Follicular dendritic cell differentiation is associated with distinct synovial pathotype signatures in rheumatoid arthritis.

Follicular dendritic cells (FDCs) fundamentally contribute to the formation of synovial ectopic lymphoid-like structures in rheumatoid arthritis (RA) which is associated with poor clinical prognosis. Despite this critical role, regulation of FDC development in the RA synovium and its correlation with synovial pathotype differentiation remained largely unknown. Here, we demonstrate that CNA.42+ FDCs distinctively express the pericyte/fibroblast-associated markers PDGFR-ß, NG2, and Thy-1 in the synovial perivascular space but not in established follicles. In addition, synovial RNA-Seq analysis revealed that expression of the perivascular FDC markers was strongly correlated with PDGF-BB and fibroid synovitis, whereas TNF-α/LT-ß was significantly associated with lymphoid synovitis and expression of CR1, CR2, and FcγRIIB characteristic of mature FDCs in lymphoid follicles. Moreover, PDGF-BB induced CNA.42+ FDC differentiation and CXCL13 secretion from NG2+ synovial pericytes, and together with TNF-α/LT-ß conversely regulated early and late FDC differentiation genes in unsorted RA synovial fibroblasts (RASF) and this was confirmed in flow sorted stromal cell subsets. Furthermore, RASF TNF-αR expression was upregulated by TNF-α/LT-ß and PDGF-BB; and TNF-α/LT-ß-activated RASF retained ICs and induced B cell activation in in vitro germinal center reactions typical of FDCs. Additionally, FDCs trapped peptidyl citrulline, and strongly correlated with IL-6 expression, and plasma cell, B cell, and T cell infiltration of the RA synovium. Moreover, synovial FDCs were significantly associated with RA disease activity and radiographic features of tissue damage. To the best of our knowledge, this is the first report describing the reciprocal interaction between PDGF-BB and TNF-α/LT-ß in synovial FDC development and evolution of RA histological pathotypes. Selective targeting of this interplay could inhibit FDC differentiation and potentially ameliorate RA in clinically severe and drug-resistant patients.

Extracellular traps and PAD4 released by macrophages induce citrullination and auto-antibody production in autoimmune arthritis.

The mechanisms underlying the transition of rheumatoid arthritis (RA) systemic autoimmunity to the joints remain largely unknown. Here, we demonstrate that macrophages in the secondary lymphoid organs (SLOs) and synovial ectopic lymphoid-like structures (ELSs) express peptidylarginine deiminase 4 (PAD4) in murine collagen induced arthritis (CIA) and synovial biopsies from RA patients. Moreover, peptidyl citrulline colocalized with macrophages in SLOs and ELSs, and depletion of macrophages in CIA decreased lymphoid tissue citrullination and serum anti-citrullinated protein/peptide antibody (ACPA) levels. Furthermore, PAD was released from activated murine and RA synovial tissue and fluid (SF) macrophages which functionally deiminated extracellular proteins/peptides in vitro. Additionally, activated murine and SF macrophages displayed macrophage extracellular trap formation (METosis) and release of intracellular citrullinated histones. Moreover, presentation of citrullinated proteins induced ACPA production in vitro. Thus, lymphoid tissue macrophages contribute to self-antigen citrullination and ACPA production, indicating that their selective targeting would potentially ameliorate citrullination-dependent autoimmune disorders.

Mast cells in early rheumatoid arthritis associate with disease severity and support B cell autoantibody production.

OBJECTIVES: Mast cells (MCs) are involved in the pathogenesis of rheumatoid arthritis (RA). However, their contribution remains controversial. To establish their role in RA, we analysed their presence in the synovium of treatment-naïve patients with early RA and their association and functional relationship with histological features of synovitis. METHODS: Synovial tissue was obtained by ultrasound-guided biopsy from treatment-naïve patients with early RA (n=99). Immune cells (CD3/CD20/CD138/CD68) and their relationship with CD117+MCs in synovial tissue were analysed by immunohistochemistry (IHC) and immunofluorescence (IF). The functional involvement of MCs in ectopic lymphoid structures (ELS) was investigated in vitro, by coculturing MCs with naïve B cells and anticitrullinated protein antibodies (ACPA)-producing B cell clones, and in vivo in interleukin-27 receptor alpha (IL27ra)-deficient and control mice during antigen-induced arthritis (AIA). RESULTS: High synovial MC counts are associated with local and systemic inflammation, autoantibody positivity and high disease activity. IHC/IF showed that MCs reside at the outer border of lymphoid aggregates. Furthermore, human MCs promote the activation and differentiation of naïve B cells and induce the production of ACPA, mainly via contact-dependent interactions. In AIA, synovial MC numbers increase in IL27ra deficient mice, in association with ELS and worse disease activity. CONCLUSIONS: Synovial MCs identify early RA patients with a severe clinical form of synovitis characterised by the presence of ELS.

ADAM10-Mediated ICOS Ligand Shedding on B Cells Is Necessary for Proper T Cell ICOS Regulation and T Follicular Helper Responses.

The proper regulation of ICOS and ICOS ligand (ICOSL) has been shown to be essential for maintaining proper immune homeostasis. Loss of either protein results in defective humoral immunity, and overexpression of ICOS results in aberrant Ab production resembling lupus. How ICOSL is regulated in response to ICOS interaction is still unclear. We demonstrate that a disintegrin and metalloproteinase (ADAM)10 is the primary physiological sheddase of ICOSL in mice and humans. Using an in vivo system in which ADAM10 is deleted only on B cells, elevated levels of ICOSL were seen. This increase is also seen when ADAM10 is deleted from human B cell lines. Identification of the primary sheddase has allowed the characterization of a novel mechanism of ICOS regulation. In wild-type mice, interaction of ICOS/ICOSL results in ADAM10-induced shedding of ICOSL on B cells and moderate ICOS internalization on T cells. When this shedding is blocked, excessive ICOS internalization occurs. This results in severe defects in T follicular helper development and TH2 polarization, as seen in a house dust mite exposure model. In addition, enhanced TH1 and TH17 immune responses are seen in experimental autoimmune encephalomyelitis. Blockade of ICOSL rescues T cell ICOS surface expression and rescues, at least in part, T follicular helper numbers and the abnormal Ab production previously reported in these mice. Overall, we propose a novel regulation of the ICOS/ICOSL axis, with ADAM10 playing a direct role in regulating ICOSL, as well as indirectly regulating ICOS, thus controlling ICOS/ICOSL-dependent responses.

Isolation and Characterization of Mouse and Human Follicular Dendritic Cells.

Follicular dendritic cells (FDCs) reside in the B cell follicles of secondary and tertiary lymphoid tissues where they play key roles in the development and maintenance of lymphoid tissue architecture and function. FDCs trap native antigens for extended periods of time in the form of immune complexes which critcally regulate germinal center reactions in health and disease. Here, we describe how to isolate and characterize FDCs from murine and human lymphoid tissues.

Follicular dendritic cells in health and disease.

Follicular dendritic cells (FDCs) are unique immune cells that contribute to the regulation of humoral immune responses. These cells are located in the B-cell follicles of secondary lymphoid tissues where they trap and retain antigens (Ags) in the form of highly immunogenic immune complexes (ICs) consisting of Ag plus specific antibody (Ab) and/or complement proteins. FDCs multimerize Ags and present them polyvalently to B-cells in periodically arranged arrays that extensively crosslink the B-cell receptors for Ag (BCRs). FDC-FcγRIIB mediates IC periodicity, and FDC-Ag presentation combined with other soluble and membrane bound signals contributed by FDCs, like FDC-BAFF, -IL-6, and -C4bBP, are essential for the induction of the germinal center (GC) reaction, the maintenance of serological memory, and the remarkable ability of FDC-Ags to induce specific Ab responses in the absence of cognate T-cell help. On the other hand, FDCs play a negative role in several disease conditions including chronic inflammatory diseases, autoimmune diseases, HIV/AIDS, prion diseases, and follicular lymphomas. Compared to other accessory immune cells, FDCs have received little attention, and their functions have not been fully elucidated. This review gives an overview of FDC structure, and recapitulates our current knowledge on the immunoregulatory functions of FDCs in health and disease. A better understanding of FDCs should permit better regulation of Ab responses to suit the therapeutic manipulation of regulated and dysregulated immune responses.

Activation of B cells by antigens on follicular dendritic cells.

A need for antigen-processing and presentation to B cells is not widely appreciated. However, cross-linking of multiple B cell receptors (BCRs) by T-independent antigens delivers a potent signal that induces antibody responses. Such BCR cross-linking also occurs in germinal centers where follicular dendritic cells (FDCs) present multimerized antigens as periodically arranged antigen-antibody complexes (ICs). Unlike T cells that recognize antigens as peptide-MHC complexes, optimal B cell-responses are induced by multimerized FDC-ICs that simultaneously engage multiple BCRs. FDC-FcgammaRIIB mediates IC-periodicity and FDC-BAFF, FDC-IL-6 and FDC-C4bBP are co-stimulators. Remarkably, specific antibody responses can be induced by FDC-ICs in the absence of T cells, opening up the exciting possibility that people with T cell insufficiencies may be immunized with T-dependent vaccines via FDC-ICs.

IL-6 produced by immune complex-activated follicular dendritic cells promotes germinal center reactions, IgG responses and somatic hypermutation.

Reports that follicular dendritic cells (FDCs) produce IL-6 prompted the hypotheses that immune complexes (ICs) induce FDCs to produce IL-6 and that FDC-IL-6 promotes germinal center (GC) reactions, somatic hypermutation (SHM) and IgG production. FDCs were activated in vitro by addition of ICs and FDC-IL-6 production was determined. Wild-type (WT) and IL-6 knockout (KO) mice, as well as chimeras with WT and IL-6 KO cells, were immunized with (4-hydroxy-3-nitrophenyl)-acetyl (NP)-chicken gamma globulin (CGG) and used to study anti-(4-hydroxy-3-iodo-5-nitrophenyl) acetyl (NIP) responses, GC formation and SHM in the VH186.2 gene segment in Ig-gamma. FDC-IL-6 increased when FDCs encountered ICs. At low immunogen dose, 1 microg NP-CGG per mouse, the IgG anti-NIP response in IL-6 KO mice was low and immunohistochemistry revealed a reduction in both the number and size of GCs. The physiological relevance of FDC-IL-6 was apparent in the chimeric mice where total splenocytes from WT mice were unable to provide the IL-6 needed for normal IgG and GC responses in IL-6 KO animals with IL-6-defective FDCs. Moreover, the rate of mutation decreased from 18 to 8.9 mutations per 1000 bases (P < 0.001) in WT versus IL-6 KO mice. Addition of anti-IL-6 to GC reactions in vitro reduced antibody levels and SHM from 3.5 to 0.65 mutations per 1000 bases (P < 0.02). Thus, the absence of FDC-IL-6 correlated with a reduction in SHM that coincided with the reduction in GCs and specific anti-NIP. This is the first study to document that ICs induce FDC-IL-6 and that FDC-derived IL-6 is physiologically relevant in generating optimal GC reactions, SHM and IgG levels.

T-independent antibody responses to T-dependent antigens: a novel follicular dendritic cell-dependent activity.

Follicular dendritic cells (FDCs) periodically arrange membrane-bound immune complexes (ICs) of T-dependent Ags 200-500A apart, and in addition to Ag, they provide B cells with costimulatory signals. This prompted the hypothesis that Ag in FDC-ICs can simultaneously cross-link multiple BCRs and induce T cell-independent (TI) B cell activation. TI responses are characterized by rapid IgM production. OVA-IC-bearing FDCs induced OVA-specific IgM in anti-Thy-1-pretreated nude mice and by purified murine and human B cells in vitro within just 48 h. Moreover, nude mice immunized with OVA-ICs exhibited well-developed GL-7(+) germinal centers with IC-retaining FDC-reticula and Blimp-1(+) plasmablasts within 48 h. In contrast, FDCs with unbound-OVA, which would have free access to BCRs, induced no germinal centers, plasmablasts, or IgM. Engagement of BCRs with rat-anti-mouse IgD (clone 11-26) does not activate B cells even when cross-linked. However, B cells were activated when anti-IgD-ICs, formed with Fc-specific rabbit anti-rat IgG, were loaded on FDCs. B cell activation was indicated by high phosphotyrosine levels in caps and patches, expression of GL-7 and Blimp-1, and B cell proliferation within 48 h after stimulation with IC-bearing FDCs. Moreover, anti-IgD-IC-loaded FDCs induced strong polyclonal IgM responses within 48 h. Blockade of FDC-FcgammaRIIB inhibited the ability of FDC-ICs to induce T-independent IgM responses. Similarly, neutralizing FDC-C4BP or -BAFF, to minimize these FDC-costimulatory signals, also inhibited this FDC-dependent IgM response. This is the first report of FDC-dependent but TI responses to T cell-dependent Ags.

TLR4 on follicular dendritic cells: an activation pathway that promotes accessory activity.

Microbial molecular patterns engage TLRs and activate dendritic cells and other accessory cells. Follicular dendritic cells (FDCs) exist in resting and activated states, but are activated in germinal centers, where they provide accessory function. We reasoned that FDCs might express TLRs and that engagement might activate FDCs by up-regulating molecules important for accessory activity. To test this hypothesis, TLR4 expression on FDCs was studied in situ with immunohistochemistry, followed by flow cytometry and RT-PCR analysis. TLR4 was expressed on FDC reticula in situ, and flow cytometry indicated that TLR4 was expressed on surface membranes and TLR4 message was readily apparent in FDCs by RT-PCR. Injecting mice or treating purified FDCs with LPS up-regulated molecules important for accessory activity including, FDC-Fc gammaRIIB, FDC-ICAM-1, and FDC-VCAM-1. Treatment of purified FDCs with LPS also induced intracellular phospho-IkappaB-alpha, indicating NF-kappaB activation, and that correlated with increased Fc gammaRIIB, ICAM-1, and VCAM-1. FDCs in C3H/HeJ mice were not activated with LPS even when mice were reconstituted with C3H/HeN leukocytes, suggesting that engagement of FDC-TLR4 is necessary for activation. Moreover, activated FDCs exhibited increased accessory activity in anti-OVA recall responses in vitro, and the FDC number could be reduced 4-fold if they were activated. In short, we report expression of TLR4 on FDCs for the first time and that engagement of FDC-TLR4 activated NF-kappaB, up-regulated expression of molecules important in FDC accessory function, including Fc gammaRIIB, ICAM-1, and VCAM-1, as well as FDC accessory activity in promoting recall IgG responses.