Extracellular traps and PAD4 released by macrophages induce citrullination and auto-antibody production in autoimmune arthritis

Barts Charity grant number MRC0177

Investigation of genetically regulated gene expression and response to treatment in rheumatoid arthritis highlights an association between IL18RAP expression and treatment response.

This article has been accepted for publication in Annals of the Rheumatic Diseases, 2020 following peer review, and the Version of Record can be accessed online at http://dx.doi.org/10.1136/annrheumdis-2020-217204OBJECTIVES: In this study, we sought to investigate whether there was any association between genetically regulated gene expression (as predicted using various reference panels) and anti-tumour necrosis factor (anti-TNF) treatment response (change in erythrocyte sedimentation rate (ESR)) using 3158 European ancestry patients with rheumatoid arthritis. METHODS: The genetically regulated portion of gene expression was estimated in the full cohort of 3158 subjects (as well as within a subcohort consisting of 1575 UK patients) using the PrediXcan software package with three different reference panels. Estimated expression was tested for association with anti-TNF treatment response. As a replication/validation experiment, we also investigated the correlation between change in ESR with measured gene expression at the Interleukin 18 Receptor Accessory Protein (IL18RAP) gene in whole blood and synovial tissue, using an independent replication data set of patients receiving conventional synthetic disease modifying anti-rheumatic drugs, with directly measured (via RNA sequencing) gene expression. RESULTS: We found that predicted expression of IL18RAP showed a consistent signal of association with treatment response across the reference panels. In our independent replication data set, IL18RAP expression in whole blood showed correlation with the change in ESR between baseline and follow-up (r=-0.35, p=0.0091). Change in ESR was also correlated with the expression of IL18RAP in synovial tissue (r=-0.28, p=0.02). CONCLUSION: Our results suggest that IL18RAP expression is worthy of further investigation as a potential predictor of treatment response in rheumatoid arthritis that is not specific to a particular drug type

Circulating and Synovial Pentraxin-3 (PTX3) Expression Levels Correlate With Rheumatoid Arthritis Severity and Tissue Infiltration Independently of Conventional Treatments Response

International audienceAims To determine the relationship between PTX3 systemic and synovial levels and the clinical features of rheumatoid arthritis (RA) in a cohort of early, treatment naïve patients and to explore the relevance of PTX3 expression in predicting response to conventional-synthetic (cs) Disease-Modifying-Anti-Rheumatic-Drugs (DMARDs) treatment. Methods PTX3 expression was analyzed in 119 baseline serum samples from early naïve RA patients, 95 paired samples obtained 6-months following the initiation of cs-DMARDs treatment and 43 healthy donors. RNA-sequencing analysis and immunohistochemistry for PTX3 were performed on a subpopulation of 79 and 58 synovial samples, respectively, to assess PTX3 gene and protein expression. Immunofluorescence staining was performed to characterize PTX3 expressing cells within the synovium. Results Circulating levels of PTX3 were significantly higher in early RA compared to healthy donors and correlated with disease activity at baseline and with the degree of structural damages at 12-months. Six-months after commencing cs-DMARDs, a high level of PTX3, proportional to the baseline value, was still detectable in the serum of patients, regardless of their response status. RNA-seq analysis confirmed that synovial transcript levels of PTX3 correlated with disease activity and the presence of mediators of inflammation, tissue remodeling and bone destruction at baseline. PTX3 expression in the synovium was strongly linked to the degree of immune cell infiltration, the presence of ectopic lymphoid structures and seropositivity for autoantibodies. Accordingly, PTX3 was found to be expressed by numerous synovial cell types such as plasma cells, fibroblasts, vascular and lymphatic endothelial cells, macrophages, and neutrophils. The percentage of PTX3-positive synovial cells, although significantly reduced at 6-months post-treatment as a result of global decreased cellularity, was similar in cs-DMARDs responders and non-responders. Conclusion This study demonstrates that, early in the disease and prior to treatment modification, the level of circulating PTX3 is a reliable marker of RA activity and predicts a high degree of structural damages at 12-months. In the joint, PTX3 associates with immune cell infiltration and the presence of ectopic lymphoid structures. High synovial and peripheral blood levels of PTX3 are associated with chronic inflammation characteristic of RA. Additional studies to determine the mechanistic link are required

Role of synovial fibroblast subsets across synovial pathotypes in rheumatoid arthritis: a deconvolution analysis

OBJECTIVES: To integrate published single-cell RNA sequencing (scRNA-seq) data and assess the contribution of synovial fibroblast (SF) subsets to synovial pathotypes and respective clinical characteristics in treatment-naïve early arthritis. METHODS: In this in silico study, we integrated scRNA-seq data from published studies with additional unpublished in-house data. Standard Seurat, Harmony and Liger workflow was performed for integration and differential gene expression analysis. We estimated single cell type proportions in bulk RNA-seq data (deconvolution) from synovial tissue from 87 treatment-naïve early arthritis patients in the Pathobiology of Early Arthritis Cohort using MuSiC. SF proportions across synovial pathotypes (fibroid, lymphoid and myeloid) and relationship of disease activity measurements across different synovial pathotypes were assessed. RESULTS: We identified four SF clusters with respective marker genes: PRG4(+) SF (CD55, MMP3, PRG4, THY1(neg)); CXCL12(+) SF (CXCL12, CCL2, ADAMTS1, THY1(low)); POSTN(+) SF (POSTN, collagen genes, THY1); CXCL14(+) SF (CXCL14, C3, CD34, ASPN, THY1) that correspond to lining (PRG4(+) SF) and sublining (CXCL12(+) SF, POSTN(+) + and CXCL14(+) SF) SF subsets. CXCL12(+) SF and POSTN(+) + were most prominent in the fibroid while PRG4(+) SF appeared highest in the myeloid pathotype. Corresponding, lining assessed by histology (assessed by Krenn-Score) was thicker in the myeloid, but also in the lymphoid pathotype + the fibroid pathotype. PRG4(+) SF correlated positively with disease severity parameters in the fibroid, POSTN(+) SF in the lymphoid pathotype whereas CXCL14(+) SF showed negative association with disease severity in all pathotypes. CONCLUSION: This study shows a so far unexplored association between distinct synovial pathologies and SF subtypes defined by scRNA-seq. The knowledge of the diverse interplay of SF with immune cells will advance opportunities for tailored targeted treatments

Integration of molecular characterization of microorganisms in a global antimicrobial resistance surveillance program

© 2001 by the Infectious Diseases Society of America. All rights reserved.The SENTRY Antimicrobial Surveillance Program has incorporated molecular strain typing and resistance genotyping as a means of providing additional information that may be useful for understanding pathogenic microorganisms worldwide. Resistance phenotypes of interest include multidrug-resistant pathogens, extended-spectrum β-lactamase (ESBL)–producing Enterobacteriaceae, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci, and fluoroquinolone-resistant (FQR) strains of gram-negative bacilli and Streptococcus pneumoniae. Clusters of 2 isolates within a given resistance profile that are linked temporally and by hospital location are flagged for DNA fingerprinting. Further characterization of organisms with respect to resistance genotype is accomplished with use of polymerase chain reaction and DNA sequencing. This process has been highly successful in identifying clonal spread within clusters of multiresistant pathogens. Between 50% and 90% of MRSA clusters identified by phenotypic screening contained evidence of clonal spread. Among the Enterobacteriaceae, ESBL-producing strains of Escherichia coli and Klebsiella pneumoniae are the most common pathogens causing clusters of infection, and 50% of recognized clusters demonstrate clonal spread. Clusters of Pseudomonas aeruginosa, Acinetobacter species, and Stenotrophomonas maltophilia have been noted with clonal spread among patients with urinary tract, respiratory, and bloodstream infections. Characterization of mutations in the FQR-determining region of phenotypically susceptible isolates of E. coli and S. pneumoniae has identified first-stage mutants among as many as 40% of isolates. The ability to characterize organisms phenotypically and genotypically is extremely powerful and provides unique information that is important in a global antimicrobial surveillance program.M. A. Pfaller, J. Acar, R. N. Jones, J. Verhoef, J. Turnidge, and H. S. Sade

Axl and MerTK regulate synovial inflammation and are modulated by IL-6 inhibition in rheumatoid arthritis.

The TAM tyrosine kinases, Axl and MerTK, play an important role in rheumatoid arthritis (RA). Here, using a unique synovial tissue bioresource of patients with RA matched for disease stage and treatment exposure, we assessed how Axl and MerTK relate to synovial histopathology and disease activity, and their topographical expression and longitudinal modulation by targeted treatments. We show that in treatment-naive patients, high AXL levels are associated with pauci-immune histology and low disease activity and inversely correlate with the expression levels of pro-inflammatory genes. We define the location of Axl/MerTK in rheumatoid synovium using immunohistochemistry/fluorescence and digital spatial profiling and show that Axl is preferentially expressed in the lining layer. Moreover, its ectodomain, released in the synovial fluid, is associated with synovial histopathology. We also show that Toll-like-receptor 4-stimulated synovial fibroblasts from patients with RA modulate MerTK shedding by macrophages. Lastly, Axl/MerTK synovial expression is influenced by disease stage and therapeutic intervention, notably by IL-6 inhibition. These findings suggest that Axl/MerTK are a dynamic axis modulated by synovial cellular features, disease stage and treatment

Interferon-γ-producing immature myeloid cells confer protection against severe invasive group A Streptococcus infections

Cytokine-activated neutrophils are known to be essential for protection against group A Streptococcus infections. However, during severe invasive group A Streptococcus infections that are accompanied by neutropenia, it remains unclear which factors are protective against such infections, and which cell population is the source of them. Here we show that mice infected with severe invasive group A Streptococcus isolates, but not with non-invasive group A Streptococcus isolates, exhibit high concentrations of plasma interferon-γ during the early stage of infection. Interferon-γ is necessary to protect mice, and is produced by a novel population of granulocyte–macrophage colony-stimulating factor-dependent immature myeloid cells with ring-shaped nuclei. These interferon-γ-producing immature myeloid cells express monocyte and granulocyte markers, and also produce nitric oxide. The adoptive transfer of interferon-γ-producing immature myeloid cells ameliorates infection in wild-type and interferon-γ-deficient mice. Our results indicate that interferon-γ-producing immature myeloid cells have a protective role during the early stage of severe invasive group A Streptococcus infections

Nosocomial infection in a newborn intensive care unit (NICU), South Korea

BACKGROUND: This study aimed to determine the occurrence of nosocomial infections (NIs), including infection rates, main infection sites, and common microorganisms. Patients included in the study were taken from a newborn intensive care unit (NICU), in a hospital in South Korea. METHODS: A retrospective cohort study was performed by reviewing chart. The subjects were 489 neonates who were admitted to the NICU, survived longer than 72 hours, and not transferred to another unit, between Jan. 1. 1995 to Sep. 30, 1999. NIs were identified according to the NNIS definition. Data were analyzed with descriptive statistics. RESULTS: Cumulative incidence rate for NIs was 30.3 neonates out of 100 admissions, with a total of 44.6 infections. The incidence density was average 10.2 neonates and 15.1 infections per 1000 patient days. The most common infections were pneumonia (28%), bloodstream infection (26%), and conjunctivitis (22%). Major pathogens were Gram-positives such as Staphylococcus aureus and coagulase-negative staphylococci. The factors associated with NI was less than 1500 g of birth weight, less than 32 weeks of gestational age, and less than 8 of apgar score. There's no statistical difference in discharge status between two groups, but hospital stay was longer in subjects with nosocomial infection than those without infection. CONCLUSION: Although the distribution of pathogens was similar to previous reports, a high rate of nosocomial infection and in particular conjunctivitis was observed in this study that merits further evaluation

16S rRNA Gene-based Analysis of Fecal Microbiota from Preterm Infants with and without Necrotizing Enterocolitis

Neonatal necrotizing enterocolitis (NEC) is an inflammatory intestinal disorder affecting preterm infants. Intestinal bacteria play a key role; however no causative pathogen has been identified. The purpose of this study was to determine if there are differences in microbial patterns which may be critical to the development of this disease. Fecal samples from twenty preterm infants, ten with NEC and ten matched controls (including four twin pairs) were obtained from patients in a single site Level III neonatal intensive care unit. Bacterial DNA from individual fecal samples were PCR amplified and subjected to terminal restriction fragment length polymorphism analysis and library sequencing of the 16S rRNA gene to characterize diversity and structure of the enteric microbiota. The distribution of samples from NEC patients distinctly clustered separately from controls. Intestinal bacterial colonization in all preterm infants was notable for low diversity. Patients with NEC had even less diversity, an increase in abundance of Gammaproteobacteria, a decrease in other bacteria species, and had received a higher mean number of previous days of antibiotics. Our results suggest that NEC is associated with severe lack of microbiota diversity which may accentuate the impact of single dominant microorganisms favored by empiric and wide-spread use of antibiotics