Sandra M.J. Paulissen

The role and modulation of CCR6+ Th17 cell populations in rheumatoid arthritis

The IL-17A producing T-helper-17 (Th17) cell population plays a major role in rheumatoid arthritis (RA) pathogenesis and has gained wide interest as treatment target. IL-17A expressing Th cells are characterized by the expression of the chemokine receptor CCR6 and the transcription factor RORC. In RA, CCR6+ Th cells were identified in peripheral blood, synovial fluid and inflamed synovial tissue. CCR6+ Th cells might drive the progression of an early inflammation towards a persistent arthritis. The CCR6+ Th cell population is heterogeneous and several subpopulations can be distinguished, including Th17, Th22, Th17.1 (also called non-classic Th1 cells), and unclassified or intermediate populations. Interestingly, some of these populations produce low levels of IL-17A but are still very pathogenic. Furthermore, the CCR6+ Th cells phenotype is unstable and plasticity exists between CCR6+ Th cells and T-regulatory (Treg) cells and within the CCR6+ Th cell subpopulations. In this review, characteristics of the different CCR6+ Th cell populations, their plasticity, and their potential impact on rheumatoid arthritis are discussed. Moreover, current approaches to target CCR6+ Th cells and future directions of research to find specific CCR6+ Th cell targets in the treatment of patients with RA and other CCR6+ Th cell mediated autoimmune diseases are highlighted.

T-helper 17 cell cytokines and interferon type I: partners in crime in systemic lupus erythematosus?

IntroductionA hallmark of systemic autoimmune diseases like systemic lupus erythematosus (SLE) is the increased expression of interferon (IFN) type I inducible genes, so-called IFN type I signature. Recently, T-helper 17 subset (Th17 cells), which produces IL-17A, IL-17F, IL-21, and IL-22, has been implicated in SLE. As CCR6 enriches for Th17 cells, we used this approach to investigate whether CCR6+ memory T-helper cells producing IL-17A, IL-17F, IL-21, and/or IL-22 are increased in SLE patients and whether this increase is related to the presence of IFN type I signature.MethodsIn total, 25 SLE patients and 15 healthy controls (HCs) were included. SLE patients were divided into IFN type I signature-positive (IFN+) (n = 16) and negative (IFN-) (n = 9) patients, as assessed by mRNA expression of IFN-inducible genes (IFIGs) in monocytes. Expression of IL-17A, IL-17F, IL-21, and IL-22 by CD4+CD45RO+CCR6+ T cells (CCR6+ cells) was measured with flow cytometry and compared between IFN+, IFN- patients and HCs.ResultsIncreased percentages of IL-17A and IL-17A/IL-17F double-producing CCR6+ cells were observed in IFN+ patients compared with IFN- patients and HCs. IL-17A and IL-17F expression within CCR6+ cells correlated significantly with IFIG expression. In addition, we found significant correlation between B-cell activating factor of the tumor necrosis family (BAFF)–a factor strongly correlating with IFN type I - and IL-21 producing CCR6+ cells.ConclusionsWe show for the first time higher percentages of IL-17A and IL-17A/IL-17F double-producing CCR6+ memory T-helper cells in IFN+ SLE patients, supporting the hypothesis that IFN type I co-acts with Th17 cytokines in SLE pathogenesis.

CCR6(+) Th cell populations distinguish ACPA positive from ACPA negative rheumatoid arthritis.

IntroductionPatients with rheumatoid arthritis (RA) can be separated into two major subpopulations based on the absence or presence of serum anti-citrullinated protein antibodies (ACPAs). The more severe disease course in ACPA+ RA and differences in treatment outcome between these subpopulations suggest that ACPA+ and ACPA− RA are different disease subsets. The identification of T-helper (Th) cells specifically recognizing citrullinated peptides, combined with the strong association between HLA-DRB1 and ACPA positivity, point toward a pathogenic role of Th cells in ACPA+ RA. In this context we recently identified a potential pathogenic role for CCR6+ Th cells in RA. Therefore, we examined whether Th cell population distributions differ by ACPA status.MethodsWe performed a nested matched case–control study including 27 ACPA+ and 27 ACPA− treatment-naive early RA patients matched for disease activity score in 44 joints, presence of rheumatoid factor, sex, age, duration of complaints and presence of erosions. CD4+CD45RO+ (memory) Th cell distribution profiles from these patients were generated based on differential chemokine receptor expression and related with disease duration.ResultsACPA status was not related to differences in total CD4+ T cell or memory Th cell proportions. However, ACPA+ patients had significantly higher proportions of Th cells expressing the chemokine receptors CCR6 and CXCR3. Similar proportions of CCR4+ and CCR10+ Th cells were found. Within the CCR6+ cell population, four Th subpopulations were distinguished based on differential chemokine receptor expression: Th17 (CCR4+CCR10−), Th17.1 (CXCR3+), Th22 (CCR4+CCR10+) and CCR4/CXCR3 double-positive (DP) cells. In particular, higher proportions of Th22 (p = 0.02), Th17.1 (p = 0.03) and CCR4/CXCR3 DP (p = 0.01) cells were present in ACPA+ patients. In contrast, ACPA status was not associated with differences in Th1 (CCR6−CXCR3+; p = 0.90), Th2 (CCR6−CCR4+; p = 0.27) and T-regulatory (CD25hiFOXP3+; p = 0.06) cell proportions. Interestingly, CCR6+ Th cells were inversely correlated with disease duration in ACPA− patients (R2 = −0.35; p < 0.01) but not in ACPA+ (R2 < 0.01; p = 0.94) patients.ConclusionsThese findings demonstrate that increased peripheral blood CCR6+ Th cells proportions distinguish ACPA+ RA from ACPA− RA. This suggests that CCR6+ Th cells are involved in the differences in disease severity and treatment outcome between ACPA+ and ACPA− RA.

Enhanced Bruton's Tyrosine Kinase Activity in Peripheral Blood B Lymphocytes From Patients With Autoimmune Disease

Brutons tyrosine kinase (BTK) transmits crucial survival signals from the B cell receptor (BCR) in B cells. Pharmacologic BTK inhibition effectively diminishes disease symptoms in mouse models of autoimmunity; conversely, transgenic BTK overexpression induces systemic autoimmunity in mice. We undertook this study to investigate BTK expression and activity in human B cells in the context of autoimmune disease.

Enhanced Bruton's tyrosine kinase activity in peripheral blood B lymphocytes of autoimmune disease patients

Brutons tyrosine kinase (BTK) transmits crucial survival signals from the B cell receptor (BCR) in B cells. Pharmacologic BTK inhibition effectively diminishes disease symptoms in mouse models of autoimmunity; conversely, transgenic BTK overexpression induces systemic autoimmunity in mice. We undertook this study to investigate BTK expression and activity in human B cells in the context of autoimmune disease.

Association of Increased Treg Cell Levels With Elevated Indoleamine 2,3-Dioxygenase Activity and an Imbalanced Kynurenine Pathway in Interferon-Positive Primary Sjögren's Syndrome.

Indoleamine 2,3‐dioxygenase (IDO), the rate‐limiting enzyme that converts tryptophan to kynurenine, is driven in part by type I and type II interferons (IFNs). Naive T cells are polarized into FoxP3+ Treg cells upon exposure to either IDO+ cells or kynurenine. Recent studies have suggested that the kynurenine pathway reflects a crucial interface between the immune and nervous system. The aims of the present study were to evaluate whether Treg cell levels are elevated, in conjunction with increased IDO activity, in patients with primary Sjögrens syndrome (SS) who are positive for the IFN gene expression signature, and to investigate the downstream kynurenine pathway in these patients.

Increased Tregs associated with elevated Indoleamine-2,3-dioxygenase activity and an imbalanced Kynurenine pathway in IFNpositive primary Sjögren's syndrome

Indoleamine 2,3‐dioxygenase (IDO), the rate‐limiting enzyme that converts tryptophan to kynurenine, is driven in part by type I and type II interferons (IFNs). Naive T cells are polarized into FoxP3+ Treg cells upon exposure to either IDO+ cells or kynurenine. Recent studies have suggested that the kynurenine pathway reflects a crucial interface between the immune and nervous system. The aims of the present study were to evaluate whether Treg cell levels are elevated, in conjunction with increased IDO activity, in patients with primary Sjögrens syndrome (SS) who are positive for the IFN gene expression signature, and to investigate the downstream kynurenine pathway in these patients.

The COX/prostaglandin-E2 pathway is critical for autocrine IL-17A production by TH17 cells upon synovial fibroblast interaction

Backgroundand objectives Recently, the authors have shown that Th17, but not Th1 cells, from patients with early rheumatoid arthritis (RA) are potent activators of RA synovial fibroblasts (RASF) resulting in autocrine IL-17A production. This autocrine IL-17A production by Th17 cells is critical for the perseverance of the pro-inflammatory loop, but the mechanism underlying the autocrine IL-17A induction is unclear. Objective To investigate the mechanism responsible for the autocrine IL-17A induction upon Th17-RASF interaction. Materials and methods CD4+CD45RO+CCR6+ (Th17) and CD4+CD45RO+CCR6- (Th1) cells were isolated by FACS sorting from healthy controls and early RA patients. These cells were co-cultured with RASF, in the presence of neutralising antibodies directed against soluble IL-6R (anti-sIL-6R) and/or IL-1β, and celecoxib. Gene expression profiles were generated and supernatant was collected for cytokine analyses by ELISA. Results Gene expression analyses revealed that the genes encoding for IL-6 and IL-1β were up-regulated in Th17-RASF cultures. These data were confirmed by ELISA and Q-PCR, respectively. Since IL-1β and IL-6 may be involved in IL-17/Th17 polarisation the authors examined the contribution of these cytokines on the autocrine IL-17A loop. Blockade of IL-1β and IL-6 activity, but not TNFα significantly suppressed IL-17A production in the co-culture, but the inhibitory effects were limited. Of note, the combination of anti-IL-1β and anti-sIL-6R markedly suppressed IL-17F. No effects were found on IFNγ. Interestingly, the genes encoding for cyclo-oxygenase-2 (COX-2) and prostaglandin-E-synthase (PTGES), which are involved in prostaglandin-E2 (PGE2) synthesis, were also up-regulated in Th17-RASF cultures. This was associated with a dramatic increase of PGE2 production in Th17-RASF cultures compared to Th1-RASF cultures. Treatment of Th17-RASF cultures with celecoxib, an inhibitor of cyclo-oxygenase-2 (COX-2) activity, resulted in a significant decrease of IL-17A, but not of IFNγ and TNFα production. Furthermore, the decrease in IL-17A production caused by celecoxib treatment was much more pronounced compared to neutralising IL-1 and IL-6 activity. Flow cytometry revealed a decrease in Th17 cells in celecoxib treated co-cultures, while no effects on Th1 or Th2 cells were found. Moreover, celecoxib treatment significantly inhibited the pro-inflammatory loop as less induction of IL-6, IL-8 and matrix metalloproteinase-3 production was observed in the Th17-RASF co-cultures. Conclusions These findings show the critical role of the COX/PGE2 pathway in the autocrine IL-17A production upon Th17 and synovial fibroblast interaction. Inhibition of this pathway down-regulates the pro-inflammatory feedback loop induced by Th17-RASF interaction leading to less production of pro-inflammatory cytokines and destructive mediators.

Increased T-helper 17.1 cells in sarcoidosis mediastinal lymph nodes

The lung-draining mediastinal lymph nodes (MLNs) are currently widely used to diagnose sarcoidosis. We previously reported that T-helper (Th) 17.1 cells are responsible for the exaggerated interferon-γ production in sarcoidosis lungs. In this study, we aimed to investigate 1) whether Th17.1 cells are also increased in the MLNs of sarcoidosis patients and 2) whether frequencies of the Th17.1 cells at diagnosis may correlate with disease progression. MLN cells from treatment-naive pulmonary sarcoidosis patients (n=17) and healthy controls (n=22) and peripheral blood mononuclear cells (n=34) and bronchoalveolar lavage fluid (BALF) (n=36) from sarcoidosis patients were examined for CD4+ T-cell subset proportions using flow cytometry. Higher proportions of Th17.1 cells were detected in sarcoidosis MLNs than in control MLNs. Higher Th17.1 cell proportions were found in sarcoidosis BALF compared with MLNs and peripheral blood. Furthermore, BALF Th17.1 cell proportions were significantly higher in patients developing chronic disease than in patients undergoing resolution within 2 years of clinical follow-up. These data suggest that Th17.1 cell proportions in pulmonary sarcoidosis can be evaluated as a diagnostic and/or prognostic marker in clinical practice and could serve as a new therapeutic target. Th17-lineage cells are increased in sarcoidosis MLNs and BALF Th17.1 cell proportions correlate with disease progression http://ow.ly/hBb730hSEv7

A1.76 Higher proportions of pathogenic CCR6+ T cell subpopulations in ACPA+ compared to ACPA- patients with early rheumatoid arthritis

Background and Objectives Presence of serum anti-citrullinated protein antibodies (ACPAs) in patients with rheumatoid arthritis (RA) predicts worse disease course and a more erosive disease. In the pathogenesis of RA, inflammatory T cells play a central role. In this context, we recently found increased proportions of pathogenic peripheral CCR6+ memory T helper cells in patients with early RA compared to healthy individuals. However, it is unclear how T cell proportions are distributed between ACPA+ and ACPA- patients. Therefore alterations in peripheral T cell populations and their pathogenic potential were examined in ACPA+ and ACPA- patient groups. Materials and Methods A nested matched case control study was performed including 27 ACPA+ and 27 ACPA- patients with early RA. T cell profiles (Treg, Th1, Th2, Th17, Th22 and various less well classified populations) from these ACPA+ and ACPA- patients were generated based on chemokine receptor, cytokine and transcription factor expression. Differentially present T cell populations were isolated from peripheral blood and analysed for their pathologic potential in a co-culture system with RA derived synovial fibroblasts (RASF). Results In comparison to ACPA- patients, ACPA+ patients have higher proportions of regulatory T cells (Treg) and CD4+ memory CCR6+ T cells. Since the CCR6+ T cell population is still a heterogeneous population, four CCR6+ T cell subpopulations were distinguished by differential expression of CXCR3 and CCR4. All four CCR6+ subpopulations shared Th17 cell characteristics such as Rorγt and CCL20 expression, but IL-17A, IL-17F, IL-22 and IFN-γ expression differed greatly between these subpopulations. However, even the population with lowest expression of these cytokines showed high pathological potential as shown by stimulating IL-1β, IL-6, IL-8, COX-2 and MMP-3 expression upon co-culture with RASF. Indeed, despite dissimilar Th17 and Th1 characteristics between the CCR6+ subpopulations, all four showed highly increased pathological potential in co-culture compared to naive and Th1 cells. Conclusions ACPA+ and ACPA- patients can be distinguished by the distribution of Treg and CCR6+ T cell subpopulations. These CCR6+ subpopulations exhibit dissimilar Th17 and Th1 characteristics, but all possess high pathological potential, including the population that has low IL-17A/F and IL-22 expression. These findings indicate a prominent role of CCR6+ T cells in the pathogenesis of ACPA+ patients with early RA and may contribute to the worse disease outcome in ACPA+ patients.