Helen Baldwin

An investigation of the inflammatory cytokine and chemokine network in systemic sclerosis

Objectives Systemic sclerosis (SSc) is characterised by vasculopathy, an aberrantly activated immune system and excessive extracellular matrix deposition. Inflammatory chemokines control migration of cells to sites of tissue damage; their removal from inflamed sites is essential for resolution of the inflammatory response. The atypical chemokine receptor D6 has a critical role in this physiological balance. To explore potential deregulation of this system in SSc, inflammatory chemokine and D6 expression were compared with that in healthy controls (HC). Methods Serum levels of inflammatory mediators were assessed by luminex analysis. Peripheral blood mononuclear cells (PBMCs) were used in molecular and immunocytochemical analysis. Platelet-rich plasma was collected and assessed by western blotting for D6 expression levels. Sex-matched HC were used for comparison. Results 72 patients with SSc and 30 HC were enrolled in the study. The chemokines MCP-1/CCL2, MIP-1α/CCL3, MIP-1β/CCL4 and IL-8/CXCL8 were significantly increased in patients with SSc, regardless of disease subtype and phase. Quantitative PCR analysis revealed a significant 10-fold upregulation of D6 transcripts in patients with SSc compared with controls, and this was paralleled by increased D6 protein expression in the PBMCs of patients with SSc. Platelet lysates also showed strong D6 expression in patients with SSc but not in controls. Importantly, high levels of D6 expression correlated with reduced levels of its ligands in serum. Conclusions Inflammatory chemokines and the regulatory receptor D6 are significantly upregulated in SSc and high D6 levels are associated with lower systemic chemokine levels, indicating that some patients control systemic chemokine levels using D6. These results suggest that chemokines may represent a therapeutic target in SSc.

Tumour necrosis factor alpha blockade impairs dendritic cell survival and function in rheumatoid arthritis

Objectives Tumour necrosis factor alpha (TNFα) blockade is an effective therapy for rheumatoid arthritis (RA). The immunomodulatory effects of TNFα antagonists are thought to contribute to their therapeutic action. This study investigated whether anti-TNFα therapeutics exerted their immunoregulatory effects through modulation of dendritic cell (DC) function. Methods Two complementary approaches were taken: in the first ‘in vitro’ approach monocyte-derived DC from healthy donors were matured with lipopolysaccharide and treated with TNFα antagonists in vitro for 48 h. In the second ‘ex vivo’ approach monocyte-derived DC were generated from RA patients before and 8–12 weeks into anti-TNFα treatment. DC were analysed for survival, phenotype, cytokine production and T-cell stimulatory capacity. Results TNFα blockade during DC maturation in vitro induced approximately 40% of DC to undergo apoptosis. Importantly, the surviving DC displayed a semimature phenotype with reduced levels of HLA-DR, CD80, CD83, CD86 and CCR7, and their production of IL-10 was enhanced compared with DC matured without TNFα antagonists. Furthermore, anti-TNFα-treated DC were poor stimulators of T-cell proliferation and polarised T-cell development towards a higher IL-10/lower IFNγ cytokine profile. Similarly, DC derived from RA patients after anti-TNFα treatment showed impaired upregulation of CD80 and CD86 upon lipopolysaccharide activation and displayed poor T-cell stimulatory activity. Conclusions The data show that TNFα blockade has profound effects on DC function with downstream, potentially immunoregulatory, effects on T cells. These data provide an interesting new insight into the potential mechanism by which anti-TNFα drugs contribute to the restoration of immunoregulation in RA patients.

Microarray Analyses Demonstrate the Involvement of Type I Interferons in Psoriasiform Pathology Development in D6-deficient Mice

Background: D6 regulates resolution of inflammatory responses. Its mode of action has not been molecularly defined. Results: Microarray analysis of inflamed D6-deficient mouse skin identifies dysregulated type I interferon responses as underpinning exaggerated inflammatory responses in D6-deficient mice. Conclusion: D6 is important for regulating type I interferon-based responses in inflammation. Significance: The study provides novel insights into roles for D6 in the resolution of inflammatory responses. The inflammatory response is normally limited by mechanisms regulating its resolution. In the absence of resolution, inflammatory pathologies can emerge, resulting in substantial morbidity and mortality. We have been studying the D6 chemokine scavenging receptor, which played an indispensable role in the resolution phase of inflammatory responses and does so by facilitating removal of inflammatory CC chemokines. In D6-deficient mice, otherwise innocuous cutaneous inflammatory stimuli induce a grossly exaggerated inflammatory response that bears many similarities to human psoriasis. In the present study, we have used transcriptomic approaches to define the molecular make up of this response. The data presented highlight potential roles for a number of cytokines in initiating and maintaining the psoriasis-like pathology. Most compellingly, we provide data indicating a key role for the type I interferon pathway in the emergence of this pathology. Neutralizing antibodies to type I interferons are able to ameliorate the psoriasis-like pathology, confirming a role in its development. Comparison of transcriptional data generated from this mouse model with equivalent data obtained from human psoriasis further demonstrates the strong similarities between the experimental and clinical systems. As such, the transcriptional data obtained in this preclinical model provide insights into the cytokine network active in exaggerated inflammatory responses and offer an excellent tool to evaluate the efficacy of compounds designed to therapeutically interfere with inflammatory processes.

Elevated ACKR2 expression is a common feature of inflammatory arthropathies.

Abstract Objectives Chemokines are essential contributors to leucocyte accumulation at sites of inflammatory pathology. Interfering with chemokine or chemokine receptor function therefore represents a plausible therapeutic option. However, our currently limited understanding of chemokine orchestration of inflammatory responses means that such therapies have not yet been fully developed. We have a particular interest in the family of atypical chemokine receptors that fine-tune, or resolve, chemokine-driven responses. In particular we are interested in atypical chemokine receptor 2 (ACKR2), which is a scavenging receptor for inflammatory CC-chemokines and that therefore helps to resolve in vivo inflammatory responses. The objective of the current study was to examine ACKR2 expression in common arthropathies. Methods ACKR2 expression was measured by a combination of qPCR and immuno-histochemistry. In addition, circulating cytokine and chemokine levels in patient plasma were assessed using multiplexing approaches. Results Expression of ACKR2 was elevated on peripheral blood cells as well as on leucocytes and stromal cells in synovial tissue. Expression on peripheral blood leucocytes correlated with, and could be regulated by, circulating cytokines with particularly strong associations being seen with IL-6 and hepatocyte growth factor. In addition, expression within the synovium was coincident with aggregates of lymphocytes, potentially atopic follicles and sites of high inflammatory chemokine expression. Similarly increased levels of ACKR2 have been reported in psoriasis and SSc. Conclusion Our data clearly show increased ACKR2 in a variety of arthropathies and taking into account our, and others’, previous data we now propose that elevated ACKR2 expression is a common feature of inflammatory pathologies.

SAT0037 Local expression of the atypical chemokine receptor D6 in systemic sclerosis

Background The atypical chemokine receptor D6 is an inflammatory CC-chemokine scavenger. It is involved in resolution of inflammatory responses and leukocyte trafficking from sites of injury to secondary lymphoid organs through its expression on lymphatic endothelial cells and leukocytes. Peripheral blood mononuclear cells (PBMC) from Systemic Sclerosis (SSc) patients show a marked upregulation of D6 transcript and protein, functionally relevant in buffering CC-chemokines in the circulation. Objectives To investigate D6 expression and its potential roles in the skin of SSc patients Methods Patients with diagnosis of SSc according to Leroy’s criteria for limited cutaneous (lc), diffuse cutaneous (dc) or early disease, and healthy controls (HC), both consenting to the study according to local ethic committees, were enrolled. They underwent complete clinical evaluation, paired PBMC extraction and a skin punch biopsy on the forearm. Both cells and tissues were used for transcriptional analysis by real-time qPCR with standard curve quantification method. Transcripts were normalised to TATA-Binding Protein expression in PBMCs, to beta-actin in skin. Skin sections were double stained in immunofluorescence with anti-human D6 (R&D or Sigma) and markers of lymphatic endothelial cells (anti-human podoplanin, Sigma), or of hematopoietic derived cells (anti-CD45, Dako) and analysed by microscopy. Results The study enrolled a total of 19 patients (mean age 57.6±15 years, 18 Females, 4 early-SSc, 12 lc-SSc and 3dc-SSc). D6 transcripts in skin were significantly upregulated in SSc patients (3443±1761 vs 1035±327 in control skin, n=9, p<0.001), with no significant differences between disease subsets and independently from D6 expression on paired PBMCs. Patients with high D6 expression showed significantly lower modified Rodnan’s skin scores (mRSS) (8±7 vs 19±9 in those with no or low D6 expression) but were comparable for Valentini’s disease activity scores and systemic inflammatory markers (e.g. ESR, CRP). While there was a trend for higher lymphatics/mm2 in SSc patients, D6 protein expression was detected with no significant difference on a mean percentage of 63±15% podoplanin+ lymphatic endothelial cells in patients and HC, while D6+ leukocytes were found only on a subset of patients and HC and their presence was apparently not related to any clinical feature. D6+ leukocytes were significantly more frequent in tissues of higher CD45+ infiltration. There was a trend toward an inverse relationship between D6 expression on lymphatics and CD45+ leukocyte infiltration on the skin, being the latter higher where there was less D6 on lymphatics. Conclusions Expression of the atypical CC-chemokine receptor D6 is upregulated in SSc skin and even in early phases of the disease. Its expression is higher in patients with lower mRSS scores indicating that it could represent an attempt to restrain local pathological activity. Inverse relationship between presence of D6 on lymphatics and leukocyte infiltration confirms this is a critical molecule in the regulation of trafficking to secondary lymphoid organs. Disclosure of Interest None Declared