Robert Lafyatis

Cloning by Polymerase Chain Reaction of a New Mouse TGF-β, mTGF-β3

Two TGF-beta s, TGF-beta 1 and 2, have previously been isolated from the mouse. Here we report the isolation of a murine TGF-beta 3 cDNA. RNAs extracted from 15-day-old mouse embryos and several mouse cell lines were reverse-transcribed. These cDNA mixtures were used as substrates for polymerase chain-reaction amplifications, using oligonucleotides designed on the basis of known human and chicken TGF-beta 3 sequences, including the initiation and stop codons. Several overlapping cDNAs containing either the amino-terminal domain or the carboxy-terminal domain, as well as the complete 1.2-kb coding region of the mouse TGF-beta 3 cDNA were obtained. The mouse TGF-beta 3 coding region is 1230 nucleotides long and codes for a 410 amino acid polypeptide very similar to its human counterpart. This cDNA hybridizes to a unique 3.5-kb RNA and is differentially expressed in various mouse tissues and at different embryonic stages.

Enhanced interleukin-10 production by dendritic cells upon stimulation with Toll-like receptor 4 agonists in systemic sclerosis that is possibly implicated in CCL18 secretion

Background: It has been suggested that the T‐cell attracting and profibrotic chemokine CCL18 might play a role in the pathogenesis of systemic sclerosis (SSc). However, it is unclear what underlies the higher CCL18 levels in SSc. The aim of our study was to determine whether Toll‐like receptor (TLR)‐mediated stimulation of monocytes and dendritic cells (DCs) contributes to the higher levels of CCL18 in SSc. Methods: CCL18 levels were measured in 40 patients with SSc, primary Raynauds phenomenon (RP) and healthy controls. The presence of TLR4 agonists in the circulation of SSc patients was investigated using TLR4 transgenic Chinese hamster ovary (CHO) cells. CCL18 and interleukin (IL)‐10 secretion by monocytes/macrophages and monocyte‐derived DCs (moDCs) was measured in the supernatant. The indirect effect of lipopolysaccharide (LPS)‐stimulated moDCs on CCL18 secretion by monocytes/macrophages was investigated using a transwell system. Results: CCL18 levels were significantly elevated in SSc patients compared to patients with RP and healthy controls. SSc sera strongly induced CD25 expression on CHO cells genetically modified to express TLR4 but not on those expressing CD14 only. By contrast, serum from systemic lupus erythematosus (SLE) patients or healthy individuals did not have an effect. Neither monocytes/macrophages nor moDCs from SSc patients secreted higher levels of CCL18 compared to healthy controls. However, moDCs matured with the TLR4 ligand LPS from patients with SSc did secrete significantly higher amounts of IL‐10 compared to those from healthy counterparts, which were IL‐10 dependent. Conclusions: Our results suggest that elevated CCL18 levels in SSc are not caused by an intrinsically enhanced CCL18 secretion by monocytes/macrophages but are, at least partly, orchestrated by an enhanced IL‐10 secretion by TLR4‐stimulated DCs. These observations suggest a role for TLR4 ligands and DCs in the pathogenesis of SSc, a topic that warrants further investigation.

Type I Interferons Inhibition of Inflammatory T Helper Cell Responses in Systemic Lupus Erythematosus

Abstract:  T helper (Th) cells play a central role in systemic lupus erythematosus (SLE). Activated autoreactive Th cells provide the help required for autoreactive B cells to differentiate and produce pathogenic autoAbs. Both autoAb‐containing immune complexes and direct effects of inflammatory Th cells promote tissue injury and organ damage. In SLE, triggering of plasmacytoid dendritic cell (pDC) Toll‐like receptors by autoimmune complexes containing nucleic acid autoantigens stimulates pDC secretion of high levels of type I interferons (IFN‐α/β). Study of SLE patients and murine disease models implicate these type I IFNs as key disease effectors. However, the role of pDC‐derived type I IFNs in regulating the inflammatory function of Th cells in SLE is unknown. Although, type I IFNs are classically considered to promote Th1‐mediated inflammation, they can also act as potent inhibitors of both Th1 and Th17 inflammatory cell responses. Work of ourselves and others leads us to hypothesize that if initiated during stages of SLE when Th cell‐mediated tissue inflammation is absent or minimal, such as early in the disease or during periods of remission, type I IFN neutralization will disrupt the cycle of systemic autoimmune induction and disease. However, if initiated during advanced stages of disease when there is substantial ongoing Th1 (and possibly Th17) cell‐mediated inflammation, targeting type I IFNs will exacerbate the Th cell‐mediated inflammatory disease and thus potentiate end‐organ damage and destruction. This has important implications for the application of the numerous anti‐type I IFN therapies currently under development for SLE treatment.

The -2518A>G promoter polymorphism in the CCL2 gene is not associated with systemic sclerosis susceptibility or phenotype: Results from a multicenter study of European Caucasian patients

A single nucleotide polymorphism (SNP) of the gene encoding monocyte chemoattractant protein-1 (MCP-1, CCL2) has previously been suggested to be involved in the susceptibility of systemic sclerosis (SSc). Here we have tested whether the -2518A>G CCL2 variant is associated with SSc susceptibility and/or phenotype using a cohort of SSc patients (n = 345). Clinical data from SSc patients attending rheumatology clinics in the Netherlands and Germany was collected DNA was obtained after informed consent. The control group used (n = 272) was randomly recruited from comparable geographic regions. The -2518A>G SNP in CCL2 (rs1024611) was determined using a Taqman SNP Genotyping assay. The genotype distribution was found to be similarly distributed among SSc patients and healthy controls. In addition, no association could be detected between the genotype and the presence of antinuclear antibodies, anticentromere antibodies, and antitopoisomerase antibodies or pulmonary involvement. Our results demonstrate that the functional variant -2518A>G of CCL2 is not implicated in the susceptibility or phenotype of SSc.

Additional file 1: Table S1. of Stress granules and RNA processing bodies are novel autoantibody targets in systemic sclerosis

Complete list of peptides identified in this analysis. TP number of total peptides mapping to a protein, UP number of unique peptides mapping to a protein, UM number of non-redundant peptides mapping exclusively to a protein, MW molecular weight, Length protein length in amino acids. (XLS 639 kb)

Telomere biology is differently affected within clinical subsets of systemic sclerosis and points towards different downstream defects

Results We observed a significant age related telomere attrition in healthy controls and lcSSc patients (Both p< 0.001), but not in dcSSc patients. In the immune cell subset specific analysis we observed significant shorter telomeres in B cells and myeloid dendritic cells of both lcSSc and dcSSc patients (B-Cells p=0.014, p=0.002 & myDCs p=0.019, p=0.004 respectively). PDCs and T cells were significantly shorter in dcSSc patients only (p=0.001 and p=0.003 respectively). In addition, we observed in early SSc, that B cells exhibit a significant upregulation of the telosome genes SIRT6, RIF1 and WRN (after correction for multiple testing p=0.03, 0.006 and 0.048 respectively). In later disease there is a significant higher expression of HDAC9 in monocytes from dcSSc compared to lcSSc patients. Intriguingly, in PDCs of diffuse SSc patients, regardless whether it is early or progressed disease the expression of SIRT1 is significantly lower (p=0.002 in all comparisons).

Biomarkers inform clinical trials and pathogenesis in systemic sclerosis

Biomarkers of disease are becoming increasingly recognized as central in design of clinical trials. Biomarkers allow us to link what “might be” as defined by in vitro and animal model studies with what “is” happening in disease pathogenesis. In scleroderma or systemic sclerosis (SSc) this is particularly important for implementing new strategies for clinical trials and understanding pathogenesis.

Skewed X-chromosomal inactivation patterns are present in systemic sclerosis and associated with foxp3 expression

To investigate the possible role of X-chromosomal inactivation (XCI) in systemic sclerosis (SSc) and clinical subtypes. The authors also examined the presence of skewing in immune cell subsets and the effects of XCI on Foxp3 expression in CD4CD25 cells. A total of 217 women with SSc and 107 healthy female controls were included in the study. …