Stromal cell states are altered in active Crohn's disease (CD), but their origin and phenotypic stability are unknown. Using single-cell spatial transcriptomics, RNA sequencing and ATAC sequencing of ~2,500,000 cells, we map 18 distinct stromal cell states within their cellular and cytokine signaling environments in human full-thickness CD bowel. Inflammatory fibroblasts (IFs) reside in immune cell-rich mucosal ulcers and are induced through combinatorial cytokine exposure suppressing submucosal universal fibroblast programs. The IF state is stabilized through transcription factor (TF) activity of GLI3, TWIST1, ETV4, PRDM1 and RELB, and does not spontaneously revert; however, histone deacetylase inhibition destabilizes the IF state, preventing IF secretome-induced epithelial transmigration and activation of neutrophils. The IF open chromatin configuration is distinct from that of fibrotic contractile stroma, which populate adjoining immune-depleted submucosal fibrotic niches. These findings show that microenvironments in pathological tissue niches shape open chromatin configuration of stromal states that are amenable to modulation by epigenetic modifiers.

This paper describes an end-to-end workflow for highly multiplexed fluorescence imaging with the Cell DIVE platform, allowing simultaneous detection of 40+ markers at single-cell resolution. Combining whole-slide multiplexed imaging with a dedicated analysis pipeline provides a powerful approach to investigate immune cell interactions with stromal and vascular networks within human tissue microenvironments. With a focus on spatial investigation of human immune niches, here we provide a complete framework for tissue preparation, autofluorescence reduction, multiplex panel design and whole-slide image analysis. For complete details on the use and execution of this protocol, please refer to Korsunsky et al. (Med, 2022) [[1][1]]. Highlights ![Figure][2] ### Competing Interest Statement F.G., A.C., E.M., and C.S. are employees at GE HealthCare. C.D.B. and M.C.C. are founders of Mestag and hold equity in the company. All other authors declare no competing interests Research into Inflammatory Arthritis Centre Versus Arthritis UK, 22072 F. Hoffmann-La Roche (Roche) Medical Research Council, MR/S025308/1, MR/X012093/1, MR/S035850/1, MR/W025981/1 Wellcome Trust Collaborative Award in Science, HMR05310 Kennedy Trust for Rheumatology Research, KENN161704, KENN222310 [1]: #ref-1 [2]: pending:yes

BACKGROUND AND AIMS:Obesity is a risk factor for both Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). However, it is unclear whether obesity drives the malignant progression of BE. We aimed to assess whether obesity is associated with high-grade dysplasia (HGD) or cancer in patients with BE. METHODS:We searched MEDLINE and EMBASE from inception through April 2024 for studies reporting the effect of body mass index (BMI) on the progression of nondysplastic BE or low-grade dysplasia (LGD) to HGD or EAC. A 2-stage dose-response meta-analysis was performed to estimate the dose-response relationship between BMI with malignant progression. Study quality was appraised using a modified Newcastle-Ottawa scale. RESULTS:Twenty studies reported data on 38,565 patients (74.4% male) in total, of whom 1684 patients were diagnosed with HGD/cancer. Nineteen studies were considered moderate to high quality. Eight cohort studies reported data on 6647 male patients with baseline nondysplastic BE/LGD, of whom 555 progressed to HGD/EAC (pooled annual rate of progression, 0.02%; 95% confidence interval [CI], 0.01%-0.03%), and 1992 female patients with baseline nondysplastic BE/LGD, with 110 progressors (pooled annual rate of progression, 0.01%; 95% CI, 0.01%-0.02%). There was no significant difference in pooled annual rate of progression between males and females (P = .15). Each 5-kg/m2 increase in BMI was associated with a 6% increase in the risk of malignant progression (adjusted odds ratio, 1.06; 95% CI, 1.02-1.10; P < .001; I2= 0%). CONCLUSION:Our meta-analysis provides some evidence that obesity as measured by BMI is associated with malignant progression of BE with a dose-response relationship. This finding requires confirmation in future high-quality cohort studies. Future risk prediction models could incorporate measures of obesity to potentially improve risk stratification in patients with BE. PROSPERO, Number: CRD42017051046.

Immune-mediated inflammatory diseases (IMIDs) are responsible for substantial global disease burden and associated health-care costs. Traditional models of research and service delivery silo their management within organ-based medical disciplines. Very often patients with disease in one organ have comorbid involvement in another, suggesting shared pathogenic pathways. Moreover, different IMIDs are often treated with the same drugs (including glucocorticoids, immunoregulators and biologics). Unlocking the cellular basis of these diseases remains a major challenge, leading us to ask why, if these diseases have so much in common, they are not investigated in a common manner. A tissue-based, cellular understanding of inflammation might pave the way for cross-disease, cross-discipline basket trials (testing one drug across two or more diseases) to reduce the risk of failure of early-phase drug development in IMIDs. This new approach will enable rapid assessment of the efficacy of new therapeutic agents in cross-disease translational research in humans.

Single-cell multiomic analysis of the epigenome, transcriptome, and proteome allows for comprehensive characterization of the molecular circuitry that underpins cell identity and state. However, the holistic interpretation of such datasets presents a challenge given a paucity of approaches for systematic, joint evaluation of different modalities. Here, we present Panpipes, a set of computational workflows designed to automate multimodal single-cell and spatial transcriptomic analyses by incorporating widely-used Python-based tools to perform quality control, preprocessing, integration, clustering, and reference mapping at scale. Panpipes allows reliable and customizable analysis and evaluation of individual and integrated modalities, thereby empowering decision-making before downstream investigations.

Precision medicine in immune-mediated inflammatory diseases (IMIDs) requires an understanding of how cellular networks change following therapy. We describe a therapeutic atlas for Crohn’s disease (CD) and ulcerative colitis (UC) following anti-tumour necrosis factor (TNF) therapy. We generated ~1 million single-cell transcriptomes, organised into 109 cell states, from 216 gut biopsies from 38 patients and three controls, revealing disease- and therapy-specific differences. A systems-biology analysis identified distinct spatially-resolved cellular microenvironments: granuloma signatures in CD and interferon (IFN)-response signatures localising to T-cell aggregates and epithelial damage in CD and UC. Longitudinal comparisons demonstrated that disease progression in non-responders associated with myeloid and stromal cell perturbations in CD and increased multi-cellular IFN signalling in UC. IFN signalling was also observed in rheumatoid arthritis (RA) synovium with a lymphoid pathotype. Our therapeutic atlas informs drug positioning across IMIDs, and suggests a rationale for the use of janus kinase (JAK) inhibition following anti-TNF resistance.

CD4 T helper cells (TH cells) play a vital role in coordinating and amplifying the immune response to specific pathogens. They constitutively produce different kinds of extracellular vesicles (EVs), which mediate cell-to cell communication and play diverse roles in immune regulation and inflammatory processes. Here we provide a resource documenting the composition of activated TH cell EVs and demonstrating their ability to instigate pro-inflammatory response in antigen-presenting cells (APCs). EVs were characterized by lipidomics, proteomics, and NanoFCM. The activated TH cells derived EVs (act-EVs) were found to be enriched in TH cell-specific proteins, transmembrane and cytosolic EV marker proteins, and HLA proteins relative to resting CD4 T cells EVs (rest-EVs). The pro-inflammatory effect of act-EVs vs rest-EVs on donor matched APCs were characterized by chemokine and cytokine profiling and flow cytometry analysis. There was no discernible contrast in endotoxin levels between act-EVs and rest-EVs. Functional distinctions were seen to arise from variations in the content and composition of these EVs. Moreover, we validated our findings with an in-vivo investigation in mice, demonstrating the recruitment of monocytes, dendritic cells (DCs), neutrophils, and NK cells in the spleen, accompanied by the release of pro-inflammatory cytokines in the serum after administering act-EVs. In summary, this study sheds light on the role of TH cell released EVs in modulating the immune response during pro-inflammatory responses and this resource provides a foundation for development of novel therapeutics on EV based scaffolds. ### Competing Interest Statement Ashwin K. Jainarayanan and Michael L. Dustin are founders at Granza Bio Limited. Mariana Conceicao is an Associate Research Director (AAV) at Evox Therapeutics Ltd.

Ulcerative colitis (UC) and Crohn’s disease (CD) are complex and heterogenous diseases characterized by chronic and progressive inflammation of the digestive tract. Single cell RNA sequencing combined with computational biology provides unprecedented insights into disease biology, informing novel therapeutic targets and precision medicine approaches. To identify potential mediators of persistent inflammation in inflammatory bowel disease (IBD), we performed single cell RNA sequencing on tissue samples collected from UC and CD patients before and after beginning adalimumab. In this study, 41 participants, including 21 UC patients, 17 CD patients and 3 healthy individuals, were enrolled at the University of Oxford’s Translational Gastroenterology Unit and Kennedy Institute of Rheumatology. Patients had moderate-to-severe disease based on symptoms and endoscopy, were biologic naïve, and planned to initiate adalimumab. Mucosal biopsies for single cell RNA sequencing were collected from multiple anatomic locations for each subject before beginning and after a minimum of 3 months of adalimumab, at which time treatment response was assessed. During the study, clinical response as defined by composite measures was observed in 8 UC and 9 CD patients. Differential cell composition and gene expression analysis of the single cell data revealed inflammatory monocyte subtypes as highly enriched in inflamed relative to uninflamed samples, and this cellular subset remained highly elevated in inflamed samples from adalimumab non-responders. These cells were characterized by expression of multiple cell-surface receptors with potential to drive pathology, including TREM1 (Triggering Receptor Expressed on Myeloid Cells 1), a transmembrane receptor that amplifies anti-bacterial neutrophil and monocyte mediated responses. TREM1+ inflammatory monocytes were highly enriched in UC and CD inflamed samples (Fig 1), both prior to adalimumab and in adalimumab non-responders. The enrichment of TREM1+ cells was specific to inflamed IBD tissue and was not observed in non-inflamed tissue from IBD patients or healthy subjects; enrichment of TREM1+ cells was also not observed in circulating peripheral blood mononuclear cells. TREM1+ neutrophils and monocytes in the gut mucosa amplify innate immune responses to bacteria in the context of epithelial barrier dysfunction and bacterial dysbiosis in IBD. When activated, TREM1 signaling induces multiple soluble mediators relevant for IBD pathology. Our single cell RNA sequencing data provide further evidence that TREM1 is an exciting therapeutic target for UC and CD, with inhibition of TREM1 signaling expected to address pathology while maintaining an anti-bacterial response. We therefore developed a highly potent and selective monoclonal antibody inhibitor of TREM-1, CEL383, with the Phase 1 clinical trial expected to start in 2023. Figure 1. tSNE plot of myeloid cells from inflamed IBD tissue samples demonstrating high TREM1 expression across inflammatory monocytes

Abstract Background Ulcerative colitis (UC) and Crohn’s disease (CD) are complex and heterogenous diseases characterized by chronic, progressive inflammation of the digestive tract. Single cell RNA sequencing combined with computational biology provides unprecedented insights into disease biology, informing novel therapeutic targets and precision medicine approaches. To identify potential mediators of persistent inflammation in inflammatory bowel disease (IBD), we performed single cell RNA sequencing on tissue samples collected from UC and CD patients before and after beginning adalimumab. Methods Forty-one participants, including 21 UC patients, 17 CD patients and 3 healthy individuals, were enrolled at the University of Oxford’s Translational Gastroenterology Unit and Kennedy Institute of Rheumatology. Patients had moderate-to-severe disease based on symptoms and endoscopy, were biologic naïve, and planned to initiate adalimumab. Mucosal biopsies for single cell RNA sequencing were collected for each subject before beginning and after a minimum of 3 months of adalimumab, at which time treatment response was assessed. During the study, clinical response as defined by composite measures was observed in 8 UC and 9 CD patients. Results Differential cell composition and gene expression analysis of single cell data revealed inflammatory monocyte subtypes as highly enriched in inflamed relative to uninflamed samples. This cellular subset remained highly elevated in inflamed samples from adalimumab non-responders. These cells were characterized by expression of multiple cell-surface receptors with potential to drive pathology, including TREM1 (Triggering Receptor Expressed on Myeloid Cells 1), a transmembrane receptor that amplifies anti-bacterial neutrophil and monocyte mediated responses. TREM1+ inflammatory monocytes were highly enriched in UC and CD inflamed samples (Fig. 1), both prior to adalimumab and in adalimumab non-responders. The enrichment of TREM1+ cells was specific to inflamed IBD tissue and was not observed in non-inflamed tissue from IBD patients or healthy subjects; enrichment of TREM1+ cells was also not observed in circulating peripheral blood mononuclear cells. Conclusion TREM1+ neutrophils and monocytes in the gut mucosa amplify innate immune responses to bacteria in the context of epithelial barrier dysfunction and bacterial dysbiosis in IBD. When activated, TREM1 signaling induces multiple soluble mediators relevant for IBD pathology. Our single cell RNA sequencing data provide further evidence that TREM1 is an exciting therapeutic target for UC and CD. We therefore developed a highly potent and selective monoclonal antibody inhibitor of TREM-1, CEL383, with the Phase 1 clinical trial expected to start in 2023.

CD8+ T lymphocytes play vital roles in killing infected or deranged host cells, recruiting innate immune cells, and regulating other aspects of immune responses. Like any other cell, CD8+ T cells also produce extracellular particles. These include extracellular vesicles (EVs) and non-vesicular extracellular particles (NVEPs). T cell-derived EVs are proposed to mediate cell-to-cell signalling, especially in the context of inflammatory responses, autoimmunity, and infectious diseases. CD8+ T cells also produce supramolecular attack particles (SMAPs), which are in the same size range as EVs and mediate a component of T cell mediated killing. The isolation technique selected will have a profound effect on yield, purity, biochemical properties and function of T cell-derived particles; making it important to directly compare different approaches. In this study, we compared commonly used techniques (membrane spin filtration, ultracentrifugation, or size exclusion liquid chromatography) to isolate particles from activated human CD8+ T cells and validated our results by single-particle methods, including nanoparticle tracking analysis, flow cytometry, electron microscopy and super-resolution microscopy of the purified sample as well as bulk proteomics and lipidomics analyses to evaluate the quality and nature of enriched T cell-derived particles. Our results show that there is a trade-off between the yield and the quality of T cell-derived particles. Furthermore, the protein and lipid composition of the particles is dramatically impacted by the isolation technique applied. We conclude that from the techniques evaluated, size exclusion liquid chromatography offers the highest quality of T cell derived EVs and SMAPs with acceptable yields for compositional and functional studies.

Foxp3+ regulatory T (Treg) cells are essential for maintaining immune homeostasis. Breakdown of homeostasis can lead to inflammatory bowel disease (IBD), a condition characterised by chronic inflammation of the gastrointestinal tract. Changes in the composition and function of Treg subsets can underlie IBD in some patients. Moreover, anti-TNF, the most common first-line biologic in the treatment of IBD, may mediate its anti-inflammatory effects through modulating Treg responses. However, up to 40% of patients with IBD exhibit primary non-responsiveness to anti-TNF therapy. The molecular and cellular mechanisms associated with anti-TNF resistance have yet to be fully elucidated. Furthermore, it is not clear whether Treg responses contribute to this state. Herein, we used multi-modal profiling of Tregs from both the tissue and blood of patients with IBD before and after anti-TNF therapy. In contrast to non-responders to therapy, we found increased PD-1 expression on circulating HLA-DRB1hiTregs from patients responding to anti-TNF. Anti-TNF induced IL-27 production by circulating and tissue mononuclear phagocytes (MNPs) through Fc gamma receptor activation, and in turn increased the expression of PD-1 on Tregs in a STAT3-dependent manner. Expression of selected genes known to be up-regulated upon IL-27 stimulation was increased after anti-TNF therapy in circulating Tregs from responders. We further demonstrated that IL-27 augmented the functional suppressive capacity of Tregs from responders but not from non-responders. Together, these data reveal anti-TNF therapy-induced MNP-Treg interaction and provide evidence that the IL-27/PD-1+Treg axis is associated with anti-TNF response in IBD.ONE SENTENCE SUMMARY Anti-TNF response in IBD is linked to an increase of PD-1 expression on circulating Tregs mediated by IL-27 induction via Fc gamma receptor activation.### Competing Interest StatementFMP received research support from Roche and Janssen, and consulting fees from GSK, Novartis and Genentech. CDB received research support from Roche, Janssen, Celsius Therapeutics, and consulting fees from GSK. HHU received research support or consultancy fees from Janssen, Eli Lilly, UCB Pharma, BMS/Celgene, MiroBio, Mestag and Omass. TT received research support from Celsius Therapeutics and consulting fees from AbbVie and ZuraBio. ST received research support from AbbVie, Buhlmann, Celgene, IOIBD, Janssen, Lilly, Pfizer, Takeda, UCB, Vifor and the Norman Collisson Foundation; consulting fees from AbbVie, Allergan, Αbiomics, Amgen, Arena, Asahi, Astellas, Biocare, Biogen, Boehringer Ingelheim, Bristol-Myers Squibb, Buhlmann, Celgene, Chemocentryx, Cosmo, Enterome, Ferring, Giuliani SpA, GSK, Genentech, Immunocore, Immunometabolism, Indigo, Janssen, Lexicon, Lilly, Merck, MSD, Neovacs, Novartis, NovoNordisk, NPS Pharmaceuticals, Pfizer, Proximagen, Receptos, Roche, Sensyne, Shire, Sigmoid Pharma, SynDermix, Takeda, Theravance, Tillotts, Topivert, UCB, Vhsquared, Vifor and Zeria; and speaker fees from AbbVie, Amgen, Biogen, Ferring, Janssen, Pfizer, Shire, Takeda and UCB (no stocks or share options). All other authors declare no competing interests.

Progressive multiple sclerosis (MS) is driven by demyelination, neuroaxonal loss, and mitochondrial damage occurring behind a closed blood-brain barrier (BBB). 1,2 Patients with progressive MS typically fail to respond to available immunomodulatory drugs that reduce relapses in early disease. 2 This indicates a dire need to identify non-canonical therapeutic avenues to limit neurodegeneration and promote protection and repair. 3 Here, we have employed high-resolution multiomic profiling to characterise the biochemical and metabolic adaptations underpinning MS pathology, as these have been incompletely described but critically, may be amenable to BBB-permeable drug targeting. Using synchrotron radiation (SR)- and focal plane array (FPA)-based Fourier transform infrared microspectroscopy (μFTIR), we spatially mapped the biochemical features present in human progressive MS and control post-mortem brain and rare spinal cord tissue. By employing single-nuclear RNA sequencing (snRNA-seq), 10x Genomics Visium spatial transcriptomics and spatial proteomics to resolve their cellular context, we found that these biochemical features provide a uniquely and highly disease-specific barcode for distinct pathological niches within the tissue. Characterisation of the metabolic processes underpinning these niches revealed an associated re-organisation of the astrocytic landscape in the grey and white matter, with implications for the treatment of progressive MS.

ObjectivesOphthalmic conditions including anterior uveitis (AU), episcleritis and scleritis may occur in association with the inflammatory bowel diseases (IBD) as ophthalmic extraintestinal manifestations. The aim of this study was to assess the risk of a later IBD diagnosis in those presenting with IBD associated ocular inflammation (IAOI).DesignRetrospective cohort study.SettingPrimary care UK database.Participants38 805 subjects with an IAOI were identified (median age 51 (38–65), 57% women) and matched to 153 018 subjects without IAOI.MeasuresThe risk of a subsequent diagnosis of IBD in subjects with IAOIs compared with age/sex matched subjects without IAOI. HRs were adjusted for age, sex, body mass index, deprivation, comorbidity, smoking, baseline axial arthropathy, diarrhoea, loperamide prescription, anaemia, lower gastrointestinal bleeding and abdominal pain.Logistic regression was used to produce a prediction model for a diagnosis of IBD within 3 years of an AU diagnosis.Results213 (0.6%) subsequent IBD diagnoses (102 ulcerative colitis (UC) and 111 Crohn’s disease (CD)) were recorded in those with IAOIs and 329 (0.2%) (215 UC and 114 CD) in those without. Median time to IBD diagnosis was 882 (IQR 365–2043) days in those with IAOI and 1403 (IQR 623–2516) in those without. The adjusted HR for a subsequent diagnosis of IBD was 2.25 (95% CI 1.89 to 2.68), p<0.001; for UC 1.65 (95% CI 1.30 to 2.09), p<0.001; and for CD 3.37 (95% CI 2.59 to 4.40), p<0.001 in subjects with IAOI compared with those without.Within 3 years of an AU diagnosis, 84 (0.5%) subjects had a recorded diagnosis of IBD. The prediction model performed well with a C-statistic of 0.75 (95% CI 0.69 to 0.80).ConclusionsSubjects with IAOI have a twofold increased risk of a subsequent IBD diagnosis. Healthcare professionals should be alert for potential signs and symptoms of IBD in those presenting with ophthalmic conditions associated with IBD.

Current inflammatory bowel disease (IBD) therapies are ineffective in a high proportion of patients. Combining bulk and single-cell transcriptomics, quantitative histopathology and in situ localization across three cohorts of patients with IBD (total n = 376), we identify coexpressed gene modules within the heterogeneous tissular inflammatory response in IBD that map to distinct histopathological and cellular features (pathotypes). One of these pathotypes is defined by high neutrophil infiltration, activation of fibroblasts and vascular remodeling at sites of deep ulceration. Activated fibroblasts in the ulcer bed display neutrophil-chemoattractant properties that are IL-1R, but not TNF, dependent. Pathotype-associated neutrophil and fibroblast signatures are increased in nonresponders to several therapies across four independent cohorts (total n = 343). The identification of distinct, localized, tissular pathotypes will aid precision targeting of current therapeutics and provides a biological rationale for IL-1 signaling blockade in ulcerating disease.