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Abstract Introduction Primary angiosarcoma of the brain is extremely rare; only 15 cases have been reported in adults over the last 25 years. Case presentations We describe two cases of primary angiosarcoma of the brain that are well characterized by imaging, histopathology, and immunohistochemistry. Case 1: our first patient was a 35-year-old woman who developed exophthalmos. Subtotal resection of a left extra-axial retro-orbital mass was performed. Case 2: our second patient was a 47-year-old man who presented to our facility with acute visual loss, word-finding difficulty and subtle memory loss. A heterogeneously-enhancing left sphenoid wing mass was removed. We also review the literature aiming at developing a rational approach to diagnosis and treatment, given the rarity of this entity. Conclusions Gross total resection is the standard of care for primary angiosarcoma of the brain. Adjuvant radiation and chemotherapy are playing increasingly recognized roles in the therapy of these rare tumors.

Abstract Background In higher eukaryotes DNA methylation regulates important biological functions including silencing of gene expression and protection from adverse effects of retrotransposons. In the protozoan parasite Entamoeba histolytica, a DNA methyltransferase has been identified and treatment with 5-azacytidine (5-AzaC), a potent inhibitor of DNA methyltransferase, has been reported to attenuate parasite virulence. However, the overall extent of DNA methylation and its subsequent effects on global gene expression in this parasite are currently unknown. Results In order to identify the genome-wide effects of DNA methylation in E. histolytica, we used a short oligonucleotide microarray representing 9,435 genes (~95% of all annotated amebic genes) and compared the expression profile of E. histolytica HM-1:IMSS parasites with those treated with 23 μM 5-AzaC for up to one week. Overall, 2.1% of genes tested were transcriptionally modulated under these conditions. 68 genes were upregulated and 131 genes down regulated (2-fold change; p-value < 0.05). Sodium-bisulfite treatment and sequencing of genes indicated that there were at least two subsets of genes with genomic DNA methylation in E. histolytica: (i) genes that were endogenously silenced by genomic DNA methylation and for which 5-AzaC treatment induced transcriptional de-repression, and (ii) genes that have genomic DNA methylation, but which were not endogenously silenced by the methylation. We identified among the genes down regulated by 5-AzaC treatment a cysteine proteinase (2.m00545) and lysozyme (52.m00148) both of which have known roles in amebic pathogenesis. Decreased expression of these genes in the 5-AzaC treated E. histolytica may account in part for the parasites reduced cytolytic abilities. Conclusion This work represents the first genome-wide analysis of DNA-methylation in Entamoeba histolytica and indicates that DNA methylation has relatively limited effects on gene expression in this parasite.

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., (© 2024 Wiley‐VCH GmbH.)

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., Competing Interests: At the time of the study, all authors were employees of Roche. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 (Ago134 Δ ) 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., Competing Interests: Declaration of interests All authors, except J.G., S.E.H., and Y.J.Z., are current or past employees of Genentech, Inc., and members of the Roche group and may hold Roche stock or stock options., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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-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., Competing Interests: J.A.H., H.S., J.V.H., C.O., L.O., X.G., N.W., A.Q., D.C., A.C, D.F.C., T.R., J.M.M., F.C., A.T., M.B., L.T., A.R., S.B.K., R.N.B., and C.M.R. were employees of Genentech, Inc. at the time of this study and own equity in Roche. The COMET study was supported in part by Genentech funding. C.J.Y. is a scientific advisory board member for and holds equity in Related Sciences and ImmunAI, a consultant for and holds equity in Maze Therapeutics, and a consultant for TRex Bio. C.J.Y. has received research support from Chan Zuckerberg Initiative, Chan Zuckerberg Biohub, and Genentech. C.S.C. has received research funding from Roche-Genentech for an unrelated project as well as from NIH, DOD, and Quantum Leap Healthcare Collaborative. C.S.C. is a consultant for Vasomune, Quark, and GEn1E Lifesciences. C.H. is a consultant for Spring Discovery but does not have any financial interest in the company nor is the work related to what is covered in this manuscript. A.R. is a co-founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas Therapeutics and, until 31 July 2020, was a scientific advisory board member of Thermo Fisher Scientific, Syros Pharmaceuticals, Asimov, and Neogene Therapeutics. A.R. is a named inventor on multiple patents related to single-cell and spatial genomics filed by or issued to the Broad Institute., (© 2023 The Authors.)

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., Competing Interests: Declaration of interests All authors except D.A.F. are current or past employees of Genentech, a member of the Roche group, and may hold Roche stock or stock options., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: H.S., J.A.H., C.M.R., A.Q., O.O., J.M., F.C., M.B., L.T., A.R., and R.N.B. are employees of Roche/Genentech and hold stock and/or stock options in Roche/Genentech. A.T. is an employee of Roche/Genentech. F.C. has a patent pending to Genentech for biomarkers for predicting a response to an interleukin (IL)-6 antagonist (P36367-US). L.T. is an author of a patent Method for Treating Pneumonia, including COVID-19 Pneumonia with an IL-6 Antagonist pending, owned by Genentech/Roche. A.R. is a co-founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas Therapeutics and, until July 31, 2020, was a scientific advisory board member of ThermoFisher Scientific, Syros Pharmaceuticals, Asimov, and Neogene Therapeutics. A.R. is a named inventor on multiple patents related to single cell and spatial genomics filed by or issued to the Broad Institute. I.O.R. has nothing to declare., (© 2023 The Authors.)

Bacille Calmette-Guerin (BCG), the only approved Mycobacterium tuberculosis (Mtb) vaccine, provides limited durable protection when administered intradermally. However, recent work revealed that intravenous (i.v.) BCG administration yielded greater protection in macaques. Here, we perform a dose-ranging study of i.v. BCG vaccination in macaques to generate a range of immune responses and define correlates of protection. Seventeen of 34 macaques had no detectable infection after Mtb challenge. Multivariate analysis incorporating longitudinal cellular and humoral immune parameters uncovered an extensive and highly coordinated immune response from the bronchoalveolar lavage (BAL). A minimal signature predicting protection contained four BAL immune features, of which three remained significant after dose correction: frequency of CD4 T cells producing TNF with interferon γ (IFNγ), frequency of those producing TNF with IL-17, and the number of NK cells. Blood immune features were less predictive of protection. We conclude that CD4 T cell immunity and NK cells in the airway correlate with protection following i.v. BCG., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

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., Competing Interests: Authors are/were employees of Genentech when the studies were conducted., (© 2023 The Authors.)

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.

B cells, which are critical for intestinal homeostasis, remain understudied in ulcerative colitis (UC). In this study, we recruited three cohorts of patients with UC (primary cohort, n = 145; validation cohort 1, n = 664; and validation cohort 2, n = 143) to comprehensively define the landscape of B cells during UC-associated intestinal inflammation. Using single-cell RNA sequencing, single-cell IgH gene sequencing and protein-level validation, we mapped the compositional, transcriptional and clonotypic landscape of mucosal and circulating B cells. We found major perturbations within the mucosal B cell compartment, including an expansion of naive B cells and IgG + plasma cells with curtailed diversity and maturation. Furthermore, we isolated an auto-reactive plasma cell clone targeting integrin αvβ6 from inflamed UC intestines. We also identified a subset of intestinal CXCL13-expressing TFH-like T peripheral helper cells that were associated with the pathogenic B cell response. Finally, across all three cohorts, we confirmed that changes in intestinal humoral immunity are reflected in circulation by the expansion of gut-homing plasmablasts that correlates with disease activity and predicts disease complications. Our data demonstrate a highly dysregulated B cell response in UC and highlight a potential role of B cells in disease pathogenesis., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Targeting interactions between α4β7 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 αEβ7 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. β1 and β7 integrin expression on circulating lymphocytes was similar across groups. TGF-β1 treatment induced expression of αE on both β7 + and β7 - T cells, suggesting that cells entering the mucosa independently of MAdCAM-1/α4β7 can become αEβ7 + ITGAE gene polymorphisms did not alter protein induction following TGF-β1 stimulation. Increased phospho-SMAD3, which is directly downstream of TGF-β, and increased TGF-β-responsive gene expression were observed in the colonic mucosa of IBD patients. Finally, in vitro stimulation experiments showed that baseline β7 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 α4β7 - , and α4β7 + T cells may upregulate αEβ7 in response to TGF-β once within the gut mucosa., (Copyright © 2021 The Authors.)

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., (© 2021, American College of Rheumatology.)

Anti-integrins are therapeutically effective for inflammatory bowel disease, yet the relative contribution of α4β7 and αEβ7 to gut lymphocyte trafficking is not fully elucidated. Here, we evaluate the effect of α4β7 and αEβ7 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 α4β7 and αEβ7 reduces CD8 + T cell accumulation in the gut to a greater extent than blockade of either integrin alone. Anti-αEβ7 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 αEβ7 + T cells. Etrolizumab-treated patients with CD display a treatment-specific reduction in inflammatory and cytotoxic intraepithelial lymphocytes (IEL) genes. Concurrent blockade of α4β7 and αEβ7 promotes reduction of cytotoxic IELs and inflammatory T cells in the gut mucosa through a stepwise inhibition of intestinal tissue entry and retention., Competing Interests: All authors, except J.G., A.H., M.D., M.S., and W.A.F., are current or past employees of Genentech, a member of the Roche group, and may hold Roche stock or stock options., (© 2021 The Authors.)

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., Competing Interests: HT, IR, JH, SC, ZM employee of Genentech Inc, MP, TR, JK, HM, BE, HJ No competing interests declared, (© 2021, Tauc et al.)

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 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&#95;F1 hybrid) models of lupus development. Our findings suggest that kinase inhibitors of IRAK4 might be a therapeutic in patients with SLE., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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., (This article is protected by copyright. All rights reserved.)

Age-related macular degeneration (AMD) is a leading cause of vision loss. To better understand disease pathogenesis and identify causal genes in GWAS loci for AMD risk, we present a comprehensive database of human retina and retinal pigment epithelium (RPE). Our database comprises macular and non-macular RNA sequencing (RNA-seq) profiles from 129 donors, a genome-wide expression quantitative trait loci (eQTL) dataset that includes macula-specific retina and RPE/choroid, and single-nucleus RNA-seq (NucSeq) from human retina and RPE with subtype resolution from more than 100,000 cells. Using NucSeq, we find enriched expression of AMD candidate genes in RPE cells. We identify 15 putative causal genes for AMD on the basis of co-localization of genetic association signals for AMD risk and eye eQTL, including the genes TSPAN10 and TRPM1. These results demonstrate the value of our human eye database for elucidating genetic pathways and potential therapeutic targets for ocular diseases., Competing Interests: Declaration of Interests L.D.O., H.-H.C., C. Cox, K.J.K., R.A., C.E., P.C., S.S.N., Y.-J.C., Z.M., A.D., L.D.G., C. Clarke, T.B., B.Y., M.J., M.J.T., M.v.L.C., and J.A.H. were employees of Genentech at the time these studies were carried out., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Mycobacterium tuberculosis (Mtb) is the leading cause of death from infection worldwide 1 . The only available vaccine, BCG (Bacillus Calmette-Guérin), is given intradermally and has variable efficacy against pulmonary tuberculosis, the major cause of mortality and disease transmission 1,2 . Here we show that intravenous administration of BCG profoundly alters the protective outcome of Mtb challenge in non-human primates (Macaca mulatta). Compared with intradermal or aerosol delivery, intravenous immunization induced substantially more antigen-responsive CD4 and CD8 T cell responses in blood, spleen, bronchoalveolar lavage and lung lymph nodes. Moreover, intravenous immunization induced a high frequency of antigen-responsive T cells across all lung parenchymal tissues. Six months after BCG vaccination, macaques were challenged with virulent Mtb. Notably, nine out of ten macaques that received intravenous BCG vaccination were highly protected, with six macaques showing no detectable levels of infection, as determined by positron emission tomography-computed tomography imaging, mycobacterial growth, pathology and granuloma formation. The finding that intravenous BCG prevents or substantially limits Mtb infection in highly susceptible rhesus macaques has important implications for vaccine delivery and clinical development, and provides a model for defining immune correlates and mechanisms of vaccine-elicited protection against tuberculosis.

Objective: To establish whether synovial pathobiology improves current clinical classification and prognostic algorithms in early inflammatory arthritis and identify predictors of subsequent biological therapy requirement., Methods: 200 treatment-naïve patients with early arthritis were classified as fulfilling RA1987 American College of Rheumatology (ACR) criteria (RA1987) or as undifferentiated arthritis (UA) and patients with UA further classified into those fulfilling RA2010 ACR/European League Against Rheumatism (EULAR) criteria. Treatment requirements at 12 months (Conventional Synthetic Disease Modifying Antirheumatic Drugs (csDMARDs) vs biologics vs no-csDMARDs treatment) were determined. Synovial tissue was retrieved by minimally invasive, ultrasound-guided biopsy and underwent processing for immunohistochemical (IHC) and molecular characterisation. Samples were analysed for macrophage, plasma-cell and B-cells and T-cells markers, pathotype classification (lympho-myeloid, diffuse-myeloid or pauci-immune) by IHC and gene expression profiling by Nanostring., Results: 128/200 patients were classified as RA1987, 25 as RA2010 and 47 as UA. Patients classified as RA1987 criteria had significantly higher levels of disease activity, histological synovitis, degree of immune cell infiltration and differential upregulation of genes involved in B and T cell activation/function compared with RA2010 or UA, which shared similar clinical and pathobiological features. At 12-month follow-up, a significantly higher proportion of patients classified as lympho-myeloid pathotype required biological therapy. Performance of a clinical prediction model for biological therapy requirement was improved by the integration of synovial pathobiological markers from 78.8% to 89%-90%., Conclusion: The capacity to refine early clinical classification criteria through synovial pathobiological markers offers the potential to predict disease outcome and stratify therapeutic intervention to patients most in need., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active β-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human β-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a β-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma., (Copyright © 2019 Elsevier Inc. All rights reserved.)

There is a current imperative to unravel the hierarchy of molecular pathways that drive the transition of early to established disease in rheumatoid arthritis (RA). Herein, we report a comprehensive RNA sequencing analysis of the molecular pathways that drive early RA progression in the disease tissue (synovium), comparing matched peripheral blood RNA-seq in a large cohort of early treatment-naive patients, namely, the Pathobiology of Early Arthritis Cohort (PEAC). We developed a data exploration website (https://peac.hpc.qmul.ac.uk/) to dissect gene signatures across synovial and blood compartments, integrated with deep phenotypic profiling. We identified transcriptional subgroups in synovium linked to three distinct pathotypes: fibroblastic pauci-immune pathotype, macrophage-rich diffuse-myeloid pathotype, and a lympho-myeloid pathotype characterized by infiltration of lymphocytes and myeloid cells. This is suggestive of divergent pathogenic pathways or activation disease states. Pro-myeloid inflammatory synovial gene signatures correlated with clinical response to initial drug therapy, whereas plasma cell genes identified a poor prognosis subgroup with progressive structural damage., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Invariant NKT (iNKT) cells in both humans and non-human primates are activated by the glycolipid antigen, α-galactosylceramide (α-GalCer). However, the extent to which the molecular mechanisms of antigen recognition and in vivo phenotypes of iNKT cells are conserved among primate species has not been determined. Using an evolutionary genetic approach, we found a lack of diversifying selection in CD1 genes over 45 million years of evolution, which stands in stark contrast to the history of the MHC system for presenting peptide antigens to T cells. The invariant T cell receptor (TCR)-α chain was strictly conserved across all seven primate clades. Invariant NKT cells from rhesus macaques (Macaca mulatta) bind human CD1D-α-GalCer tetramer and are activated by α-GalCer-loaded human CD1D transfectants. The dominant TCR-β chain cloned from a rhesus-derived iNKT cell line is nearly identical to that found in the human iNKT TCR, and transduction of the rhesus iNKT TCR into human Jurkat cells show that it is sufficient for binding human CD1D-α-GalCer tetramer. Finally, we used a 20-color flow cytometry panel to probe tissue phenotypes of iNKT cells in a cohort of rhesus macaques. We discovered several tissue-resident iNKT populations that have not been previously described in non-human primates but are known in humans, such as TCR-γδ iNKTs. These data reveal a diversity of iNKT cell phenotypes despite convergent evolution of the genes required for lipid antigen presentation and recognition in humans and non-human primates.

Objectives: To unravel the hierarchy of cellular/molecular pathways in the disease tissue of early, treatment-naïve rheumatoid arthritis (RA) patients and determine their relationship with clinical phenotypes and treatment response/outcomes longitudinally., Methods: 144 consecutive treatment-naïve early RA patients (<12 months symptoms duration) underwent ultrasound-guided synovial biopsy before and 6 months after disease-modifying antirheumatic drug (DMARD) initiation. Synovial biopsies were analysed for cellular (immunohistology) and molecular (NanoString) characteristics and results compared with clinical and imaging outcomes. Differential gene expression analysis and logistic regression were applied to define variables correlating with treatment response and predicting radiographic progression., Results: Cellular and molecular analyses of synovial tissue demonstrated for the first time in early RA the presence of three pathology groups: (1) lympho-myeloid dominated by the presence of B cells in addition to myeloid cells; (2) d iffuse-myeloid with myeloid lineage predominance but poor in B cells nd (3) pauci-immune characterised by scanty immune cells and prevalent stromal cells. Longitudinal correlation of molecular signatures demonstrated that elevation of myeloid- and lymphoid-associated gene expression strongly correlated with disease activity, acute phase reactants and DMARD response at 6 months. Furthermore, elevation of synovial lymphoid-associated genes correlated with autoantibody positivity and elevation of osteoclast-targeting genes predicting radiographic joint damage progression at 12 months. Patients with predominant pauci-immune pathology showed less severe disease activity and radiographic progression., Conclusions: We demonstrate at disease presentation, prior to pathology modulation by therapy, the presence of specific cellular/molecular synovial signatures that delineate disease severity/progression and therapeutic response and may pave the way to more precise definition of RA taxonomy, therapeutic targeting and improved outcomes., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY. Published by BMJ.)

Tuberculosis (TB) is the leading cause of death from infection worldwide. The only approved vaccine, BCG, has variable protective efficacy against pulmonary TB, the transmissible form of the disease. Therefore, improving this efficacy is an urgent priority. This study assessed whether heterologous prime-boost vaccine regimens in which BCG priming is boosted with either (i) protein and adjuvant (M72 plus AS01 E or H56 plus CAF01) delivered intramuscularly (IM), or (ii) replication-defective recombinant adenovirus serotype 5 (Ad5) expressing various Mycobacterium tuberculosis (Mtb) antigens (Ad5(TB): M72, ESAT-6/Ag85b, or ESAT-6/Rv1733/Rv2626/RpfD) administered simultaneously by IM and aerosol (AE) routes, could enhance blood- and lung-localized T-cell immunity and improve protection in a nonhuman primate (NHP) model of TB infection. Ad5(TB) vaccines administered by AE/IM routes following BCG priming elicited ~10-30% antigen-specific CD4 and CD8 T-cell multifunctional cytokine responses in bronchoalveolar lavage (BAL) but did not provide additional protection compared to BCG alone. Moreover, AE administration of an Ad5(empty) control vector after BCG priming appeared to diminish protection induced by BCG. Boosting BCG by IM immunization of M72/AS01 E or H56:CAF01 elicited ~0.1-0.3% antigen-specific CD4 cytokine responses in blood with only a transient increase of ~0.5-1% in BAL; these vaccine regimens also failed to enhance BCG-induced protection. Taken together, this study shows that boosting BCG with protein/adjuvant or Ad-based vaccines using these antigens, by IM or IM/AE routes, respectively, do not enhance protection against primary infection compared with BCG alone, in the highly susceptible rhesus macaque model of tuberculosis., Competing Interests: Competing interestsThe authors from University of Pittsburgh, NIH and Aeras have no competing interests. At Statens Serum Institut, Peter Andersen is a coinventor of patents relating to cationic liposomes as vaccine adjuvants and of patents relating to TB fusion protein Ag85B-ESAT-6-Rv2660 (H56). All rights have been assigned to Statens Serum Institut (SSI).

Foxp3 + regulatory T cells (T reg cells) are crucial for the maintenance of immune homeostasis both in lymphoid tissues and in non-lymphoid tissues. Here we demonstrate that the ability of intestinal T reg cells to constrain microbiota-dependent interleukin (IL)-17-producing helper T cell (T H 17 cell) and immunoglobulin A responses critically required expression of the transcription factor c-Maf. The terminal differentiation and function of several intestinal T reg cell populations, including RORγt + T reg cells and follicular regulatory T cells, were c-Maf dependent. c-Maf controlled T reg cell-derived IL-10 production and prevented excessive signaling via the kinases PI(3)K (phosphatidylinositol-3-OH kinase) and Akt and the metabolic checkpoint kinase complex mTORC1 (mammalian target of rapamycin) and expression of inflammatory cytokines in intestinal T reg cells. c-Maf deficiency in T reg cells led to profound dysbiosis of the intestinal microbiota, which when transferred to germ-free mice was sufficient to induce exacerbated intestinal T H 17 responses, even in a c-Maf-competent environment. Thus, c-Maf acts to preserve the identity and function of intestinal T reg cells, which is essential for the establishment of host-microbe symbiosis.

Defective autophagy is linked to diseases such as rheumatoid arthritis, lupus and inflammatory bowel disease (IBD). However, the mechanisms by which autophagy limits inflammation remain poorly understood. Here we found that loss of the autophagy-related gene Atg16l1 promoted accumulation of the adaptor TRIF and downstream signaling in macrophages. Multiplex proteomic profiling identified SQSTM1 and Tax1BP1 as selective autophagy-related receptors that mediated the turnover of TRIF. Knockdown of Tax1bp1 increased production of the cytokines IFN-β and IL-1β. Mice lacking Atg16l1 in myeloid cells succumbed to lipopolysaccharide-mediated sepsis but enhanced their clearance of intestinal Salmonella typhimurium in an interferon receptor-dependent manner. Human macrophages with the Crohn's disease-associated Atg16l1 variant T300A exhibited more production of IFN-β and IL-1β. An elevated interferon-response gene signature was observed in patients with IBD who were resistant to treatment with an antibody to the cytokine TNF. These findings identify selective autophagy as a key regulator of signaling via the innate immune system.

NF-κB-inducing kinase (NIK) mediates non-canonical NF-κB signaling downstream of multiple TNF family members, including BAFF, TWEAK, CD40, and OX40, which are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Here, we show that experimental lupus in NZB/W F1 mice can be treated with a highly selective and potent NIK small molecule inhibitor. Both in vitro as well as in vivo, NIK inhibition recapitulates the pharmacological effects of BAFF blockade, which is clinically efficacious in SLE. Furthermore, NIK inhibition also affects T cell parameters in the spleen and proinflammatory gene expression in the kidney, which may be attributable to inhibition of OX40 and TWEAK signaling, respectively. As a consequence, NIK inhibition results in improved survival, reduced renal pathology, and lower proteinuria scores. Collectively, our data suggest that NIK inhibition is a potential therapeutic approach for SLE.

This corrects the article DOI: 10.1038/ncomms11473.

Microglia and other tissue-resident macrophages within the central nervous system (CNS) have essential roles in neural development, inflammation and homeostasis. However, the molecular pathways underlying their development and function remain poorly understood. Here we report that mice deficient in NRROS, a myeloid-expressed transmembrane protein in the endoplasmic reticulum, develop spontaneous neurological disorders. NRROS-deficient (Nrros -/- ) mice show defects in motor functions and die before 6 months of age. Nrros -/- mice display astrogliosis and lack normal CD11b hi CD45 lo microglia, but they show no detectable demyelination or neuronal loss. Instead, perivascular macrophage-like myeloid cells populate the Nrros -/- CNS. Cx3cr1-driven deletion of Nrros shows its crucial role in microglial establishment during early embryonic stages. NRROS is required for normal expression of Sall1 and other microglial genes that are important for microglial development and function. Our study reveals a NRROS-mediated pathway that controls CNS-resident macrophage development and affects neurological function.

Tumor progression locus 2 (TPL2; also known as MAP3K8) is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) that phosphorylates the MAPK kinases MEK1 and MEK2 (MEK1/2), which, in turn, activate the MAPKs extracellular signal-regulated kinase 1 (ERK1) and ERK2 (ERK1/2) in macrophages stimulated through the interleukin-1 receptor (IL-1R), Toll-like receptors (TLRs), or the tumor necrosis factor receptor (TNFR). We describe a conserved and critical role for TPL2 in mediating the effector functions of neutrophils through the activation of the p38 MAPK signaling pathway. Gene expression profiling and functional studies of neutrophils and monocytes revealed a MEK1/2-independent branch point downstream of TPL2 in neutrophils. Biochemical analyses identified the MAPK kinases MEK3 and MEK6 and the MAPKs p38α and p38δ as downstream effectors of TPL2 in these cells. Genetic ablation of the catalytic activity of TPL2 or therapeutic intervention with a TPL2-specific inhibitor reduced the production of inflammatory mediators by neutrophils in response to stimulation with the TLR4 agonist lipopolysaccharide (LPS) in vitro, as well as in rodent models of inflammatory disease. Together, these data suggest that TPL2 is a drug target that activates not only MEK1/2-dependent but also MEK3/6-dependent signaling to promote inflammatory responses., (Copyright © 2017, American Association for the Advancement of Science.)

Systemic lupus erythematosus (SLE) is often associated with exaggerated B cell activation promoting plasma cell generation, immune-complex deposition in the kidney, renal infiltration of myeloid cells, and glomerular nephritis. Type-I IFNs amplify these autoimmune processes and promote severe disease. Bruton's tyrosine kinase (Btk) inhibitors are considered novel therapies for SLE. We describe the characterization of a highly selective reversible Btk inhibitor, G-744. G-744 is efficacious, and superior to blocking BAFF and Syk, in ameliorating severe lupus nephritis in both spontaneous and IFNα-accelerated lupus in NZB/W&#95;F1 mice in therapeutic regimens. Selective Btk inhibition ablated plasmablast generation, reduced autoantibodies, and - similar to cyclophosphamide - improved renal pathology in IFNα-accelerated lupus. Employing global transcriptional profiling of spleen and kidney coupled with cross-species human modular repertoire analyses, we identify similarities in the inflammatory process between mice and humans, and we demonstrate that G-744 reduced gene expression signatures essential for splenic B cell terminal differentiation, particularly the secretory pathway, as well as renal transcriptional profiles coupled with myeloid cell-mediated pathology and glomerular plus tubulointerstitial disease in human glomerulonephritis patients. These findings reveal the mechanism through which a selective Btk inhibitor blocks murine autoimmune kidney disease, highlighting pathway activity that may translate to human SLE., Competing Interests: Conflict of interest: All authors (except KC, PB, and JD) are or were employed by Genentech Inc. at the time of the study and hold equity in the Roche Group. KC, PB, and JD were employed by CGI Pharmaceuticals. In addition, the research in this manuscript was fully funded by Genentech Inc.

Dendritic cells (DCs) promote either tolerogenic or immunogenic T cell responses, the latter upon sensing microbes. Using an in vitro system, we analyzed transcriptional determinants that enable mature DCs to direct these opposing T cell outcomes. In the absence of microbial products, the transcription factor interferon regulatory factor 4 (IRF4) promotes regulatory T cell (T reg ) generation by enhancing expression of genes required for antigen presentation along with those for T cell tolerance. IRF4-deficient DCs were impaired for T reg generation in vivo. When exposed to microbial stimuli, DCs activated nuclear factor (NF)-κB, which induced expression of a proinflammatory cytokine module that, along with the antigen presentation module, promoted the generation of effector T cells. NF-κB was, however, dispensable for T reg development. Chromatin profiling revealed transcriptional motifs associated with the divergent DC programs. Thus, DCs modulate their ability to prime tolerogenic or immunogenic T cells by expressing a core antigen presentation module that is overlaid by distinctive regulatory modules to promote either tolerance or immunity., (© 2017 Genentech.)

Diabetic foot ulcers (DFU) are one of the major complications in type II diabetes patients and can result in amputation and morbidity. Although multiple approaches are used clinically to help wound closure, many patients still lack adequate treatment. Here we show that IL-20 subfamily cytokines are upregulated during normal wound healing. While there is a redundant role for each individual cytokine in this subfamily in wound healing, mice deficient in IL-22R, the common receptor chain for IL-20, IL-22, and IL-24, display a significant delay in wound healing. Furthermore, IL-20, IL-22 and IL-24 are all able to promote wound healing in type II diabetic db/db mice. Mechanistically, when compared to other growth factors such as VEGF and PDGF that accelerate wound healing in this model, IL-22 uniquely induced genes involved in reepithelialization, tissue remodeling and innate host defense mechanisms from wounded skin. Interestingly, IL-22 treatment showed superior efficacy compared to PDGF or VEGF in an infectious diabetic wound model. Taken together, our data suggest that IL-20 subfamily cytokines, particularly IL-20, IL-22, and IL-24, might provide therapeutic benefit for patients with DFU., Competing Interests: We have read the journal’s policy and the authors of this manuscript have the following competing interests: All authors are current or previous employees of Genentech. Genentech has reviewed the manuscript, and may potentially file patents and develop therapies based on part of the data in the manuscript. However, this does not alter our adherence to PLOS ONE policies on sharing data and materials.

Viruses encode secreted and cell-surface expressed proteins essential to modulate host immune defenses and establish productive infections. However, to date there has been no systematic study of the extracellular interactome of any human virus. Here we utilize the E3 proteins, diverse and rapidly evolving transmembrane-containing proteins encoded by human adenoviruses, as a model system to survey the extracellular immunomodulatory landscape. From a large-scale protein interaction screen against a microarray of more than 1,500 human proteins, we find and validate 51 previously unidentified virus-host interactions. Our results uncover conserved strategies as well as substantial diversity and multifunctionality in host targeting within and between viral species. Prominent modulation of the leukocyte immunoglobulin-like and signalling lymphocyte activation molecule families and a number of inhibitory receptors were identified as hubs for viral perturbation, suggesting unrecognized immunoregulatory strategies. We describe a virus-host extracellular interaction map of unprecedented scale that provides new insights into viral immunomodulation.

We analyzed transcriptomes (n = 211), whole exomes (n = 99) and targeted exomes (n = 103) from 216 malignant pleural mesothelioma (MPM) tumors. Using RNA-seq data, we identified four distinct molecular subtypes: sarcomatoid, epithelioid, biphasic-epithelioid (biphasic-E) and biphasic-sarcomatoid (biphasic-S). Through exome analysis, we found BAP1, NF2, TP53, SETD2, DDX3X, ULK2, RYR2, CFAP45, SETDB1 and DDX51 to be significantly mutated (q-score ≥ 0.8) in MPMs. We identified recurrent mutations in several genes, including SF3B1 (∼2%; 4/216) and TRAF7 (∼2%; 5/216). SF3B1-mutant samples showed a splicing profile distinct from that of wild-type tumors. TRAF7 alterations occurred primarily in the WD40 domain and were, except in one case, mutually exclusive with NF2 alterations. We found recurrent gene fusions and splice alterations to be frequent mechanisms for inactivation of NF2, BAP1 and SETD2. Through integrated analyses, we identified alterations in Hippo, mTOR, histone methylation, RNA helicase and p53 signaling pathways in MPMs.

Age-related macular degeneration (AMD), a leading cause of vision impairment in the ageing population, is characterized by irreversible loss of retinal pigment epithelial (RPE) cells and photoreceptors and can be associated with choroidal neovascularization. Mononuclear phagocytes are often present in AMD lesions, but the processes that direct myeloid cell recruitment remain unclear. Here, we identify IL-33 as a key regulator of inflammation and photoreceptor degeneration after retina stress or injury. IL-33(+) Müller cells were more abundant and IL-33 cytokine was elevated in advanced AMD cases compared with age-matched controls with no AMD. In rodents, retina stress resulted in release of bioactive IL-33 that in turn increased inflammatory chemokine and cytokine expression in activated Müller cells. Deletion of ST2, the IL-33 receptor α chain, or treatment with a soluble IL-33 decoy receptor significantly reduced release of inflammatory mediators from Müller cells, inhibited accumulation of mononuclear phagocytes in the outer retina, and protected photoreceptor rods and cones after a retina insult. This study demonstrates a central role for IL-33 in regulating mononuclear phagocyte recruitment to the photoreceptor layer and positions IL-33 signaling as a potential therapeutic target in macular degenerative diseases., (© 2016 Xi et al.)

Background & Aims: Etrolizumab is a humanized monoclonal antibody against the β7 integrin subunit that has shown efficacy vs placebo in patients with moderate to severely active ulcerative colitis (UC). Patients with colon tissues that expressed high levels of the integrin αE gene (ITGAE) appeared to have the best response. We compared differences in colonic expression of ITGAE and other genes between patients who achieved clinical remission with etrolizumab vs those who did., Methods: We performed a retrospective analysis of data collected from 110 patients with UC who participated in a phase 2 placebo-controlled trial of etrolizumab, as well as from 21 patients with UC or without inflammatory bowel disease (controls) enrolled in an observational study at a separate site. Colon biopsies were collected from patients in both studies and analyzed by immunohistochemistry and gene expression profiling. Mononuclear cells were isolated and analyzed by flow cytometry. We identified biomarkers associated with response to etrolizumab. In the placebo-controlled trial, clinical remission was defined as total Mayo Clinic Score ≤2, with no individual subscore >1, and mucosal healing was defined as endoscopic score ≤1., Results: Colon tissues collected at baseline from patients who had a clinical response to etrolizumab expressed higher levels of T-cell-associated genes than patients who did not respond (P < .05). Colonic CD4(+) integrin αE(+) cells from patients with UC expressed higher levels of granzyme A messenger RNA (GZMA mRNA) than CD4(+) αE(-) cells (P < .0001); granzyme A and integrin αE protein were detected in the same cells. Of patients receiving 100 mg etrolizumab, a higher proportion of those with high levels of GZMA mRNA (41%) or ITGAE mRNA (38%) than those with low levels of GZMA (6%) or ITGAE mRNA (13%) achieved clinical remission (P < .05) and mucosal healing (41% GZMA(high) vs 19% GZMA(low) and 44% ITGAE(high) vs 19% ITGAE(low)). Compared with ITGAE(low) and GZMA(low) patients, patients with ITGAE(high) and GZMA(high) had higher baseline numbers of epithelial crypt-associated integrin αE(+) cells (P < .01 for both), but a smaller number of crypt-associated integrin αE(+) cells after etrolizumab treatment (P < .05 for both). After 10 weeks of etrolizumab treatment, expression of genes associated with T-cell activation and genes encoding inflammatory cytokines decreased by 40%-80% from baseline (P < .05) in patients with colon tissues expressing high levels of GZMA at baseline., Conclusions: Levels of GZMA and ITGAE mRNAs in colon tissues can identify patients with UC who are most likely to benefit from etrolizumab; expression levels decrease with etrolizumab administration in biomarker(high) patients. Larger, prospective studies of markers are needed to assess their clinical value., (Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.)

CD11b(+) dendritic cells (DCs) seem to be specialized for presenting antigens via major histocompatibility (MHC) class II complexes to stimulate helper T cells, but the genetic and regulatory basis for this is not established. Conditional deletion of Irf4 resulted in loss of CD11b(+) DCs, impaired formation of peptide-MHC class II complexes and defective priming of helper T cells but not of cytotoxic T lymphocyte (CTL) responses. Gene expression and chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) analyses delineated an IRF4-dependent regulatory module that programs enhanced MHC class II antigen presentation. Expression of the transcription factor IRF4 but not of IRF8 restored the ability of IRF4-deficient DCs to efficiently process and present antigen to MHC class II-restricted T cells and promote helper T cell responses. We propose that the evolutionary divergence of IRF4 and IRF8 facilitated the specialization of DC subsets for distinct modes of antigen presentation and priming of helper T cell versus CTL responses.

Introduction: Rheumatoid arthritis (RA) is a complex and clinically heterogeneous autoimmune disease. Currently, the relationship between pathogenic molecular drivers of disease in RA and therapeutic response is poorly understood., Methods: We analyzed synovial tissue samples from two RA cohorts of 49 and 20 patients using a combination of global gene expression, histologic and cellular analyses, and analysis of gene expression data from two further publicly available RA cohorts. To identify candidate serum biomarkers that correspond to differential synovial biology and clinical response to targeted therapies, we performed pre-treatment biomarker analysis compared with therapeutic outcome at week 24 in serum samples from 198 patients from the ADACTA (ADalimumab ACTemrA) phase 4 trial of tocilizumab (anti-IL-6R) monotherapy versus adalimumab (anti-TNFα) monotherapy., Results: We documented evidence for four major phenotypes of RA synovium - lymphoid, myeloid, low inflammatory, and fibroid - each with distinct underlying gene expression signatures. We observed that baseline synovial myeloid, but not lymphoid, gene signature expression was higher in patients with good compared with poor European league against rheumatism (EULAR) clinical response to anti-TNFα therapy at week 16 (P =0.011). We observed that high baseline serum soluble intercellular adhesion molecule 1 (sICAM1), associated with the myeloid phenotype, and high serum C-X-C motif chemokine 13 (CXCL13), associated with the lymphoid phenotype, had differential relationships with clinical response to anti-TNFα compared with anti-IL6R treatment. sICAM1-high/CXCL13-low patients showed the highest week 24 American College of Rheumatology (ACR) 50 response rate to anti-TNFα treatment as compared with sICAM1-low/CXCL13-high patients (42% versus 13%, respectively, P =0.05) while anti-IL-6R patients showed the opposite relationship with these biomarker subgroups (ACR50 20% versus 69%, P =0.004)., Conclusions: These data demonstrate that underlying molecular and cellular heterogeneity in RA impacts clinical outcome to therapies targeting different biological pathways, with patients with the myeloid phenotype exhibiting the most robust response to anti-TNFα. These data suggest a path to identify and validate serum biomarkers that predict response to targeted therapies in rheumatoid arthritis and possibly other autoimmune diseases., Trial Registration: ClinicalTrials.gov NCT01119859

Unlabelled: It is common for computational analyses to generate large amounts of complex data that are difficult to process and share with collaborators. Standard methods are needed to transform such data into a more useful and intuitive format. We present ReportingTools, a Bioconductor package, that automatically recognizes and transforms the output of many common Bioconductor packages into rich, interactive, HTML-based reports. Reports are not generic, but have been individually designed to reflect content specific to the result type detected. Tabular output included in reports is sortable, filterable and searchable and contains context-relevant hyperlinks to external databases. Additionally, in-line graphics have been developed for specific analysis types and are embedded by default within table rows, providing a useful visual summary of underlying raw data. ReportingTools is highly flexible and reports can be easily customized for specific applications using the well-defined API., Availability: The ReportingTools package is implemented in R and available from Bioconductor (version ≥ 2.11) at the URL: http://bioconductor.org/packages/release/bioc/html/ReportingTools.html. Installation instructions and usage documentation can also be found at the above URL.

Objective: Personalised healthcare is contingent on the identification of biomarkers that represent disease relevant pathways and predict drug response. The authors aimed to develop a gene expression signature in synovial tissue that could enrich clinical response of rheumatoid arthritis (RA) patients to rituximab., Methods: The authors studied synovial gene expression using high-throughput quantitative real-time-PCR in 20 RA patients who underwent arthroscopy before and after treatment with rituximab. Several objective approaches were used to explore patterns in the data and to find genes associated with changes in disease activity due to treatment., Results: This analysis revealed two patient populations associated with distinct clinical, laboratory and histological features and, importantly, showed enrichment for response (60% non-responders vs 90% responders). A composite baseline gene score (GS) correlated with change in disease activity score (ΔDAS) between baseline and month 3 (r=0.74, p=0.0002), but also with ΔDAS at later time-points (month 9, r=0.54, p=0.016; month 15, r=0.45, p=0.06; month 21, r=0.72, p=0.003). Notably, the GS significantly correlated with baseline erythrocyte sedimentation rate (r=0.69, p=0.0008), but not with other DAS components. The GS genes represented T cell, macrophage, remodelling and interferon-α biology. Responders demonstrated higher expression of macrophage and T cell genes, while non-responders showed higher expression of interferon-α and remodelling genes., Conclusions: This study reveals a baseline synovial GS that correlates with early and late clinical responses to rituximab. The GS biology suggests that T cells and macrophages are important for response to B cell depleting therapy, while expression of remodelling and interferon-α genes correlates with poor response.

Genome-wide association studies (GWAS) in several populations have demonstrated significant association of the IL23R gene with IBD (Crohn's disease (CD) and ulcerative colitis (UC)) and psoriasis, suggesting that perturbation of the IL-23 signaling pathway is relevant to the pathophysiology of these diseases. One particular variant, R381Q (rs11209026), confers strong protection against development of CD. We investigated the effects of this variant in primary T cells from healthy donors carrying IL23R(R381) and IL23R(Q381) haplotypes. Using a proprietary anti-IL23R antibody, ELISA, flow cytometry, phosphoflow and real-time RT-PCR methods, we examined IL23R expression and STAT3 phosphorylation and activation in response to IL-23. IL23R(Q381) was associated with reduced STAT3 phosphorylation upon stimulation with IL-23 and decreased number of IL-23 responsive T-cells. We also observed slightly reduced levels of proinflammatory cytokine secretion in IL23R(Q381) positive donors. Our study shows conclusively that IL23R(Q381) is a loss-of-function allele, further strengthening the implication from GWAS results that the IL-23 pathway is pathogenic in human disease. This data provides an explanation for the protective role of R381Q in CD and may lead to the development of improved therapeutics for autoimmune disorders like CD.