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.

The role of lymphoid tissue SPARC in the pathogenesis and response to treatment of multiple myeloma.

Background: Despite the significant progress in the treatment of multiple myeloma (MM), the disease remains untreatable and its cure is still an unmet clinical need. Neoplastic transformation in MM is initiated in the germinal centers (GCs) of secondary lymphoid tissue (SLT) where B cells experience extensive somatic hypermutation induced by follicular dendritic cells (FDCs) and T-cell signals. Objective: We reason that secreted protein acidic and rich in cysteine (SPARC), a common stromal motif expressed by FDCs at the origin (SLTs) and the destination (BM) of MM, plays a role in the pathogenesis of MM, and, here, we sought to investigate this role. Methods: There were 107 BM biopsies from 57 MM patients (taken at different time points) together with 13 control specimens assessed for SPARC gene and protein expression and compared with tonsillar tissues. In addition, regulation of myeloma-promoting genes by SPARC-secreting FDCs was assessed in in vitro GC reactions (GCRs). Results: SPARC gene expression was confirmed in both human primary (BM) and secondary (tonsils) lymphoid tissues, and the expression was significantly higher in the BM. Sparc was detectable in the BM and tonsillar lysates, co-localized with the FDC markers in both tissues, and stimulation of FDCs in vitro induced significantly higher levels of SPARC expression than unstimulated controls. In addition, SPARC inversely correlated with BM PC infiltration, ISS staging, and ECOG performance of the MM patients, and in vitro addition of FDCs to lymphocytes inhibited the expression of several oncogenes associated with malignant transformation of PCs. Conclusion: FDC-SPARC inhibits several myelomagenic gene expression and inversely correlates with PC infiltration and MM progression. Therapeutic induction of SPARC expression through combinations of the current MM drugs, repositioning of non-MM drugs, or novel drug discovery could pave the way to better control MM 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.

Symmetric molecules with 1,4-triazole moieties as potent inhibitors of tumour-associated lactate dehydrogenase-A.

A series of symmetric molecules incorporating aryl or pyridyl moieties as central core and 1,4-substituted triazoles as a side bridge was synthesised. The new compounds were investigated as lactate dehydro-genase (LDH, EC 1.1.1.27) inhibitors. The cancer associated LDHA isoform was inhibited with IC50 = 117-174 µM. Seven compounds exhibited better LDHA inhibition (IC50 117-136 µM) compared to known LDH inhibitor - galloflavin (IC50 157 µM).

Impaired immunological synapse in sperm associated antigen 6 (SPAG6) deficient mice.

Sperm associated antigen 6 (SPAG6), a component of the central apparatus of the "9 + 2" axoneme, plays a central role in ciliary and flagellar motility; but, its contribution to adaptive immunity and immune system development is completely unknown. While immune cells lack a cilium, the immunological synapse is a surrogate cilium as it utilizes the same machinery as ciliogenesis including the nucleation of microtubules at the centrosome. This prompted our hypothesis that SPAG6 critically regulates the formation and function of immunological synapses. Using bone marrow reconstitution studies of adult WT mice, we demonstrate that SPAG6 is expressed in primary and secondary lymphoid tissues, is associated with the centrosome in lymphocytes, and its deficiency results in synapse disruption due to loss of centrosome polarization and actin clearance at the synaptic cleft. Improper synapse formation in Spag6KO mice was associated with defective CTL functions and impaired humoral immunity as indicated by reduced germinal centers reactions, follicular CD4 T cells, and production of class-switched antibody, together with expansion of B1 B cells. This novel report demonstrates the requirement of SPAG6 for optimal synapse formation and function, its direct role in immune cell function, and provides a novel mechanism for infertility disorders related to SPAG6.

LLT1 and CD161 Expression in Human Germinal Centers Promotes B Cell Activation and CXCR4 Downregulation.

Germinal centers (GCs) are microanatomical structures critical for the development of high-affinity Abs and B cell memory. They are organized into two zones, light and dark, with coordinated roles, controlled by local signaling. The innate lectin-like transcript 1 (LLT1) is known to be expressed on B cells, but its functional role in the GC reaction has not been explored. In this study, we report high expression of LLT1 on GC-associated B cells, early plasmablasts, and GC-derived lymphomas. LLT1 expression was readily induced via BCR, CD40, and CpG stimulation on B cells. Unexpectedly, we found high expression of the LLT1 ligand, CD161, on follicular dendritic cells. Triggering of LLT1 supported B cell activation, CD83 upregulation, and CXCR4 downregulation. Overall, these data suggest that LLT1-CD161 interactions play a novel and important role in B cell maturation within the GC in humans.

Increased B Cell ADAM10 in Allergic Patients and Th2 Prone Mice.

ADAM10, as the sheddase of the low affinity IgE receptor (CD23), promotes IgE production and thus is a unique target for attenuating allergic disease. Herein, we describe that B cell levels of ADAM10, specifically, are increased in allergic patients and Th2 prone WT mouse strains (Balb/c and A/J). While T cell help augments ADAM10 expression, Balb WT B cells exhibit increased ADAM10 in the naïve state and even more dramatically increased ADAM10 after anti-CD40/IL4 stimulation compared C57 (Th1 prone) WT B cells. Furthermore, ADAM17 and TNF are reduced in allergic patients and Th2 prone mouse strains (Balb/c and A/J) compared to Th1 prone controls. To further understand this regulation, ADAM17 and TNF were studied in C57Bl/6 and Balb/c mice deficient in ADAM10. C57-ADAM10B-/- were more adept at increasing ADAM17 levels and thus TNF cleavage resulting in excess follicular TNF levels and abnormal secondary lymphoid tissue architecture not noted in Balb-ADAM10B-/-. Moreover, the level of B cell ADAM10 as well as Th context is critical for determining IgE production potential. Using a murine house dust mite airway hypersensitivity model, we describe that high B cell ADAM10 level in a Th2 context (Balb/c WT) is optimal for disease induction including bronchoconstriction, goblet cell metaplasia, mucus, inflammatory cellular infiltration, and IgE production. Balb/c mice deficient in B cell ADAM10 have attenuated lung and airway symptoms compared to Balb WT and are actually most similar to C57 WT (Th1 prone). C57-ADAM10B-/- have even further reduced symptomology. Taken together, it is critical to consider both innate B cell levels of ADAM10 and ADAM17 as well as Th context when determining host susceptibility to allergic disease. High B cell ADAM10 and low ADAM17 levels would help diagnostically in predicting Th2 disease susceptibility; and, we provide support for the use ADAM10 inhibitors in treating Th2 disease.

Disturbed follicular architecture in B cell A disintegrin and metalloproteinase (ADAM)10 knockouts is mediated by compensatory increases in ADAM17 and TNF-α shedding.

B cell A disintegrin and metalloproteinase 10 (ADAM10) is required for the development and maintenance of proper secondary lymphoid tissue architecture; however, the underlying mechanism remains unclear. In this study, we show disturbances in naive lymph node architecture from B cell-specific ADAM10-deficient mice (ADAM10(B-/-)) including loss of B lymphocyte/T lymphocyte compartmentalization, attenuation of follicular dendritic cell reticula, excessive collagen deposition, and increased high endothelial venule formation. Because TNF-α signaling is critical for secondary lymphoid tissue architecture, we examined compensatory changes in ADAM17 and TNF-α in ADAM10(B-/-) B cells. Surprisingly, defective follicular development in these mice was associated with increased rather than decreased TNF-α expression. In this article, we describe an increase in TNF-α message, mRNA stability, soluble protein release, and membrane expression in ADAM10(B-/-) B cells compared with wild type (WT), which coincides with increased ADAM17 message and protein. To assess the mechanistic contribution of excessive TNF-α to abnormal lymphoid architecture in ADAM10(B-/-) mice, we performed a bone marrow reconstitution study. Rectification of WT architecture was noted only in irradiated WT mice reconstituted with ADAM10(B-/-) + TNF knockout bone marrow because of normalization of TNF-α levels not seen in ADAM10(B-/-) alone. We conclude that ADAM17 overcompensation causes excessive TNF-α shedding and further upregulation of TNF-α expression, creating an aberrant signaling environment within B cell cortical regions of ADAM10(B-/-) lymph nodes, highlighting a key interplay between B cell ADAM10 and ADAM17 with respect to TNF-α homeostasis.

Multi-therapeutic potential of autoantibodies induced by immune complexes trapped on follicular dendritic cells.

Induction of autoantibodies (autoAbs) targeting disease drivers / mediators is emerging as a potential immunotherapeutic strategy. Auto-immune complex (IC)-retaining follicular dendritic cells (FDCs) critically regulate pathogenic autoAb production in autoreactive germinal centers (GCs); however, their ability to induce potentially therapeutic autoAbs has not been explored. We hypothesized that deliberate display of clinically targeted antigens (Ags) in the form of ICs on FDC membranes induces target-specific autoreactive GCs and autoAbs that may be exploited therapeutically. To test our hypothesis, three therapeutically relevant Ags: TNF-α, HER2/neu and IgE, were investigated. Our results indicated that TNF-α-, HER2/neu- and IgE-specific autoAbs associated with strong GC reactions were induced by TNF-α-, HER2/neu- and IgE-IC retention on FDCs. Moreover, the induced anti-TNF-α autoAbs neutralized mouse and human TNF-α with half maximal Inhibitory Concentration (IC50) of 7.1 and 1.6 nM respectively. In addition, we demonstrated that FDC-induced Ab production could be non-specifically inhibited by the IgG-specific Endo-S that accessed the light zones of GCs and interfered with FDC-IC retention. In conclusion, the ability of FDCs to productively present autoAgs raises the potential for a novel immunotherapeutic platform targeting mediators of autoimmune disorders, allergic diseases, and Ab responsive cancers.

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.

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.

RAPD typing of Aspergillus chevalieri, Aspergillus nidulans, Aspergillus tetrazonus (quadrilineatus) and their teleomorphs using 5'-d[AACGCGCAAC]-3' and 5'-d[CCCGTCAGCA]-3' primers.

Sexuality in fungi has long been a matter of concerns and debates that always necessitated extensive analysis of the relationship between organisms assumed to represent different developmental forms of the same organism. Moreover, mating-virulence correlation and the growing worry of opportunistic fungal infections in immunocompromised states associated with AIDS, cancer chemotherapy and organ transplantation protocols have been critically addressed nowadays. In view of that, we genetically characterized Aspergillus chevalieri, Aspergillus nidulans, Aspergillus tetrazonus (quadrilineatus) and their teleomorphs with RAPD analysis using 5'-d[AACGCGCAAC]-3' and 5'-d[CCCGTCAGCA]-3' primers. The reported similarities between the sexual and the asexual forms of the tested species, using Dice coefficient, ranged between 40% and 70% which holds pretty much with the current systematics of the genus. The study presents a rapid consistent method for identification of A. chevalieri, A. nidulans, A. tetrazonus (quadrilineatus) and their teleomorphs based on the banding pattern of RAPD-generated fragments that can be reliably used ahead of further applications on these species.

Follicular dendritic cell (FDC)-FcgammaRIIB engagement via immune complexes induces the activated FDC phenotype associated with secondary follicle development.

Follicular dendritic cell (FDC)-FcgammaRIIB levels are up-regulated 1-3 days after challenge of actively immunized mice with Ag. This kinetics suggested that memory cells are not driving this response, prompting the hypothesis that immune complex (IC)-FDC interactions lead to FDC activation. To test this, mice passively immunized with anti-OVA Ab were OVA challenged to produce IC. After 3 days, levels of IC, FcgammaRIIB, ICAM-1, and VCAM-1 on FDC were analyzed. FDC were also stimulated with IC in vitro, and mRNA for FcgammaRIIB, ICAM-1, and VCAM-1 was quantified by quantitative RT-PCR. IC labeling in passively immunized WT and FcgammaRIIB-/- mice revealed five to six FDC-reticula per LN midsagittal section. In WT mice, these IC-bearing FDC-reticula corresponded with FDC-reticula labeling for FcgammaRIIB, ICAM-1, and VCAM-1. Increases in these molecules on IC-stimulated FDC were confirmed by flow cytometry. In marked contrast, in FcgammaRIIB-/- mice, no increased VCAM-1 or ICAM-1 was seen on IC-bearing FDC-reticula or on purified FDC. Addition of IC in vitro resulted in dramatic increases in mRNA for FcgammaRIIB, ICAM-1 and VCAM-1 in WT FDC, but not in FDC from FcgammaRIIB-/- mice, 2.4G2-pretreated WT FDC, B cells, or macrophages. Thus, although FDC-FcgammaRIIB was not essential for IC trapping, engagement of FDC-FcgammaRIIB with IC initiated an FDC activation pathway.