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.

Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn’s disease (CD), is characterized by chronic gastrointestinal inflammation. A high unmet need exists for non-invasive biomarkers in IBD to detect mucosal inflammation to monitor changes in disease severity and guide treatment decisions. Fecal proteomics has the potential to allow frequent, non-invasive monitoring of biomarkers in stool of IBD patients, however the fecal proteome remains under explored. Here a data-independent acquisition LC-MS/MS approach was used to profile the human fecal proteome in two independent cohorts of IBD patients and healthy controls (HC) to identify non-invasive biomarkers reflective of disease activity. 688 human proteins were quantified, with 523 measured in both cohorts. In UC stool 96 proteins were differentially abundant and in CD stool 126 proteins were differentially abundant compared to HC stool (absolute log2 fold change >1, p-value <0.05). Many of these fecal proteins are associated with infiltrating immune cells and ulceration/rectal bleeding, which are hallmarks of IBD pathobiology. Mapping of the identified fecal proteins to a whole blood single-cell RNA sequencing data set revealed the involvement of various immune cell subsets to the IBD fecal proteome. Findings from this study not only confirmed the presence of established fecal biomarkers for IBD, such as calprotectin and lactoferrin, but also revealed new fecal proteins from multiple pathways known to be dysregulated in IBD. These novel proteins could serve as potential non-invasive biomarkers to monitor specific aspects of IBD disease activity which could expedite clinical development of novel therapeutic targets.

Tryptase, the most abundant mast cell granule protein, is elevated in severe asthma patients independent of type 2 inflammation status. Higher active β tryptase allele counts are associated with higher levels of peripheral tryptase and lower clinical benefit from anti-IgE therapies. Tryptase is a therapeutic target of interest in severe asthma and chronic spontaneous urticaria. Active and inactive allele counts may enable stratification to assess response to therapies in asthmatic patient subpopulations. Tryptase gene loci TPSAB1 and TPSB2 have high levels of sequence identity, which makes genotyping a challenging task. Here, we report a targeted next-generation sequencing (NGS) assay and downstream bioinformatics analysis for determining polymorphisms at tryptase TPSAB1 and TPSB2 loci. Machine learning modeling using multiple polymorphisms in the tryptase loci was used to improve the accuracy of genotyping calls. The assay was tested and qualified on DNA extracted from whole blood of healthy donors and asthma patients, achieving accuracy of 96%, 96% and 94% for estimation of inactive α and βΙΙΙFS tryptase alleles and α duplication on TPSAB1, respectively. The reported NGS assay is a cost-effective method that is more efficient than Sanger sequencing and provides coverage to evaluate known as well as unreported tryptase polymorphisms.

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.

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.

Argonaute (AGO) proteins execute microRNA (miRNA)-mediated gene silencing. However, it is unclear whether all 4 mammalian AGO proteins (AGO1, AGO2, AGO3, and AGO4) are required for miRNA activity. We generate Ago1, Ago3, and Ago4-deficient mice (Ago134Δ) and find AGO1/3/4 to be redundant for miRNA biogenesis, homeostasis, or function, a role that is carried out by AGO2. Instead, AGO1/3/4 regulate the expansion of type 2 immunity via precursor mRNA splicing in CD4+ T helper (Th) lymphocytes. Gain- and loss-of-function experiments demonstrate that nuclear AGO3 interacts directly with SF3B3, a component of the U2 spliceosome complex, to aid global mRNA splicing, and in particular the isoforms of the gene Nisch, resulting in a dysregulated Nisch isoform ratio. This work uncouples AGO1, AGO3, and AGO4 from miRNA-mediated RNA interference, identifies an AGO3:SF3B3 complex in the nucleus, and reveals a mechanism by which AGO proteins regulate inflammatory diseases.

High interleukin (IL)-6 levels are associated with greater COVID-19 severity. IL-6 receptor blockade by tocilizumab (anti-IL6R; Actemra) is used globally for the treatment of severe COVID-19, yet a molecular understanding of the therapeutic benefit remains unclear. We characterized the immune profile and identified cellular and molecular pathways modified by tocilizumab in peripheral blood samples from patients enrolled in the COVACTA study, a phase 3, randomized, double-blind, placebo-controlled trial of the efficacy and safety of tocilizumab in hospitalized patients with severe COVID-19. We identified markers of inflammation, lymphopenia, myeloid dysregulation, and organ injury that predict disease severity and clinical outcomes. Proteomic analysis confirmed a pharmacodynamic effect for tocilizumab and identified novel pharmacodynamic biomarkers. Transcriptomic analysis revealed that tocilizumab treatment leads to faster resolution of lymphopenia and myeloid dysregulation associated with severe COVID-19, indicating greater anti-inflammatory activity relative to placebo and potentially leading to faster recovery in patients hospitalized with COVID-19.

Ulcerative colitis (UC) is an idiopathic chronic inflammatory disease of the colon with sharply rising global prevalence. Dysfunctional epithelial compartment (EC) dynamics are implicated in UC pathogenesis although EC-specific studies are sparse. Applying orthogonal high-dimensional EC profiling to a Primary Cohort (PC; n=222), we detail major epithelial and immune cell perturbations in active UC. Prominently, reduced frequencies of mature BEST4+OTOP2+ absorptive and BEST2+WFDC2+ secretory epithelial enterocytes were associated with the replacement of homeostatic, resident TRDC+KLRD1+HOPX+ γδ+ T cells with RORA+CCL20+S100A4+ TH17 cells and the influx of inflammatory myeloid cells. The EC transcriptome (exemplified by S100A8, HIF1A, TREM1, CXCR1) correlated with clinical, endoscopic, and histological severity of UC in an independent validation cohort (n=649). Furthermore, therapeutic relevance of the observed cellular and transcriptomic changes was investigated in 3 additional published UC cohorts (n=23, 48 and 204 respectively) to reveal that non-response to anti-Tumor Necrosis Factor (anti-TNF) therapy was associated with EC related myeloid cell perturbations. Altogether, these data provide high resolution mapping of the EC to facilitate therapeutic decision-making and personalization of therapy in patients with UC.

Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the up regulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGE(hi) myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS.

The ability of stem cells to rapidly proliferate and differentiate is integral to the steady-state maintenance of tissues with high turnover such as the blood and intestine. Mutations that alter these processes can cause primary immunodeficiencies, malignancies and defects in barrier function. The Rho-kinases, Rock1 and Rock2, regulate cell shape and cytoskeletal rearrangement, activities essential to mitosis. Here, we use inducible gene targeting to ablate Rock1 and Rock2 in adult mice, and identify an obligate requirement for these enzymes in the preservation of the hematopoietic and gastrointestinal systems. Hematopoietic cell progenitors devoid of Rho-kinases display cell cycle arrest, blocking the differentiation to mature blood lineages. Similarly, these mice exhibit impaired epithelial cell renewal in the small intestine, which is ultimately fatal. Our data reveal a novel role for these kinases in the proliferation and viability of stem cells and their progenitors, which is vital to maintaining the steady-state integrity of these organ systems.

Abstract Background While the majority of Ulcerative Colitis (UC)-related mucosal studies have focused on whole intestinal tissues or the lamina propria (LP), epithelial compartment (EC)-specific studies are largely lacking. Here, we have defined EC-associated molecular and cellular dynamics during inflammation and studied their response to anti-tumor necrosis factor inhibitor (TNFi) therapy. Methods EC-focused analyses that included total RNA sequencing (RNAseq), single-cell (sc) RNAseq, spatial transcriptomics (ST), microscopy and flow cytometry (FC) were performed in a cohort of UC patients (n=103) and healthy controls (HC, n=116); Primary Cohort (PC). Inflammation-associated signatures were validated in an internal validation cohort (VC-1; UC, n= 401; HC, n = 243). Additionally, 3 distinct validation cohorts (VC-2a, n=23; VC-3b, n=48; VC-4c, n=214) were used to determine cellular and molecular phenotypes that were associated with TNFi-treatment response. a. Gut, 2018; PMID-27802155 b. Am J Gastroenterol., 2011; PMID- 21448149 c. Lancet Gastroenterol Hepatol., 2022; PMID: 34798036 Results Total RNAseq and scRNA seq analyses and FC revealed distinct immune perturbations in the EC of patients with UC, including a major increase in neutrophils, monocyte-macrophages (MoMac) and inflammatory macrophages, while EC-resident, homeostatic gd T cells were significantly reduced (Fig 1A, B). ST identified significantly reduced frequencies of mature epithelial subtypes in UC and significantly increased co-localization between multiple cell types, including epithelial cells and myeloid cells (Fig 1C), that was confirmed by microscopy (Fig 1D). A signature of 255 EC inflammation-associated genes was derived that reversed with TNFi. This included treatment associated reduced expression of genes such as CSF3R, FCGR3B, MZB1, PDPN, TREM, FPR2 with a concomitant increased expression of genes like BEST4, CA2, SLC16A1, UGT1A10 (Fig 1E). Interrogation of UC-associated inflammatory cell types within VC-2, VC-3, and VC-4 demonstrated that reduction in neutrophils, MoMac, macrophages, DCs and plasma cells and an increase in epithelial cells was associated with TNFi response. Furthermore, early (W8-W10) reductions in myeloid cell- and plasma cell-associated genes was associated with TNFi response (Fig 1F, G). Conclusion Detailed multiomic characterization of the EC in UC reveals myeloid cell-, plasma cell- and epithelial cell-associated modules that are associated with non-response to TNFi and charts a course to define rational drug sequencing and combinations in UC.