R. M. R. Pereira

Splenic Macrophage Subsets and Their Function during Blood-Borne Infections.

The spleen is one of the major immunological sites for maintaining blood homeostasis. Previous studies showed that heterogeneous splenic macrophage populations contribute in complimentary ways to control blood-borne infections and induce effective immune responses. Marginal metallophilic macrophages (MMMΦs) and marginal zone macrophages (MZMΦs) are cells with great ability to internalize blood-borne pathogens such as virus or bacteria. Their localization adjacent to T- and B-cell-rich splenic areas favors the rapid contact between these macrophages and cells from adaptive immunity. Indeed, MMMΦs and MZMΦs are considered important bridges between innate and adaptive immunity. Although red pulp macrophages (RpMΦs) are mainly considered scavengers for senescent erythrocytes, several data indicate a role for RpMΦs in control of infections such as blood-stage malaria as well as in the induction of innate and adaptive immunity. Here, we review current data on how different macrophage subsets recognize and help eliminate blood-borne pathogens, and, in turn, how the inflammatory microenvironment in different phases of infection (acute, chronic, and after pathogen clearance) influences macrophage function and survival.

Metformin exerts antitumor activity via induction of multiple death pathways in tumor cells and activation of a protective immune response

The antitumor effect of metformin has been demonstrated in several types of cancer; however, the mechanisms involved are incompletely understood. In this study, we showed that metformin acts directly on melanoma cells as well as on the tumor microenvironment, particularly in the context of the immune response. In vitro, metformin induces a complex interplay between apoptosis and autophagy in melanoma cells. The anti-metastatic activity of metformin in vivo was assessed in several mouse models challenged with B16F10 cells. Metformins activity was, in part, immune system-dependent, whereas its antitumor properties were abrogated in immunodeficient (NSG) mice. Metformin treatment increased the number of lung CD8-effector-memory T and CD4+Foxp3+IL-10+ T cells in B16F10-transplanted mice. It also decreased the levels of Gr-1+CD11b+ and RORγ+ IL17+CD4+ cells in B16F10-injected mice and the anti-metastatic effect was impaired in RAG-1−/− mice challenged with B16F10 cells, suggesting an important role for T cells in the protection induced by metformin. Finally, metformin in combination with the clinical metabolic agents rapamycin and sitagliptin showed a higher antitumor effect. The metformin/sitagliptin combination was effective in a BRAFV600E/PTEN tamoxifen-inducible murine melanoma model. Taken together, these results suggest that metformin has a pronounced effect on melanoma cells, including the induction of a strong protective immune response in the tumor microenvironment, leading to tumor growth control, and the combination with other metabolic agents may increase this effect.

Lysosomal Cathepsin Release Is Required for NLRP3-Inflammasome Activation by Mycobacterium tuberculosis in Infected Macrophages

Lysosomal cathepsin B (CTSB) has been proposed to play a role in the induction of acute inflammation. We hypothesised that the presence of active CTSB in the cytosol is crucial for NLRP3-inflammasome assembly and, consequently, for mature IL-1β generation after mycobacterial infection in vitro. Elevated levels of CTSB was observed in the lungs of mice and rabbits following infection with Mycobacterium tuberculosis (Mtb) H37Rv as well as in plasma from acute tuberculosis patients. H37Rv-infected murine bone marrow-derived macrophages (BMDMs) displayed both lysosomal leakage, with release of CTSB into the cytosol, as well as increased levels of mature IL-1β. These responses were diminished in BMDM infected with a mutant H37Rv deficient in ESAT-6 expression. Pharmacological inhibition of cathepsin activity with CA074-Me resulted in a substantial reduction of both mature IL-1β production and caspase-1 activation in infected macrophages. Moreover, cathepsin inhibition abolished the interaction between NLRP3 and ASC, measured by immunofluorescence imaging in H37Rv-infected macrophages, demonstrating a critical role of the enzyme in NLRP3-inflammasome activation. These observations suggest that during Mtb infection, lysosomal release of activated CTSB and possibly other cathepsins inhibitable by CA07-Me is critical for the induction of inflammasome-mediated IL-1β processing by regulating NLRP3-inflammasome assembly in the cytosol.

128 Outcomes of 847 childhood-onset systemic lupus erythematousus patients in three age groups

Background and aims To assess outcomes of childhood systemic lupus erythematosus (cSLE) in three different age groups evaluated at last visit: group A early-onset disease(<6 years), group B school age(≥6and<12 years) and group C adolescent(≥12and<18 years). Methods Observational cohort study of 10 Paediatric Rheumatology centres, including 847 cSLE patients. Results Group A had 39 (4%), B 395 (47%) and C 413 (49%). Median disease duration was higher in group A compared to groups B and C[8.3 (0.1–23.4) vs. 6.2 (0–17) vs. 3.3 (0–14.6) years, p<0.0001]. The median SLICC/ACR-DI[0 (0–9) vs. 0 (0–6) vs. 0 (0–7), p=0.065] was comparable in all groups. Further analysis of organ/system damage revealed that frequencies of neuropsychiatric(21% vs. 10% vs. 7%, p=0.007), skin (10% vs. 1% vs. 3%, p=0.002) and peripheral vascular involvements(5% vs. 3% vs. 0.3%, p=0.008) were more frequent in group A compared to B and C. Frequencies of severe cumulative lupus manifestations such as nephritis, thrombocytopenia and autoimmune hemolytic anaemia were similar in all groups(p>0.05). Mortality rate was higher in group A compared to groups B and C(15% vs. 10% vs. 6%, p=0.028). Out of 69 deaths, 33/69 (48%) occurred within the first two years after diagnosis. Infections accounted for 54/69 (78%) of the deaths and 38/54 (70%) had concomitant disease activity. Conclusions This large multicenter study provided evidence that early-onset cSLE group had distinct outcomes, with higher mortality rate and neuropsychiatric/vascular/skin organ damages in spite of comparable frequencies of severe cumulative lupus manifestations. We also identified that overall death in cSLE patients was an early event mainly attributed to infection associated with disease activity.

129 Panniculitis in childhood-onset systemic lupus erythematosus: a multicentric cohort study

Background and Aims To evaluate prevalence, clinical manifestations, laboratory abnormalities, treatment and outcome in a multicenter cohort of childhood-onset systemic lupus erythematosus(cSLE) patients with and without panniculitis. Methods Panniculitis was diagnosed due to painful subcutaneous nodules and/or plaques in deep dermis/subcutaneous tissues and lobular/mixed panniculitis with lymphocytic lobular inflammatory infiltrate in skin biopsy. Statistical analysis was performed using Bonferroni correction(p<0.004). Results Panniculitis was observed in 6/847 (0.7%) cSLE. Painful subcutaneous erythematosus and indurated nodules were observed in 6/6 panniculitis patients and painful subcutaneous plaques in 4/6. Generalised distribution was evidenced in 3/6 and localised in upper limbs in 2/6 and face in 1/6. Histopathology features showed lobular panniculitis without vasculitis in 5/6(one of them had concomitant obliterative vasculopathy due to antiphospholipid syndrome) and panniculitis with vasculitis in 1/6. Comparison between cSLE with panniculitis and 60 cSLE without panniculitis with same disease duration [2.75 (0–11.4) vs. 2.83 (0–11.8) years, p=0.297], showed higher frequencies of constitutional involvement (67% vs. 10%,p=0.003), leukopenia (67% vs. 7%, p=0.002) and median C-reactive protein (10.5 vs. 0.5 mg/L, p=0.001). Cutaneous atrophy and hyperpigmentation occurred in 83% of patients. Conclusions Panniculitis is a rare skin manifestation of cSLE occurring in the first three years of disease with considerable sequelae. The majority of patients have concomitant mild lupus manifestations.

TLR4-mediated immunomodulatory properties of the bacterial metalloprotease arazyme in preclinical tumor models

ABSTRACT Despite the recent approval of new agents for metastatic melanoma, its treatment remains challenging. Moreover, few available immunotherapies induce a strong cellular immune response, and selection of the correct immunoadjuvant is crucial for overcoming this obstacle. Here, we studied the immunomodulatory properties of arazyme, a bacterial metalloprotease, which was previously shown to control metastasis in a murine melanoma B16F10-Nex2 model. The antitumor activity of arazyme was independent of its proteolytic activity, since heat-inactivated protease showed comparable properties to the active enzyme; however, the effect was dependent on an intact immune system, as antitumor properties were lost in immunodeficient mice. The protective response was IFNγ-dependent, and CD8+ T lymphocytes were the main effector antitumor population, although B and CD4+ T lymphocytes were also induced. Macrophages and dendritic cells were involved in the induction of the antitumor response, as arazyme activation of these cells increased both the expression of surface activation markers and proinflammatory cytokine secretion through TLR4-MyD88-TRIF-dependent, but also MAPK-dependent pathways. Arazyme was also effective in the murine breast adenocarcinoma 4T1 model, reducing primary and metastatic tumor development, and prolonging survival. To our knowledge, this is the first report of a bacterial metalloprotease interaction with TLR4 and subsequent receptor activation that promotes a proinflammatory and tumor protective response. Our results show that arazyme has immunomodulatory properties, and could be a promising novel alternative for metastatic melanoma treatment.

SAT0253 Evans Syndrome at Childhood-Onset Systemic Lupus Erythematosus Diagnosis: A Large Multicenter Study

Background Evans syndrome (ES) is an uncommon manifestation characterized by autoimmune destruction of red cells and platelets and concomitant or sequential appearance of immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA). This involvement has been associated to severe disease activity in adult patients with systemic lupus erythematosus (SLE), particularly at disease onset. However, ES studies in childhood-onset SLE (cSLE) patients have been rarely reported and limited to small populations. Objectives The objective of the present multicenter study was to assess ES in a large cSLE cohort at diagnosis evaluating prevalence, clinical features, laboratory findings and outcomes. Methods A retrospective multicenter cohort study (Brazilian cSLE group) was performed in 10 Pediatric Rheumatology services including 850 patients with cSLE (ACR criteria). None of them had secondary etiologies of autoimmune cytopenias, such as infections, primary immunodeficiencies and malignancies. Patients were divided in two groups for the assessment of lupus manifestations, laboratory exams and treatment at cSLE diagnosis: patients with ES and patients without ES. Results ES was observed in 11/850 (1.3%) cSLE patients at diagnosis. The majority of them had hemorrhagic manifestations (58%) and active disease (82%). All patients with ES were hospitalized and none of them died. Comparisons of cSLE patients with and without ES at diagnosis revealed similar frequencies of female gender, multi-organ involvement, autoantibody profile and low complement levels (p>0.05). Patients with ES had a lower frequency of malar rash (9% vs. 53%, p=0.003) and musculoskeletal involvement (18% vs. 69%, p=0.001) than those without this complication. The median of hemoglobin [7.4 (5.4–9.4) vs. 10.3 (3.5–16.4)g/dL, p<0.001] and platelets [27 (15–54) vs. 231 (2–761)x103/mm3, p=0.005] were significantly lower in ES compared to non-ES patients, whereas lymphocytes were significantly higher in ES patients [1.8 (1–2.38) vs. 1.16 (0.07–7)x103/mm3, p<0.001]. The frequencies of intravenous methylprednisolone (82% vs. 43%, p=0.013) and intravenous immunoglobulin use (64% vs. 3%, p<0.0001) were significantly higher in the former group. Current prednisone dose between the two groups was similar [1.1 (0.76–1.5) vs. 1.0 mg/kg/day (0–30), 0.195]. Conclusions Our large multicenter study identified that ES was a rare and severe cSLE manifestation with a difficult diagnosis due to the absence of typical lupus manifestations, often requiring hospitalization and intravenous treatment. Disclosure of Interest G. Lube: None declared, M. Ferriani: None declared, L. Campos: None declared, M. Terreri: None declared, E. Bonfá: None declared, C. Magalhães: None declared, N. Aikawa: None declared, D. Piotto: None declared, O. Peracchi: None declared, M. C. Santos: None declared, S. Appenzeller: None declared, V. Ferriani: None declared, R. Pereira: None declared, C. Silva Grant/research support from: Conselho Nacional de Desenvolvimento Científico e Tecnolόgico (CNPq 302724/2011–7 to CAS), Federico Foundation (to CAS) and by Núcleo de Apoio à Pesquisa “Saúde da Criança e do Adolescente” da USP (NAP-CriAd) to CAS

FRI0515 Quantification and Impact of Secondary Osteoarthritis in Patients with Anti-Citrullinated Protein Antibodies Positive Rheumatoid Arthritis

Background Erosions have been described as the hallmark of bone damage in RA, resembling catabolic changes [1]. Usually bone erosions in RA are not accompanied by proliferative bone changes, which are typically found in psoriatic arthritis and spondyloarthritis [2]. Although such changes are much less pronounced in RA than in other diseases, RA patients may not be completely spared from bony proliferations, which likely resemble the development of secondary osteoarthritis. Objectives To search for evidence of secondary osteoarthritis in patients with rheumatoid arthritis (RA) in a cross-sectional and longitudinal setting and to relate osteophyte formation to functional outcome. Methods Anti-citrullinated protein antibody positive (ACPA+) RA patients underwent high-resolution peripheral quantitative computed tomography (HR-pQCT) of the hand. Cross-sectional and longitudinal measurements were performed. Number and size of osteophytes as well as bone erosions were documented. Relation of osteophytes to bone erosions and to demographic and disease specific data was evaluated by multiple logistic regression models. Results 202 ACPA+ RA patients were enrolled in the cross-sectional and 77 subjects in the longitudinal analysis (interval: 1.5 years). The mean number and size of osteophytes was 1.3±2.3 and 9.4±23.9 mm3 respectively. Osteophyte number was significantly (p<0.001) correlated to erosion number and osteophyte size to erosion size (p<0.001). Moreover, presence and size of osteophytes were related to age (p<0.001), disease duration (p<0.001) and health assessment questionnaire (HAQ) (p=0.013). Multivariate regression analyses showed an independent association between osteophytes and erosions. In the longitudinal analysis the mean number (p=0.033) and size (p<0.001) of osteophytes significantly increased in RA patients during their disease course (Figure 1). Conclusions Age, disease duration and bone erosions are associated with osteophytes indicating development of secondary osteoarthritis in patients with RA. References McInnes IB, Schett G. The pathogenesis of rheumatoid arthritis. N Engl J Med. 2011;365(23):2205–19. Diarra D, Stolina M, Polzer K, Zwerina J, Ominsky MS, Dwyer D, et al. Dickkopf-1 is a master regulator of joint remodeling. Nat Med. 2007;13(2):156–63. Disclosure of Interest None declared

OP0004 Visceral Fat Measured by Dual-Energy X-Ray Absorptiometry is Associated with Increased Risk of Non-Spine Fractures in Nonobese Elderly Women: A Population-Based Prospective Cohort Analysis from The São Paulo Ageing & Health (Spah) Study

Background The protective role of obesity on bone health has currently been questioned, since it has been demonstrated that visceral fat have a deleterious effect on bone. However, there are no studies evaluating the association between visceral fat measured by DXA with fracture risk. Objectives The aim of this study was to investigate the association of visceral fat with incident non-spine fractures in community-dwelling elderly women. Methods This is a longitudinal prospective population-based cohort study evaluating 433 community-dwelling women aged 65 years or older. Specific questionnaire (clinical and anthropometric data), including personal history of fragility fracture in non-spine osteoporotic sites (hip, humerus, wrist, rib) was performed at baseline and after an average of 4.3 years. All incident fractures during the study period were confirmed by affected site radiography. Bone mineral density (BMD) and laboratory tests were also performed at baseline. Visceral fat was measured by a new software of dual-energy X-ray absorptiometry (DXA) in the android region of a total body DXA scan. Logistic regression models were used to estimate the relationship between visceral fat and non-spine fractures. Results The mean age was 72.8±4.7 years and 28 incident non-spine osteoporotic fractures were identified after a mean follow-up time of 4.3±0.8 years. According the Lipschitz classification for nutritional status in elderly, 61.4% of women were considered obese/overweigth (BMI >27 kg/m2) and 38.6% were nonobese (7.4% underweight- BMI <22 kg/m2 and 31.2% normal weight- BMI ≥22 and ≤27 kg/m2). After adjusting for age, previous fracture and BMD (parameters with significance at univariate analysis), visceral fat area had a significant association with incident non-spine fractures in nonobese (BMI ≤27 kg/m2) elderly women (p=0.009). Conclusions Higher visceral fat was associated with the risk for non-spine fractures in nonobese elderly women. This study supports a potential negative effect of visceral adiposity on bone health. References Gower BA, Casazza K. Divergent effects of obesity on bone health. J Clin Densitom. 2013;16(4):450-454. Cohen A, Dempster DW, Recker RR, et al. Abdominal fat is associated with lower bone formation and inferior bone quality in healthy premenopausal women: a transiliac bone biopsy study. J Clin Endocrinol Metab. 2013;98(6):2562-2572. Sheu Y, Marshall LM, Holton KF, et al. Abdominal body composition measured by quantitative computed tomography and risk of non-spine fractures: the Osteoporotic Fractures in Men (MrOS) Study. Osteoporos Int. 2013;24(8):2231-2241. Lipschitz DA. Screening for nutritional status in the elderly. Prim Care. 1994; 21(1):55-67. Disclosure of Interest None declared

THU0517 Invasive Fungal Infections Survey in 852 Childhood-Onset Systemic Lupus Erythematosus: A Multicenter Cohort

Background The majority of infections in childhood-onset systemic lupus erythematosus (cSLE) patients are caused by virus and bacteria, and less frequently by opportunistic agents, such as fungi.1 However, studies evaluating solely invasive fungal infections (IFI) in cSLE patients are restricted to case reports or case series1 without any systematic evaluation of the possible associated risk factors and outcomes in pediatric lupus population. Objectives To study the prevalence, risk factors and mortality of IFI in cSLE patients. Methods A retrospective multicenter cohort study was performed in 852 cSLE patients from 10 Pediatric Rheumatology services of São Paulo state, Brazil. An investigator meeting was held and all participants received database training. IFI were diagnosed according to EORTC/MSG Consensus Group (proven, probable and possible).2 Demographic data, clinical, laboratorial, SLEDAI-2K, SLICC/ACR-DI, treatment and outcome were also evaluated. Results IFI were recorded in 33/852 (3.9%) cSLE patients. Proven IFI was evidenced in 22 cSLE patients, probable IFI in 5 and possible IFI in 6. The most frequent types of IFI were candidiasis (n=20) and aspergillosis (n=9). The median of disease duration was lower (1.0 vs. 4.7 years, p<0.0001), with a higher current SLEDAI-2K [19.5 (0-44) vs. 2 (0-45), p<0.0001] and current prednisone dose [50 (10-60) vs. 10 (2-90) mg/day, p<0.0001] in patients with IFI compared to those without IFI. The frequency of death was higher in the former group (51% vs. 6%, p<0.0001). Logistic regression analysis revealed that SLEDAI-2K (OR=1.108; 95%CI=1.057-1.163; p<0.0001), current prednisone dose (OR=1.046; 95%CI=1.021-1.071; p<0.0001) and disease duration (OR=0.984; 95%CI=0.969-0.998; p=0.030) were independent risk factors for IFI (R2 Nagelkerke 0.425). Conclusions This was the first study that characterized IFI in a large cSLE population. We identified that disease activity and glucocorticoid use were the main risk factors for these life-threatening infections, mainly in early disease course and with a high rate of fatal outcome. References Silva MF, Ribeiro AS, Fiorot FJ, et al. Invasive aspergillosis: a severe infection in juvenile systemic lupus erythematosus patients. Lupus 2012;21:1011-6. De Pauw B, Walsh TJ, Donnelly JP, et al. Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group. Clin Infect Dis 2008;46:1813-21. Disclosure of Interest None declared