Krisztina Szabó

Follicular helper T cells may play an important role in the severity of primary Sjögren's syndrome

The aim of this study was to investigate the possible role of follicular helper T (TFH) cells in the pathogenesis of primary Sjögrens syndrome (pSS) by analyzing immune-competent cells and serological markers with special emphasis on clinical symptoms. We enrolled 50 pSS patients and 16 healthy individuals in the study. Patients had elevated ratio of peripheral TFH cells, however, when dividing patients into two groups defined by the presence of extraglandular manifestations (EGMs), only patients with EGMs differed from controls significantly. Moreover, TFH cell percentages correlated positively with both activated T cell and Tr1 cell values. On the contrary, TFH cell percentages showed negative correlation with both IgM and IgG memory B cell proportions. Elevated TFH percent\ages were observed in the anti-SSA/SSB positive patients, and also in patients with higher IL-12, IL-21 levels and focus score values. Increased TFH cell proportions seem to have an important role in disease development.

A comprehensive investigation on the distribution of circulating follicular T helper cells and B cell subsets in primary Sjögren's syndrome and systemic lupus erythematosus.

Follicular T helper (Tfh) cells have a crucial role in regulating immune responses within secondary lymphoid follicles by directing B cell differentiation towards memory B cells and plasma cells. Because abnormal humoral responses are key features in both primary Sjögrens syndrome (pSS) and systemic lupus erythematosus (SLE), the aim of this study was to profile the pathological connection between peripheral Tfh cells and B cells in the two diseases. Twenty‐five pSS patients, 25 SLE patients and 21 healthy controls were enrolled into the study. We determined the ratio of circulating Tfh‐like cells, their interleukin (IL)‐21 production and different B cell subsets by flow cytometry. We observed higher percentages of naive B cells in both diseases, while non‐switched and switched memory B cells showed decreased frequencies. The proportions of double‐negative B cells and plasmablasts were elevated in SLE and decreased in pSS. The percentages of transitional B cells and mature‐naive B cells were higher in SLE. Patients with more severe disease course had an elevated ratio of TFH‐like cells and increased IL‐21 production. Moreover, expansion of Tfh‐like cells correlated positively with parameters related to antibody secretion, including serum immunoglobulin (Ig)G, immune complexes (ICs) and autoantibodies. Correlation analysis between Tfh‐like cells and certain B cell subsets revealed possible defects during B cell selection. In conclusion, our observations on the profound expansion of circulating Tfh‐like cells and their IL‐21 production, along with the characteristic aberrant peripheral B cell distribution in both pSS and SLE, indicate the prominent role of Tfh cell in the regulation of B cell selection.

Follicular helper T cells in autoimmune diseases

In autoimmune diseases, the breakdown of immune tolerance leads to the development of autoreactive immune responses targeting self-structures, and subsequent tissue and organ damage. Although the complex immunobiological mechanisms of autoimmune processes are still not fully clear, altered B cell function and autoantibody production seem to play a special role in the development of numerous autoimmune diseases. Understanding the process of B cell activation and autoantibody production is particularly important not only for early diagnosis, but also for development of novel effective treatments. Follicular T helper (TFH) cells are special CD4 + T cells mediating antigen-specific naive or memory B cell activation within the B cell follicles of secondary lymphoid organs. TFH cells are generated from peripheral naive CD4 T cells in the T cell zone of these lymphoid organs. The differentiation of TFH cells begins with their migration to the border of T cell zone and B cell follicles. This follicular homing process is directed by B cell lymphoma 6 protein (Bcl-6), by coordinating the downregulation of CCR7, a receptor for certain T zone chemokines, and the up-regulation of CXCR5, the receptor for CXCL13. CXCL13 is a chemokine ligand secreted by follicular stromal cells in B cell follicles, which attracts primed T cells to the follicle edge, where they interact with antigen-primed B cells and differentiate into TFH cells. Interplay of TFH and activated B cells is essential for the generation of extrafollicular short-lived plasma cells producing low-affinity antibodies, and for germinal centre (GC) responses as well. Within GCs, TFH cells promote the development of high-affinity memory B cells and long-lived plasma cells by providing survival signals to centrocytes, which have undergone somatic hypermutation. Regarding the critical role of TFH cells in B cell activation and antibody production, their failure to maintain self-tolerance and potential contribution to autoimmunity has drawn much attention. Lessons learned from animal models, mainly murine models of SLE, shed light on altered TFH profiles in autoimmune conditions. First, the significance of IL-21, now considered the hallmark cytokine of TFH cells, was recognized in autoimmunity. Ozaki et al. [1] demonstrated enhanced IL-21 production in BXSB-Yaa mice, a model exhibiting lupus-like disease. IL-21 blockade or IL-21 receptor deficiency in lupus-prone MRL-Fas mice resulted in diminished lupus-associated features, including IgG deposits in glomeruli, circulating dsDNA autoantibodies, total IgG1 and IgG2a production, lymphadenopathy and spontaneous GC formation [2, 3]. Further studies examining TFH cells directly showed an aberrantly expanded TFH population. Interestingly, when Wu et al. [4] investigated the effect of nasal anti-CD3 on TFH cells in NZB/WF1 mice, CD4/ICOS/CXCR5 TFH cells obtained from anti-CD3treated mice showed decreased IL-21 and IL-17 expression and induced less IgG, IgG1 or IgG2a anti-dsDNA antibody production in an in vitro co-culture with naive CD19 B cells. Recent observations in autoimmune animal models have further enriched our knowledge about the development and function of TFH cells. In an elegant study, Linterman et al. [5] investigated the deletion of Sap (Sh2d1a) and the loss of one Bcl-6 allele in Roquin (sanroque) mice. They found that deletion of one Bcl-6 allele diminished spontaneous GC formation and the lupus phenotype. Moreover, the deficiency of the Sap molecule caused a dramatic reduction in CD4/ CXCR5/PD-1 TFH cells, IL-21 production, renal pathology, formation of GC and autoantibodies. Moreover, adoptive transfer of sanroque TFH cells into wild-type recipients resulted in spontaneous GC formation, underscoring the direct role of TFH cells in the development of lupus-associated autoimmunity. Investigations of human autoimmune diseases also suggest that aberrant TFH cell development and function can drive autoimmunity. Simpson et al. were the first to demonstrate altered TFH proportions in SLE patients. By determining CD4CXCR5ICOS and CD4CXCR5 PD-1 TFH cells in peripheral blood, they observed an overrepresented population of CD4ICOS and CD4CXCR5ICOS TFH cells, which showed an association with the high autoantibody titre and the presence of glomerulonephritis [6]. Subsequently expansion of circulating TFH cells has been reported in patients with various autoimmune diseases, such as SS, RA, JDM and autoimmune thyroid disorders. In primary SS, our group demonstrated that the elevated circulating CD4/ CXCR5/ICOS/PD-1 TFH cell percentages are associated with the presence of systemic extraglandular manifestations and anti-SSA/SSB positivity. Patients with higher TFH cell proportions also had elevated serum levels of IL-12 and IL-21 [7]. Maehara et al. [8] investigated the selective localization of Th1, Th2, Th17, regulatory T and TFH cells in labial salivary gland biopsies. They found that the expression of TFH and Th2-related molecules in infiltrating lymphocytes with ectopic GCs was higher than in those without ectopic GCs. An elevated percentage of circulating TFH cells was found in SLE. High TFH cell

Exposure to inhomogeneous static magnetic field beneficially affects allergic inflammation in a murine model

Previous observations suggest that static magnetic field (SMF)-exposure acts on living organisms partly through reactive oxygen species (ROS) reactions. In this study, we aimed to define the impact of SMF-exposure on ragweed pollen extract (RWPE)-induced allergic inflammation closely associated with oxidative stress. Inhomogeneous SMF was generated with an apparatus validated previously providing a peak-to-peak magnetic induction of the dominant SMF component 389 mT by 39 T m−1 lateral gradient in the in vivo and in vitro experiments, and 192 mT by 19 T m−1 in the human study at the 3 mm target distance. Effects of SMF-exposure were studied in a murine model of allergic inflammation and also in human provoked skin allergy. We found that even a single 30-min exposure of mice to SMF immediately following intranasal RWPE challenge significantly lowered the increase in the total antioxidant capacity of the airways and decreased allergic inflammation. Repeated (on 3 consecutive days) or prolonged (60 min) exposure to SMF after RWPE challenge decreased the severity of allergic responses more efficiently than a single 30-min treatment. SMF-exposure did not alter ROS production by RWPE under cell-free conditions, while diminished RWPE-induced increase in the ROS levels in A549 epithelial cells. Results of the human skin prick tests indicated that SMF-exposure had no significant direct effect on provoked mast cell degranulation. The observed beneficial effects of SMF are likely owing to the mobilization of cellular ROS-eliminating mechanisms rather than direct modulation of ROS production by pollen NAD(P)H oxidases.

MicroRNA expression profiles identify disease-specific alterations in systemic lupus erythematosus and primary Sjögren's syndrome

The discovery of microRNAs (miRNAs) and their critical role in genetic control opened new avenues in understanding of various biological processes including immune cell lineage commitment, differentiation, proliferation and apoptosis. However, a given miRNA may have hundreds of different mRNA targets and a target might be regulated by multiple miRNAs, thus the characterisation of dysregulated miRNA expression profiles could give a better insight into the development of immunological disturbances in autoimmune diseases. The aim of our study was to examine the changes in miRNA expression profiles in patients with systemic lupus erythematosus (SLE) and primary Sjögrens syndrome (pSS). Eight SLE patients, 8 pSS patients and 7 healthy subjects were enrolled in the investigation. MiRNAs were isolated from peripheral blood mononuclear cells, and expression patterns were determined with Illumina next-generation sequencing technology. Since the immunopathogenesis of pSS and SLE encompasses pronounced B cell hyperactivity along with specific autoantibody production, we paid a special attention on the association between miRNA expression levels and altered peripheral B cell distribution. In SLE patients 135, while in pSS patients 26 miRNAs showed altered expression. Interestingly, the 25 miRNAs including miR-146a, miR-16 and miR-21, which were over-expressed in pSS patients, were found to be elevated in SLE group, as well. On the contrary, we observed the down-regulation of miR-150-5p, which is a novel and unique finding in pSS. Levels of several miRNAs over-expressed in SLE, were not changed in pSS, such as miR-148a-3p, miR-152, miR-155, miR-223, miR-224, miR-326 and miR-342. Expression levels of miR-223-5p, miR-150-5p, miR-155-5p and miR-342-3p, which miRNAs are potentially linked to B cell functions, showed associations with the B cell proportions within peripheral blood mononuclear cells. The observed differences in miRNA expression profiles and the better understanding of immune regulatory mechanisms of miRNAs may help to elucidate the pathogenesis of SLE and pSS.

Increased IL-21 Expression Induces Granzyme B in Peripheral CD5(+) B Cells as a Potential Counter-Regulatory Effect in Primary Sjögren's Syndrome.

Recently, we reported elevated proportions of circulating follicular T helper cells and higher levels of interleukin- (IL-) 21 in primary Sjögrens syndrome (pSS). Interaction of invariant natural killer T (iNKT) cells with B cells and granzyme B (GrB) production may be also important in pSS. Thirty-two pSS patients and 24 healthy controls were enrolled in our study. We investigated the expression of intracellular GrB and IL-21 receptor (IL-21R) of CD19+CD5+ and CD19+CD5− B cells; furthermore, we determined the IL-21 expression of iNKT cells as well. We also assessed the proportion of transitional (CD19+CD24highCD38high), mature (CD19+CD24intCD38int) and primarily memory (CD19+CD24highCD38−) B cells. CD5+ but not CD5− B cells showed elevated GrB and IL-21R expression in pSS; additionally IL-21 expression of iNKT cells was also elevated. The ratios of transitional and mature B cells were elevated in pSS, while primarily memory B cell percentages were decreased, which correlated with GrB and IL-21R expression of CD19+ B cells. Our results suggest that enhanced IL-21R expression of CD19+CD5+ B cells and production of IL-21 by iNKT cells may play an important role in the pathogenesis of pSS by regulating CD19+CD5+ B cell functions and increasing GrB production, presumably leading to a counter-regulatory effect in the disease.

Meeting Abstracts from The 13th International Symposium on Sjögren's Syndrome Bergen, Norway, May 19-22, 2015

Background: Cognitive impairment in primary Sjogren’s syndrome (PSS) has been identified in several small studies using self-reported measures. Objectives: To quantify cognitive impairment symptoms in a large cohort of 150 PSS patients compared with controls and to explore the relationship between cognitive impairment with fatigue, pain and mood symptoms. Methods: PSS patients fulfilling the American European Consensus Criteria were recruited from 12 sites in England. They completed the Cognitive Failures Questionnaire (CFQ) as well as measures of mood (Hospital Anxiety and Depression Scale), fatigue (visual analogue scale (VAS)), dryness (VAS) and pain (VAS). CFQ scores were compared with data from controls. Completion of the CFQ yields a possible score between 0 and 100. The higher the score the greater the impairment. Results: One hundred and fifty PSS patients and 198 controls completed the CFQ. Cognitive symptoms were worse in the PSS group (43.7 ± 17.8 vs 35.9 ± 12.9; P < 0.001). This difference persisted (P < 0.001) following analysis of covariance adjusting for age and gender. There were significant correlations with pain, fatigue, anxiety, depression and subjective dryness scores with CFQ scores. In order to partition the variability in CFQ scores into its component parts, we performed a multiple regression analysis. This confirmed that anxiety was the most important predictor of CFQ scores (P = 0.004). Conclusion: Cognitive symptoms are common in PSS and independently associate with anxiety. Clinicians should give consideration to cognitive failure and anxiety in the management of PSS patients.

Proteomic analysis of protein phosphatase Z1 from Candida albicans

Protein phosphatase Z is a “novel type” fungus specific serine/threonine protein phosphatase. Previously our research group identified the CaPPZ1 gene in the opportunistic pathogen Candida albicans and reported that the gene deletion had several important physiological consequences. In order to reveal the protein targets and the associated mechanisms behind the functions of the phosphatase a proteomic method was adopted for the comparison of the cappz1 deletion mutant and the genetically matching QMY23 control strain. Proteins extracted from the control and deletion mutant strains were separated by two-dimensional gel electrophoresis and the protein spots were stained with RuBPS and Pro-Q Diamond in order to visualize the total proteome and the phosphoproteome, respectively. The alterations in spot intensities were determined by densitometry and were analysed with the Delta2D (Decodon) software. Spots showing significantly different intensities between the mutant and control strains were excised from the gels and were digested with trypsin. The resulting peptides were identified by LC-MS/MS mass spectrometry. As many as 15 protein spots were found that exhibited significant changes in their intensity upon the deletion of the phosphatase and 20 phosphoproteins were identified in which the level of phosphorylation was modified significantly in the mutant. In agreement with previous findings we found that the affected proteins function in protein synthesis, oxidative stress response, regulation of morphology and metabolism. Among these proteins we identified two potential CaPpz1 substrates (Eft2 and Rpp0) that may regulate the elongation step of translation. RT-qPCR experiments revealed that the expression of the genes coding for the affected proteins was not altered significantly. Thus, the absence of CaPpz1 exerted its effects via protein synthesis/degradation and phosphorylation/dephosphorylation. In addition, our proteomics data strongly suggested a role for CaPpz1 in biofilm formation, was confirmed experimentally. Thus our unbiased proteomic approach lead to the discovery of a novel function for this phosphatase in C. albicans.

Basic and clinical immunology – 3036. Investigation of peripheral follicular helper T-cells in primary Sjögren's syndrome

Background The exact mechanisms behind the immune disturbances in primary Sjogrens syndrome (pSS) are still not known in details, but evidence suggests that abnormal T- and B-cell activation plays a critical role in the disease develop- ment. Recent studies emphasized the important role of follicular helper T (TFH) cells, which contribute to B-cell proliferation and differentiation, as well as antibody pro- duction. The aim of this study was to investigate the possi- ble role of TFH cells in the pathogenesis of pSS, by analyzing a wide spectrum of immune-competent cells and serological markers with a special emphasis on clinical symptoms of the disease. Methods Results Patients with pSS showed elevated ratio of peripheral TFH cells. However, when we divided patients into two groups defined by the presence or absence of EGMs, only patients with EGMs had significant differences, while values of patients without EGMs were similar to healthy controls. We found a significant positive correlation between acti- vated Ta nd TFH cell percentages as well as between the Tr1 and TFH cells. Significant negative correlations were observed between IgM memory B and TFH cell values and between IgG memory B and TFH cell proportions. Elevated TFH percentages were observed in the anti-SSA positive patients, compared to autoantibody negative patients and healthy controls. Moreover, the percentages of TFH cells were significantly elevated in patients with higher IL-12 and IL-21 levels. Conclusions Our data suggest that percentages of peripheral TFH cells are increased in pSS, especially in patients suffering from a systemic, more pronounced course of disease (pSS with EGMs). Moreover, the elevated TFH cell percentages cor- relates with activation of immune system, proportions of memory B cells and titers of autoantibodies, which implies that TFH cells may play an important role in the disease development.