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Objective Primary Sjögren syndrome (pSS) is characterized by T and B cell infiltration of exocrine glands. The cysteine protease cathepsin S (CatS) is crucially involved in MHCII processing and T cell stimulation, and elevated levels have been found in patients with RA, psoriasis and pSS. However, little is known about the functional characteristics and mechanisms of SS-A- and SS-B-specific T cells in pSS patients. We herein investigated the inhibition of CatS activity in different biocompartments of pSS patients including antigen-specific T cell responses. Methods Ex vivo CatS activity was assessed in tears, plasma and saliva of 15 pSS patients and 13 healthy controls (HC) and in the presence or absence of the specific CatS inhibitor RO5459072. In addition, antigen (SS-A (60kD), SS-B, influenza H3N2, tetanus toxoid and SEB)-specific T cell responses were examined using ex vivo IFN-γ/IL-17 Dual ELISPOT and Bromdesoxyuridin (BrdU) proliferation assays in the presence or absence of RO5459072. Supernatants were analysed for IL-1β, IL-6, IL-10, TNF-α, IL-21, IL-22 and IL-23, using conventional ELISA. Results CatS activity was significantly elevated in tear fluid, but not other biocompartments, was inversely associated with exocrinic function in pSS patients and could significantly be suppressed by RO5459072. Moreover, CatS inhibition by RO5459072 led to strong and dose-dependent suppression of SS-A/SS-B-specific T cell effector functions and cytokine secretion by CD14+ monocytes. However, RO5459072 was incapable of suppressing SS-A/SS-B-induced secretion of cytokines in CD14+ monocytes when T cells were absent, confirming a CatS/MHCII-mediated mechanism of suppression. Conclusion CatS activity in tear fluid seems to be a relevant biomarker for pSS disease activity. Conversely, CatS inhibition diminishes T cell and associated monokine responses towards relevant autoantigens in pSS. Thus, CatS inhibition may represent a promising novel treatment strategy in pSS. Electronic supplementary material The online version of this article (10.1186/s13075-019-1955-2) contains supplementary material, which is available to authorized users.

Aims Impairments of retinal vessel diameter are associated with major adverse cardiovascular (CV) events. Promoter DNA methylation is a repressor of the mitochondrial adaptor p66Shc gene transcription, a key driver of ageing-induced reactive oxygen species. The study aimed to investigate whether high-intensity interval training (HIIT) affects retinal microvascular phenotype as well as p66Shc expression and oxidative stress in ageing subjects with increased CV risk from the EXAMIN AGE cohort. Methods and results Eighty-four sedentary subjects (mean age 59.4 ± 7.0 years) with ≥2 CV risk factors were randomized into either a 12-week HIIT or standard physical activity recommendations. Retinal arteriolar and venular diameters were measured by use of a retinal vessel analyser. As a marker of oxidative stress plasma 3-nitrotyrosine (3-NT) level was determined by ELISA. Gene expression of p66Shc and DNA methylation were assessed in mononuclear cells by RT-qPCR and methylated-DNA capture (MethylMiner Enrichment Kit) coupled with qPCR, respectively. High-intensity interval training reduced body mass index, fat mass, low-density lipoprotein and increased muscle mass, as well as maximal oxygen uptake (VO2max). Moreover, HIIT restored microvascular phenotype by inducing retinal arteriolar widening (pre: 175 ± 14 µm vs. post: 181 ± 13 µm, P = 0.001) and venular narrowing (pre: 222 ± 14 µm vs. post: 220 ± 14 µm, P = 0.007). After HIIT, restoration of p66Shc promoter methylation (P = 0.034) reduced p66Shc gene expression (P = 0.037) and, in turn, blunted 3-NT plasma levels (P = 0.002). Conclusion High-intensity interval training rescues microvascular dysfunction in ageing subjects at increased CV risk. Exercise-induced reprogramming of DNA methylation of p66Shc gene may represent a putative mechanistic link whereby exercise protects against age-related oxidative stress. Clinical trial registration ClinicalTrials.gov: NCT02796976 (https://clinicaltrials.gov/ct2/show/NCT02796976).

Background Narrower retinal arterioles and wider venules are linked to adverse cardiovascular outcomes. The mitochondrial adaptor p66Shc is a major source of ageing-induced generation of reactive oxygen species. Promoter DNA methylation inhibits p66Shc gene transcription. This cross-sectional study was designed to investigate the link between physical activity, retinal vessel diameters and p66Shc expression in active and sedentary ageing subjects. Design/methods Altogether 158 subjects were included in the study (mean age 59.4 ± 7.0 years). Thirty-eight subjects were healthy active, 36 were healthy sedentary and 84 were sedentary with ≥2 cardiovascular risk factors. Retinal arteriolar and venular diameters were measured by means of a retinal vessel analyser. As a marker of oxidative stress, plasma 3-nitrotyrosine was determined by enzyme-linked immunosorbent assay. Gene expression of p66Shc and DNA methylation were assessed in mononuclear cells by real-time quantitative polymerase chain reaction and methylated-DNA capture (MethylMiner Enrichment kit) coupled with quantitative polymerase chain reaction, respectively. Results Wider retinal arterioles (179 ± 14 vs 172 ± 11 and 171 ± 14 µm; p Shc expression and lower 3-nitrotyrosine plasma levels compared with healthy sedentary and sedentary subjects with ≥2 cardiovascular risk factors. Accordingly, hypomethylation of p66Shc promoter observed in healthy sedentary and sedentary subjects with ≥2 cardiovascular risk factors was not found in healthy active subjects. Conclusion Long-term physical activity-induced DNA methylation of p66Shc may represent a putative mechanistic link whereby active lifestyle promotes healthy microvascular ageing.

Career situation of first and presenting author Post-doctoral fellow. Introduction Recurrent activation of synovial macrophages is considered as intrinsic factor that precedes rheumatoid arthritis (RA)-related autonomous cytokine cascades. microRNAs (miRs) and Toll-like receptors (TLRs) play a pivotal role in regulating the inflammatory response in innate immune cells and are at dysregulated levels in RA patients. Objectives Here we explored TLR-regulated miRs that tune the inflammatory response of M2-type macrophages. Methods Based on a miR-screen in TLR-stimulated human M2-macrophages we selected miR-221-3p to analyze its expression profile in clinical samples from RA, other inflammatory arthritis (OIA), osteoarthritis (OA) and healthy donors (HD) by qPCR. We challenged M1- and M2-macrophages with elevated miR-221-3p and/or miR-155-5p expression thereby mimicking RA-related conditions. Cytokine/chemokine secretion of IL-6, IL-10, IL-12, IL-8 and CXCL13 was measured using ELISA. Changes of inflammatory pathways including JAK/STAT were evaluated by Western Blot. Results TLR4-stimulation of M2-macrophages results in suppression of miR-221-3p, while in vivo expression in synovial tissue and fluids is significantly increased in RA versus OA or OIA. M2-macrophages with high levels of miR-221-3p lose their anti-inflammatory response and support a M1-like profile. miR-221-3p in combination with miR-155-5p leads to the secretion of M1-specific IL-12 and lowered IL-10. miR-221-3p is affecting the JAK/STAT pathway in M2-macrophages by downregulating JAK3 protein leading to decreased STAT3 activation. Treatment of M2-macrophages using JAK3 inhibitors display the same shift in cytokine/chemokine pattern as increased miR-221-expression. Conclusions TLR4-activated M2-macrophages exposed to elevated miR-221-3p expression diminished anti-inflammatory response partly by suppressing JAK3/STAT3 pathway. Thus, miR-221-3p is a homeostatic regulator of M2-M1 inflammatory function, which is dysregulated in RA condition. Acknowledgements Tissue RNA from RA and OA patients was kindly provided by Caroline Ospelt, University of Zurich, Center of Experimental Rheumatology, Switzerland. Disclosure of Interest None declared.

Background Primary Sjogren syndrome (pSS) is an autoimmune disease characterised by an infiltration of T and B cells into exocrine gland tissue and its subsequent destruction 1 . Antigen presenting cells, including B cells, foster T cell activation and anti-SS-A/SS-B producing plasma cells, eventually leading to disease progression and systemic complications 2 . Objectives Cathepsin S (CatS) is crucially involved in MHCII processing in pSS mouse models 3 and patients 4 . In this translational study we investigated the ex vivo effects of the CatS inhibitor RO5459072 in different bio-compartments, including specific T cells, of pSS patients and healthy controls. Methods Ex vivo CatS activity was assessed in different bio-compartments of 15 pSS patients and 13 healthy controls and in presence or absence of RO5459072 using commercial activity and quantification assays. In addition, antigen (5 µg/mL SS-A, 5 µg/mL SS-B, 5 µg/mL Influenza H 3 N 2 ; 2 µg/mL Tetanus Toxoid and 100 ng/mL SEB) specific T cell responses were examined using 2 × 10 5 PBMC/well IFN-g/IL-17 Dual ELISPOT (48 hour incubation) and 5 × 10 4 PBMC/well BrdU proliferation assays after (72 hour incubation) in presence or absence of RO5459072. Results pSS patients showed significantly higher CatS activity in tear fluid than healthy controls (two-tailed t-test p ex vivo derived pSS patient cells in Elispot and BrdU assays (table 1). Table 1 Suppression of antigen specific T cell responses (Elispot) and suppression of cell proliferation (BrdU) in the absence or presence of RO5450972 (0–100 µM). One-tailed t-test: *p Conclusions CatS activity in tear fluid seems to be a relevant biomarker for pSS disease activity. RO5459072 is a potent inhibitor of CatS and the pSS associated relevant antigen specific T cell responses. References [1] Voulgarelis M, Tzioufas AG. Pathogenetic mechanisms in the initiation and perpetuation of Sjogren’s syndrome. Nat. Rev. 2010;6:529–537. [2] Brito-Zeron P, Baldini C, Bootsma H, et al. Sjogren syndrome. Nat Rev Dis Primers 2016;2:16047. [3] Saegusa K, Ishimaru N, Yanagi K, et al. Cathepsin S inhibitor prevents autoantigen presentation and autoimmunity. J Clin Invest2002;110(3):361–9. [4] Hamm-Alvarez SF, Janga SR, Edman MC, et al. Tear cathepsin S as a candidate biomarker for Sjogren’s syndrome. Arthritis Rheumatol 2014;66(7):1872–81. Acknowledgements We would like to thank Ms. Evelyn Fischer and Dr. Bettina Bannert for their valuable contribution of patient samples, technical assistance and clinical information. Disclosure of Interest P. Hargreaves Grant/research support from: Roche, M. Theron Employee of: Roche, F. Kolb Employee of: Roche, M. Manchester Employee of: Roche, B. Reis Employee of: Roche, A. Tiaden: None declared, D. Kyburz Grant/research support from: Roche, T. Manigold Grant/research support from: Roche

Background Toll-like receptors (TLRs) and macrophages play an important role in rheumatoid arthritis (RA). Currently, it is not clear whether inflammatory M1 or anti-inflammatory M2 predominate among the resident macrophages in the synovium. In the present study, we set out to investigate the impact of TLR stimulation on monocyte-derived M1 and M2 macrophage function and phenotype by mimicking the exposure to abundant TLR agonists as occurs in the context of RA. The response of macrophage subsets to TLR2 and TLR4 activation was evaluated on cluster of differentiation (CD) marker profile; cytokine secretion; gene expression; and NF-κB, interferon regulatory factors 3 and 7 (IRF3/7), and mitogen-activated protein kinase (MAPK) activation. Methods Human monocytes were isolated from peripheral blood of healthy individuals and patients with RA and differentiated into M1-like and M2-like macrophages by granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), respectively. Cells were either (1) stimulated with TLR ligands Pam3 or lipopolysaccharide (LPS) or (2) classically activated via interferon (IFN)-γ/LPS. Cytokine production was measured by enzyme-linked immunosorbent assay, and gene expression was measured by qPCR. Cells were stained for CD markers and analyzed by fluorescence-activated cell sorting. NF-κB, IRF3/7, and MAPKs were detected by Western blotting. Results Monocyte-derived macrophages of healthy donors (HD) or patients with RA displayed comparable subset-specific phenotypes upon exposure to TLR agonists. CD14 and CD163 marker expression on M2 macrophages did not change upon TLR2 and TLR4 engagement. By contrast, M2 gene markers HMOX1, FOLR2, and SLC40A1 were decreased. Importantly, M2 macrophages derived from HD or patients with RA showed both a decreased ratio of interleukin (IL)-10/IL-6 and IL-10/IL-8 upon stimulation with TLR2 ligand Pam3 compared with TLR4 ligand LPS. Gene expression of TLR2 was increased, whereas TLR4 expression was decreased, by TLR ligand stimulation. MAPKs p38, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase were activated more strongly in M2 than in M1 macrophages by Pam3 or LPS. Conclusions We show that the anti-inflammatory activity of M2 macrophages is reduced in the presence of abundant TLR2 ligands without significant changes in cell surface markers. Thus, the classical M1/M2 paradigm based on cellular markers does not apply to macrophage functions in inflammatory conditions such as RA. Electronic supplementary material The online version of this article (doi:10.1186/s13075-017-1447-1) contains supplementary material, which is available to authorized users.

BackgroundNatural immunity to Plasmodium falciparum has been widely studied, but its effects on parasite dynamics are poorly understood. Acquisition and clearance rates of untreated infections are key elements of the dynamics of malaria, but estimating these parameters is challenging because of frequent super-infection and imperfect detectability of parasites. Consequently, information on effects of host immune status or age on infection dynamics is fragmentary.MethodsAn age-stratified cohort of 347 individuals from Northern Ghana was sampled six times at 2 month intervals. High-throughput capillary electrophoresis was used to genotype the msp-2 locus of all P. falciparum infections detected by PCR. Force of infection (FOI) and duration were estimated for each age group using an immigration-death model that allows for imperfect detection of circulating parasites.ResultsAllowing for imperfect detection substantially increased estimates of FOI and duration. Effects of naturally acquired immunity on the FOI and duration would be reflected in age dependence in these indices, but in our cohort data FOI tended to increase with age in children. Persistence of individual parasite clones was characteristic of all age-groups. Duration peaked in 5-9 year old children (average duration 319 days, 95% confidence interval 318;320).ConclusionsThe main age-dependence is on parasite densities, with only small age-variations in the FOI and persistence of infections. This supports the hypothesis that acquired immunity controls transmission mainly by limiting blood-stage parasite densities rather than changing rates of acquisition or clearance of infections.