing immune cell infiltration, pro-inflammatory cytokines (e.g., IL-1β, IFNγ, IL-17A, CCL5), and improving colon histopathology. PK/PD modeling revealed that a 200 mg/kg BID dosing regimen of NIK-smi provided optimal pathway inhibition and therapeutic efficacy.ConclusionsNIK is a key regulator of gut inflammation through its multi-cellular contributions in IBD pathogenesis. Pharmacologic inhibition of NIK offers a promising therapeutic strategy for IBD, with PK/PD modeling providing a translational framework to optimize dosing and efficacy.

BACKGROUND:Tumor necrosis factor (TNF) is a pleiotropic cytokine that plays a critical role in the pathogenesis of immune-mediated diseases including inflammatory bowel disease (IBD). The stability of its mRNA transcript, determined in part by destabilizing sequences in its AAUU repeats (ARE) gene region, is an important regulator of its tissue and systemic levels. A deletion in the ARE region of the gene resulted in IBD and arthritis in mice and pigs, supporting a critical role for the cytokine in human IBD and several human arthritides. A mutation in the same area of the mouse genome by Genentech scientists (T.Y., M.K.) resulted in a similar but not identical phenotype. METHODS:Here, we compare histopathological, cellular, and molecular features of the strains and propose reasons for their distinct phenotypes. First, while homozygous TNFΔARE mice develop severe arthritis and die after weaning, homozygous Genentech TNFΔARE (ΔG/ΔG) mice have normal lifespans, and males are often fertile. RESULTS:We found that while the ileitic phenotype had peaked at 12 weeks of age in all mice, colitis progressed mostly after 20 weeks of age in heterozygous mice. Their variably penetrant arthritic phenotype progressed mostly after 20 weeks, also in heterozygous mice from both strains. There was expansion of central memory T and B cells in lymphoid organs of TNF-overproducing strains and their transcriptional profile shared well-known pathogenetic pathways with human IBD. Finally, we found differences in the mutated sequences within the ARE regions of the TNF gene and in their microbiota composition and genetic background. These differences likely explain their phenotypic differences. CONCLUSIONS:In summary, we describe a different strain of TNF-overproducing mice with an overlapping, yet not identical phenotype, which may have differential applications than the original strain.

BACKGROUND & AIMS: Efmarodocokin alfa is an interleukin (IL)-22 agonist, with favorable pharmacokinetic properties and an acceptable safety profile. This study further explored the therapeutic potential of efmarodocokin alfa compared with vedolizumab in patients with ulcerative colitis (UC). METHODS: This randomized phase II trial evaluated the efficacy, safety, pharmacokinetics, and pharmacodynamics of 3 doses of efmarodocokin alfa administered intravenously every 4 weeks (30 mu g/kg [n = 43], 60 mu g/kg [n = 44], and 90 mu g/kg [n = 43]) compared with placebo (n = 22) and with vedolizumab (n = 43) in the treatment of moderate to severe UC. Key clinical outcomes were assessed through the modified Mayo Clinic Score, and endoscopic evaluations by a central reader. RESULTS: Efmarodocokin alfa was adequately tolerated with an acceptable safety profile. Although efmarodocokin alfa did not show statistically significant improvement in clinical remission, clinical response, endoscopic healing, or endoscopic remission at week 8 compared with placebo, vedolizumab demonstrated some efficacy. Clinical remission was achieved by 12%, 9%, and 12% of patients in the 30, 60, and 90 mu g/kg dose arms, respectively, compared with 9% and 26% of patients in the placebo and vedolizumab arms at week 8. Similarly, endoscopic healing at week 8 was achieved by 14%, 14%, and 12% of patients in the 30, 60, and 90 mu g/kg dose arms, respectively, compared with 14% and 33% of patients in the placebo and vedolizumab arms. A dose-dependent increase in pharmacodynamic biomarkers was observed (regenerating islet-derived protein 3-alpha and C-reactive protein levels). CONCLUSION: Efmarodocokin alfa did not demonstrate efficacy compared with placebo, and this phase II study was ended early for futility; however, there was evidence of target engagement (skin adverse events, regenerating islet-derived protein 3-alpha levels). ClinicalTrials.gov, Number: NCT03558152.

While ulcerative colitis (UC)-associated inflammation primarily occurs in the colon, subclinical terminal ileum inflammation has been previously observed. Given that colonic microbial dysbiosis is common in UC, we profiled the terminal ileum microbiota to determine the extent of gastrointestinal dysbiosis in UC.

Background and Aims This study aimed to identify microbial drivers of inflammatory bowel disease [IBD], by investigating mucosal-associated bacteria and their detrimental products in IBD patients.Methods We directly cultured bacterial communities from mucosal biopsies from paediatric gastrointestinal patients and examined for pathogenicity-associated traits. Upon identifying Clostridium perfringens as toxigenic bacteria present in mucosal biopsies, we isolated strains and further characterized toxicity and prevalence.Results Mucosal biopsy microbial composition differed from corresponding stool samples. C. perfringens was present in eight of nine patients' mucosal biopsies, correlating with haemolytic activity, but was not present in all corresponding stool samples. Large IBD datasets showed higher C. perfringens prevalence in stool samples of IBD adults [18.7-27.1%] versus healthy controls [5.1%]. In vitro, C. perfringens supernatants were toxic to cell types beneath the intestinal epithelial barrier, including endothelial cells, neuroblasts, and neutrophils, while the impact on epithelial cells was less pronounced, suggesting C. perfringens may be particularly damaging when barrier integrity is compromised. Further characterization using purified toxins and genetic insertion mutants confirmed perfringolysin O [PFO] toxin was sufficient for toxicity. Toxin RNA signatures were found in the original patient biopsies by PCR, suggesting intestinal production. C. perfringens supernatants also induced activation of neuroblast and dorsal root ganglion neurons in vitro, suggesting C. perfringens in inflamed mucosal tissue may directly contribute to abdominal pain, a frequent IBD symptom.Conclusions Gastrointestinal carriage of certain toxigenic C. perfringens may have an important pathogenic impact on IBD patients. These findings support routine monitoring of C. perfringens and PFO toxins and potential treatment in patients.

Small bowel strictures remain a debilitating consequence of Crohn’s disease and contribute to poor outcomes for patients. Recently, TGFβ has been identified as an important driver of intestinal fibrosis. We studied the localization of TGFβ isoforms in ileal strictures of patients with Crohn’s disease using in situ hybridization to understand TGFβ’s role in stricture formation. The mucosa of strictures was characterized by higher TGFβ1 while the stricture submucosa showed higher TGFβ3 compared to normal ileum from patients without Crohn’s disease (p = 0.02 and p = 0.044, respectively). We correlated these findings with single-cell transcriptomics which demonstrated that TGFβ3 transcripts overall are very rare, which may partially explain why its role in intestinal fibrosis has remained unclear to date. There were no significant differences in fibroblast or B cell TGFβ1 and/or TGFβ3 expression in inflamed vs. noninflamed ileum. We discuss the implications of these findings for therapeutic development strategies to treat patients with fibrostenotic Crohn’s disease.

Macrophages exhibit remarkable functional plasticity, a requirement for their central role in tissue homeostasis. During chronic inflammation, macrophages acquire sustained inflammatory 'states' that contribute to disease, but there is limited understanding of the regulatory mechanisms that drive their generation. Here we describe a systematic functional genomics approach that combines genome-wide phenotypic screening in primary murine macrophages with transcriptional and cytokine profiling of genetic perturbations in primary human macrophages to uncover regulatory circuits of inflammatory states. This process identifies regulators of five distinct states associated with key features of macrophage function. Among these regulators, loss of the N-6-methyladenosine (m6A) writer components abolishes m6A modification of TNF transcripts, thereby enhancing mRNA stability and TNF production associated with multiple inflammatory pathologies. Thus, phenotypic characterization of primary murine and human macrophages describes the regulatory circuits underlying distinct inflammatory states, revealing post-transcriptional control of TNF mRNA stability as an immunosuppressive mechanism in innate immunity.

BACKGROUND AND AIMS:The gut microbiota contributes to aberrant inflammation in inflammatory bowel disease, but the bacterial factors causing or exacerbating inflammation are not fully understood. Further, the predictive or prognostic value of gut microbial biomarkers for remission in response to biologic therapy is unclear. METHODS:We perform whole metagenomic sequencing of 550 stool samples from 287 ulcerative colitis patients from a large, phase 3, head-to-head study of infliximab and etrolizumab. RESULTS:We identify several bacterial species in baseline and/or post-treatment samples that associate with clinical remission. These include previously described associations [Faecalibacterium prausnitzii_F] as well as new associations with remission to biologic therapy [Flavonifractor plautii]. We build multivariate models and find that gut microbial species are better predictors for remission than clinical variables alone. Finally, we describe patient groups that differ in microbiome composition and remission rate after induction therapy, suggesting the potential utility of microbiome-based endotyping. CONCLUSIONS:In this large study of ulcerative colitis patients, we show that few individual species associate strongly with clinical remission, but multivariate models including microbiome can predict clinical remission and have better predictive power compared with clinical data alone.

Ubiquitination is a posttranslational modification that is crucial for the dynamic regulation of diverse signaling pathways. To enhance our understanding of ubiquitination-mediated signaling, we generated a new class of bispecific antibodies that combine recognition of ubiquitination substrates and specific polyubiquitin linkages. RIP1-K63 and RIP1–linear (Lin) linkage polyubiquitin bispecific antibodies detected linkage-specific ubiquitination of the proinflammatory kinase RIP1 in cells and in tissues and revealed RIP1 ubiquitination by immunofluorescence. Similarly, ubiquitination of the RIP1-related kinase RIP2 with K63 or linear linkages was specifically detected with the RIP2-K63 and RIP2-Lin bispecific antibodies, respectively. Furthermore, using the RIP2-K63 and RIP2-Lin bispecific antibodies, we found prominent K63-linked and linear RIP2 ubiquitination in samples from patients with ulcerative colitis and Crohn’s disease. We also developed a bispecific antibody (K63-Lin) that simultaneously recognizes K63-linked and linear ubiquitination of components of various signaling pathways. Together, these bispecific antibodies represent a new class of reagents with the potential to be developed for the detection of inflammatory biomarkers.

Ulcerative colitis (UC) is a chronic inflammatory large bowel disease characterized by immune cell infiltration and continuous erosion of intestinal crypts, causing severe ulceration and abdominal pain. In the etrolizumab Phase 3 studies, transcriptional analyses of colonic biopsies revealed reductions in genes associated with aEb7+ intraepithelial lymphocytes with etrolizumab but not adalimumab. Both treatments significantly reduced stromal and myeloid cell-associated genes, with changes associated with MCS remission status. Generation of a single-cell atlas from inflamed and uninflamed colonic biopsies from UC patients led to the identification of thirty-six discrete cell populations, including cells of the myeloid compartment. The UC atlas was used to generate cell-specific signatures, allowing for cellular deconvolution of the Phase 3 datasets. It revealed significant reductions in neutrophil subsets, monocyte-derived macrophages, and inflammatory fibroblasts, as well as increases in colonic epithelial cells common to both etrolizumab and adalimumab. Pseudo-time trajectory analyses identified four unique neutrophil subsets with unique cell phenotypes reflecting changes in cell state or differentiation from PADI4hi, OSMhi, MX1hi, and ultimately to CXCR4hi populations. PADI4hi and OSMhi neutrophils exhibited high levels of proteases (MMP9, LYZ), inflammatory cytokines (CXCL1, IL1B, OSM), and abundant cytokine or chemokine receptors (CXCR1, CXCR2). MX1 populations expressed markers indicating prior IFN exposure (MX1, IFIT1). In contrast, more differentiated or mature neutrophils exhibited high levels of CXCL2, TNF-a, and CXCR4, as well as angiogenic factors like VEGFA. PADI4hi and OSMhi neutrophils, we predict, have abundant cytokine and chemokine interactions with inflammatory fibroblasts within the inflamed colon, such as OSM: OSMR and IL1B: IL1R1 interactions. Changes in PADI4hi and OSMhi neutrophils were closely associated with MCS remission in both etrolizumab and adalimumab-treated patients. In contrast, only minor changes in CXCR4hi neutrophils were observed and not associated with clinical outcomes. Our results suggest that neutrophils are not only heterogeneous in phenotype but have abundant cell-cell interactions in inflamed colonic tissue that are likely implicated in maintaining chronic disease activity. We hypothesize that limiting the interactions between neutrophils and other myeloid cells with resident cells such as inflammatory fibroblasts may reduce the production of inflammatory mediators and limit activation and infiltration of neutrophils, which may be necessary for achieving greater rates of clinical remission in response to interventional agents. ### Competing Interest Statement All employees of Genentech, Inc. are shareholders of F. Hoffman-LaRoche, Ltd.

Background The interleukin-22 cytokine (IL-22) has demonstrated efficacy in preclinical colitis models with non-immunosuppressive mechanism of action. Efmarodocokin alfa (UTTR1147A) is a fusion protein agonist that links IL-22 to the crystallisable fragment (Fc) of human IgG4 for improved pharmacokinetic characteristics, but with a mutation to minimise Fc effector functions. Methods This randomised, phase 1b study evaluated the safety, tolerability, pharmacokinetics and pharmacodynamics of repeat intravenous dosing of efmarodocokin alfa in healthy volunteers (HVs; n=32) and patients with ulcerative colitis (n=24) at 30–90 µg/kg doses given once every 2 weeks or monthly (every 4 weeks) for 12 weeks (6:2 active:placebo per cohort). Results The most common adverse events (AEs) were on-target, reversible, dermatological effects (dry skin, erythema and pruritus). Dose-limiting non-serious dermatological AEs (severe dry skin, erythema, exfoliation and discomfort) were seen at 90 μg/kg once every 2 weeks (HVs, n=2; patients, n=1). Pharmacokinetics were generally dose-proportional across the dose levels, but patients demonstrated lower drug exposures relative to HVs at the same dose. IL-22 serum biomarkers and IL-22-responsive genes in colon biopsies were induced with active treatment, and microbiota composition changed consistent with a reversal in baseline dysbiosis. As a phase 1b study, efficacy endpoints were exploratory only. Clinical response was observed in 7/18 active-treated and 1/6 placebo-treated patients; clinical remission was observed in 5/18 active-treated and 0/6 placebo-treated patients. Conclusion Efmarodocokin alfa had an adequate safety and pharmacokinetic profile in HVs and patients. Biomarker data confirmed IL-22R pathway activation in the colonic epithelium. Results support further investigation of this non-immunosuppressive potential inflammatory bowel disease therapeutic. Trial registration number NCT02749630.

Signal regulatory protein (SIRPα) is an immune inhibitory receptor expressed by myeloid cells to inhibit immune cell phagocytosis, migration, and activation. Despite the progress of SIRPα and CD47 antagonist antibodies to promote anti-cancer immunity, it is not yet known whether SIRPα receptor agonism could restrain excessive autoimmune tissue inflammation. Here, we report that neutrophil- and monocyte-associated genes including SIRPA are increased in inflamed tissue biopsies from patients with rheumatoid arthritis and inflammatory bowel diseases, and elevated SIRPA is associated with treatment-refractory ulcerative colitis. We next identify an agonistic anti-SIRPα antibody that exhibits potent anti-inflammatory effects in reducing neutrophil and monocyte chemotaxis and tissue infiltration. In preclinical models of arthritis and colitis, anti-SIRPα agonistic antibody ameliorates autoimmune joint inflammation and inflammatory colitis by reducing neutrophils and monocytes in tissues. Our work provides a proof of concept for SIRPα receptor agonism for suppressing excessive innate immune activation and chronic inflammatory disease treatment.

Background:Children with congenital heart disease (CHD) are living longer than ever before. This growing cohort of adults with CHD has high medical and psychosocial needs. Also, patients and advocacy groups are justifiably demanding that their voices be heard in all phases of clinical and health services research.Methods:We conducted a first of its kind research priority-setting exercise with teens and adults with moderate-to-complex CHD. Focus groups were held using a fixed, mixed methods, exploratory sequential design. Objectives were to include the patient voice in all phases of the research process, determine the key needs of patients living with CHD, to guide health services research, and identify the "top 10" research priorities of teens and adults living with CHD.Results:Thirty-five patients participated in one of nine 3-hour focus groups where they shared their experiences living with CHD. They expressed a desire for connection with others living with CHD and altruistic motives for participating. Patients with CHD identified a need for information about their disease and prognosis, a need for connection through physical activity and mentorship programmes, and a need for advanced communication with health care teams. Qualitative results correlated well with quantitative ratings to create a patient-derived "top 10" research priorities list.Conclusions:Patients affected by a chronic disease like CHD want to be included in all phases of research. Our research priority-setting exercise in teens and adults with CHD has created a roadmap for clinicians and researchers to investigate issues most important to those living with CHD.

IL-22 is induced by aryl hydrocarbon receptor (AhR) signaling and plays a critical role in gastrointestinal barrier function through effects on antimicrobial protein production, mucus secretion, and epithelial cell differentiation and proliferation, giving it the potential to modulate the microbiome through these direct and indirect effects. Furthermore, the microbiome can in turn influence IL-22 production through the synthesis of L-tryptophan (L-Trp)-derived AhR ligands, creating the prospect of a host-microbiome feedback loop. We evaluated the impact IL-22 may have on the gut microbiome and its ability to activate host AhR signaling by observing changes in gut microbiome composition, function, and AhR ligand production following exogenous IL-22 treatment in both mice and humans. Microbiome alterations were observed across the gastrointestinal tract of IL-22-treated mice, accompanied by an increased microbial functional capacity for L-Trp metabolism. Bacterially derived indole derivatives were increased in stool from IL-22-treated mice and correlated with increased fecal AhR activity. In humans, reduced fecal concentrations of indole derivatives in ulcerative colitis (UC) patients compared to healthy volunteers were accompanied by a trend towards reduced fecal AhR activity. Following exogenous IL-22 treatment in UC patients, both fecal AhR activity and concentrations of indole derivatives increased over time compared to placebo-treated UC patients. Overall, our findings indicate IL-22 shapes gut microbiome composition and function, which leads to increased AhR signaling and suggests exogenous IL-22 modulation of the microbiome may have functional significance in a disease setting.

INTRODUCTION:Magnetic resonance enterography (MRE) is useful for detecting bowel strictures, whereas a number of imaging biomarkers may reflect severity of fibrosis burden in Crohn's disease (CD). This study aimed to verify the association of MRE metrics with histologic fibrosis independent of inflammation.METHODS:This prospective European multicenter study performed MRE imaging on 60 patients with CD with bowel strictures before surgical resection. Locations of 61 histological samples were annotated on MRE examinations, followed by central readings using the Chiorean score and measurement of delayed gain of enhancement (DGE), magnetization transfer ratio, T2-weighted MRI sequences (T2R), apparent diffusion coefficient (ADC), and the magnetic resonance index of activity (MaRIA). Correlations of histology and MRE metrics were assessed. Least Absolute Shrinkage and Selection Operator and receiver operator characteristic (ROC) curve analyses were used to select composite MRE scores predictive of histology and to estimate their predictive value.RESULTS:ADC and MaRIA correlated with fibrosis (R = -0.71, P < 0.0001, and 0.59, P < 0.001) and more moderately with inflammation (R = -0.35, P < 0.01, and R = 0.53, P < 0.001). Lower or no correlations of fibrosis or inflammation were found with DGE, magnetization transfer ratio, or T2R. Least Absolute Shrinkage and Selection Operator and ROC identified a composite score of MaRIA, ADC, and DGE as a very good predictor of histologic fibrosis (ROC area under the curve = 0.910). MaRIA alone was the best predictor of histologic inflammation with excellent performance in identifying active histologic inflammation (ROC area under the curve = 0.966).Discussion:MRE-based scores for histologic fibrosis and inflammation may assist in the characterization of CD stenosis and enable development of fibrosis-targeted therapies and clinical treatment of stenotic patients.

Patients with adult congenital heart disease (ACHD) are at increased risk for poor outcomes when compared to the age matched non-ACHD population and require specialist care to optimize outcomes including well-being and survival. The COVID-19 pandemic significantly impacted healthcare provision across Canada with reduction on in person evaluations. The effect of the COVID-19 restrictions on ACHD care including clinic evaluation, diagnostic and procedures in Canada has not been well characterized. All Canadian Adult Congenital Heart Network affiliated ACHD centers were contacted and asked to collect data on outpatient clinic and procedural volumes for the 2019 and 2020 calendar years. A survey was sent to each site detailing questions on clinic and procedural volumes and wait times pre and post pandemic restrictions. Descriptive statistics were used with student t test to compare groups. Pre-pandemic (2019) there were 19326 ACHD clinic visits across Canada with 296 (1.5%) being virtual. During the first year of the pandemic (2020) there were a similar number of total clinic visits 20532, however 11412 (56%) visits were virtual p< 0.0001. Total procedural volumes for ACHD care are presented in figure 1. Pre-pandemic mean estimated clinic waiting times (in months) for non-urgent consults were: 5.4 + 2.57 vs. pandemic wait time 6.5 + 4.22, p=0.65, for elective ACHD cardiac surgery 6.0 + 3.46 vs.7.3 + 4.59, p=0.47, for ACHD electrophysiology procedures 6.3 + 3.33 vs 6.7 + 3.27 p=0.72, for ACHD percutaneous intervention 4.6 + 3.89 vs 4.4 + 2.33 p=0.74. During the pandemic, despite social distancing restrictions, the use of virtual clinics visits have helped to maintain continuity in ACHD clinical care. The procedural volumes and wait times for consultation, percutaneous and surgical interventions were not delayed.