Human Genetics in Rheumatoid Arthritis Guides a High-Throughput Drug Screen of the CD40 Signaling Pathway

Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10(−9)). Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10(−9)), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA–approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in complex traits such as RA

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

How to understand the complexity of product quality and the challenges in differentiating between organically and conventionally grown products—exemplified by fresh and heat-processed carrots (Daucus carota L.)

Quality traits are highly focused upon in the marketing of organic food products. There is a need to define and measure quality as consumers seem to have preconceived notions about the superior health value and taste of organic compared to non-organic products. A commonly held opinion among many consumer groups is that organic farming guarantees optimum quality, despite the fact that this remains unproven. The aim of this paper is to contribute to a better understanding of the complexity of quality traits in a plant-based food product, using carrots as an example. Selected designated quality aspects are presented to describe the complexity of quality and discuss the challenges of using these aspects in differentiating between organic and conventional products. The paper concludes we have insufficient tools to be able to adequately authenticate organically produced carrots. The same may be the case for most vegetables and fruit products. Suggestions for further studies include the soil and location aspect (terroir), in order to trace a product back to its origin in an organically or conventionally farmed field by finding a unique fingerprint for chemical constituents of sample

New data and an old puzzle: the negative association between schizophrenia and rheumatoid arthritis

A long-standing epidemiological puzzle is the reduced rate of rheumatoid arthritis (RA) in those with schizophrenia (SZ) and vice versa. Traditional epidemiological approaches to determine if this negative association is underpinned by genetic factors would test for reduced rates of one disorder in relatives of the other, but sufficiently powered data sets are difficult to achieve. The genomics era presents an alternative paradigm for investigating the genetic relationship between two uncommon disorders.status: publishe

New data and an old puzzle: the negative association between schizophrenia and rheumatoid arthritis

A long-standing epidemiological puzzle is the reduced rate of rheumatoid arthritis (RA) in those with schizophrenia (SZ) and vice versa. Traditional epidemiological approaches to determine if this negative association is underpinned by genetic factors would test for reduced rates of one disorder in relatives of the other, but sufficiently powered data sets are difficult to achieve. The genomics era presents an alternative paradigm for investigating the genetic relationship between two uncommon disorders.We use genome-wide common single nucleotide polymorphism (SNP) data from independently collected SZ and RA case-control cohorts to estimate the SNP correlation between the disorders. We test a genotype X environment (GxE) hypothesis for SZ with environment defined as winter- vs summer-born.We estimate a small but significant negative SNP-genetic correlation between SZ and RA (-0.046, s.e. 0.026, P = 0.036). The negative correlation was stronger for the SNP set attributed to coding or regulatory regions (-0.174, s.e. 0.071, P = 0.0075). Our analyses led us to hypothesize a gene-environment interaction for SZ in the form of immune challenge. We used month of birth as a proxy for environmental immune challenge and estimated the genetic correlation between winter-born and non-winter born SZ to be significantly less than 1 for coding/regulatory region SNPs (0.56, s.e. 0.14, P  = 0.00090).Our results are consistent with epidemiological observations of a negative relationship between SZ and RA reflecting, at least in part, genetic factors. Results of the month of birth analysis are consistent with pleiotropic effects of genetic variants dependent on environmental context