Search

Conditional mutation of protein geranylgeranyltransferase type I (GGTase-I) in macrophages (GLC) activates Rho-GTPases and causes arthritis in mice. Knocking out Rag1 in GLC mice alleviates arthritis which indicates that lymphocytes are required for arthritis development in those mice. To study GLC dependent changes in the adaptive immunity, we isolated CD4

Background:Geranylgeranyltransferase type I (GGTaseI) is the enzyme responsible for the prenylation/ lipidation of the RhoA family proteins, which keeps them attached to the cell membrane. We reported that GGTaseI-deficient (GLC) mice develop a spontaneous and age-dependent arthritis, reproducing the pathology of RA1. Targeting GGTaseI activates RhoA proteins.Objectives:To study which of the activated Rho proteins is responsible for development of arthritis, we deleted individual RhoA, Rac1 or Cdc42 genes in GLC mice. We study consequences of GGTaseI deficiency for lymphocyte function.Methods:Double deficient mice that lack Rac1 (GLC Rac1fl/fl), RhoA (GLC RhoAfl/fl) and Cdc42 (GLC Cdc42fl/fl) were developed by Cre-technology using the LysM-promotor, and were on a mixed genetic background (129Ola/Hsd-C57BL/6)2. Joints of the hind paws were assessed for signs of arthritis histologically and by micro CT at age of 16 weeks. Phenotype of spleen CD4 and CD8 T cells was analysis by flow cytometry. Proliferation and cytokine production was assessed in spleen cultures by ELISA. Gene expression profile was analyzed by RT-PCR.Results:Deletion of Rho proteins had divergent effect on development of arthritis in GLC mice. We observed a reduction of the arthritis index in GLC Rac1fl/fl (n=19, p=0.027) and GLC RhoAfl/fl (n=4, p=0.007) mice compared to GLC (n=16), while GLC Cdc42fl/fl (n=4) had no change in arthritis development. GLC RhoAfl/fl mice increased the bone mass compared to GLC (p=0.029).Flow cytometry analysis showed that RA-prone GLC and GLC Cdc42fl/fl mice had lower number of CD4 cells in spleen. CD4 cells of RA-prone GLC and GLC Cdc42 mice had significantly higher subsets of the regulatory FoxP3+ and FOXp3+CD25+ cells (p=0.016-0.029 and p=0.016-0.029 respectively) compared to control and GLC RhoAfl/fl mice. Additionally, RA-prone mice had higher expression of receptors to extracellular matrix proteins collagen (α2β2) and fibronectin (α5β1) compared to control mice (p=0.016 and p=0.011 resp) and to RA-protected mice (GLC Rac1fl/fl and GLC RhoAfl/fl, p=0.0004 and p=0.011, resp). In total, both the number of FoxP3+ CD4 cells and the expression of α5β1 receptors on CD4 cells correlated strongly with the synovitis score (r=0.72, p=0.0017 and r=0.59, p=0.012, respectively).GGTaseI gene lays under the control of HOX proteins essential for cell homing. Importantly, HOX regulate the expression of integrins. Studying the expression of HoxA genes in spleen, we found that RA prone GLC and GLC Cdc42 mice tended to have lower expression of HoxA2 and higher expression of HoxA9 compared to RA-protected GLC Rac1 and GLC RhoA and to control mice. The Hoxa9/Hoxa2 ratio was significantly higher in RA prone mice compared to RA-protected mice (p=0.0085) and control mice (p=0.019). This ratio correlated with α5β1 receptors (r=0.55, p=0.0084), FOXP3+ CD4 cells (r=0.50, p=0.017), and the arthritis index (r=0.50, p=0.033).Conclusion:Taken together this study shows that Rho proteins play divergent role in development of arthritis. Activation of Rac1 and RhoA by GGTaseI deletion changes the pattern of HOXA proteins and increases expression of integrin receptors, which facilitates leukocyte influx in the paw joints. Deletion of Rac1 and RhoA has RA-protective effect in GLC mice.References:[1]Khan, O.M., et al.J Clin Invest121, 628 (2011).[2]Akula, M.K., et al.Nat Commun10, 3975 (2019).Disclosure of Interests:None declared

Background:Deficiency in geranylgeranyltransferase type I (GGTase-I) results in accumulation of active Rho family proteins RhoA, Rac1 and Cdc42, responsible for cell communication and migration. We reported that mice with GGTase-I deficient macrophages (GLC mice) develop a spontaneous and age-dependent arthritis, reproducing pathology of RA [1].Objectives:We study how GGTase-I deficiency in Mø changes T cell phenotype to facilitate their translocation to joints and the development of arthritis.Methods:GLC mice were developed on a mixed genetic background (129Ola/Hsd-C57BL/6) by Cre-technology using LysM-promotor to knockout the Pggt1b gene in Mø[2]. CD4+ cells were isolated from spleen and lymph node (LN) of 16 weeks-old mice (GLC n=7, wt n=5) expected to have high prevalence of arthritis. RNA was extracted to measure expression of the Rho proteins and signature genes to characterize differences in Th-subtypes and migration abilities of CD4+ cells between GLC and wt mice. Furthermore, Illumina RNAseq analyzed the transcriptome of LN CD4+ cells. In a separate experiment we treated GLC mice with CTLA4-FP (n=12) or PBS (n=11) for 20 weeks from the age of 5 weeks. Rationale was to disrupt Mø/T cell contact to prevent arthritis. To study Rho-protein dependent phenotype in human RA, we performed RNAseq of sorted CD4+ cells of RA patients.Results:RNAseq showed that CD4+ cells in LN of GLC mice had IFN-γ dependent cytotoxic profile and upregulated numerous pro-inflammatory genes including Eomes, Cxcr3, Tigit, Tnfsf10, Il-1rl1, Stat1, Jak3, Irf7, Irf5, Ptpn13. Furthermore, the over-represented genes often depended on the IRF family in their transcription.GLC mice overexpressed Cdc42 and Rac1 in spleen CD4+ compared to wt (p=0.005 and p=0.048 resp.). Spleen GLC CD4+ cells had higher levels of α5β1 and α2β2 integrins, strongly correlating to Cdc42 (r= 0.61 p=0.0027 and r=0.50, p=0.018) and arthritis (r=0.64, p=0.0015 and r=0.69, p=0.0004). Importantly, Cdc42, Rac1, and RhoA were higher expressed in LN CD4+ compared to spleen (p=0.016, p=0.031 and p=0.016). In addition, Itgb1 coding for β1 integrin, was upregulated in GLC CD4+ cells of both spleen and LN (p=0.003 p=0.03, resp.), suggesting Rho proteins are important for migration of CD4+ cells to the joint draining LN and for arthritis development. CD4+ cells that migrated to the LN had high proportion of Foxp3+ cells. This also correlated to the expression of Itgb1 (r=0.84, p=0.0012) presenting a plausible mechanism for increased influx of Tregs into joints. Several observations are in favor of this notion. First, GLC mice expressed more Foxp3 in LN compared to spleen CD4+ cells (p=0.016). Second, transcription of Foxp3 in LN CD4+ cells was higher in GLC mice compared to wt (p=0.015). Third, this high Foxp3 coexisted with low transcription of Lef1 (p=0.03), required for Treg immunosuppression. Last, Foxp3 correlated negatively to both Lef1 (r=-0.72, p=0.017), and its cofactor Tcf1 (r=-0.75, p=0.01).CTLA4-FP reduced inflammation in GLC mice evident as lower IFN-γ, IL-6 and TNF-α production (p=0.0002, p+CD4+cells in spleen (p=0.027). In contrast, we observed increased IL-17A production (p=0.056). However, CTLA4-FP treatment did not affect migration of CD4+ cells enriched with Rho-protein into draining LN nor alleviate arthritis.Similar to the GLC mice, CD4+ cells of RA patients with high expression of RhoA, Rac1 and Cdc42 demonstrated enrichment for Th1 signature genes including IFNG, TBX21, Eomes, IL2RA, IL2RB, IL12RB2, TNF, IL18RAP (all, adj. pConclusion:This study shows that accumulation of Rho-proteins in CD4+ cells results in pro-inflammatory IFN-γ dependent phenotype in mice and human RA. Accumulation of RhoA, Rac1 and Cdc42 proteins trigger the migration of CD4+ cells into joint draining LN and facilitates arthritis. Inhibiting Mø/T cell contact in GLC mice did not suffice to prevent migration of Rho-protein expressing cells and arthritisReferences:[1]Khan, O.M., et al. J Clin Invest, 2011. 121(2): p. 628-39.[2]Akula, M.K., et al. Nat Commun, 2019. 10(1): p. 3975.Disclosure of Interests:None declared

Specific serum antibodies mediating humoral immunity and autoimmunity are provided by mature plasma cells (PC) residing in the bone marrow (BM), yet their dynamics and composition are largely unclear. We here characterize distinct subsets of human PC differing by CD19 expression. Unlike CD19 + PC, CD19 − PC were restricted to BM, expressed predominantly IgG, and they carried a prosurvival, distinctly mature phenotype, that is, HLA-DR low Ki-67 − CD95 low CD28 + CD56 +/− , with increased BCL2 and they resisted their mobilization from the BM after systemic vaccination. Fewer mutations within immunoglobulin V H rearrangements of CD19 − BMPC may indicate their differentiation in early life. Their resistance to in vivo B-cell depletion, that is, their independency from supply with new plasmablasts, is consistent with long-term stability of this PC subset in the BM. Moreover, CD19 − PC were detectable in chronically inflamed tissues and secreted autoantibodies. We propose a multilayer model of PC memory in which CD19 + and CD19 − PC represent dynamic and static components, respectively, permitting both adaptation and stability of humoral immune protection.

Background Rheumatoid arthritis (RA) has recently been linked to an exhausted state of CD4+ T cells in peripheral blood of patients [1]. Exhaustion of CD4+ T cells limits their proliferation and increases cell death. In CD8+ T cells smoking counteract exhaustion, which may lead to increased cytotoxic activity exemplified by targeting of cells with high expression of the anti-apoptotic protein survivin [2]. Exhaustion of CD4+ T cells coincides with expression of interferon (IFN) response genes [3], referred to as the IFN signature. The development of the IFN signature has been suggested to predate RA [4]. Objectives We investigated how smoking affect the CD4+ T cell population in peripheral blood of RA patients with focus on the exhaustion marker programmed cell death-1 (PD-1). Methods Blood samples were collected from RA patients and healthy women with different smoking status and analysed for PD-1 and survivin expression using flow cytometry and qPCR. Sorted Th17 cells from peripheral blood were analysed for expression of 18 genes up regulated during exhaustion [3], herein referred to as the exhaustion set, and serum levels of survivin were assessed by ELISA. Peripheral blood CD4+ cells were analysed for their expression of seven IFN response genes [4]. The role of survivin in the formation of exhausted CD4+ T cells was studied in collagen-induced arthritis (CIA), where mice were treated with nicotine or vaccinated with survivin peptides. Results High frequency of exhausted PD-1+CD4+ cells was found in smoking RA patients. The numbers of PD1+CD4+ cells correlated inversely with the PD-1 expression by cytotoxic CD8+CD107+ cells (r=-0.62, p=0.01). Additionally, the frequency of PD-1+CD4+ cells increased with reduction of the CD4+ population (r=-0.71, p=0.002). The IFN signature was found exclusively among smoking RA patients. The patients with the IFN signature all had CD4+ cells with low survivin production. Th17 cells from RA patients with high serum survivin were enriched in genes of the exhaustion set. CD4+ cells with high survivin expression were negative for PD-1, while PD-1hi cells had low expression of survivin. In CIA mice the survivinhiPD-1- CD4+ cells were reduced by nicotine treatment (p=0.03) or survivin vaccination (p=0.009). Conclusions Smoking associates with exhaustion of CD4+ T cells in RA by increasing the frequency of PD-1+CD4+ cells and supporting the IFN signature. Balancing T cell exhaustion and preventing the IFN signature are potential future treatment strategies for RA. References Frenz T, et al. J Allergy Clin Immunol 2016. 138(2): 586–589. Wasen C, et al. J Autoimmun 2017. DOI: 10.1016/j.jaut.2016.12.00. Crawford A, et al. Immunity 2014. 40(2): 289–302. Lubbers J, et al. Ann Rheum Dis 2013. 72(5): 776–780. Disclosure of Interest None declared

Survivin is a protein functionally important for cell division, apoptosis, and possibly, for micro-RNA biogenesis. It is an established marker of malignant cell transformation. In non-malignant conditions, the unique properties of survivin make it indispensable for homeostasis of the immune system. Indeed, it is required for the innate and adaptive immune responses, controlling differentiation and maintenance of CD4+ and CD8+ memory T-cells, and in B cell maturation. Recently, survivin has emerged as an important player in the pathogenesis of autoimmune diseases. Under the conditions of unreserved inflammation, survivin enhances antigen presentation, maintains persistence of autoreactive cells, and supports production of autoantibodies. In this context, survivin takes its place as a diagnostic and prognostic marker in rheumatoid arthritis, psoriasis, systemic sclerosis and pulmonary arterial hypertension, neuropathology and multiple sclerosis, inflammatory bowel diseases and oral lichen planus. In this review, we summarise the knowledge about non-malignant properties of survivin and focus on its engagement in cellular and molecular pathology of autoimmune diseases. The review highlights utility of survivin measures for clinical applications. It provides rational for the survivin inhibiting strategies and presents results of recent reports on survivin inhibition in modern therapies of cancers and autoimmune diseases.

Background Exhausted CD4+ T cells are recognised by low proliferation, increased cell death and high expression the inhibitory co-receptor programmed cell death-1 (PD-1). CD4+ T cells in patients with rheumatoid arthritis (RA) have recently been reported to show signs of exhaustion.1 Exhaustion of CD4+ in experimental infection was associated with expression of interferon (IFN) response genes,2 referred to as the IFN signature. In human, the IFN signature has been suggested to predict rheumatoid arthritis (RA).3 We investigated if CD4+ T cells with an exhausted phenotype and IFN signature are accumulating in peripheral blood of RA patients. We study a connexion between CD4+ T cell exhaustion and survivin expression in RA. Materials and methods IFN response genes, PD-1 and survivin expression were analysed in peripheral blood of RA patients and healthy women with different smoking status using flow cytometry and qPCR. The role of survivin in the formation of exhausted CD4+ T cells was studied in the collagen-induced arthritis model, where mice where treated with nicotine or vaccinated with survivin peptides. Results High frequency of exhausted PD-1+CD4+ cells was found in smoking RA patients. The numbers of PD1+CD4+ cells correlated inversely with the PD-1 expression by cytotoxic CD8+CD107+ cells (r=−0.62, p=0.01). Additionally, the frequency of PD-1+CD4+ cells increased with reduction of the CD4+ population (r=−0.71, p=0.002). The IFN signature was found exclusively among smoking RA patients. The patients with the IFN signature all had CD4+ cells with low survivin production. Sorted Th17 cells from RA patients with high serum levels of survivin had higher transcription of exhaustion related genes compared to healthy controls. CD4+ cells with high survivin expression were negative for PD-1. In mice, the survivinhiPD-1- population of CD4+ cells was reduced in nicotine-treated mice (p=0.03) and in response to activation of survivin targeting cells through vaccination (p=0.009). Conclusions We show that smoking associates with exhaustion of CD4+ T cells in RA. Smoking increases the frequency of PD-1+CD4+ cells with the IFN signature by activating survivin targeting mechanisms. References Frenz, T, et al, CD4+ T cells in patients with chronic inflammatory rheumatic disorders show distinct levels of exhaustion. Journal of Allergy and Clinical Immunology, 2016;138(2):p.586–589.e10. Crawford, A, et al, Molecular and Transcriptional Basis of CD4+ T Cell Dysfunction during Chronic Infection. Immunity2014;40(2):p.289–302. Lubbers, J, et al, The type i IFN signature as a biomarker of preclinical rheumatoid arthritis. Annals of the Rheumatic Diseases2013;72(5):p.776–780.

Epstein-Barr virus (EBV) infection may initiate production of autoantibodies and development of cancer and autoimmune diseases. Here we outline phenotypic and functional changes in B cells of patients with rheumatoid arthritis (RA) related to EBV infection. The B-cell phenotype was analysed in blood and bone marrow (BM) of RA patients who had EBV transcripts in BM (EBV(+) , n = 13) and in EBV(-) (n = 22) patients with RA. The functional effect of EBV was studied in the sorted CD25(+) and CD25(-) peripheral B cells of RA patients (n = 18) and healthy controls (n = 9). Rituximab treatment results in enrichment of CD25(+) B cells in peripheral blood (PB) of EBV(+) RA patients. The CD25(+) B-cell subset displayed a more mature phenotype accumulating IgG-expressing cells. It was also enriched with CD27(+) and CD95(+) cells in PB and BM. EBV stimulation of the sorted CD25(+) B cells in vitro induced a polyclonal IgG and IgM secretion in RA patients, while CD25(+) B cells of healthy subjects did not respond to EBV stimulation. CD25(+) B cells were enriched in PB and synovial fluid of RA patients. EBV infection affects the B-cell phenotype in RA patients by increasing the CD25(+) subset and by inducing their immunoglobulin production. These findings clearly link CD25(+) B cells to the EBV-dependent sequence of reactions in the pathogenesis of RA.

CD8

Rationale: The innate immune system and in particular the pattern-recognition receptors Toll-like receptors have recently been linked to atherosclerosis. Consequently, inhibition of various signaling molecules downstream of the Toll-like receptors has been tested as a strategy to prevent progression of atherosclerosis. Receptor-interacting protein 2 (Rip2) is a serine/threonine kinase that is involved in multiple nuclear factor-κB (NFκB) activation pathways, including Toll-like receptors, and is therefore an interesting potential target for pharmaceutical intervention. Objective: We hypothesized that inhibition of Rip2 would protect against development of atherosclerosis. Methods and Results: Surprisingly, and contrary to our hypothesis, we found that mice transplanted with Rip2 −/− bone marrow displayed markedly increased atherosclerotic lesions despite impaired local and systemic inflammation. Moreover, lipid uptake was increased whereas immune signaling was reduced in Rip2 −/− macrophages. Further analysis in Rip2 −/− macrophages showed that the lipid accumulation was scavenger-receptor independent and mediated by Toll-like receptor 4 (TLR4)–dependent lipid uptake. Conclusions: Our data show that lipid accumulation and inflammation are dissociated in the vessel wall in mice with Rip2 −/− macrophages. These results for the first time identify Rip2 as a key regulator of cellular lipid metabolism and cardiovascular disease.

BACKGROUND Efficient host defense mechanisms are crucial for survival in sepsis and septic arthritis. S100 proteins are reported to have proinflammatory and bactericidal properties. The aim of this study was to investigate the role of S100A4 in staphylococcal arthritis. METHODS S100A4 knockout mice (S100A4KO) and wild-type counterparts (WT) were intravenously and intra-articularly challenged with Staphylococcus aureus strain LS-1. Clinical and morphological signs of arthritis and sepsis, phagocytosis, bone mineral density (BMD), and bone metabolism were then monitored in S100A4 and WT mice. RESULTS S100A4KO mice had a lower bacterial load in the kidneys than WT mice (P < .05) but developed more severe clinical signs of arthritis (P < .001) and had higher levels of interleukin 6 and L-selectin (P = .002). S100A4KO mice had fewer morphological signs of synovitis and cartilage/bone destruction following intra-articular instillation of bacteria. S100A4KO mice were protected from loss of BMD and had lower levels of RANKL, MMP3, and MMP9 (P < .05). S100A4 was not bactericidal in vitro. CONCLUSIONS In staphylococcal infection, S100A4 regulates bacterial clearance as well as systemic and local inflammatory responses.

RHO family proteins are important for the function of inflammatory cells. They are modified with a 20-carbon geranylgeranyl lipid in a process catalyzed by protein geranylgeranyltransferase type I (GGTase-I). Geranylgeranylation is viewed as essential for the membrane targeting and activity of RHO proteins. Consequently, inhibiting GGTase-I to interfere with RHO protein activity has been proposed as a strategy to treat inflammatory disorders. However, here we show that mice lacking GGTase-I in macrophages develop severe joint inflammation resembling erosive rheumatoid arthritis. The disease was initiated by the GGTase-I-deficient macrophages and was transplantable and reversible in bone marrow transplantation experiments. The cells accumulated high levels of active GTP-bound RAC1, CDC42, and RHOA, and RAC1 remained associated with the plasma membrane. Moreover, GGTase-I deficiency activated p38 and NF-κB and increased the production of proinflammatory cytokines. The results challenge the view that geranylgeranylation is essential for the activity and localization of RHO family proteins and suggest that reduced geranylgeranylation in macrophages can initiate erosive arthritis.

The posttranslational modification of proteins has the potential to generate neoepitopes that may subsequently trigger immune responses. The carbamylation of lysine residues to form homocitrulline may be a key mechanism triggering inflammatory responses. We evaluated the role of carbamylation in triggering immune responses and report a new role for this process in the induction of arthritis. Immunization of mice with homocitrulline-containing peptides induced chemotaxis, T cell activation, and Ab production. The mice also developed erosive arthritis following intra-articular injection of peptides derived from homocitrulline and citrulline. Adoptive transfer of T and B cells from homocitrulline-immunized mice into normal recipients induced arthritis, whereas systemic injection of homocitrulline-specific Abs or intra-articular injection of homocitrulline-Ab/citrulline-peptide mixture did not. Thus, the T cell response to homocitrulline-derived peptides, as well as the subsequent production of anti-homocitrulline Abs, is critical for the induction of autoimmune reactions against citrulline-derived peptides and provides a novel mechanism for the pathogenesis of arthritis.

We have shown that human CD20(+)25(+) B cells display immunomodulatory properties. The aim of this study was to investigate if CD25(+) B cells are found within the CD27 memory B cell population, and to analyse pattern of their cytokine production. B cells isolated from healthy subjects, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients were analysed regarding the frequency of CD25(+) B cells within certain B cell subsets. Purified CD25(+) B cells from healthy subject were used in vitro to evaluate their production of immunomodulatory cytokines. In healthy subjects the majority (60%) of memory B cells (CD20(+)27(+)) also co-expressed CD25 while only 10-20% of the naive B cells (CD20(+)27(-)) and plasmablasts (CD20-27(+)) expressed CD25. In RA and SLE patients, we found that 51% and 48%, respectively, co-expressed CD25 in the memory population, whereas only 11% and 9% co-expressed CD25 in the naive B cell population. Phenotypic analysis of the CD20(+)25(+)27(+) and CD20(+)25(+)27(-) cells using CD10, CD24, CD38, CD45, CD71, CD80, CD86, CD95, CD138, BAFF-R, TACI, IgA, IgD, IgG and IgM showed that CD20(+)25(+)27(+) B cells preferentially represent highly activated, Ig class switched memory B cells. Cytokine profile analysis showed that CD25(+) B cells secreted significantly higher levels of IL-10 versus CD25(-) B cells. In contrast, TGF-beta1 secretion was similar between the CD25(+) and CD25(-) sub-populations. In conclusion, CD20(+)25(+) B cells constitute a unique subpopulation preferentially occurring among CD20(+)27(+) memory B cells. We suggest that CD25 can be used as a marker for a memory B cell subset.

B cells play an important role in the development of autoimmune diseases due to their production of autoantibodies, antigen-presenting capacity and production of pro-inflammatory cytokines. The purpose of the present study was to analyse B cells from rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients, with respect to their expression of the IL-2 receptor (IL-2R) subunit CD25. Using flow cytometry, we found that CD25(+) B cells from RA patients expressed significantly higher frequencies of CD122 and CD132 than CD25(+) B cells from control subjects, indicating a fully functional IL-2R. These CD25(+) B cells also expressed higher frequencies of the co-stimulatory molecule CD80, whereas IgM and IgA expression was decreased compared with CD25(+) B cells from healthy controls. In addition B cells from SLE patients co-expressed CD25 together with CD80, CD122, and CD132, but to a lower degree IgD and IgM, when compared with healthy controls. Taken together, our results indicate that CD25(+) B cells from RA and SLE patients are in a highly activated state, display a more mature phenotype and suggest that this B cell subset may be involved in the pathogenesis of RA and SLE.

Switched antibody classes are important for efficient immune responses. Aberrant antibody production to otherwise harmless antigens may result in autoimmunity. The protein kinase fms-like tyrosine kinase 3 receptor (Flt3) has an important role during early B-cell development, but the role of Flt3 in peripheral B cells has not been assessed before. Herein we describe a previously unappreciated role for Flt3 in IgG1 class-switch recombination (CSR) and production. We show that Flt3 is reexpressed on B-cell lymphoma 6(+) germinal center B cells in vivo and following LPS activation of peripheral B cells in vitro. Absence of Flt3 signaling in Flt3 ligand-deficient mice results in impaired IgG1 CSR and accumulation of IgM-secreting plasma cells. On activated B cells, Flt3 is coexpressed and functions in synergy with the common-gamma chain receptor family. B cells from Flt3 ligand-deficient mice have impaired IL-4R signaling, with reduced phosphorylation of signal transducer and activator of transcription (Stat) 6, and demonstrate a failure to initiate CSR to IgG1 with low expression of γ1 germ-line transcripts, resulting in impaired IgG1 production. Thus, functional synergy between Flt3 and IL-4R signaling is critical for Stat-mediated regulation of sterile γ1 germ-line transcripts and CSR to IgG1.

Introduction Alternative splicing distinguishes normal and pathologic cells. High levels of oncoprotein survivin recognise patients with severe rheumatoid arthritis (RA). Here, we assess clinical relevance of alternative splicing of survivin in leukocytes of peripheral blood (PBMC) and bone marrow (BM) in RA patients. Method Transcription of survivin wild-type (survivin-WT), survivin-2B and survivin-ΔEx3 was measured in 67 randomly selected RA patients and in 23 patients before and after B cell depletion with rituximab. Analysis was done in relation to disease activity, anti-rheumatic treatment and serum levels of rheumatoid factor (RF) and survivin. Results Survivin-WT was the dominant splice variant equally expressed in T and B cells, while survivin-2B and survivin-ΔEx3 were higher in B cells. High disease activity (DAS28>5.1) was associated with an excess of survivin-WT and low ratios between survivin-2B/WT (p=0.035) and survivin-ΔEx3/WT in PBMC. Depletion of B cells by rituximab caused a decrease in survivin-WT (p=0.005) in PBMC, increasing the ratio between survivin-2B/WT (p=0.009) and survivin-ΔEx3/WT (p=0.001) in BM. This increase in survivin-2B/WT was associated with reduction in CD19+ BM cells (r=0.929, p=0.007), RF (IgM, r=0.857, p=0.024; IgA, r=0.739, p=0.021), and DAS28 (0.636, p=0.054). The increase in survivin-ΔEx3 in BM was associated with a reduction of CD19+ BM cells (r=0.714, p=0.058) and DAS28 (r=0.648, p=0.049), while survivin-ΔEx3/WT was associated with RF (IgG, r=0.882, p=0.016). Conclusion This study demonstrates that the suppressed diversity of survivin splicing in leukocytes may attribute to adverse self-recognition in RA. Depletion of autoantibody producing B cells improves the balance of survivin splicing.

T-helper cells producing interleukin (IL)-17A and IL-17F cytokines (Th17 cells) are considered the source of autoimmunity in rheumatoid arthritis (RA). In this study, we characterized specific pathogenic features of Th17 cells in RA. By using nano-string technology, we analyzed transcription of 419 genes in the peripheral blood CCR6(+)CXCR3(-) CD4(+) cells of 14 RA patients and 6 healthy controls and identified 109 genes discriminating Th17 cells of RA patients from the controls. Th17 cells of RA patients had an aggressive pathogenic profile and in addition to signature cytokines IL-17, IL-23 and IL-21, and transcriptional regulators RAR-related orphan receptor gamma of T cells (RORγt) and Janus kinase 2 (JAK2), they produced high levels of IL-23R, C-C chemokine ligand type 20 (CCL20), granulocyte-monocyte colony-stimulating factor (GM-CSF ) and transcription factor Tbet required for synovial homing. We showed that Th17 cells are enriched with Helios-producing Foxp3- and IL2RA-deficient cells, indicating altered regulatory profile. The follicular T-helper (Tfh) cells presented a functional profile of adaptor molecules, transcriptional regulator Bcl-6 and B-cell activating cytokines IL-21, IL-31 and leukemia inhibitory factor (LIF ). We observed that anti-tumor necrosis factor (TNF) treatment had a limited effect on the transcription signature of Th17 cells. Patients in remission retained the machinery of receptors (IL-23R and IL-1R1), proinflammatory cytokines (IL-17F, IL-23, IL-21 and TNF ) and adaptor molecules (C-X-C chemokine receptor 5 [CXCR5] and cytotoxic T-lymphocyte-associated protein 4 [CTLA-4]), essential for efficient transdifferentiation and accumulation of Th17 cells. This study convincingly shows that the peripheral blood CCR6(+)CXCR3(-) CD4(+) cells of RA patients harbor pathogenic subsets of Th17 and Tfh cells, which may transdifferentiate from Tregs and contribute to perpetuation of the disease.

We demonstrate that humans have a phenotypically and functionally distinct subset of B lymphocytes that express the interleukin (IL)-2 receptor (IL-2R) α-chain, cluster of differentiation (CD) 25. We found that one-third of the circulating CD20 + B cells expressed CD25 and, using fluorescence-activated cell sorter (FACS) analysis, that these cells were significantly larger and more granulated than B cells not expressing CD25. The simultaneous expression of the other two subunits (CD122 and CD132) and the proliferative responses of cells expressing CD25 to IL-2 suggested that, in addition to CD25, functional IL-2 receptors were expressed on this cell population. CD25 expression on B cells was selectively up-regulated by Toll-like receptor 2 (TLR2), TLR4, and TLR9 ligands but not by a TLR3 ligand or Epstein-Barr virus (EBV) stimulation. Blockade of the nuclear factor (NF)-KB pathway completely abolished CD25 up-regulation by these B cells. Interestingly, CD25 + B cells expressed significantly higher levels of surface immunoglobulins but lacked the ability to secrete immunoglobulin (Ig), as compared with CD25 - B cells. Furthermore, CD25 + B cells performed significantly better as antigen-presenting cells in allogeneic mixed lymphocyte reactions (MLR), which may be a result of their expression of high levels of the costimulatory molecules CD27 and CD80. Finally, blocking of CD25 on B cells led to an almost total abrogation of MLR. Our results indicate that CD25 + B cells have distinct phenotypic and functional properties, including the ability to contribute to antigen presentation, which is linked to their expression of CD25. Finally, the differential regulation of CD25 expression via selective TLR ligands suggests a role for CD25 + B cells in bridging innate and acquired immune responses.

Helicobacter pyloriinfection is associated with pronounced infiltration of granulocytes and lymphocytes into the gastric mucosa, resulting in active chronic gastritis that may develop into duodenal ulcer disease or gastric adenocarcinoma. Infiltrating T cells play a major role in the pathology of these diseases, but the signals involved in recruitment of T cells from blood toH. pylori-infected tissues are not well understood. We therefore examinedH. pylori-induced T-cell transendothelial migration (TEM). The Transwell system, employing a monolayer of human umbilical vein endothelial cells, was used as a model to study TEM.H. pyloriinduced a significant T-cell migration, compared to spontaneous migration. CD4+and CD8+T cells migrated to the same extent in response toH. pylori, whereas there was significantly larger transmigration of memory T cells compared to naive T cells. BothH. pyloriculture filtrate and urease induced migration, and the presence of theH. pylori cagpathogenicity island increased TEM. T-cell TEM was mediated by LFA-1-ICAM-1 interactions in accordance with an increased ICAM-1 expression on the endothelial cells after contact withH. pylori. Migrating T cells had increased expression of activation marker CD69 and chemokine receptors CXCR3, CCR4, and CCR9. Furthermore, T cells migrating in response toH. pylorisecreted Th1 but not Th2 cytokines upon stimulation. In conclusion, our data indicate that liveH. pyloriand its secreted products contribute to T-cell recruitment to the gastric mucosa and that the responding T cells have an activated memory Th1 phenotype.

Continuous recruitment of neutrophils into the inflamed gastric mucosal tissue is a hallmark of Helicobacter pylori infection in humans. In this study, we examined the ability of H. pylori to induce transendothelial migration of neutrophils using a transwell system consisting of a cultured monolayer of human endothelial cells as barrier between two chambers. We showed for the first time that live H. pylori, but not formalin-killed bacteria, induced a significantly increased transendothelial migration of neutrophils. H. pylori conditioned culture medium also induced significantly increased transendothelial migration, whereas heat-inactivated culture filtrates had no effect, suggesting that the chemotactic factor was proteinaceous. Depletion of H. pylori-neutrophil activating protein (HP-NAP) from the culture filtrates resulted in significant reduction of the transmigration. Culture filtrates from isogenic HP-NAP deficient mutant bacteria also induced significantly less neutrophil migration than culture filtrates obtained from wild-type bacteria. HP-NAP did not induce endothelial cell activation, suggesting that HP-NAP acts directly on the neutrophils. In conclusion, our results demonstrate that secreted HP-NAP is one of the factors resulting in H. pylori induced neutrophil transendothelial migration. We propose that HP-NAP contributes to the continuous recruitment of neutrophils to the gastric mucosa of H. pylori infected individuals.

Background Smoking is the most extensively studied environmental risk factor of rheumatoid arthritis (RA). We have recently shown that smoking is associated with high serum levels of survivin (Svensson, Hafstrom et al. 2014). Survivin is a novel biomarker for RA that can predict severe joint destruction as well as treatment outcome for RA patients (Levitski et al, 2015). We hypothesized cytotoxic activity to be the reason for the increased serum levels of survivin observed in RA patients and we wished to further investigate possible molecular mechanisms behind cytotoxicity in RA. Objectives We have studied the effect of nicotine on cytotoxic activity of T lymphocytes during RA paying special attention to the expression of PD-1, a receptor that inhibits activation of T cells. Methods We used female Balb/c mice that received 0.03% nicotine in drinking water or regular tap water as controls, after 18 days mice were immunized with collagen and subsequently developed arthritis (CIA). At the end of experiment bone marrow, lymph nodes and serum were collected and analyzed using flow cytometry (FACS), qPCR and ELISA. To confirm nicotine-dependent nature of experimental findings, we analyzed blood samples of female RA patients and healthy women with known smoking status. Results The proportion of CD8+ cells was 1.9 times larger in nicotine treated mice compared to control (p=0.00020). Meanwhile, the nicotine treated mice had a 2.3 times less PD-1 expressing CD8+ cells in the bone marrow (p=0.032), but PD-1 expression in the lymph nodes was not affected by nicotine. This resulted in an accumulation of the CD8+PD-1- population in the bone marrow of the nicotine treated mice (p=0.0074). The nicotine treated mice had higher levels of serum survivin (0.0 vs 0.29 ng/ml, p=0.017) which also correlated to the size of CD8+PD-1- population in the bone marrow (r=0.52, p=0.024). The decrease in PD-1 expression of CD8+ T cells caused by nicotine was confirmed in smoking women. Smokers had a 1.7 times less PD-1 positive CD8+ T cells compared to non-smokers (p=0.041). In patients, the serum levels of survivin correlated to the expression of the cytotoxic marker CD107 on CD8+ T cells (r=0.52, p=0.047). Conclusions Nicotine exposure supports cytotoxic phenotype of CD8+ T cells characterized by high CD107 and low PD-1 expression in experimental arthritis and RA patients. Increased cytotoxic activity of CD8+ T cells is a plausible mechanism for the enrichment of survivin in serum in RA patients. See graphical abstract. References Svensson B, Hafstrom I, Erlandsson MC, Forslind K, Bokarewa MI. Smoking in combination with antibodies to cyclic citrullinated peptides is associated with persistently high levels of survivin in early rheumatoid arthritis: a prospective cohort study. Arthritis Res Ther. 2014;16(1):R12. doi: 10.1186/ar4438. Levitsky A, Erlandsson MC, van Vollenhoven RF, Bokarewa MI, Serum survivin predicts responses to treatment in active rheumatoid arthritis: a post hoc analysis from the SWEFOT trial. BMC Med. 2015;13:247. doi: 10.1186/s12916-015-0485-2. Disclosure of Interest None declared

Receptor for advanced glycation end products (RAGE) is a pattern recognition receptor that binds a variety of pro-inflammatory ligands. Its soluble form, sRAGE, can compete for ligand binding and thereby have an anti-inflammatory effect. We have recently reported that sRAGE also exerts pro-inflammatory and chemotactic properties suggesting a dual role for sRAGE in immune modulation. Our present aim was to analyse the immunomodulatory properties of sRAGE in vivo with respect to acquired immunity. Naive mice were treated intra-peritoneally with sRAGE and cells from peritoneal lavage, spleens and bone marrow were examined. Mice treated with sRAGE displayed an increased leucocyte count in the peritoneal cavity, enlarged spleens and increased cellularity compared with vehicle-treated animals. Furthermore, sRAGE-treated mice had a significantly increased frequency and number of CD19(+) B cells in spleen and a reduced frequency of CD19(+) B cells in bone marrow compared with controls. Functionally, splenocytes from sRAGE-treated mice showed elevated IgG production and up to a four-fold increased IgM secretion compared with control animals and produced significantly higher levels of interleukin-10, interferon-γ and interleukin-6 in response to lipopolysaccharide stimulation. Our results suggest that sRAGE has immunomodulatory properties, since intra-peritoneal administration of sRAGE into healthy mice leads to rearrangements in cellular composition in the bone marrow and spleen. Moreover, the administration of sRAGE directs B cells into the spleen and towards differentiation. Our novel findings indicate that sRAGE exerts an effect on the cells of adaptive immunity.

TKs are intracellular signaling molecules essential for cell homeostasis. Inhibition of TKs is used in treatment of malignancies and diabetes mellitus. The present study evaluated the role of Flt3 in antigen-induced arthritis. Mice were immunized with mBSA, and arthritis was induced by an i.a. injection of mBSA. Treatment with the Flt3 inhibitor sunitinib was started together with mBSA immunization or together with the induction of arthritis. The mBSA-injected joints were evaluated morphologically for signs of synovitis and bone/cartilage destruction. Markers of bone metabolism and antibody responses were measured by ELISA. Maturation of DCs in the bone marrow and spleen was evaluated by flow cytometry. Sunitinib treatment reduced the intensity of synovitis and the incidence of bone destruction. The reduction in bone destruction was seen when the treatment was started at the time of immunization or at the time of arthritis induction. The antiarthritic effect was achieved by inhibition of DCs, reduction of antibody production, and bone metabolism. Inhibition of Flt3 is a potent antiarthritic mechanism reducing antigen presentation, synovial inflammation, and bone resorption. Down-regulation of TKs may be a useful tool in the treatment of human RA.

Introduction Activated fibroblast-like synoviocytes (FLSs) in rheumatoid arthritis (RA) share many characteristics with tumour cells and are key mediators of synovial tissue transformation and joint destruction. The glycoprotein podoplanin is upregulated in the invasive front of several human cancers and has been associated with epithelial-mesenchymal transition, increased cell migration and tissue invasion. The aim of this study was to investigate whether podoplanin is expressed in areas of synovial transformation in RA and especially in promigratory RA-FLS. Methods Podoplanin expression in human synovial tissue from 18 RA patients and nine osteoarthritis (OA) patients was assessed by immunohistochemistry and confirmed by Western blot analysis. The expression was related to markers of synoviocytes and myofibroblasts detected by using confocal immunofluoresence microscopy. Expression of podoplanin, with or without the addition of proinflammatory cytokines and growth factors, in primary human FLS was evaluated by using flow cytometry. Results Podoplanin was highly expressed in cadherin-11-positive cells throughout the synovial lining layer in RA. The expression was most pronounced in areas with lining layer hyperplasia and high matrix metalloproteinase 9 expression, where it coincided with upregulation of α-smooth muscle actin (α-sma). The synovium in OA was predominantly podoplanin-negative. Podoplanin was expressed in 50% of cultured primary FLSs, and the expression was increased by interleukin 1β, tumour necrosis factor α and transforming growth factor β receptor 1. Conclusions Here we show that podoplanin is highly expressed in FLSs of the invading synovial tissue in RA. The concomitant upregulation of α-sma and podoplanin in a subpopulation of FLSs indicates a myofibroblast phenotype. Proinflammatory mediators increased the podoplanin expression in cultured RA-FLS. We conclude that podoplanin might be involved in the synovial tissue transformation and increased migratory potential of activated FLSs in RA.

B cells are an important part of both innate and adaptive immune system. Their ability to produce antibodies, cytokines and to present antigen makes them a crucial part in defence against pathogens. In this study, we have in naive Naval Medical Research Institute mice functionally characterized a subpopulation of splenic B cells expressing CD25, which comprise about 1% of the total B cell compartment. Murine spleen cells were sorted into two highly purified B cell populations either CD19(+) CD25(+) or CD19(+) CD25(-). We found that CD25(+) B cells secreted higher levels of IL-6, IL-10 and INFgamma in response to different TLR-agonists, and were better at presenting alloantigen to CD4(+) T cells. CD25 expressing B cells spontaneously secreted immunoglobulins of IgA, IgG and IgM subclass and had better migratory ability when compared with CD25(-) B cells. In conclusion, our results demonstrate that CD25(+) B cells are highly activated and functionally mature. Therefore, we suggest that this population plays a major role in the immune system and may belong to the memory B-cell population.

Objectives. Viruses may contribute to RA. This prompted us to monitor viral load and response to anti-CD20 therapy in RA patients. Methods. Blood and bone marrow from 35 RA patients were analysed for CMV, EBV, HSV-1, HSV-2, parvovirus B19 and polyomavirus using real-time PCR before and 3 months after rituximab (RTX) treatment and related to the levels of autoantibodies and B-cell depletion. Clinical response to RTX was defined as decrease in the 28-joint disease activity score (DAS-28) >1.3 at 6 months. Results. Before RTX treatment, EBV was identified in 15 out of 35 patients (EBV-positive group), of which 4 expressed parvovirus. Parvovirus was further detected in eight patients (parvo-positive group). Twelve patients were negative for the analysed viruses. Following RTX, EBV was cleared, whereas parvovirus was unaffected. Eighteen patients were responders, of which 12 were EBV positive. The decrease in the DAS-28 was significantly higher in EBV-positive group compared with parvo-positive group (P = 0.002) and virus-negative patients (P = 0.04). Most of EBV-negative patients that responded to RTX (75%) required retreatment within the following 11 months compared with only 8% of responding EBV-positive patients. A decrease of RF, Ig-producing cells and CD19+ B cells was observed following RTX but did not distinguish between viral infections. However, EBV-infected patients had significantly higher levels of Fas-expressing B cells at baseline as compared with EBV-negative groups. Conclusions. EBV and parvovirus genomes are frequently found in bone marrow of RA patients. The presence of EBV genome was associated with a better clinical response to RTX. Thus, presence of EBV genome may predict clinical response to RTX.

Introduction: Rheumatoid arthritis (RA) is frequently complicated with infections. The aim of our study was to evaluate vaccination response in patients with RA after B-cell depletion by using rituximab. Methods: Influenza (Afluria) and pneumococcal polysaccharides (Pneumo23) vaccines were given 6 months after rituximab (post-RTX group, n = 11) or 6 days before rituximab treatment (pre-RTX group; n = 8). RA patients never exposed to RTX composed the control group (n = 10). Vaccine-specific cellular responses were evaluated on day 6 after vaccination, and vaccine-specific humoral responses, on day 21. Results: On day 6 after vaccination, formation of influenza-specific B cells was lower in post-RTX group as compared with the pre-RTX group and controls (P = 0.04). Polysaccharide-specific B cells were found in 27% to 50%, being equally distributed between the groups. On day 21, the impairment of humoral responses was more pronounced with respect to influenza as compared with the pneumococcal vaccine and affected both IgG and light-chain production. Total absence of influenza-specific IgG production was observed in 55% of the post-RTX group. Conclusions: RTX compromises cellular and humoral vaccine responses in RA patients. However, repeated RTX treatment or previous anti-tumor necrosis factor (anti-TNF) treatment did not accentuate these defects.

Environmental factors are thought to play a major role in the development of rheumatoid arthritis. Because the use of ethanol is widespread, we assessed the role of ethanol intake on the propensity to develop chronic arthritis. Collagen type II-immunized mice were given water or water containing 10% (vol/vol) ethanol or its metabolite acetaldehyde. Their development of arthritis was assessed, as well as the impact of ethanol on leukocyte migration and activation of intracellular transcription factors. Mice exposed daily to this dose of ethanol did not display any liver toxicity, and the development of erosive arthritis was almost totally abrogated. In contrast, the antibody-mediated effector phase of collagen-induced arthritis was not influenced by ethanol exposure. Also, the major ethanol metabolite, acetaldehyde, prevented the development of arthritis. This antiinflammatory and antidestructive property of ethanol was mediated by ( i ) down-regulation of leukocyte migration and ( ii ) up-regulation of testosterone secretion, with the latter leading to decreased NF-κB activation. We conclude that low but persistent ethanol consumption delays the onset and halts the progression of collagen-induced arthritis by interaction with innate immune responsiveness.

Objective Receptor for advanced glycation end products (RAGE) is a cell surface molecule that binds a variety of ligands, including high mobility group box chromosomal protein 1 (HMGB-1), a potent proinflammatory cytokine. RAGE–ligand interaction leads to an inflammatory response. A truncated form of the receptor, soluble RAGE (sRAGE), has been suggested to function as a decoy abrogating cellular activation, but its endogenous activity is not fully understood. We undertook this study to assess the properties of sRAGE in vivo and in vitro and to analyze the role of sRAGE in HMGB-1–induced arthritis. Methods Mice were injected intraarticularly with HMGB-1 and treated systemically with sRAGE prior to histologic joint evaluation. All animals were subjected to peritoneal lavage to assess the local effect of sRAGE treatment. For in vitro studies, mouse splenocytes were incubated with sRAGE followed by assessment of NF-κB activation and cytokine production. The chemotactic properties of sRAGE were investigated using in vitro migration assay. Results Soluble RAGE was determined to have proinflammatory properties since it gave rise to production of interleukin-6, tumor necrosis factor α, and macrophage inflammatory protein 2. This effect was triggered by interaction with leukocyte β2 integrin Mac-1 and was mediated via NF-κB. Systemic treatment with sRAGE significantly down-regulated HMGB-1–triggered arthritis, but the observed effect was due to a deviation of the inflammatory response from the joint to the peritoneal cavity rather than a genuine antiinflammatory effect. Apart from its proinflammatory properties, sRAGE was proven to act as a chemotactic stimulus for neutrophils. Conclusion We conclude that sRAGE interacts with Mac-1, thereby acting as an important proinflammatory and chemotactic molecule.

B cells are in analogy with T cells capable of expressing functional IL-2 receptors. IL-2R alpha-chain (CD25) positive T cells have been studied in detail but not much is known about CD25 positive B cells. The aim of this study was to examine the phenotypic properties of the CD25 expressing B cells collected from different lymphoid organs in mice. Samples were stained for various cell surface markers and analysed using flow cytometry. We found that approximately 49% of B cells in bone marrow, 16% in peritoneal cavity, 2% in spleen and 1% in lymph nodes express CD25. In contrast, CD25 expressing B cells were not found in the blood or in Peyer's patches. Phenotypic characterization showed that CD25+ B cells in spleen, lymph nodes and peritoneal cavity have higher expression of AA4.1, CD5, CD69, CD80, CD86, CD122, CD132, IgA, IgG and IgM on their surface in comparison with CD25- B cells. In contrast, expression of IgD and IA-IE was lower on CD25+ B cells in spleen and lymph nodes. In bone marrow, the expression of CD5, CD80, CD86, CD122, CD132, IgA, IgD and IgM was lower, while the expression of AA4.1, IgG and IA-IE was increased on CD25+ B cells compared with CD25- B cells. In conclusion, our results indicate that B cells expressing CD25 are phenotypically distinctly different from those that are CD25 negative. Our findings suggest that CD25+ B cells are more prone to efficient antigen presentation and display a more mature phenotype.

Uric acid, the naturally occurring degradation product of purine metabolism, is a danger signal, driving maturation of dendritic cells. It is well known that uric acid crystals display potent proinflammatory properties—the cause of gout—whereas the biological properties of soluble uric acid are less well documented. We have demonstrated previously that nucleic acids of endogenous and exogenous origin display proinflammatory properties. The aim of the present study was to assess the impact of soluble uric acid on in vivo inflammatory responses. Mice were administered with uric acid suspension in saline or saline alone prior to induction of neutrophil-mediated inflammation, delayed-type hypersensitivity, histamin-induced edema (measure of vasodilation capacity), as well as double-stranded (ds)RNA-triggered arthritis. Frequency and severity of arthritis were decreased significantly in mice exposed to dsRNA and simultaneously treated with uric acid as compared with saline-treated controls. Also, granulocyte-mediated inflammatory response and vasodilation capacity were reduced significantly in mice treated with uric acid as compared with their control group. The data suggest that down-regulation of inflammation was mediated by skewing the inflammatory response from the peripheral sites to the peritoneal cavity and down-regulating vasodilatatory capacity and thereby affecting leukocyte migration. In contrast, the T cell-mediated delayed-type hypersensitivity reaction was not affected significantly in mice exposed to uric acid. These findings demonstrate that uric acid displays a potent, distant anti-inflammatory effect in vivo. This property seems to be mediated by down-regulation of neutrophil influx to the site of inflammatory insult.

Background Survivin is recognized as a marker of persistently active and erosive RA. The findings in experimental arthritis suggested that survivin co-ordinates the antigen-dependent leukocyte maturation and function by regulating transcriptional activity of Bcl-6, essential factor of germinal center formation and a factor involved neoplastic transformation to lymphoma. Objectives We studied intracellular expression of survivin and Bcl-6 in the peripheral lymphocytes and its relation to clinical features and treatment of RA patients. Methods The intracellular expression of survivin and Bcl-6 was studied on the effector and memory subsets of CD4+ and CD8+ T-cells and in CD19+ B-cells in 18 RA patients using flow cytometry. Serum and mRNA levels of survivin were analysed in 144 RA patients (age 21-71 years, disease duration 1-49 years) using ELISA and qPCR, respectively. Patients with serum levels of survivin >0.45 ng/mL comprised the serum survivin-positive (sSurv+) group. Results The sSurv+ group (n=76) was characterized by higher frequency of RF+ and aCCP+ patients, as well as by higher levels of aCCP and of differentiation factor Flt3-ligand compared to sSurv – (n=68). The sSurv+ and sSurv- patients were similar with respect to age, disease duration, DAS28 and anti-rheumatic treatment. Intracellular mRNA levels of Bcl-6 were lower in sSurv + compared to sSurv – patients, while RNA of survivin were similar. Flow cytometry showed that survivin was present in >85% of non-stimulated T-cells and B-cells lymphocytes of RA patients. The survivin hi subset comprised 2-14% of T-cells and was enriched in the effector (CD62L neg ) population of CD4+ T-cells compared to central memory and naive CD4+ T-cells. The expression of Bcl-6 was found within 7-38% of survivin + effector CD4+ T-cells and in 21-69% of CD19+ B-cells. The subsets of survivin+ and of survivin+Bcl-6+ T-cells was smaller in the sSurv+ patients compared to sSurv – patients, and was inversely correlated to the levels of serum survivin in these patients. The subset of survivin+Bcl-6+ B-cells was similar in sSurv+ and sSurv- patients. Conclusions In this pilot study we demonstrate that RA patients have co-expression of the two oncoproteins involved in neoplastic transformation to lymphoma, survivin and Bcl-6. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5490

Background A calcium-binding protein S100A4 regulates non-muscle myosin assembly and activity and plays important part in cell motility and differentiation. The binding of S100A4 to calcium drives conformation changes and permits S100A4 to regulate the activity of its multiple intracellular protein partners placing S100A4 at the crossroad of several intracellular transduction mechanisms. In RA, S100A4 is abundantly expressed in synovial fibroblasts, macrophages and vascular endothelial cells of the inflamed joints and may be measured in synovial fluid and in blood. The clinical consequences of the high levels of S100A4 in RA patients are associated with resistant joint inflammation and high skeletal damage. Objectives In the present study we evaluated the role of intracellular functions of S100A4 for T-cell responses in rheumatoid arthritis. Methods A preclinical model of the methylated BSA induced arthritis was induced in S100A4-deficent mice lacking the entire S100A4 protein (S100A4KO) and in wild-type (WT) counterparts where the S100A4 gene was inhibited with a specific shRNA-lentiviral constructs (S100A4-shRNA). The subsets of Th-cell in synovial infiltrates were analysed by immunohistology and in spleens by flow cytometry and qPCR. Phosphorylation of STAT3 (pTy5705), CD5 (pTyr453), Fyn (pTyr530) and ZA70 (pTyr319/Syk, pTyr352) were analysed by flow cytometry in spleen cell cultures. Kinase activity of Fyn and Lck was analysed by phosphorylation of CD5 peptide. Results Histological analysis of the inflamed joints demonstrated that S100A4-deficient mice had significantly alleviated joint inflammation and damage. Leukocyte infiltrates in the arthritic joints of S100A4-deficient mice presented accumulation of Foxp3 + Treg, while the effector T-cell population and the number of RORγt + and pSTAT3 + cells were reduced. T-cells of S100A4-deficient mice were characterized by a limited formation of Th17-cells, which was a result of low mRNA levels of RORγt and STAT3 (pTyr705) activity in spleen and low production of IL17 and IFNg. In vitro experiments provide evidence that S100A4 directly binds Lck and Fyn and reciprocally regulates their kinase activity towards TCR inhibitor CD5. Nuclear magnetic resonance analysis demonstrated an interaction between CD5 cytoplasmic domain and EF2-binding sites of S100A4. This suggests that S100A4 is able to disrupt intracellular interactions of CD5 and could modify its inhibition of TCR. Consequently, S100A4-deficiency was translated in low expression of CD5, and increased lymphocyte proliferation and phosphorylation of ZAP-70. Conclusions Here we provide experimental evidence that S100A4 directly binds the Src-kinases Lck and Fyn and reciprocally regulates their kinase activity. S100A4-deficiency results in reduced activity of STAT3 suppressing transcription of RORgt and lineage differentiation of Th17-cells. The immunological events controlled by S100A4 are functionally important for the pathogenesis of arthritis, since the deficiency in S100A4 alleviates experimental arthritis. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5414

Background Plasma cells (PC) are the unique source of protective and autoreactive immunoglobulin. Mature PC are resting and have downregulated CD20, rendering them refractory to almost all conventional and modern antirheumatic therapies including rituximab. Consequently, the failure to eridacte autoantibody production by plasma cells appears as a therapeutical hurdle in patients with autoimmune diseases. We have identified two distinct subsets of plasma cells based on differing expression of CD19 in the human BM which are comparatively characterised here against the background of the development of anti-CD19 directed immunotherapies. Objectives To characterise CD19+ vs. CD19-negative PC subsets in normal human bone marrow. Methods BM samples from iliac crest, femural heads and sternum as well as blood, spleen and tonsil specimen were analyzed for CD38hi plasma cells by FACS and the expression of CD19, CD138, Ki-67, HLA-DR, CD95, CD28 and CD56 wa studied. Transcriptional analyses of sort-purified plasma cell subsets were performed on an Affymetrix platform. The antibodies expressed by individual plasma cells were characterized using Elispot, cytoplasmic Ig staining and single cell RT-PCR. Results CD19+ and CD19-negative plasma cells were readily detectable in all human BM samples irrespective of the anatomic site analyzed and co-expressed CD138. At average, 60-70% of the plasma cells expressed CD19 in the BM, while 85% were CD19+ in spleen and tonsils and 95-100% in the blood, respectively. Transcriptional and phenotypical analyses of CD19-negative BMPC suggest their advanced maturity, inasmuch they express CD56 and CD28 while HLA-DR, CD95 and Ki-67 are downregulated compared to CD19+ BMPC. CD19-negative BMPC were enriched for IgG-secreting cells which showed suprisingly low mutational load within their VH gene rearrangements. Treatment of RA patients with rituximab significantly reduced numbers of CD19+ BMPC but not CD19-negative BMPC. Conclusions Our data indicate an unexpected heterogeneity within the mature plasma cell compartment in the human bone marrow. Detailled characterisation according to CD19 expression suggests that CD19-negative plasma cells aquired a more mature differentiation state and represents a rather static population as compared to CD19+ BMPC. A potential anti-CD19 directed B cell depletion approach for therapy of autoimmunity will likely target many but not all plasma cells, while the relationship betwen CD19 expression by plasma cells and autoantibody production remains to be analysed in order to estimate targeting of autoantibody production. Our data provides new insight into the homeostasis of normal mature plasma cells which opens new perspectives for studies on plasma cells as sources of auto-antibodies in patients with autoimmune diseases. Disclosure of Interest None Declared

Despite the promise of RNA interference (RNAi) and its potential, e.g. for use in cancer therapy, several technical obstacles must first be overcome. The major hurdle of RNAi-based therapeutics is to deliver nucleic acids across the cell's plasma membrane. This study demonstrates that exosome vesicles derived from humans can deliver short interfering RNA (siRNA) to human mononuclear blood cells. Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication, i.e. antigen presentation, tolerance development and shuttle RNA (mainly mRNA and microRNA). Having tested different strategies, an optimized method (electroporation) was used to introduce siRNA into human exosomes of various origins. Plasma exosomes (exosomes from peripheral blood) were used as gene delivery vector (GDV) to transport exogenous siRNA to human blood cells. The vesicles effectively delivered the administered siRNA into monocytes and lymphocytes, causing selective gene silencing of mitogen-activated protein kinase 1. These data suggest that human exosomes can be used as a GDV to provide cells with heterologous nucleic acids such as therapeutic siRNAs.

Introduction In the present study we evaluated changes in the B cell phenotype in peripheral blood and bone marrow (BM) of patients with rheumatoid arthritis (RA) following anti-CD20 treatment using rituximab. Methods Blood and BM samples were obtained from 37 patients with RA prior to rituximab treatment. Ten of these patients were resampled 1 month following rituximab, 14 patients after 3 months and the remaining 13 patients were included in the long-term follow up. B cell populations were characterized by CD27/IgD/CD38/CD24 expression. Results One and three months following rituximab BM retained up to 30% of B cells while circulation was totally depleted of B cells. Analysis of the remaining BM B cells showed prevalence of immature and/or transitional B cells (CD38++CD24++) and CD27 + IgD - memory cells, while IgD + cells were completely depleted. A significant reduction of CD27+ cells in BM and in circulation was observed long after rituximab treatment (mean 22 months), while levels of naive B cells in BM and in circulation were increased. The levels of rheumatoid factor decline after rituximab treatment but returned to baseline levels at the time of retreatment. Conclusions Anti-CD20 treatment achieves a depletion of IgD + B cells shortly after the treatment. At the long term follow up, a reduction of CD27 + B cells was observed in blood and BM. The prolonged inability to up-regulate CD27 may inhibit the renewal of memory B cells. This reduction of CD27 + B cells does not prevent autoantibody production suggesting that mechanisms regulating the formation of auto reactive clones are not disrupted by rituximab.

Objectives: To evaluate the role of S100A4, a calcium-binding regulator of nonmuscle myosin assembly, for T-cell responses in rheumatoid arthritis. Methods: Arthritis was induced in the methylated bovine serum albumin (mBSA)-immunized mice lacking the entire S100A4 protein (S100A4KO) and in wild-type counterparts treated with short hairpin ribonucleic acid (shRNA)-lentiviral constructs targeting S100A4 (S100A4-shRNA). The severity of arthritis was evaluated morphologically. T-cell subsets were characterized by the expression of master transcription factors, and functionally by proliferation activity and cytokine production. The activity of the Scr-kinases Fyn and Lck was assessed by the autophosphorylation of C-terminal thyrosine and by the phosphorylation of the CD5 cytodomain. The interaction between S100A4 and the CD5 cytodomain was analysed by nuclear magnetic resonance spectrophotometry. Results: S100A4-deficient mice (S100A4KO and S100A4-shRNA) had significantly alleviated morphological signs of arthritis and joint damage. Leukocyte infiltrates in the arthritic joints of S100A4-deficient mice accumulated Foxp3+ Treg cells, while the number of RORγt+ and (pTyr705)STAT3+ cells was reduced. S100A4-deficient mice had a limited formation of Th17-cells with low retinoic acid orphan receptor gamma t (RORγt) mRNA and IL17 production in T-cell cultures. S100A4-deficient mice had a low expression and activity of T-cell receptor (TCR) inhibitor CD5 and low (pTyr705)STAT3 (signal transducer and activator of transcription 3), which led to increased (pTyr352)ZAP-70 (theta-chain associated protein kinase of 70 kDa), lymphocyte proliferation and production of IL2. In vitro experiments showed that S100A4 directly binds Lck and Fyn and reciprocally regulates their kinase activity towards the CD5 cytodomain. Spectrometry demonstrates an interaction between the CD5 cytodomain and EF2-binding sites of S100A4. Conclusion: The present study demonstrates that S100A4 plays an important part in the pathogenesis of arthritis. It controls CD5-dependent differentiation of Th17 cells by regulating the activity of the Src-family kinases Lck and Fyn.