Impacts of GFP-FoxP3+ regulatory T cells on lupus hallmarks differ by genetic background and type of GFP knock-in.

FoxP3 reporter mice expressing green fluorescence protein (GFP) have been used as a very convenient tool to investigate the impact of regulatory T (Treg) cells on pathogenesis in autoimmune diseases. Here, we found that GFP-FoxP3+ knock-in (KI) mice showed alterations in the production of anti-nuclear autoantibodies (ANAs) and nephritis with different extent, depending on the presence or absence of lupus susceptibility gene locus 1 (Sle1) and KI method: contrasting with B6.Sle1.fGFP-FoxP3 mice, expressing GFP via N-terminal insertion, B6.Sle1.iGFP-FoxP3, expressing GFP via bicistronic internal ribosome entry site-driven promotion, exhibited significantly lower penetrance of serum ANA, comparing to control B6.Sle1 mice. Moreover, B6.Sle1.GFP-FoxP3+ mice reduced the Sle1-induced splenomegaly and B-cell expansion independently of the KI method employed, mainly by reducing the numbers of transitional 1 (T1) B cells and CD21-CD23- B cells, including plasmablasts and plasma cells. The absolute numbers of both splenic CD4+ T cells and Treg cells from B6.Sle1.GFP-FoxP3 KI mice were significantly reduced but their proportion was not changed, compared to B6.Sle1 mice. Although the glomerular basement membranes were thickened in both B6.Sle1 and B6.Sle1.iGFP-FoxP3 mice, they were thinner in B6.Sle1.fGFP-FoxP3 mice. The latter mice expressed more nephrophilic autoantibodies and deposited more complement component 3 in glomeruli compared to B6.iGFP-FoxP3 mice. FoxP3+ Treg cells may modulate B-cell tolerance in lupus-prone B6.Sle1 mice, presumably by modulating pathogenic, nephrophilic autoantibody production and nephritis.

Autoregulatory function of interleukin-10-producing pre-naïve B cells is defective in systemic lupus erythematosus.

INTRODUCTION: Pre-naïve B cells represent an intermediate stage in human B-cell development with some functions of mature cells, but their involvement in immune responses is unknown. The aim of this study was to determine the functional role of normal pre-naïve B cells during immune responses and possible abnormalities in systemic lupus erythematosus (SLE) that might contribute to disease pathogenesis. METHODS: Pre-naïve, naïve, and memory B cells from healthy individuals and SLE patients were stimulated through CD40 and were analyzed for interleukin-10 (IL-10) production and co-stimulatory molecule expression and their regulation of T-cell activation. Autoreactivity of antibodies produced by pre-naïve B cells was tested by measuring immunoglobulin M (IgM) autoantibodies in culture supernatants after differentiation. RESULTS: CD40-stimulated pre-naïve B cells produce larger amounts of IL-10 but did not suppress CD4(+) T-cell cytokine production. Activated pre-naïve B cells demonstrated IL-10-mediated ineffective promotion of CD4(+) T-cell proliferation and induction of CD4(+)FoxP3(+) T cells and IL-10 independent impairment of co-stimulatory molecule expression and tumor necrosis factor-alpha (TNF-α) and IL-6 production. IgM antibodies produced by differentiated pre-naïve B cells were reactive to single-stranded deoxyribonucleic acid. SLE pre-naïve B cells were defective in producing IL-10, and co-stimulatory molecule expression was enhanced, resulting in promotion of robust CD4(+) T-cell proliferation. CONCLUSIONS: There is an inherent and IL-10-mediated mechanism that limits the capacity of normal pre-naïve B cells from participating in cellular immune response, but these cells can differentiate into autoantibody-secreting plasma cells. In SLE, defects in IL-10 secretion permit pre-naïve B cells to promote CD4(+) T-cell activation and may thereby enhance the development of autoimmunity.

The lupus susceptibility locus Sle1 facilitates the peripheral development and selection of anti-DNA B cells through impaired receptor editing.

Systemic lupus erythematosus is characterized by the spontaneous production of IgG autoantibodies in patients and lupus-prone mice. In this study, we investigated the effect of the Sle1 lupus susceptibility locus on the peripheral development of 56R(+) anti-DNA transgenic B cells by tracking 56R(+) B cells in mice without (B6.56R) or with (B6.Sle1.56R) the Sle1 locus. Compared with B6.56R mice, B6.Sle1.56R mice exhibited increased class-switched IgG2a anti-DNA Abs in their serum, encoded by the transgene. Interestingly, within the spleen, Sle1 facilitated the development of these cells into clusters of IgG2a class-switched B cells juxtaposed to CD4(+) T cells within extrafollicular sites. Through sequence analysis of B cell hybridomas, we also found that B cells from B6.Sle1.56R mice are inefficient at Ig H and L chain editing. Thus, the Ig H chains in Sle1.56R(+) B cells are partnered more often with cationic L chains that facilitate DNA binding. Taken together, these findings indicate that the Sle1 lupus-susceptibility locus may facilitate the emergence of anti-DNA B cells by subduing BCR revision and possibly by shaping the extrafollicular development of effector B cells, although the precise molecular mechanisms await further study.

Indoleamine 2,3-dioxygenase-expressing dendritic cells are involved in the generation of CD4+CD25+ regulatory T cells in Peyer's patches in an orally tolerized, collagen-induced arthritis mouse model.

INTRODUCTION: The present study was devised to understand the role of systemic indoleamine 2,3-dioxygenase (IDO) in the tolerance induction for orally tolerized mice in collagen-induced arthritis (CIA). We examined whether IDO-expressing dendritic cells (DCs) are involved in the generation of CD4+CD25+ regulatory T cells during the induction of oral tolerance in a murine CIA model. METHODS: Type II collagen was fed six times to DBA/1 mice beginning 2 weeks before immunization, and the effect on arthritis was assessed. To examine the IDO expression, the DCs of messenger RNA and protein were analyzed by RT-PCR and Flow cytometry. In addition, a proliferative response assay was also carried out to determine the suppressive effects of DCs through IDO. The ability of DCs expressing IDO to induce CD4+CD25+ T regulatory cells was examined. RESULTS: CD11c+ DCs in Peyer's patches from orally tolerized mice expressed a higher level of IDO than DCs from nontolerized CIA mice. IDO-expressing CD11c+ DCs were involved in the suppression of type II collagen-specific T-cell proliferation and in the downregulation of proinflammatory T helper 1 cytokine production. The suppressive effect of IDO-expressing CD11c+ DCs was mediated by Foxp3+CD4+CD25+ regulatory T cells. CONCLUSION: Our data suggest that tolerogenic CD11c+ DCs are closely linked with the induction of oral tolerance through an IDO-dependent mechanism and that this pathway may provide a new therapeutic modality to treat autoimmune arthritis.

Transforming growth factor beta 1(TGF-beta1) down-regulates TNFalpha-induced RANTES production in rheumatoid synovial fibroblasts through NF-kappaB-mediated transcriptional repression.

Transforming growth factor (TGF)-beta1 is a pleiotropic cytokine with many functions, including those related to growth modulation, immunosuppression, and pro-inflammation, in a wide variety of cell types. In this study, we investigated the ability of TGF-beta1 to regulate RANTES production by activated rheumatoid synovial fibroblasts. Fibroblast-like synoviocytes (FLS) were cultured in the presence of TGF-beta1 and IL-1beta, IL-15, TNFalpha, or IL-17, and the secretion of RANTES into culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA). Expression of RANTES encoded mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR), and NF-kappaB binding activity for RANTES transcription was determined by electrophoretic mobility shift assay (EMSA). We found that the concentrations of RANTES in synovial fluid (SF) from rheumatoid arthritis (RA) patients were lower than in SF from osteoarthritis (OA) patients, whereas the concentrations of TGF-beta1 were higher in RA SF than in OA SF. TGF-beta1 dose-dependently inhibited TNFalpha-induced production of RANTES protein and mRNA from RA FLS. Addition of RA SF with high-level TGF-beta1 mimicked the effect of TGF-beta1 on TNFalpha-induced RANTES production, which was inhibited by treatment with anti-TGF-beta1 neutralizing antibody. TGF-beta1 blocked the degradation of cytosolic IkappaB-alpha and the translocation of activated NF-kappaB to the nucleus. EMSA showed that the inhibitory effect of TGF-beta1 was associated with decreased binding of NF-kappaB to the RANTES promoter. These results suggest that elevated TGF-beta1 in rheumatoid synovial tissue may suppress joint inflammation by inhibiting RANTES secretion from synovial fibroblasts, thus blocking the infiltration of immune cells. These findings may provide an explanation for the mechanism by which TGF-beta1 regulates immune function in RA.