Introduction

Treatment response in inflammatory bowel disease (IBD) is highly variable, with an urgent need for biomarkers to guide therapeutic decisions. Circulating monocytes, key players in intestinal immunity, act as precursors to tissue macrophages and contribute to inflammation and mucosal healing. The transcriptional programs that govern monocyte fate and their association with treatment outcomes remain poorly defined. Ikaros family proteins regulate gene expression through distinct mechanisms in lymphocytes, but little is known about their role in monocyte-macrophage function in IBD. We aimed to investigate the role of Ikaros family (Ikaros, Helios, Aiolos, Eos, Pegasus) in shaping monocyte function linked to advanced therapies response.

Aims & Methods

Peripheral blood samples were collected at baseline (W0) and after 8-12 weeks of induction therapy (W8/12) from IBD patients who initiated or switched advanced therapy [biologics, oral synthetics] and from healthy controls (HC) (n=8). We included patients achieving clinical remission after induction [Super-Responders] SR (n=18) and those showing no response [Non-Responders] NR (n=16). Immune phenotype and expression of Ikaros proteins were assessed via multiparametric flow cytometry (FACS) and correlated with clinical outcomes. To assess functional roles of Ikaros proteins, monocyte-derived macrophages were generated in vitro from buffy coat (n=6). Cells were polarised using cytokine cocktails to induce pro-inflammatory (LPS+IFNγ or TNF+PGE2+Pam3CSK4; TPP), alternative (IL1β+LPS), or regulatory (IL10) phenotypes. siRNA-loaded nanoparticles were used to silence Ikaros or Helios prior to polarisation. Phenotypic changes were analysed by FACS; cytokines secretion by ELISA; genes expression by qPCR; and phagocytosis by fluorescent beads uptake.

Results

At W0, IBD patients exhibited a significant increase in circulating monocyte counts (vs HC, p<0.001). At W8/12, monocyte levels significantly decreased in the SR group, while remaining elevated in the NR group. FACS analysis revealed an increased proportion of non-classical monocytes (CD14⁻CD16⁺) in the IBD group at W0 (vs HC; p< 0.05), persisting in NR at W8/12 (vs SR; p<0.05). IBD monocytes also showed higher expression of CCR2 and CX3CR1 compared to HC. Helios expression was absent in monocytes from IBD patients at W0, while the mean expression of Helios was 47.31% in monocytes from the HC group. At W8/12, Helios expression re-emerged in 34.14% of monocytes derived from the SR patients but remained undetectable in the NR. In vitro, macrophages treated with LPS+IFNγ or TPP upregulated Ikaros, STAT1, and CXCL10 gene expression (p< 0.01), while Helios, TGM2, and MCR1 were upregulated in response to IL10 (p< 0.05). Helios expression was absent in TNF-stimulated macrophages. Ikaros silencing reduced pro-inflammatory markers, IL6 and IL1β cytokines secretion, and inhibited phagocytosis in response to LPS, whereas Helios silencing increased macrophages activation, and pro-inflammatory markers in response to IL10.

Conclusion

Monocyte subsets distribution and Helios, Ikaros expression dynamics clearly distinguished SR from NR in IBD. Sustained absence of Helios and persistence of non-classical monocytes in NR suggest a failure to transition toward a regulatory phenotype. This may further contribute to altered migratory capacity and influence their functional fate within intestinal tissue. Functional assays confirm opposing roles of Ikaros and Helios in the macrophages activation, underscoring their unique potential as biomarkers or therapeutic targets to modulate inflammation in IBD.

Introduction

Inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis, are characterized by dysregulated T-cell responses, causing chronic intestinal inflammation.¹ While anti-TNF therapy is established in IBD, targeting additional pathways may improve outcomes for patients who do not respond adequately to current treatments.² OX40L, an immune co-stimulator, has been implicated in the pathogenesis of several inflammatory conditions.³ Our study provides a comprehensive analysis of the effects of combined TNF and OX40L blockade in a murine adoptive T cell transfer (ATC) model of colitis, examining body weight, detailed histopathological parameters, and a broad range of inflammatory cytokines in colon tissue.

Aims & Methods

Colitis was induced in severe combined immunodeficiency (SCID) mice by intraperitoneal transfer of CD4⁺CD45RB^high T cells isolated from healthy BALB/c donor mice. We established five treatment groups (n=8-10): 1) CD45RB^low transfer “healthy control”, 2) CD45RB^high transfer with isotype antibodies “disease control group”, 3) CD45RB^high transfer with anti-TNF antibody (XT3.11, 250 μg/mouse), 4) CD45RB^high transfer with anti-OX40L antibody (RM134L, 250 μg/mouse), and 5) CD45RB^high transfer with both antibodies at 250 µg/mouse, each. Body weight was monitored for 46 days as a measure of disease progression and normalized to baseline weight at day 1. At study termination, we collected colon tissue for analysis of inflammatory cytokines using multiplex immunoassays and conducted histopathological examination. Histopathological scoring was performed blinded using a validated scoring system for colonic inflammation.⁴

Results

Overall, combined anti-TNF/anti-OX40L therapy was most effective in preventing body weight loss compared to the isotype disease control group, followed by anti-TNF monotherapy and anti-OX40L monotherapy (p<0.0001, p=0.0009, and p=0.03, respectively). Mean normalized body weight (MNBW) of the CD45RB^low healthy control group at termination on day 46 was 115.2 %, while CD45RB^high cell transfer in the disease control group triggered disease induction and body weight loss, with a MNBW of 93.8 % at day 46. The CD45RB^high groups treated with anti-TNF or anti OX40L monotherapy reached a MNBW of 107.6 % and 102.9%, respectively. The CD45RB^high group treated with combination therapy reached 112.6%, closest to the CD45RB^low healthy control group. Notably, only combination therapy led to a significant improvement in histopathological scoring (p=0.0176) compared to the isotype disease control. Similarly, combination treatment consistently resulted in the strongest downregulation of several inflammatory cytokines including IFNg, IL-4, or TNFa, which are increased in colon tissue in the CD45RB^high disease group compared to the CD45RB^low healthy control group.

Conclusion

Combined blockade of TNF and OX40L demonstrated significantly greater efficacy in the ATC preclinical model of IBD in prevention of weight loss, downregulation of key inflammatory cytokines, and reduction of histological damage compared to either monotherapy. These data suggest potential additive efficacy for TNF and OX40L inhibition in controlling intestinal inflammation, and warrant further exploration of dual TNF/OX40L inhibition in clinical trials, particularly among those with inadequate response to anti-TNF monotherapy.

References

1. Kaser A, Zeissig S, Blumberg RS. Inflammatory bowel disease. Annu Rev Immunol. 2010;28:573-621.
2. Vermeire S. Combination biologic therapy for ulcerative colitis. Lancet Gastroenterol Hepatol. 2023;8:288-90.
3. Fu Y, Lin Q, Zhang Z, Zhang L. Therapeutic strategies for the costimulatory molecule OX40 in T-cell-mediated immunity. Acta Pharm Sin B. 2020;10:414-33.
4. Erben U, Loddenkemper C, Doerfel K, Spieckermann S, Haller D, Heimesaat MM, Zeitz M, Siegmund B, Kühl AA. A guide to histomorphological evaluation of intestinal inflammation in mouse models. Int J Clin Exp Pathol. 2014;7:4557-76.

Disclosure

Thomas Leeuw, Rolf Keiffer, Danping Ding-Pfennigdorff, Christian Asbrand, Raaj Mehta, Matthias Herrmann are employees of Sanofi and may hold stock or equity options in Sanofi

Introduction

A dysfunctional intestinal mucosal barrier is a key pathophysiological hallmark and driver of a broad range of gastrointestinal diseases including celiac disease and inflammatory bowel disease. To date, there are no adequate treatment strategies to ameliorate impaired barrier function.
IMU-856 is an orally available and systemically acting small molecule modulator of sirtuin 6 (SIRT6), a histone/non-histone protein deacetylase which serves as a transcriptional regulator of intestinal barrier function and regeneration of bowel epithelium. IMU-856 may represent a new approach to treat gastrointestinal diseases with compromised barrier function.

Aims & Methods

This was a first-in-human, double-blind, randomized, placebo-controlled clinical trial of IMU-856 in healthy volunteers and patients with celiac disease. In the multiple ascending dose part, healthy human subjects were dosed once-daily for 14 consecutive days with IMU-856 at three different dose levels or placebo. Additionally, the Phase 1/1b study was designed to assess safety and tolerability of IMU-856 at two different dose levels in patients with celiac disease during periods of gluten-free diet and a 15-day gluten challenge. Further objectives included pharmacokinetics and the assessment of histological changes. Biomarkers reflecting intestinal enterocyte mass and function, nutrient absorption, and enteroendocrine hormones like GLP-1 were analyzed to assess potential effects on gut health.

Results

IMU-856 was safe and well-tolerated with a benign adverse event profile and pharmacokinetics that allow once-daily dosing. Treatment with IMU-856 protected against gluten-induced histologic deterioration, improved the health and metabolic function of intestinal enterocytes as measured by nutrient uptake and citrulline [1], and had positive effects on enteroendocrine cells increasing endogenous GLP-1 levels in a dose-dependent manner.

Conclusion

IMU-856 is a safe, highly selective and potent epigenetic modulator, improving the intestinal barrier function in patients with celiac disease undergoing a gluten challenge.
Positive effects of IMU-856 on gut hormones and various biomarkers for overall gut health including intestinal function and integrity were observed. Therefore, IMU-856 may offer extensive potential for treatment of gastrointestinal diseases with compromised intestinal barrier function and metabolic diseases.

References

1. Daveson AJM, Stubbs R, Polasek TM, Isola J, Anderson R, Tye-Din JA, et al. Safety, clinical activity, pharmacodynamics, and pharmacokinetics of IMU-856, a SIRT6 modulator, in coeliac disease: a first-in-human, randomised, double-blind, placebo-controlled, phase 1 trial.

Disclosure

AS, MW, EP: employees of Immunic AG, option and shareholders of Immunic, Inc.
IPO, JF, IK, SE: employees of Immunic AG, option and/or shareholders of Immunic, Inc.
HK: employee of Immunic AG, shareholder of Immunic, Inc., co-inventor of patents for the drug in investigation
DP: option holder of Immunic, Inc.
DV, AM: employees of Immunic AG and Immunic, Inc., shareholders of Immunic, Inc., co-inventors of patents for the drug in investigation

Introduction

Chronic pouchitis is a common complication following ileal pouch-anal anastomosis (IPAA), with limited effective treatment options.¹ Antibiotics remain the first-line therapy; however, in cases of chronic pouchitis, prolonged antibiotic use contributes to increased antimicrobial resistance within the ileal pouch microbiome.² Fecal microbiota transplantation (FMT) has been proposed as a means of restoring microbial balance. Nevertheless, randomized placebo-controlled trials have demonstrated that, although FMT can alter the microbiome, it does not confer clinical benefits compared to placebo.^3–5

Aims & Methods

Given the lack of efficacy of FMT using stool from healthy donors, the present study aims to characterize the microbiome of a healthy ileal pouch and to evaluate the impact of high antibiotic exposure in patients with chronic pouchitis.
In this cohort study, stool samples were collected from four groups: patients with a normally functioning IPAA, patients with chronic pouchitis, patients with ulcerative colitis, and healthy controls. Microbiome composition and antibiotic resistance genes were analyzed across these groups. Fecal samples underwent metagenomic sequencing, and metagenome-assembled genomes (MAGs) were reconstructed. For Escherichia coli MAGs, the antibiotic resistance gene (ARG) profiles and plasmid content were specifically examined.

Results

This study included 197 participants. A clear pattern emerged in the microbial composition, revealing that reduced alpha diversity is directly associated with declining gut function and clinical health. Both species richness and Shannon diversity were lowest in patients with chronic pouchitis and highest in healthy individuals, with intermediate levels observed in the other groups. Notably, the ileal pouch microbiome of many chronic pouchitis patients was dominated by E. coli. Given the potential clinical implications of high E. coli abundance, we further investigated the ARG profiles and plasmid content of E. coli MAGs. E. coli MAGs from chronic pouchitis patients exhibited a significantly higher frequency of single nucleotide polymorphisms (SNPs) associated with resistance to fluoroquinolones—an antibiotic uniformly used among this patient group—compared to MAGs from the normally functioning IPAA and healthy control cohorts. Furthermore, E. coli MAGs from chronic pouchitis patients harbored a greater number of plasmids, with an increased presence of plasmid-borne ARGs conferring resistance to antibiotics commonly administered in this group, relative to the other cohorts.

Conclusion

These findings demonstrate that the composition of the ileal pouch microbiome—both in chronic pouchitis and normally functioning IPAA—differs significantly from that of individuals with normal gut physiology. This suggests that the microbiome of a normally functioning IPAA may serve as a more appropriate baseline for comparative studies than the healthy gut microbiome. Additionally, our results indicate that antibiotic use exerts increased selective pressure on E. coli strains carrying antibiotic resistance genes, directly correlating with the types of antibiotics administered.

References

1. Hata K, Ishihara S, Nozawa H, et al. Pouchitis after ileal pouch-anal anastomosis in ulcerative colitis: Diagnosis, management, risk factors, and incidence. Dig Endosc 2017;29:26–34.
2. Shen B, Kochhar GS, Rubin DT, et al. Treatment of pouchitis, Crohn’s disease, cuffitis, and other inflammatory disorders of the pouch: consensus guidelines from the International Ileal Pouch Consortium. Lancet Gastroenterol Hepatol 2021;7:69–95.
3. Herfarth H, Barnes EL, Long MD, et al. Combined Endoscopic and Oral Fecal Microbiota Transplantation in Patients with Antibiotic-Dependent Pouchitis: Low Clinical Efficacy due to Low Donor Microbial Engraftment. Inflamm Intest Dis 2019;4:1–6.
4. Karjalainen EK, Renkonen-Sinisalo L, Satokari R, et al. Fecal Microbiota Transplantation in Chronic Pouchitis: A Randomized, Parallel, Double-Blinded Clinical Trial. Inflamm Bowel Dis 2021;27:1766–1772.
5. Kousgaard SJ, Cold F, Halkjær SI, et al. The effect of non-pooled multi-donor faecal microbiota transplantation for inducing clinical remission in patients with chronic pouchitis: Results from a multicentre randomised double-blinded placebo-controlled trial (MicroPouch). J Crohn’s Colitis 2024;18:1753–1766.