Annemarie L. Dorjée

Retraction Note: Mast cells are the main interleukin 17-positive cells in anticitrullinated protein antibody-positive and -negative rheumatoid arthritis and osteoarthritis synovium

IntroductionMast cells have been implicated to play a functional role in arthritis, especially in autoantibody-positive disease. Among the cytokines involved in rheumatoid arthritis (RA), IL-17 is an important inflammatory mediator. Recent data suggest that the synovial mast cell is a main producer of IL-17, although T cells have also been implicated as prominent IL-17 producers as well. We aimed to identify IL-17 expression by mast cells and T cells in synovium of arthritis patients.MethodsSynovial samples of anticitrullinated protein antibody-positive (ACPA+) and ACPA-negative (ACPA-) RA and osteoarthritis (OA) patients were stained for IL-17 in combination with CD117 (mast cells), CD3 (T cells) and CD68 (macrophages). Concentrations of IL-17 in synovial fluid were determined by ELISA.ResultsThe number of IL-17+ cells in synovium was comparable in all groups. Although the vast majority of IL-17+ cells are mast cells, no difference in the percentage of IL-17+ mast cells was observed. Nonetheless, levels of IL-17 in synovial fluid were increased in ACPA+ RA patients compared to ACPA- RA and OA patients.ConclusionsThe synovial mast cell is the main IL-17+ cell in all three arthritis groups analyzed. These data are relevant for studies aimed at blocking IL-17 in the treatment of arthritis.

Toll-like receptor triggering augments activation of human mast cells by anti-citrullinated protein antibodies

Objective Mast cells may play a role in rheumatoid arthritis (RA), but activation of human mast cells in autoimmune settings has been little studied. Toll-like receptors (TLR) and Fcγ receptors (FcγR) are important receptors for cellular activation in the joint, but expression and stimulation of these receptors in human mast cells or the functional interplay between these pathways is poorly understood. Here, we analysed triggering of human mast cells via these receptors in the context of anti-citrullinated protein antibody-positive (ACPA+) RA. Methods RNA and protein expression of TLRs and FcγR was quantified using PCR and flow cytometry, respectively. Mast cells were stimulated with TLR ligands (including HSP70) combined with IgG immune complexes and IgG-ACPA. Results Human mast cells expressed TLRs and produced cytokines in response to TLR ligands. Both cultured and synovial mast cells expressed FcγRIIA, and triggering of this receptor by IgG immune complexes synergised with activation by TLR ligands, leading to two- to fivefold increased cytokine levels. Mast cells produced cytokines in response to ACPA immune complexes in a citrulline-specific manner, which synergised in the presence of HSP70. Conclusions Our data show that synovial mast cells express FcγRIIA and that mast cells can be activated by IgG-ACPA and TLR ligands. Importantly, combined stimulation via TLRs and immune complexes leads to synergy in cytokine production. These findings suggest mast cells are important targets for TLR ligands and immune complexes, and that combined activation of mast cells via these pathways greatly enhances inflammation in synovial tissue of RA patients.

Communication between human mast cells and CD4(+) T cells through antigen-dependent interactions.

Mast cells (MCs) are immune cells residing in tissues where pathogens are first encountered. It has been indicated that MCs might also be involved in setting the outcome of T‐cell responses. However, little is known about the capacity of human MCs to express MHC class II and/or to capture and present antigens to CD4+ T cells. To study the T‐cell stimulatory potential of human MCs, CD34+ stem cell derived MCs were generated. These cells expressed HLA‐DR when stimulated with IFN‐γ, and, importantly, presented peptide and protein for activation of antigen‐specific CD4+ T cells. The interplay between MC and T cell led to increased HLA‐DR expression on MCs. MCs were present in close proximity to T cells in tonsil and expressed HLA‐DR and CD80, indicating their ability to present antigens to CD4+ T cells in T‐cell areas of human LNs. Our data show that MCs can present native antigens to human CD4+ T cells and that HLA‐DR expressing MCs are present in tonsil tissue, indicating that human MCs can directly activate T cells and provide a rationale to study the potential of MCs to prime and/or skew human T‐cell responses.

Differential TLR-induced cytokine production by human mast cells is amplified by FcɛRI triggering

Mast cells are mainly present in strategic locations, where they may have a role in defence against parasites and bacteria. These pathogens can be recognized by mast cells via Toll‐like receptors (TLR). Allergic symptoms are often increased in the presence of pathogens at the site of allergen exposure, but it is unknown which cytokines can mediate such an effect.

Ability of Interleukin-33– and Immune Complex–Triggered Activation of Human Mast Cells to Down-Regulate Monocyte-Mediated Immune Responses

Mast cells have been implicated in the pathogenesis of rheumatoid arthritis (RA). In particular, their activation by interleukin‐33 (IL‐33) has been linked to the development of arthritis in animal models. The aim of this study was to evaluate the functional responses of human mast cells to IL‐33 in the context of RA.

Expansion of Th17 Cells by Human Mast Cells Is Driven by Inflammasome-Independent IL-1β

Mast cells (MC) are most well known for their role in innate immune responses. However, MC are increasingly recognized as important regulators of adaptive immune responses, especially in setting the outcome of T cell responses. In this study we determined the effect of MC on cytokine production by naive and memory human Th cells. CD4+ T cells were cultured with MC supernatant or control medium, after which cytokine production by T cells was determined. Supernatant of activated MC specifically increased the number of IL-17–producing T cells. This enhancement of Th17 cell number was specifically observed for the memory CD4+ T cell population and not for the naive CD4+ T cell population. The effect of MC was inhibited for ∼80% by blocking Abs to IL-1β and the rIL-1R antagonist anakinra. Importantly, secretion of active IL-1β by MC was independent of caspase activity, indicating that Th17 cell expansion by MC occurred through inflammasome-independent IL-1β. Together, these studies reveal a role for human MC in setting the outcome of T cell responses through release of caspase-independent IL-1β, and provide evidence for a novel contribution of MC in boosting the Th17 axis in mucosal immune responses.

Rituximab in early systemic sclerosis

Objectives (1) Hypothesis testing of the potency of rituximab (RTX) in preventing fibrotic complications and (2) assessing acceptability and feasibility of RTX in early systemic sclerosis (SSc). Methods A small, 24-month, randomised, double-blind, placebo-controlled, single-centre trial in patients with SSc diagnosed <2 years was conducted. Patients received RTX or placebo infusions at t=0, t=15 days and t=6 months. Patients were clinically evaluated every 3 months, with lung function tests and high-resolution CT every other visit. Skin biopsies were taken at baseline and month 3. Immunophenotyping of peripheral blood mononuclear cells was performed at every visit, except at months 9 and 18. Adverse events, course of skin and pulmonary involvement and B cell populations in skin and peripheral blood were evaluated. Results In total 16, patients (rituximab n=8, placebo n=8) were included. Twelve patients had diffuse cutaneous SSc. Eighty-eight adverse events (RTX n=53, placebo n=35, p=0.22) and 11 serious adverse events (RTX n=7, placebo n=4, p=0.36) occurred. No unexpected RTX-related events were observed. Mean skin score over time did not differ between the groups. Over time, forced vital capacity and extent of lung involvement slightly improved with RTX, but this difference was insignificant. In peripheral blood B cells depletion was demonstrated. Conclusions No unexpected safety issues were observed with RTX in early SSc. Although this small trial could not confirm or reject potential efficacy of RTX in these patients, future placebo-controlled trials are warranted, specifically in the subgroup of patients with pulmonary involvement. Trial registration number EudraCT 2008-07180-16; Results.

The extensive glycosylation of the ACPA variable domain observed for ACPA-IgG is absent from ACPA-IgM

Recently, we described the presence of highly sialylated N -linked glycans in the antigen-binding fragment (Fab) of almost all anti-citrullinated protein antibody (ACPA) IgG molecules.1 2 These glycans could not be found on several other autoantibody systems analysed. Given the low affinity of ACPA,3 this observation raises the intriguing possibility that citrullinated antigen-specific B cells could be selected based on the presence of glycans in the variable domain, rather than on affinity for their cognate antigen. N -glycosylation requires the presence of specific consensus sequences in the amino acid backbone of proteins.4 However, only few human germline Ig variable region genes encode for such sequences.5 So far, we could identify several N -glycosylation sites in ACPA-IgG Fab-domains using mass spectrometry, but none of these were encoded in the germline sequence.1 This suggests that the extensive presence of N -glycans in ACPA-IgG Fab-domains results from somatic mutations. Moreover, it indicates that the ACPA response matures under the influence of T-cell help, presumably in germinal centres, and makes it conceivable that the introduction of N -glycosylation sites …

Human mast cells costimulate T cells through a CD28-independent interaction.

Mast cells are innate immune cells usually residing in peripheral tissues, where they are likely to activate T‐cell responses. Similar to other myeloid immune cells, mast cells can function as antigen‐presenting cells. However, little is known about the capacity of human mast cells to costimulate CD4+ T cells. Here, we studied the T‐cell stimulatory potential of human mast cells. Peripheral blood derived mast cells were generated and cocultured with isolated CD4+ T cells. In the presence of T‐cell receptor triggering using anti‐CD3, mast cells promoted strong proliferation of T cells, which was two‐ to fivefold stronger than the “T‐cell promoting capacity” of monocytes. The interplay between mast cells and T cells was dependent on cell–cell contact, suggesting that costimulatory molecules on the mast cell surface are responsible for the effect. However, in contrast to monocytes, the T‐cell costimulation by mast cells was independent of the classical costimulatory molecule CD28, or that of OX40L, ICOSL, or LIGHT. Our data show that mast cells can costimulate human CD4+ T cells to induce strong T‐cell proliferation, but that therapies aiming at disrupting the interaction of CD28 and B7 molecules do not inhibit mast cell mediated T‐cell activation.

FRI0356 Antisense long noncoding rnas are deregulated in skin tissue of ssc patients

Background Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of skin and multiple organs of which pathogenesis is poorly understood. Here we studied differentially expressed coding and non-coding genes in relation to SSc pathogenesis with a specific focus on antisense non-coding RNAs. Objectives Here we studied differentially expressed coding and non-coding genes in relation to SSc pathogenesis with a specific focus on antisense non-coding RNAs. Methods Skin biopsy-derived RNAs from fourteen early SSc patients and six healthy individuals were sequenced with ion-torrent and analysed using DEseq2. Protein-coding and non-coding genes annotated in GENCODEV7 were analysed. Significant long non-coding RNAs were independently replicated in a Northern American dataset. Results 4901 genes with a fold change >1.5 and a false discovery rate of less than 5% were detected in patients versus controls. Upregulated coding genes clustered in immunological, cell adhesion and keratin-related processes as previously found by microarray studies. Interestingly, 676 deregulated non-coding genes were detected, 257 of which were classified as antisense genes. 42% of these antisense genes had a concurrent deregulated sense gene. The majority of the sense-antisense genes had a similar effect sizes in an independent North American dataset with three genes (OTUD6B-AS1, CTBP1-AS2 and HMGN3-AS1) exceeding the study-wide Bonferroni-corrected ρ-value (PBonf<0.0024, Pcombined=1.6x10-9, 1.7x10-6, 2.6xx10-6, respectively). Intriguingly, the correlation of sense-antisense gene pairs deregulated in SSc is stronger than sense-antisense gene pairs not deregulated in SSc (p<0.001). Conclusions For the first time we highlight that together with coding genes, (antisense) long noncoding RNAs are deregulated in skin tissue of SSc patients suggesting a novel class of genes involved in pathogenesis of SSc. Disclosure of Interest None declared