IL-23 Promotes a Coordinated B Cell Germinal Center Program for Class-Switch Recombination to IgG2b in BXD2 Mice.

IL-23 promotes autoimmune disease, including Th17 CD4 T cell development and autoantibody production. In this study, we show that a deficiency of the p19 component of IL-23 in the autoimmune BXD2 (BXD2-p19-/- ) mouse leads to a shift of the follicular T helper cell program from follicular T helper (Tfh)-IL-17 to Tfh-IFN-γ. Although the germinal center (GC) size and the number of GC B cells remained the same, BXD2-p19-/- mice exhibited a lower class-switch recombination (CSR) in the GC B cells, leading to lower serum levels of IgG2b. Single-cell transcriptomics analysis of GC B cells revealed that whereas Ifngr1, Il21r, and Il4r genes exhibited a synchronized expression pattern with Cxcr5 and plasma cell program genes, Il17ra exhibited a synchronized expression pattern with Cxcr4 and GC program genes. Downregulation of Ighg2b in BXD2-p19-/- GC B cells was associated with decreased expression of CSR-related novel base excision repair genes that were otherwise predominantly expressed by Il17ra + GC B cells in BXD2 mice. Together, these results suggest that although IL-23 is dispensable for GC formation, it is essential to promote a population of Tfh-IL-17 cells. IL-23 acts indirectly on Il17ra + GC B cells to facilitate CSR-related base excision repair genes during the dark zone phase of GC B cell development.

Cutting Edge: Intracellular IFN-ß and Distinct Type I IFN Expression Patterns in Circulating Systemic Lupus Erythematosus B Cells.

In systemic lupus erythematosus (SLE), type I IFNs promote induction of type I IFN-stimulated genes (ISG) and can drive B cells to produce autoantibodies. Little is known about the expression of distinct type I IFNs in lupus, particularly high-affinity IFN-ß. Single-cell analyses of transitional B cells isolated from SLE patients revealed distinct B cell subpopulations, including type I IFN producers, IFN responders, and mixed IFN producer/responder clusters. Anti-Ig plus TLR3 stimulation of SLE B cells induced release of bioactive type I IFNs that could stimulate HEK-Blue cells. Increased levels of IFN-ß were detected in circulating B cells from SLE patients compared with controls and were significantly higher in African American patients with renal disease and in patients with autoantibodies. Together, the results identify type I IFN-producing and -responding subpopulations within the SLE B cell compartment and suggest that some patients may benefit from specific targeting of IFN-ß.

Cutting Edge: Endogenous IFN-ß Regulates Survival and Development of Transitional B Cells.

The transitional stage of B cell development is a formative stage in the spleen where autoreactive specificities are censored as B cells gain immune competence, but the intrinsic and extrinsic factors regulating survival of transitional stage 1 (T1) B cells are unknown. We report that B cell expression of IFN-ß is required for optimal survival and TLR7 responses of transitional B cells in the spleen and was overexpressed in T1 B cells from BXD2 lupus-prone mice. Single-cell gene expression analysis of B6 Ifnb+/+ versus B6 Ifnb-/- T1 B cells revealed heterogeneous expression of Ifnb in wild-type B cells and distinct gene expression patterns associated with endogenous IFN-ß. Single-cell analysis of BXD2 T1 B cells revealed that Ifnb is expressed in early T1 B cell development with subsequent upregulation of Tlr7 and Ifna1 Together, these data suggest that T1 B cell expression of IFN-ß plays a key role in regulating responsiveness to external factors.

Tamoxifen metabolite endoxifen interferes with the polyamine pathway in breast cancer.

Tamoxifen is the most widely used drug to treat women with estrogen receptor α (ERα)-positive breast cancer. Endoxifen is recognized as the active metabolite of tamoxifen in humans. We studied endoxifen effects on ERα-positive MCF-7 breast cancer cells. Estradiol increased the proliferation of MCF-7 cells by two- to threefold and endoxifen suppressed its effects. Endoxifen suppressed c-myc, c-fos and Tff1 oncogene expression, as revealed by RT-PCR. Estradiol increased the activity of ornithine decarboxylase (ODC) and adenosyl methioninedecarboxylase (AdoMetDC), whereas endoxifen suppressed these enzyme activities. Endoxifen increased activities of spermine oxidase (SMO) and acetyl polyamine oxidase (APAO) significantly, and reduced the levels of putrescine and spermidine. These data suggest a possible mechanism for the antiestrogenic effects of tamoxifen/endoxifen, involving the stimulation of polyamine oxidase enzymes. Therefore, SMO and APAO stimulation might be useful biomarkers for the efficacy of endoxifen treatment of breast cancer.

Interferon-induced mechanosensing defects impede apoptotic cell clearance in lupus.

Systemic lupus erythematosus (SLE) is a severe autoimmune disease that is associated with increased circulating apoptotic cell autoantigens (AC-Ags) as well as increased type I IFN signaling. Here, we describe a pathogenic mechanism in which follicular translocation of marginal zone (MZ) B cells in the spleens of BXD2 lupus mice disrupts marginal zone macrophages (MZMs), which normally clear AC debris and prevent follicular entry of AC-Ags. Phagocytosis of ACs by splenic MZMs required the megakaryoblastic leukemia 1 (MKL1) transcriptional coactivator-mediated mechanosensing pathway, which was maintained by MZ B cells through expression of membrane lymphotoxin-α1ß2 (mLT). Specifically, type I IFN-induced follicular shuttling of mLT-expressing MZ B cells disengaged interactions between these MZ B cells and LTß receptor-expressing MZMs, thereby downregulating MKL1 in MZMs. Loss of MKL1 expression in MZMs led to defective F-actin polymerization, inability to clear ACs, and, eventually, MZM dissipation. Aggregation of plasmacytoid DCs in the splenic perifollicular region, follicular translocation of MZ B cells, and loss of MKL1 and MZMs were also observed in an additional murine lupus model and in the spleens of patients with SLE. Collectively, the results suggest that lupus might be interrupted by strategies that maintain or enhance mechanosensing signaling in the MZM barrier to prevent follicular entry of AC-Ags.

General Approach for Tetramer-Based Identification of Autoantigen-Reactive B Cells: Characterization of La- and snRNP-Reactive B Cells in Autoimmune BXD2 Mice.

Autoreactive B cells are associated with the development of several autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. The low frequency of these cells represents a major barrier to their analysis. Ag tetramers prepared from linear epitopes represent a promising strategy for the identification of small subsets of Ag-reactive immune cells. This is challenging given the requirement for identification and validation of linear epitopes and the complexity of autoantibody responses, including the broad spectrum of autoantibody specificities and the contribution of isotype to pathogenicity. Therefore, we tested a two-tiered peptide microarray approach, coupled with epitope mapping of known autoantigens, to identify and characterize autoepitopes using the BXD2 autoimmune mouse model. Microarray results were verified through comparison with established age-associated profiles of autoantigen specificities and autoantibody class switching in BXD2 and control (C57BL/6) mice and high-throughput ELISA and ELISPOT analyses of synthetic peptides. Tetramers were prepared from two linear peptides derived from two RNA-binding proteins (RBPs): lupus La and 70-kDa U1 small nuclear ribonucleoprotein. Flow cytometric analysis of tetramer-reactive B cell subsets revealed a significantly higher frequency and greater numbers of RBP-reactive marginal zone precursor, transitional T3, and PDL-2(+)CD80(+) memory B cells, with significantly elevated CD69 and CD86 observed in RBP(+) marginal zone precursor B cells in the spleens of BXD2 mice compared with C57BL/6 mice, suggesting a regulatory defect. This study establishes a feasible strategy for the characterization of autoantigen-specific B cell subsets in different models of autoimmunity and, potentially, in humans.

IL-23 promotes TCR-mediated negative selection of thymocytes through the upregulation of IL-23 receptor and RORγt.

Transient thymic involution is frequently found during inflammation, yet the mode of action of inflammatory cytokines is not well defined. Here we report that interleukin-23 (IL-23) production by the thymic dendritic cells (DCs) promotes apoptosis of the CD4(hi)CD8(hi) double-positive (DP) thymocytes. A deficiency in IL-23 signalling interferes with negative selection in the male D(b)/H-Y T-cell receptor (TCR) transgenic mice. IL-23 plus TCR signalling results in significant upregulation of IL-23 receptor (IL-23R) expressed predominantly on CD4(hi)CD8(hi)CD3(+)αßTCR(+) DP thymocytes, and leads to RORγt-dependent apoptosis. These results extend the action of IL-23 beyond its peripheral effects to a unique role in TCR-mediated negative selection including elimination of natural T regulatory cells in the thymus.

Interleukin-21 promotes germinal center reaction by skewing the follicular regulatory T cell to follicular helper T cell balance in autoimmune BXD2 mice.

OBJECTIVE: Follicular regulatory T (Tfr) cells act as the regulatory counterpart of follicular helper T (Tfh) cells to suppress germinal center (GC) B cell differentiation. We recently showed that interleukin-21 (IL-21) promoted Tfh cell differentiation in autoimmune BXD2 mice that develop spontaneous GCs. This study was undertaken to determine the modulatory effects of IL-21 on Tfr cells and the Tfr cell to Tfh cell balance in BXD2 mice. METHODS: The percentage and phenotype of Tfr cells were determined in BXD2 and BXD2-IL21(-/-) mice. The effects of IL-21 on Tfr cells and the Tfr cell:Tfh cell ratio were evaluated. Sorted Tfr cells from BXD2-IL21(-/-) mice were cocultured with Tfh cells and B cells, or transferred into BXD2 mice to determine their function. RESULTS: The percentages and numbers of GC B cells and Tfh cells were significantly reduced, but the percentage of Tfr cells was 2-fold higher in BXD2-IL21(-/-) mice than in wild-type BXD2 mice. Administration of AdIL-21 to BXD2-IL21(-/-) mice decreased the percentages and numbers of Tfr cells and the Tfr cell:Tfh cell ratio but increased the number of GC B cells in the spleen. Recombinant murine IL-21 suppressed FoxP3 and significantly reduced Tgfb1, Il2, and Gitr but enhanced Il21, Il6, Pd1, Cxcr5, and Icos expression in Tfr cells. IL-21 also counteracted Tfr cell-mediated inhibition of antibody secretion in the Tfh cell-B cell coculture system. Transfer of Tfr cells into young BXD2 mice reduced GC size and decreased the numbers of autoantibody-producing B cells. CONCLUSION: Our findings indicate that high levels of IL-21 selectively enhance Tfh cell differentiation but inhibit Tfr cell commitment and the suppressive function of Tfr cells on Tfh cells and B cells, suggesting that IL-21 skews the balance from Tfr cells to Tfh cells to promote autoreactive GC reactions in BXD2 mice.

Inhibition of fucosylation reshapes inflammatory macrophages and suppresses type II collagen-induced arthritis.

OBJECTIVE: Fucosylation catalyzed by fucosyltransferases (FUTs) is an important posttranslational modification involved in a variety of biologic processes. This study was undertaken to determine the roles of fucosylation in rheumatoid arthritis (RA) and to assess the efficacy of reestablishing immune homeostasis with the use of 2-deoxy-d-galactose (2-d-gal), a fucosylation inhibitor. METHODS: Quantitative polymerase chain reaction was performed to determine the expression of FUT genes in synovial tissue from RA and osteoarthritis (OA) patients and in fluorescence-activated cell-sorted cells from RA synovial fluid. The in vivo inhibitory effect of 2-d-gal was evaluated in a murine collagen-induced arthritis (CIA) model. The in vitro effects of 2-d-gal on differentiation of inflammatory macrophages, production of cytokines, and antigen uptake, processing, and presentation functions were analyzed. RESULTS: FUTs that are involved in terminal or subterminal fucosylation, but not those involved in core fucosylation or O-fucosylation, were up-regulated in RA compared to OA synovial tissue. The expression of terminal FUTs was highly positively correlated with the expression of TNF (encoding for tumor necrosis factor α). Terminal FUTs were predominantly expressed in M1 macrophages. In vivo, 2-d-gal treatment of mice precluded the development of CIA by reducing inflammatory macrophages and Th17 cells in the draining lymph nodes and decreasing the levels of TNFα, interleukin-6 (IL-6), and antibodies to type II collagen in the serum. In vitro, treatment with 2-d-gal skewed the differentiation of M1 macrophages to IL-10-producing M2 macrophages. Furthermore, 2-d-gal significantly inhibited the antigen-presenting function of M1 macrophages. CONCLUSION: Terminal fucosylation is a novel hallmark of inflammatory macrophages. Inhibition of terminal FUTs reshapes the differentiation and functions of M1 macrophages, leading to resolution of inflammation in arthritis.

Death receptor 5-targeted depletion of interleukin-23-producing macrophages, Th17, and Th1/17 associated with defective tyrosine phosphatase in mice and patients with rheumatoid arthritis.

OBJECTIVE: Bidirectional interactions between granulocyte-macrophage colony-stimulating factor-positive (GM-CSF+) T cells and interferon regulatory factor 5-positive (IRF-5+) macrophages play a major role in autoimmunity. In the absence of SH2 domain-containing phosphatase 1 (SHP-1), GM-CSF-stimulated cells are resistant to death receptor (DR)-mediated apoptosis. The objective of this study was to determine whether TRA-8, an anti-DR5 agonistic antibody, can eliminate inflammatory macrophages and CD4 T cells in the SHP-1-deficient condition. METHODS: Ubiquitous Cre (Ubc.Cre) human/mouse-chimeric DR5-transgenic mice were crossed with viable SHP-1-defective motheaten (mev/mev) mice. TRA-8 was administered weekly for up to 4 weeks. The clinical scores, histopathologic severity, and macrophage and CD4 T cell phenotypes were evaluated. The role of TRA-8 in depleting inflammatory macrophages and CD4 T cells was also evaluated, using synovial fluid obtained from patients with rheumatoid arthritis (RA). RESULTS: The levels of inflammatory macrophages (interleukin-23-positive [IL-23+] IRF-5+) and CD4 T cells (IL-17+ GM-CSF+) were elevated in mev/mev mice. In DR5-transgenic mev/mev mice, DR5 expression was up-regulated in these 2 cell populations. TRA-8 treatment depleted these cell populations and resulted in a significant reduction in inflammation and in the titers of autoantibodies. In synovial cells from patients with RA, the expression of IRF5 and DR5 was negatively correlated with the expression of PTPN6. TRA-8, but not TRAIL, suppressed RA inflammatory macrophages and Th17 cells under conditions in which the expression of SHP-1 is low. CONCLUSION: In contrast to TRAIL, which lacks the capability to counteract the survival signal in the absence of SHP-1, TRA-8 eliminated both IRF-5+ IL-23+ M1 macrophages and pathogenic GM-CSF+ IL-17+ CD4 T cells in a SHP-1-independent manner. The results of the current study suggest that TRA-8 can deplete inflammatory cell populations that result from a hyperactive GM-CSF/IRF-5 axis.

Extension of the germinal center stage of B cell development promotes autoantibodies in BXD2 mice.

OBJECTIVE: Regulator of G protein signaling (RGS) proteins inhibit chemokine signaling by desensitizing G protein-coupled receptor signals. This study was undertaken to determine the mechanisms by which RGS13 promotes the generation of pathogenic autoantibodies in germinal centers (GCs), using BXD2-Rgs13-/- mice. METHODS: Confocal and light microscopy imaging techniques were used to determine the location of cells that express RGS13 and activation-induced cytidine deaminase (AID) in the mouse spleen, and the number of plasmablasts. The levels of GC and plasma cell program transcripts in GC B cells were determined by real-time quantitative polymerase chain reaction (qPCR). Differential interleukin-17 (IL-17)-mediated expression of RGS13 in GC versus non-GC B cells was analyzed using A20 and 70Z/3 B cells. RESULTS: In the spleens of BXD2 mice, RGS13 was mainly expressed by GC B cells and was stimulated by IL-17 but not IL-21. IL-17 up-regulated RGS13 in A20 GC cells but not 70Z/3 non-GC B cells. BXD2- Rgs13-/- mice exhibited smaller GCs and lower AID levels, suggesting lower somatic hypermutation and affinity maturation. However, GC B cells from BXD2- Rgs13-/- mice showed increased levels of IgMbright plasmablasts, up-regulation of the genes encoding plasma program, including interferon regulatory factor 4, B lymphocyte-induced maturation protein 1, and X-box binding protein 1 and the p-CREB target genes Fosb and Obf1, and down-regulation of the GC program genes Aid, Pax5, and Bach2 compared to BXD2 mice. BXD2-Rgs13-/- mice had lower titers of IgG autoantibodies and IgG deposits in the glomeruli, suggesting reduced autoantibody pathogenicity. CONCLUSION: RGS13 deficiency is associated with a reduction in GC program genes and the exit of fewer pathogenic IgM plasmablasts in BXD2 mice. Our findings indicate that prolonged GC program, mediated by up-regulation of RGS13, enhances AID expression and enables the generation of pathogenic autoantibodies in autoreactive GCs.

IL-17RA is essential for optimal localization of follicular Th cells in the germinal center light zone to promote autoantibody-producing B cells.

Germinal centers (GCs) provide a microenvironment that promotes and regulates the interactions of B cells with follicular Th (TFH) cells. In this study, we show that there are significantly higher frequencies of CXCR5(+)ICOS(+) TFH cells in autoimmune BXD2 mice, and these cells express both IL-21R and IL-17RA. Although IL-17 and IL-21 are both important for the formation of spontaneous GCs and development of pathogenic autoantibodies, IL-21, but not IL-17, is required for the proper development of TFH cells in BXD2 mice. The total numbers of TFH cells and their ability to induce B cell responses in vitro were not affected by a deficiency of IL-17RA in BXD2-Il17ra(-/-) mice, the majority of CXCR5(+) TFH cells from BXD2-Il17ra(-/-) mice were, however, not localized in the GC light zone (LZ). Interruption of IL-17 signaling, either acutely by AdIL-17R:Fc or chronically by Il17ra(-/-), disrupted TFH-B interactions and abrogated the generation of autoantibody-forming B cells in BXD2 mice. IL-17 upregulated the expression of regulator of G-protein signaling 16 (RGS16) to promote the ability of TFH to form conjugates with B cells, which was abolished in TFH cells from BXD2-Rgs16(-/-) mice. The results suggests that IL-17 is an extrinsic stop signal that it acts on postdifferentiated IL-17RA(+) TFH to enable its interaction with responder B cells in the LZ niche. These data suggest a novel concept that TFH differentiation and its stabilization in the LZ are two separate checkpoints and that IL-21 and IL-17 act at each checkpoint to enable pathogenic GC development.

Cutting Edge: defective follicular exclusion of apoptotic antigens due to marginal zone macrophage defects in autoimmune BXD2 mice.

Marginal zone macrophages (MZMs) act as a barrier to entry of circulating apoptotic debris into the follicles of secondary lymphoid organs. In autoimmune BXD2 mice, there is a progressive reduction in the function and numbers of MZMs. Absence of MZMs results in retention of apoptotic cell (AC) debris within the marginal zone (MZ) and increased loading of AC Ags on MZ B cells and MZ-precursor (MZ-P) B cells. The MZ-P B cells are capable of translocating the AC Ags to the follicular zone and stimulating T cells. Both MZMs and MZ-P B cells from BXD2 mice express low levels of tolerogenic signals and high levels of inflammatory signals. Thus, the current study suggests a multifaceted mechanism in which MZMs maintain tolerance to apoptotic autoantigens and suppress their translocation to follicles. Lack of clearance of apoptotic debris by MZMs drives follicular Ag-transportation by MZ-P B cells to stimulate an autoimmune response.

Increased vitamin D is associated with decline of naïve, but accumulation of effector, CD8 T cells during early aging.

Given the protective roles of 25-hydroxyvitamin D (25[OH]D or vitamin D) in musculoskeletal health and the potential beneficial effects of vitamin D supplementation in reducing the risk of various chronic diseases, intensive repletion of vitamin D has been widely advocated. Of note, CD8 T cells have the highest levels of the vitamin D receptor compared with other major immune cells. The effects of vitamin D on CD8 T cells during aging, however, remain unclear. This study determined the relationship between vitamin D levels and CD8 T-cell status in 34 healthy female subjects (all >60 years old). The CD8 T cell phenotype was defined by the surface expression of CD28 and CD95. The low-25(OH)D serum groups (≤30 ng/ml) had higher percentages of CD28+CD95-CD8+ (naïve) T cells and lower percentages of CD28+CD95+CD8+ (effector) T cells. By contrast, subjects with high levels of 25(OH)D had very low percentages of naïve CD8 T cells but very high percentages of effector CD8 T cells. There was a significant inverse correlation between 25(OH)D levels and the frequency of naïve CD8 T cells. The results show that higher levels of vitamin D are correlated with decreased frequencies of naïve CD8 T cells during early aging, suggesting that higher levels of 25(OH)D accelerate CD8 T-cell senescence. These results warrant the further evaluation of the effects of vitamin D supplementation in immune aging.

Treatment of arthritis by macrophage depletion and immunomodulation: testing an apoptosis-mediated therapy in a humanized death receptor mouse model.

OBJECTIVE: To determine the therapeutic efficacy and immunomodulatory effect of an anti-human death receptor 5 (DR5) antibody, TRA-8, in eliminating macrophage subsets in a mouse model of type II collagen-induced arthritis (CIA). METHODS: A human/mouse-chimeric DR5-transgenic mouse, under the regulation of a mouse 3-kb promoter and a loxP-flanked STOP cassette, was generated and crossed with an ubiquitous Cre (Ubc.Cre) mouse and a lysozyme M-Cre (LysM.Cre)-transgenic mouse to achieve inducible or macrophage-specific expression. Chicken type II collagen was used to induce CIA in mice, which were then treated with an anti-human DR5 antibody, TRA-8. Clinical scores, histopathologic severity, macrophage apoptosis and depletion, and T cell subset development were evaluated. RESULTS: In human/mouse DR5-transgenic Ubc.Cre mice with CIA, transgenic DR5 was most highly expressed on CD11b+ macrophages, with lower expression on CD4+ T cells. In human/mouse DR5-transgenic LysM.Cre mice, transgenic DR5 was restrictively expressed on macrophages. Both in vivo near-infrared imaging of caspase activity and TUNEL staining demonstrated that TRA-8 rapidly induced apoptosis of macrophages in inflamed synovium. Depletion of pathogenic macrophages by TRA-8 led to significantly reduced clinical scores for arthritis; decreased macrophage infiltration, synovial hyperplasia, osteoclast formation, joint destruction, cathepsin activity, and inflammatory cytokine expression in joints; reduced numbers of Th17 cells; and an increased number of Treg cells in draining lymph nodes. CONCLUSION: The anti-human DR5 antibody TRA-8 was efficacious in reducing the severity of arthritis via targeted depletion of macrophages and immunomodulation. Our data provide preclinical evidence that TRA-8 is a potential novel biologic agent for rheumatoid arthritis therapy.

A potential estrogen mimetic effect of a bis(ethyl)polyamine analogue on estrogen receptor positive MCF-7 breast cancer cells.

BE-3-3-3-3 (1,15-(ethylamino)4,8,12-triazapentadecane) is a bis(ethyl)polyamine analogue under investigation as a therapeutic agent for breast cancer. Since estradiol (E(2)) is a critical regulatory molecule in the growth of breast cancer, we examined the effect of BE-3-3-3-3 on estrogen receptor α (ERα) positive MCF-7 cells in the presence and absence of E(2). In the presence of E(2), a concentration-dependent decrease in DNA synthesis was observed using [(3)H]-thymidine incorporation assay. In the absence of E(2), low concentrations (2.5-10 µM) of BE-3-3-3-3 increased [(3)H]-thymidine incorporation at 24 and 48 h. BE-3-3-3-3 induced the expression of early response genes, c-myc and c-fos, in the absence of E(2), but not in its presence, as determined by real-time quantitative polymerase chain reaction (qPCR). BE-3-3-3-3 had no significant effect on these genes in an ERα-negative cell line, MDA-MB-231. Chromatin immunoprecipitation assay demonstrated enhanced promoter occupation by either E(2) or BE-3-3-3-3 of an estrogen-responsive gene pS2/Tff1 by ERα and its co-activator, steroid receptor co-activator 3 (SRC-3). Confocal microscopy of BE-3-3-3-3-treated cells revealed membrane localization of ERα, similar to that induced by E(2). The failure of BE-3-3-3-3 to inhibit cell proliferation was associated with autophagic vacuole formation, and the induction of Beclin 1 and MAP LC3 II. These results indicate a differential effect of BE-3-3-3-3 on MCF-7 cells in the absence and presence of E(2), and suggest that pre-clinical and clinical development of polyamine analogues might require special precautions and selection of sensitive subpopulation of patients.

Nucleosides accelerate inflammatory osteolysis, acting as distinct innate immune activators.

The innate immune system and its components play an important role in the pathogenesis of inflammatory bone destruction. Blockade of inflammatory cytokines does not completely arrest bone erosion, suggesting that other mediators also may be involved in osteolysis. Previously we showed that nucleosides promote osteoclastogenesis and bone-resorption activity in the presence of receptor activator for nuclear factor κB ligand (RANKL) in vitro. The studies described here further demonstrate that selected nucleosides and nucleoside analogues accelerate bone destruction in mice immunized with collagen II alone (CII) but also further enhance bone erosion in mice immunized by collagen II plus complete Freund's adjuvant (CII + CFA). Abundant osteoclasts are accumulated in destructive joints. These data indicate that nucleosides act as innate immune activators distinct from CFA, synergistically accelerating osteoclast formation and inflammatory osteolysis. The potential roles of the surface triggering receptor expressed on myeloid cells (TREM) and the intracellular inflammasome in nucleoside-enhanced osteoclastogenesis have been studied. These observations provide new insight into the pathogenesis and underlying mechanism of bone destruction in inflammatory autoimmune osteoarthritis.

Inhibition of the catalytic function of activation-induced cytidine deaminase promotes apoptosis of germinal center B cells in BXD2 mice.

OBJECTIVE: To determine whether functional suppression of the catalytic domain of activation-induced cytidine deaminase (AID) can suppress the hyperreactive germinal center (GC) responses in BXD2 mice. METHODS: We generated transgenic BXD2 mice expressing a dominant-negative (DN) form of Aicda at the somatic hypermutation site (BXD2-Aicda-DN-transgenic mice). Real-time quantitative reverse transcriptase-polymerase chain reaction was used to determine the expression of Aicda and DNA damage/repair genes. Enzyme-linked immunosorbent assay was used to measure serum levels of autoantibodies and immune complexes (ICs). Development of GCs and antibody-containing ICs as well as numbers of proliferative and apoptotic cells were determined using flow cytometry and/or immunohistochemical analyses. Development of arthritis and kidney disease was evaluated histologically in 6-8-month-old mice. RESULTS: Suppression of the somatic hypermutation function of AID resulted in a significant decrease in autoantibody production without affecting the expression of DNA damage-related genes in GC B cells of BXD2-Aicda-DN-transgenic mice. There was decreased proliferation, increased apoptosis, increased expression of caspase 9 messenger RNA in GC B cells, and lower numbers of GCs in the spleens of BXD2-Aicda-DN-transgenic mice. Decreased GC response was associated with lower levels of IgG-containing ICs. Anti-IgM- and anti-CD40 plus anti-Ig-induced B cell proliferative responses were decreased in BXD2-Aicda-DN-transgenic mice. CONCLUSION: Inhibition of the AID somatic hypermutation function in BXD2 mice suppressed development of spontaneous GCs, generation of autoantibody-producing B cells, and autoimmunity in BXD2 mice. Suppression of AID catalytic function to limit selection-based survival of GC B cells could become a novel therapy for the treatment of autoimmune disease.

Type I interferon-dependent CD86(high) marginal zone precursor B cells are potent T cell costimulators in mice.

OBJECTIVE: To investigate the role of CD86(high) marginal zone (MZ) precursor B cells in type I interferon (IFN)-induced T cell-dependent responses in autoimmune BXD2 mice. METHODS: Confocal microscopic imaging was used to determine the location of plasmacytoid dendritic cells (DCs), MZ precursor B cells, and CD4 T cells in the spleens of BXD2 and BXD2-Ifnar(-/-) mice. Immunohistochemical staining was used to determine IgG(bright) cells in the spleens of BXD2 and BXD2-Ifnar(-/-) mice. Enzyme-linked immunosorbent assay was used to determine serum levels of IFNα and autoantibodies, and 4-hydroxy-3-nitrophenylacetyl hapten (NP)-chicken γ-globulin (CGG) (NP-CGG)- or NP-Ficoll-induced anti-NP2 antibody titers. Real-time quantitative polymerase chain reaction was used to determine the levels of type I IFN transcripts. T cell proliferation was measured using (3) H-thymidine. The expression of CD86 and CD80 was determined by fluorescence-activated cell sorting analysis. RESULTS: The deletion of type I IFN receptor abrogated the development of IgG(bright) cells and suppressed a T cell-dependent antibody response. Type I IFN signaling was associated with the expression of CD86, but not CD80, on follicular, MZ, and MZ precursor B cells. However, MZ precursor B cells demonstrated the highest expression of CD86 and the highest capacity for T cell costimulation with intact type I IFN receptor. This effect was blocked by an antibody that neutralizes CD86. In IFN receptor-intact BXD2 mouse spleens, MZ precursor B cells clustered at the T cell-B cell border. CD86 deletion suppressed germinal center formation, autoantibody production, and development of autoimmune diseases in BXD2 mice. CONCLUSION: Type I IFN can promote autoimmune responses in BXD2 mice through up-regulation of CD86(high) expression on MZ precursor B cells and trafficking of MZ precursor B cells to the T cell-B cell border to provide costimulation of CD4 T cells.

IL-17 activates the canonical NF-kappaB signaling pathway in autoimmune B cells of BXD2 mice to upregulate the expression of regulators of G-protein signaling 16.

We previously identified that autoreactive B cells from BXD2 mice can be targeted by IL-17, leading to upregulation of the expression of regulators of G-protein signaling (Rgs) genes that facilitated the development of spontaneous germinal centers. Little is known about the signaling pathway used by IL-17 to upregulate RGS. In the current study, we found that IL-17 rapidly activates the canonical NF-kappaB signaling pathway and that BXD2 B cells exhibit higher basal and activated phosphorylated p65 levels than B6 or BXD2-Il17ra(-/-) B cells. Inhibition of p65 phosphorylation downregulated RGS16 expression and abrogated the IL-17-induced chemotactic arrest of B cells in response to CXCL12. Knockdown of TNFR-associated factor 6 or NF-kappaB activator 1 in 70Z/3 pre-B cells led to decreased Rgs16 expression, indicating that both of these two genes are involved in IL-17-mediated activation of NF-kappaB signaling in B cells. These findings identify the signaling pathway regulated by IL-17 to contribute to the development of spontaneous germinal centers in autoimmune BXD2 mice.