Comprehensive profiling of the human fecal proteome from IBD patients with DIA-MS enables evaluation of disease-relevant proteins.

PURPOSE: 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 noninvasive biomarkers in IBD to monitor changes in disease activity and guide treatment decisions. Stool is an easily accessed, disease proximal matrix in IBD, however the composition of the IBD fecal proteome remains poorly characterized. EXPERIMENTAL DESIGN: A data-independent acquisition LC-MS/MS approach was used to profile the human fecal proteome in two independent cohorts (Cohort 1: healthy n = 5, UC n = 5, CD n = 5, Cohort 2: healthy n = 20, UC n = 10, and CD n = 10) to identify noninvasive biomarkers reflective of disease activity. RESULTS: 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 healthy 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 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. CONCLUSIONS AND CLINICAL RELEVANCE: 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 noninvasive biomarkers to monitor specific aspects of IBD disease activity which could expedite clinical development of novel therapeutic targets.

A targeted amplicon next-generation sequencing assay for tryptase genotyping to support personalized therapy in mast cell-related disorders.

Tryptase, the most abundant mast cell granule protein, is elevated in severe asthma patients independent of type 2 inflammation status. Higher active ß 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 ßΙΙΙFS 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.

Argonaute3-SF3B3 complex controls pre-mRNA splicing to restrain type 2 immunity.

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.

A myeloid program associated with COVID-19 severity is decreased by therapeutic blockade of IL-6 signaling.

Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated 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-RAGEhi 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.

Transcriptomic and proteomic assessment of tocilizumab response in a randomized controlled trial of patients hospitalized with COVID-19.

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.

An agonistic anti-signal regulatory protein α antibody for chronic inflammatory diseases.

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.

Myeloid cell influx into the colonic epithelium is associated with disease severity and non-response to anti-Tumor Necrosis Factor Therapy in patients with Ulcerative Colitis.

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.

Obligate role for Rock1 and Rock2 in adult stem cell viability and function.

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.

Dual antibody inhibition of KLK5 and KLK7 for Netherton syndrome and atopic dermatitis.

The epidermis is a barrier that prevents water loss while keeping harmful substances from penetrating the host. The impermeable cornified layer of the stratum corneum is maintained by balancing continuous turnover driven by epidermal basal cell proliferation, suprabasal cell differentiation, and corneal shedding. The epidermal desquamation process is tightly regulated by balance of the activities of serine proteases of the Kallikrein-related peptidases (KLK) family and their cognate inhibitor lymphoepithelial Kazal type-related inhibitor (LEKTI), which is encoded by the serine peptidase inhibitor Kazal type 5 gene. Imbalance of proteolytic activity caused by a deficiency of LEKTI leads to excessive desquamation due to increased activities of KLK5, KLK7, and KLK14 and results in Netherton syndrome (NS), a debilitating condition with an unmet clinical need. Increased activity of KLKs may also be pathological in other dermatoses such as atopic dermatitis (AD). Here, we describe the discovery of inhibitory antibodies against murine KLK5 and KLK7 that could compensate for the deficiency of LEKTI in NS. These antibodies are protective in mouse models of NS and AD and, when combined, promote improved skin barrier integrity and reduced inflammation. To translate these findings, we engineered a humanized bispecific antibody capable of potent inhibition of human KLK5 and KLK7. A crystal structure of KLK5 bound to the inhibitory Fab revealed that the antibody binds distal to its active site and uses a relatively unappreciated allosteric inhibition mechanism. Treatment with the bispecific anti-KLK5/7 antibody represents a promising therapy for clinical development in NS and other inflammatory dermatoses.

Regulation and Role of αE Integrin and Gut Homing Integrins in Migration and Retention of Intestinal Lymphocytes during Inflammatory Bowel Disease.

Targeting interactions between α4ß7 integrin and endothelial adhesion molecule MAdCAM-1 to inhibit lymphocyte migration to the gastrointestinal tract is an effective therapy in inflammatory bowel disease (IBD). Following lymphocyte entry into the mucosa, a subset of these cells expresses αEß7 integrin, which is expressed on proinflammatory lymphocytes, to increase cell retention. The factors governing lymphocyte migration into the intestinal mucosa and αE integrin expression in healthy subjects and IBD patients remain incompletely understood. We evaluated changes in factors involved in lymphocyte migration and differentiation within tissues. Both ileal and colonic tissue from active IBD patients showed upregulation of ICAM-1, VCAM-1, and MAdCAM-1 at the gene and protein levels compared with healthy subjects and/or inactive IBD patients. ß1 and ß7 integrin expression on circulating lymphocytes was similar across groups. TGF-ß1 treatment induced expression of αE on both ß7+ and ß7- T cells, suggesting that cells entering the mucosa independently of MAdCAM-1/α4ß7 can become αEß7+ ITGAE gene polymorphisms did not alter protein induction following TGF-ß1 stimulation. Increased phospho-SMAD3, which is directly downstream of TGF-ß, and increased TGF-ß-responsive gene expression were observed in the colonic mucosa of IBD patients. Finally, in vitro stimulation experiments showed that baseline ß7 expression had little effect on cytokine, chemokine, transcription factor, and effector molecule gene expression in αE+ and αE- T cells. These findings suggest cell migration to the gut mucosa may be altered in IBD and α4ß7-, and α4ß7+ T cells may upregulate αEß7 in response to TGF-ß once within the gut mucosa.

Dual targeting of lymphocyte homing and retention through α4ß7 and αEß7 inhibition in inflammatory bowel disease.

Anti-integrins are therapeutically effective for inflammatory bowel disease, yet the relative contribution of α4ß7 and αEß7 to gut lymphocyte trafficking is not fully elucidated. Here, we evaluate the effect of α4ß7 and αEß7 blockade using a combination of murine models of gut trafficking and longitudinal gene expression analysis in etrolizumab-treated patients with Crohn's disease (CD). Dual blockade of α4ß7 and αEß7 reduces CD8+ T cell accumulation in the gut to a greater extent than blockade of either integrin alone. Anti-αEß7 reduces epithelial:T cell interactions and promotes egress of activated T cells from the mucosa into lymphatics. Inflammatory gene expression is greater in human intestinal αEß7+ T cells. Etrolizumab-treated patients with CD display a treatment-specific reduction in inflammatory and cytotoxic intraepithelial lymphocytes (IEL) genes. Concurrent blockade of α4ß7 and αEß7 promotes reduction of cytotoxic IELs and inflammatory T cells in the gut mucosa through a stepwise inhibition of intestinal tissue entry and retention.

Identification of an IL-22-Dependent Gene Signature as a Pharmacodynamic Biomarker.

Interleukin-22 (IL-22) plays a role in epithelial barrier function and repair, and may provide benefits in conditions like inflammatory bowel disease. However, limited human data are available to assess the clinical effect of IL-22 administration. This study used a human intestinal cell line to identify an IL-22-dependent gene signature that could serve as a pharmacodynamic biomarker for IL-22 therapy. The response to IL-22Fc (UTTR1147A, an Fc-stabilized version of IL-22) was assessed in HT-29 cells by microarray, and the selected responsive genes were confirmed by qPCR. HT-29 cells demonstrated dose-dependent increases in STAT3 phosphorylation and multiple gene expression changes in response to UTTR1147A. Genes were selected that were upregulated by UTTR1147A, but to a lesser extent by IL-6, which also signals via STAT3. IL-1R1 was highly upregulated by UTTR1147A, and differential gene expression patterns were observed in response to IL-22Fc in the presence of IL-1ß. An IL-22-dependent gene signature was identified that could serve as a pharmacodynamic biomarker in intestinal biopsies to support the clinical development of an IL-22 therapeutic. The differential gene expression pattern in the presence of IL-1ß suggests that an inflammatory cytokine milieu in the disease setting could influence the clinical responses to IL-22.

Efficacy, Safety, and Pharmacodynamic Effects of the Bruton's Tyrosine Kinase Inhibitor Fenebrutinib (GDC-0853) in Systemic Lupus Erythematosus: Results of a Phase II, Randomized, Double-Blind, Placebo-Controlled Trial.

OBJECTIVE: Fenebrutinib (GDC-0853) is a noncovalent, oral, and highly selective inhibitor of Bruton's tyrosine kinase (BTK). The efficacy, safety, and pharmacodynamics of fenebrutinib in systemic lupus erythematosus (SLE) were assessed in this phase II, multicenter, randomized, placebo-controlled study. METHODS: Patients who had moderately to severely active SLE while receiving background standard therapy were randomized to receive placebo, fenebrutinib 150 mg once daily, or fenebrutinib 200 mg twice daily. Glucocorticoid taper was recommended from weeks 0 to 12 and from weeks 24 to 36. The primary end point was the SLE Responder Index 4 (SRI-4) response at week 48. RESULTS: Patients (n = 260) were enrolled from 44 sites in 12 countries, with the majority from Latin America, the US, and Western Europe. The SRI-4 response rates at week 48 were 51% for fenebrutinib 150 mg once daily (P = 0.37 versus placebo), 52% for fenebrutinib 200 mg twice daily (P = 0.34 versus placebo), and 44% for placebo. British Isles Lupus Assessment Group-based Combined Lupus Assessment response rates at week 48 were 53% for fenebrutinib 150 mg once daily (P = 0.086 versus placebo), 42% for fenebrutinib 200 mg twice daily (P = 0.879 versus placebo), and 41% for placebo. Safety results were similar across all arms, although serious adverse events were more frequent with fenebrutinib 200 mg twice daily. By week 48, patients treated with fenebrutinib had reduced levels of a BTK-dependent plasmablast RNA signature, anti-double-stranded DNA autoantibodies, total IgG, and IgM, as well as increased complement C4 levels, all relative to placebo. CONCLUSION: While fenebrutinib had an acceptable safety profile, the primary end point, SRI-4 response, was not met despite evidence of strong pathway inhibition.

Age-related changes in polycomb gene regulation disrupt lineage fidelity in intestinal stem cells.

Tissue homeostasis requires long-term lineage fidelity of somatic stem cells. Whether and how age-related changes in somatic stem cells impact the faithful execution of lineage decisions remains largely unknown. Here, we address this question using genome-wide chromatin accessibility and transcriptome analysis as well as single-cell RNA-seq to explore stem-cell-intrinsic changes in the aging Drosophila intestine. These studies indicate that in stem cells of old flies, promoters of Polycomb (Pc) target genes become differentially accessible, resulting in the increased expression of enteroendocrine (EE) cell specification genes. Consistently, we find age-related changes in the composition of the EE progenitor cell population in aging intestines, as well as a significant increase in the proportion of EE-specified intestinal stem cells (ISCs) and progenitors in aging flies. We further confirm that Pc-mediated chromatin regulation is a critical determinant of EE cell specification in the Drosophila intestine. Pc is required to maintain expression of stem cell genes while ensuring repression of differentiation and specification genes. Our results identify Pc group proteins as central regulators of lineage identity in the intestinal epithelium and highlight the impact of age-related decline in chromatin regulation on tissue homeostasis.

The peptide symporter SLC15a4 is essential for the development of systemic lupus erythematosus in murine models.

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease representing a serious unmet medical need. The disease is associated with the loss of self-tolerance and exaggerated B cell activation, resulting in autoantibody production and the formation of immune complexes that accumulate in the kidney, causing glomerulonephritis. TLR7, an important mediator of the innate immune response, drives the expression of type-1 interferon (IFN), which leads to expression of type-1 IFN induced genes and aggravates lupus pathology. Because the lysosomal peptide symporter slc15a4 is critically required for type-1 interferon production by pDC, and for certain B cell functions in response to TLR7 and TLR9 signals, we considered it as a potential target for pharmacological intervention in SLE. We deleted the slc15a4 gene in C57BL/6, NZB, and NZW mice and found that pristane-challenged slc15a4-/- mice in the C57BL/6 background and lupus prone slc15a4-/- NZB/W F1 mice were both completely protected from lupus like disease. In the NZB/W F1 model, protection persisted even when disease development was accelerated with an adenovirus encoding IFNα, emphasizing a broad role of slc15a4 in disease initiation. Our results establish a non-redundant function of slc15a4 in regulating both innate and adaptive components of the immune response in SLE pathobiology and suggest that it may be an attractive drug target.

Neutrophil serine protease 4 is required for mast cell-dependent vascular leakage.

Vascular leakage, or edema, is a serious complication of acute allergic reactions. Vascular leakage is triggered by the release of histamine and serotonin from granules within tissue-resident mast cells. Here, we show that expression of Neutrophil Serine Protease 4 (NSP4) during the early stages of mast cell development regulates mast cell-mediated vascular leakage. In myeloid precursors, the granulocyte-macrophage progenitors (GMPs), loss of NSP4 results in the decrease of cellular levels of histamine, serotonin and heparin/heparan sulfate. Mast cells that are derived from NSP4-deficient GMPs have abnormal secretory granule morphology and a sustained reduction in histamine and serotonin levels. Consequently, in passive cutaneous anaphylaxis and acute arthritis models, mast cell-mediated vascular leakage in the skin and joints is substantially reduced in NSP4-deficient mice. Our findings reveal that NSP4 is required for the proper storage of vasoactive amines in mast cell granules, which impacts mast cell-dependent vascular leakage in mouse models of immune complex-mediated diseases.

The kinase IRAK4 promotes endosomal TLR and immune complex signaling in B cells and plasmacytoid dendritic cells.

The dysregulation of multiple signaling pathways, including those through endosomal Toll-like receptors (TLRs), Fc gamma receptors (FcγR), and antigen receptors in B cells (BCR), promote an autoinflammatory loop in systemic lupus erythematosus (SLE). Here, we used selective small-molecule inhibitors to assess the regulatory roles of interleukin-1 receptor (IL-1R)-associated kinase 4 (IRAK4) and Bruton's tyrosine kinase (BTK) in these pathways. The inhibition of IRAK4 repressed SLE immune complex- and TLR7-mediated activation of human plasmacytoid dendritic cells (pDCs). Correspondingly, the expression of interferon (IFN)-responsive genes (IRGs) in cells and in mice was positively regulated by the kinase activity of IRAK4. Both IRAK4 and BTK inhibition reduced the TLR7-mediated differentiation of human memory B cells into plasmablasts. TLR7-dependent inflammatory responses were differentially regulated by IRAK4 and BTK by cell type: In pDCs, IRAK4 positively regulated NF-κB and MAPK signaling, whereas in B cells, NF-κB and MAPK pathways were regulated by both BTK and IRAK4. In the pristane-induced lupus mouse model, inhibition of IRAK4 reduced the expression of IRGs during disease onset. Mice engineered to express kinase-deficient IRAK4 were protected from both chemical (pristane-induced) and genetic (NZB/W_F1 hybrid) models of lupus development. Our findings suggest that kinase inhibitors of IRAK4 might be a therapeutic in patients with SLE.

Fenebrutinib versus Placebo or Adalimumab in Rheumatoid Arthritis: A Randomized, Double-Blind, Phase II Trial (ANDES Study).

OBJECTIVE: To evaluate fenebrutinib, an oral and highly selective non-covalent inhibitor of Bruton's tyrosine kinase (BTK), in patients with active rheumatoid arthritis (RA). METHODS: Patients with RA and inadequate response to methotrexate (cohort 1, n=480) were randomized to fenebrutinib (50 mg once daily, 150 mg once daily, 200 mg twice daily), 40 mg adalimumab every other week, or placebo. Patients with RA and inadequate response to tumor necrosis factor inhibitors (cohort 2, n=98) received fenebrutinib (200 mg twice daily) or placebo. Both cohorts continued methotrexate therapy. RESULTS: In cohort 1, American College of Rheumatology scores (ACR50) at week 12 were similar for fenebrutinib 50 mg once daily and placebo, and higher for fenebrutinib 150 mg once daily (28%) and 200 mg twice daily (35%) than placebo (15%) (p=0.017; p=0.0003). Fenebrutinib 200 mg twice daily and adalimumab (36%) were comparable (p=0.81). In cohort 2, more patients achieved ACR50 with fenebrutinib 200 mg twice daily (25%) than placebo (12%) (p=0.072). The most common adverse events for fenebrutinib included nausea, headache, anemia, and upper respiratory tract infections. Fenebrutinib had significant effects on myeloid and B cell biomarkers (CCL4 and rheumatoid factor). Fenebrutinib and adalimumab caused overlapping as well as distinct changes in B cell and myeloid biomarkers. CONCLUSION: Fenebrutinib demonstrated efficacy comparable to adalimumab in patients with an inadequate response to methotrexate, and safety consistent with existing immunomodulatory therapies for RA. These data support targeting both B and myeloid cells via this novel mechanism for potential efficacy in the treatment of RA.