Maria João Salvador

Mapping and predicting mortality from systemic sclerosis

Objectives To determine the causes of death and risk factors in systemic sclerosis (SSc). Methods Between 2000 and 2011, we examined the death certificates of all French patients with SSc to determine causes of death. Then we examined causes of death and developed a score associated with all-cause mortality from the international European Scleroderma Trials and Research (EUSTAR) database. Candidate prognostic factors were tested by Cox proportional hazards regression model by single variable analysis, followed by a multiple variable model stratified by centres. The bootstrapping technique was used for internal validation. Results We identified 2719 French certificates of deaths related to SSc, mainly from cardiac (31%) and respiratory (18%) causes, and an increase in SSc-specific mortality over time. Over a median follow-up of 2.3 years, 1072 (9.6%) of 11 193 patients from the EUSTAR sample died, from cardiac disease in 27% and respiratory causes in 17%. By multiple variable analysis, a risk score was developed, which accurately predicted the 3-year mortality, with an area under the curve of 0.82. The 3-year survival of patients in the upper quartile was 53%, in contrast with 98% in the first quartile. Conclusion Combining two complementary and detailed databases enabled the collection of an unprecedented 3700 deaths, revealing the major contribution of the cardiopulmonary system to SSc mortality. We also developed a robust score to risk-stratify these patients and estimate their 3-year survival. With the emergence of new therapies, these important observations should help caregivers plan and refine the monitoring and management to prolong these patients’ survival.

Exercise‐Induced Pulmonary Hypertension in Scleroderma Patients: A Common Finding but with Elusive Pathophysiology

Background: The etiology of exercise‐induced pulmonary hypertension (exPH) in systemic sclerosis (SSc) remains a complex task, as both left ventricle (LV) diastolic dysfunction and pulmonary vascular disease can contribute to its development. We determined the incidence of exPH in SSc and examined the association between pulmonary artery systolic pressure (PASP) and tissue Doppler‐derived indexes of pulmonary capillary wedge pressure (PCWP). Methods: Thirty‐eight patients with SSc were studied, using a cycloergometer protocol; 10 were excluded due to resting PH or absence of tricuspid regurgitation (TR); TR and mitral E‐wave velocities, LV outflow tract time‐velocity integral and LV septal E′‐wave were measured before and in peak exercise to calculate cardiac output (CO), PCWP and pulmonary vascular resistance (PVR). Results: Mean age of diagnosis was 57.9 ± 8.9 years. At a mean workload of 64 ± 29 Watts, 48% of patients increased PASP ≥ 50 mmHg. PCWP, assessed by the E/e′ ratio, did not change significantly during exercise (10.2 ± 3.1–10.0 ± 5.1; P = NS). Only 3 patients had elevations of the E/e′ ratio ≥ 13 during exercise; 2 of them had an exercise PASP ≥ 50 mmHg, yielding a proportion of exPH due to elevated LV filling pressures of 2/11 (18%). Patients with exPH had lower DLCO and had more frequently the diffuse SSc. Conclusion: The elevation of PASP during exercise in most patients of this cohort seems to be related to a reduced pulmonary vascular reserve, and not to an increase in PCWP. Further studies are warranted to determine the therapeutic, as well as prognostic implications of these findings.

Increased frequencies of circulating CXCL10-, CXCL8- and CCL4-producing monocytes and Siglec-3-expressing myeloid dendritic cells in systemic sclerosis patients

ObjectiveTo investigate the ex vivo pro-inflammatory properties of classical and non-classical monocytes as well as myeloid dendritic cells (mDCs) in systemic sclerosis (SSc) patients.MethodsSpontaneous production of CXCL10, CCL4, CXCL8 and IL-6 was intracellularly evaluated in classical, non-classical monocytes and Siglec-3-expressing mDCs from peripheral blood of SSc patients and healthy controls (HC) through flow cytometry. In addition, production of these cytokines was determined upon toll-like receptor (TLR) 4 plus Interferon-γ (IFN-γ) stimulation.ResultsThe frequency of non-classical monocytes spontaneously producing CXCL10 was increased in both limited (lcSSc) and diffuse cutaneous (dcSSC) subsets of SSc patients and CCL4 was augmented in dcSSc patients. The proportion of CCL4-producing mDCs was also elevated in dcSSc patients and the percentage of mDCS producing CXCL10 only in lcSSc patients. Upon stimulation, the frequency of non-classical monocytes expressing CXCL8 was increased in both patient groups and mDCs expressing CXCL8 only in lcSSc. Moreover, these parameters in unsupervised clustering analysis identify a subset of patients which are characterized by lung fibrosis and reduced pulmonary function.ConclusionsThese data point towards a role of activated non-classical monocytes and mDCs producing enhanced levels of proinflammatory cytokines in SSc, potentially contributing to lung fibrosis.

A3.24 Distinct profile of cytokine production by th17 cells in systemic sclerosis

Background The pathogenesis of systemic sclerosis (SSc) is largely unknown, although proinflammatory cytokines are considered to play an important role. Aims Investigate the pattern of expression of proinflammatory cytokines by peripheral blood (PB) Th17 cell and IL-2 and IL-17 relative gene expression in SSc and to explore their clinical associations. Methods This study included 41 SSc patients and 20 age- and sex-matched healthy controls (HC). SSc patients were classified according to LeRoy et al. as having limited cutaneous SSc (lSSc, n = 29) or diffuse cutaneous SSc (dSSc, n = 12). A further subdivision was made based upon clinical parameters of disease duration and clinical manifestations. Intracellular expression of IL-17, IL-2, TNF-α and IFN-γ in Th17 cells was assessed by flow cytometry. A detailed functional activity characterisation of Th17 cells was performed in a single multicolour data file obtained after merging the original six-colour-staining (eight-parameter) data files from each sample using the Infinicyt (Cytogonos) software. Relative gene expression of IL-2 and IL-17 in total PB cells was determined by qRT-PCR Results No differences were observed concerning the frequency of Th17 cells or Tc17 cells between SSc patients and HC. Th17 cells from both diffuse and limited SSc showed a higher expression of IL-2 (particularly in dSSc) than Th17 cells from HC. Similar observations were made for TNF-α expression but it only reached statistical significance in lSSc. Likewise, an increased frequency of Tc17 cells expressing IL-2 in lSSc was also observed. Eight distinct Th17 cell subpopulations according to their pattern of cytokine expression (IL-17, IL-2, TNF-α and IFN-γ) were identify after merging data files. A higher frequency of Th17 cells simultaneously producing simultaneously IL17, IL-2 and TNF-α was observed in lSSc. On the other hand a significantly lower frequency of Th17 cells simultaneously producing IL-17, TNF-α and IFN-γ was also observed in the same group. The percentage of Th17 cells producing only TNF-α or IFN-γ was significantly decreased in SSc patients compared to HC group. Moreover a higher IL-17 mRNA expression in total PB cells was observed in SSc patients and no differences were found concerning IL-2 mRNA levels. Conclusions These results suggest that Th17 cells are functionally altered in SSc patients, exhibiting a higher ability to simultaneously produce IL-17, IL-2 and TNF-α. We also observed in SSc patients increased levels of IL-17 mRNA expression in total PB cells. These data suggest that Th17 cells may be involved in pathophysiology and/or disease progression of SSc.

Ultrasonography for the assessment of skin in systemic sclerosis: a systematic review

To identify and synthesize the best available evidence on the use of ultrasound to assess skin involvement in systemic sclerosis (SSc).

SAT0324 Increased frequencies of circulating CXCL10-, CXCL8- and CCL4-producing monocytes and SIGLEC-3-expressing myeloid dendritic cells in systemic sclerosis patients

Background Systemic sclerosis (SSc) is an inflammatory and fibrotic disease characterized by vascular dysfunction, excessive extracellular matrix deposition and immune dysregulation. Recent observations suggest that monocytes and dendritic cells (DCs) might be involved in SSc; including cell recruitment, trafficking, activation and an enhanced pro-fibrotic phenotype. Hence these cells might be important contributors to the disease pathogenesis. However, detailed analysis of circulating monocytes and DCs in SSc in relationship to disease activity has not been performed so far. Objectives To investigate the ex vivo pro-inflammatory properties of classical and non-classical monocytes as well as myeloid dendritic cells (mDCs) in SSc patients in relationship to disease activity. Methods This study enrolled 43 SSc patients, 30 classified as limited cutaneous SSc (lcSSc) and 13 as diffuse cutaneous (dcSSc). The healthy control group (HC) included 20 age- and gender- matched individuals. The Spontaneous production of CXCL10, CCL4, CXCL4 and IL-6 was intracellularly evaluated in classical and non-classical monocytes and Siglec-3-expressing mDCs from peripheral blood using flow cytometry. In addition, the production of these cytokines was determined upon toll like receptor 4 (TLR4) plus Interferon-γ (IFN-γ) in vitro stimulation. Results The frequency of non-classical monocytes spontaneously producing CXCL10 was increased in both lcSSc and dcSSC subsets of SSc patients (p<0.05) and CCL4 was augmented in the dcSSc patient subset (p<0.05). The proportion of CCL4 producing- mDCs were also elevated in dcSSc patients (p<0.01) and the percentage of mDCS producing CXCL10 only in lcSSc patients (p<0.05 compared to HC, but p<0.01 comparing to dcSSc). Upon in vitro stimulation the frequency of non-classical monocytes expressing CXCL8 was increased in both patient groups (p<0.01) and mDCs expressing CXCL8 only in lcSSc (p<0.01). SSc patients characterized by the presence or history of lung fibrosis, displayed a higher frequency of non-classical monocytes expressing CCL4 and CXCL10 in dcSSc patients as compared to those without this clinical manifestation (p<0.01 and p<0.05 respectively). Strikingly, the percentage of classical monocytes producing CXCL8 was augmented upon in vitro stimulation in lcSSc patients with lung fibrosis as compared to those without (p<0.01). No differences were found in the percentage of IL-6 producing cells. Conclusions These data point towards a role of activated non-classical monocytes and mDCs producing enhanced levels of proinflammatory cytokines in SSc, potentially contributing to lung fibrosis. Acknowledgements TC is supported by a grant from the Portuguese national funding agency for science, research and technology: Fundação para a Ciência e a Tecnologia [SFRH/BD/93526/2013]. Disclosure of Interest None declared

A3.25 systemic sclerosis patients exhibit an increased frequency of T cells and NK cells with effector/cytotoxic phenotype

Background Systemic sclerosis (SSc) is an inflammatory and fibrotic disease characterised by inflammation, immune dysregulation and excessive extracellular matrix deposition. T cell and NK cell disturbance have already been described in SSc and implicated in its establishment. Aims Determine the frequency of different subpopulations of T cells (CD4+, CD8+ and CD4+ /CD8+) among the functional compartments and the percentage of T cell subsets and NK cells (CD56bright and CD56dim subpopulations) expressing cytolytic markers. Materials and Methods The study enrolled 43 SSc patients, 30 with limited cutaneous subtype (lSSc) and 13 with diffuse cutaneous subtype (dSSc) according to LeRoy et al. classification and 20 healthy individuals as control group. A further subdivision was made based upon clinical parameters of disease duration and clinical manifestations. Immunophenotyping characterisation of peripheral blood T cells was performed by flow cytometry. T cells functional compartments (naïve, central memory, effector memory and terminal effector) were defined based on CD45Ra and CD27 phenotype. Cytotoxic activity was assessed by the intracellular expression of granzyme B and perforin. Results We observed a lower frequency of T cells in SSc, particularly in dSSc and in patients with pulmonary fibrosis and digital ulcers. Moreover, SSc patients presented a decreased frequency and absolute numbers of CD8+ T cells (mainly in dSSc patients, and SSc patients with pulmonary fibrosis and digital ulcers). We also observed a lower absolute value of double positive CD4+/CD8+ cells in SSc patients. Regarding NK cells, SSc patients with pulmonary fibrosis exhibit a lower absolute value of both CD56bright and CD56dim subsets. Among functional compartments, we found a higher frequency of CD8+ and CD4+ T cells with terminal effector and memory effector phenotype, closely related to a decreased frequency of cells with central memory and naïve phenotype. This reached statistical significance for CD8+ T cells. In line with these observations, we demonstrated an increased frequency of T CD4+ and CD8+ cells expressing granzyme B and perforin, particularly in patients with the disease for longer than one year. An increased frequency of CD56bright NK cells expressing perforin or granzyme B was also observed in SSc patients. Conclusions Our results indicate an increased frequency of CD4+ and CD8+ T cells and CD56bright NK cells with an effector/cytotoxic phenotype in the peripheral blood from SSc patients. These changes seem to be related with the presence of pulmonary fibrosis or digital ulcers.

A3.23 Alterations in the distribution and pro-inflammatory activity of peripheral blood monocytes and dendritic cells subpopulations in systemic sclerosis

Background Systemic sclerosis (SSc) is a connective tissue disease characterised by immune and fibrotic abnormalities. Important observations suggest monocytes and dendritic cells (DCs) involvement in SSc, including abnormalities in differentiation, recruitment, trafficking, activation and an enhanced pro-fibrotic phenotype that could be important factors in the disease pathogenesis. Objectives Evaluate the frequency of monocytes subpopulations (CD16- and CD16+ monocytes) and DCs subsets (myeloid – mDC, CD16+CD14-/low DCs and plasmacytoid DCs – pDCs) and their functional ability to produce chemokines (CXCL10, CCL4 and IL-8) in SSc patients. Materials and Methods The study enrolled 43 SSc patients, 30 with limited cutaneous subtype (lSSc) and 13 with diffuse cutaneous subtype (dSSc). A further subdivision was made based upon clinical parameters of disease duration and organ involvement. The healthy control group (HC) included 20 age and gender matched individuals. Immunophenotyping of peripheral blood DC and monocytes subpopulations was assessed by flow cytometry and their functional characterisation was performed after LPS plus IFN-g stimulation. Results We observed an increased frequency and absolute number of CD16+ monocytes in SSc patients, particularly in the lSSc group. These changes were more pronounced in patients with longer disease duration (>10 yrs). Concerning the DCs subsets, no differences were observed between the SSc subtypes and HG. Patients without pulmonary fibrosis demonstrated a higher frequency and absolute number of CD16+CD14-/low DCs than HG, while the differences were not statistically significant between patients with pulmonary fibrosis and HC. Patients with SSc for more than 10 years, demonstrated an increased frequency and absolute value of CD16+CD14-/low DCs in comparison to HG. Without in vitro stimulation, we observed an increased frequency of CD16+CD14-/low DCs and mDCs expressing CCL4 and CXCL10, particularly in dSSc subtype. After in vitro stimulus, a higher frequency of CD16+CD14-/low DCs expressing IL-8 in dSSc subtype was observed. Moreover, the frequency of monocytes, CD16+CD14-/low DCs and mDCs expressing IL-8 and the frequency of CD16+CD14-/low DCs and mDCS expressing CCL4 was higher in patients with pulmonary fibrosis than in HC. Conclusions Important alterations were observed in the frequency/absolute number of circulating monocytes and DCs subsets and their functional ability to produce chemokines in SSc patients, which seem to be related to disease duration and to pulmonary fibrosis.

AB0817 Vitamin D serum levels in a cohort of patients with systemic sclerosis: Prevalence, determinants and associations with clinical and biological aspects

Background Besides the genetic background, it is known that environmental factors play an important role in the pathogenesis and activity of autoimmune diseases and vitamin D deficiency has been associated with the risk and severity of autoimmune diseases. Apart from traditional calcium-related actions, vitamin D is now being recognized for its role in immune-homeostasis, affecting both innate and adaptative immune systems and contributing to immune-tolerance of self-structures. Objectives To compare the vitamin D serum levels in systemic sclerosis (SSc) patients with healthy controls and to explore the predictors of vitamin D serum levels in SSc patients and the association between its levels with clinical and biological aspects. Methods The levels of 25-hidroxyvitamin D (25OHD) were determined in a cohort of 45 SSc patients and in 27 healthy controls gender-, age- and season-matched, not taking vitamin D supplements. The SSc patients underwent a complete clinical and laboratorial evaluation, which included sun exposure time, 25OHD serum level and modified Rodnan skin score. Sun exposure time was estimated using a semi-quantitative scale. 25OHD levels were classified as deficient (<10ng/ml), insufficient (≥10ng/ml and <30ng/ml) and normal (≥30ng/ml). Data was analyzed using the SPSS® version 19.0 for windows. Categorical data is presented as proportions/percentages and continuous variable as median and inter-quartile range. Comparisons across groups were performed using non-parametric tests. P<0,05 is considered statistically significant. Results The mean age was 59.4+11.5 years and 53.7+11.5 years for SSc patients and controls respectively. Females represented 87.7% of the SSc group and 85.2% of the control group. We registered a statistically significant difference in 25OHD levels between SSc group and control group (Z=-4,392, p<0,001, median 16,3ng/ml versus 27ng/ml). Estimated sun exposure time was smaller in SSc patients than in controls. The difference in 25OHD levels between groups remained statistically significant even after adjusting 25OHD levels for sun exposure time. In logistic regression analysis both the disease (β=0,329, p=0,001) and sun exposure time (β=0,483, p<0,001) were considered predictors of 25OHD levels. The mean level of 25OHD among SSc patients was 16,6±8,3 ng/ml and suboptimal levels of 25OHD were observed in 40 patients (88,9%). 25OHD serum levels were lower in patients with shorter sun exposure time (χ2=2,32, p<0,001). A significant negative correlation was observed between 25OHD serum levels and Rodnan skin score (rs=-0,458, p=0,002). Significant correlation was not found between 25OHD serum concentrations and other clinical or biological aspects. Conclusions Patients with SSc have significantly lower median serum vitamin D concentrations than healthy control subjects, and a higher prevalence of vitamin D deficiency. Both disease and sun exposure time are considered good predictor of vitamin D values. Lower levels of vitamin D are observed in patients with shorter sun exposure time and a negative correlation was observed between vitamin D levels and skin involvement. References Caramaschi P, Dalla Gassa A, Ruzzenente O, Volpe A, Ravagnani V, Tinazzi I, Barausse G, Bambara LM, Biasi D. Very low levels of vitamin D in systemic sclerosis patients. Clin Rheumatol. 2010 Dec;29(12):1419-25. Disclosure of Interest None Declared