Interleukin-6 receptor blockade enhances CD39+ regulatory T cell development in rheumatoid arthritis and in experimental arthritis.

OBJECTIVE: The rationale for blocking interleukin-6 (IL-6) in rheumatoid arthritis (RA) lies chiefly in the proinflammatory effect of this cytokine. Few studies have evaluated the consequences of anti-IL-6 receptor (IL-6R) antibody treatment on Treg cells. This study was undertaken to elucidate the mechanism of action of anti-IL-6R antibody treatment by studying the effects on Treg cells in an experimental arthritis model and in patients with RA. METHODS: Mice with collagen-induced arthritis (CIA) were treated with a mouse anti-IL-6R antibody (MR16-1), and changes in Treg, Th1, and Th17 cells were assessed at key time points during the course of the disease. Peripheral blood from 15 RA patients was collected on day 0 and after 3 months of tocilizumab treatment for flow cytometry analysis of Th17 and Treg cells. RESULTS: In MR16-1-treated mice, Th17 cell frequencies were unchanged, whereas Treg cell frequencies were increased. The Treg cell phenotype showed marked changes, with an increase in the frequency of CD39+ Treg cells in the lymph nodes and spleen. Interestingly, similar CD39+ Treg cell expansion was observed in RA patients who were tocilizumab responders at 3 months, with no change in Th17 cell frequency. Moreover, fluorescence-activated cell-sorted CD39+ Treg cells from responder RA patients were functionally able to suppress the proliferation of conventional T cells. CONCLUSION: In both CIA and RA, the frequency of functionally suppressive CD39+ Treg cells is increased as a result of anti-IL-6R treatment, whereas Th17 cells are unaffected. The modification of Treg cell frequency and phenotype may be one of the mechanisms involved in the therapeutic effect of IL-6 blockade in RA.

The role of miR-155 in regulatory T cells and rheumatoid arthritis.

Recently, various micro(mi)RNAs have been found deregulated in the setting of rheumatoid arthritis (RA), but their role in the pathogenesis of this disease remains a matter of debate. In the meanwhile, increasing evidence indicates a defective function of regulatory T cells (Tregs) in RA. This review discusses relevant studies addressing the function of Tregs and Cytotoxic T-Lymphocyte Antigen 4 in RA, provides recent data on the role of miRNAs for Tregs homeostasis, and focuses on the role of miR-155 in Tregs. In a final perspective section we discuss the potential impact of therapeutic miR-155 modulation in RA.

A comparative study on intra-articular versus systemic gene electrotransfer in experimental arthritis.

BACKGROUND: Electric pulse mediated gene transfer has been applied successfully in vivo for increasing naked DNA administration in various tissues. To achieve non-viral gene transfer into arthritic joint tissue, we investigated the use of electrotransfer (ET). Because anti-inflammatory cytokine strategies have proven efficient in experimental models of arthritis, we compared the therapeutic efficiency of local versus systemic delivery of the interleukin-10 (IL-10) using in vivo ET. METHODS: A plasmid vector expressing IL-10 was transferred into DBA/1 mouse knee joints by ET with 12 pulses of variable duration and voltage. The kinetics of transgene expression were analyzed by specific enzyme-linked immunosorbent assay (ELISA) in sera and knees. Optimal conditions were then used to deliver increasing amounts of IL-10 plasmid intra-articularly (i.a.) in the collagen-induced arthritis (CIA) mouse model. The therapeutic efficiency was compared with the potency of intra-muscular (i.m.) ET. RESULTS: Following i.a. ET, local IL-10 secretion peaked on day 7 and dropped 2 weeks after. A second ET produced the same kinetics without enhancing gene transfer efficiency, while transgene was still detected in injected muscles 4 weeks after ET. Only the i.m. ET of 25 microg of IL-10 significantly inhibited all the clinical and biological features of arthritis. The i.a. ET only showed mild improvement of arthritis when 100 microg of IL-10 plasmid were electrotransfered weekly from day 18 following arthritis induction. CONCLUSIONS: The present results suggest that gene transfer into arthritic joints by ET is an effective means to deliver anti-inflammatory cytokines. However, short duration of transgene expression impedes a significant effect for the treatment of arthritis, making i.m. ET more potent than i.a. ET for clinical benefit in CIA.

Adeno-associated virus pseudotype 5 vector improves gene transfer in arthritic joints.

The potential for gene delivery to joints, using recombinant adeno-associated virus (rAAV) vectors for the treatment of rheumatoid arthritis (RA), has received much attention. Different serotypes have different virion shell proteins and, as a consequence, vary in their tropism for diverse tissues. The aim of this study was to compare the transduction efficiency of different AAV serotypes encoding murine secreted alkaline phosphatase (mSEAP) or Escherichia coli beta-galactosidase for intraarticular gene delivery in an experimental model of arthritis. The vectors contained AAV2 terminal repeats flanking the reporter gene in an AAV1, AAV2, or AAV5 capsid, producing the pseudotypes rAAV-2/1, rAAV-2/2, and rAAV-2/5. Left knee joints of mice with collagen-induced arthritis were injected and transgene expression was analyzed by chemiluminescence or direct in situ staining of frozen sections. We show for the first time that intraarticular gene transfer with AAV- 2/5 was far more efficient than with the other serotypes tested. Transgene expression was detectable as early as 7 days after injection, reached a maximum at 21 days, and was stably expressed for at least 130 days, whereas AAV-2/1- and AAV-2/2-mediated expression levels were barely detectable. These findings provide a practical application for future local AAV-mediated gene therapy trials in RA.

Specific overexpression of rheumatoid arthritis-associated HLA-DR alleles and presentation of low-affinity peptides.

OBJECTIVE: To compare levels of HLA-DR expression in rheumatoid arthritis (RA) patients and healthy controls for whom an ordered expression according to the DR alleles is demonstrated and to test the functional consequences of this expression on peptide presentation. METHODS: Using monoclonal antibodies that recognize different DRB1 alleles, DR molecules were quantitated at the surface of the peripheral blood B cells of 23 RA patients and 17 healthy subjects. The functional consequences of the level of DR surface expression was tested using a universal model of antigen presentation and mutated peptides with variable affinities for the T cell receptor. RESULTS: In healthy subjects, surface HLA-DR molecules were expressed at different levels according to allele (DR53, DR4, and DR11 less than DR1 less than DR7 less than DR15). In RA patients, this hierarchy was not conserved and, furthermore, the density of RA-associated DR4 and DR1 molecules was enhanced in patients compared with the basal density in healthy individuals. We demonstrated that an increased expression of DR molecules at the surface of antigen-presenting cells allowed a noteworthy presentation of low-affinity peptides that under normal conditions are not efficient in generating a T cell response at physiologic surface density of the DR molecules. CONCLUSION: Our results suggest that the specific overexpression of RA-associated HLA molecules could be responsible for the presentation of low-affinity autopeptides and therefore the activation of peripheral autoreactive T cells.

The down-regulation of HLA-DM gene expression in rheumatoid arthritis is not related to their promoter polymorphism.

HLA-DM molecule, a class II-like heterodimer, is a critical factor of HLA class II-dependent Ag presentation. It acts as a molecular chaperone and also functions as a peptide editor favoring the presentation of high-stability peptides. Thus, it appears to skew the peptide repertoire presented to T cells. Variation in HLA-DM expression has considerable effect on Ag presentation and regulation of these genes is likely to be a prerequisite to prevent autoimmunity. In this study, rheumatoid arthritis (RA) was chosen as a model of human autoimmune disease since its genetic susceptibility is known to be associated with the HLA-DR and -DM components. We described a limited nucleotide polymorphism in the HLA-DM promoters with functional impact on basal transcriptional activity and IFN-gamma induction as assessed in vitro. However, no difference of allele frequencies was found between controls and RA patients. Despite of this lack of association, expression of HLA-DM molecules was also investigated. Interestingly, an underexpression of HLA-DM transcripts and protein was shown in peripheral blood B cells from RA patients compared with controls or inflammatory arthritis patients. This underexpression does not affect HLA-DR genes and is responsible for a decrease of the DM:DR ratio in RA patients. This specific HLA-DM down-regulation is likely to have important consequences on Ag presentation and could participate in the autoimmune process in RA.

CREB regulates MHC class II expression in a CIITA-dependent manner.

The X2 box of MHC class II promoters is homologous to TRE/CRE elements and is required for expression of MHC class II genes. The X2 box-specific DNA binding activity, X2BP, was purified to homogeneity, sequenced, and identified as CREB. Transient transactivation experiments showed that CREB can cooperate with CIITA to enhance activation of transcription from MHC class II promoters in a dose-dependent manner. Binding of CREB to the class II promoter in vivo was demonstrated by a chromatin immunoprecipitation assay. Additionally, ICER, a dominant inhibitor of CREB function, was found to repress class II expression. These results demonstrate that CREB binds to the X2 box in vivo and cooperates with CIITA to direct MHC class II expression.

RFX-B is the gene responsible for the most common cause of the bare lymphocyte syndrome, an MHC class II immunodeficiency.

The bare lymphocyte syndrome (BLS) is characterized by the absence of MHC class II transcription and humoral- and cellular-mediated immune responses to foreign antigens. Three of the four BLS genetic complementation groups have defects in the activity of the MHC class II transcription factor RFX. We have purified the RFX complex and sequenced its three subunits. The sequence of the smallest subunit describes a novel gene, termed RFX-B. RFX-B complements the predominant BLS complementation group (group B) and was found to be mutant in cell lines from this BLS group. The protein has no known DNA-binding domain but does contain three ankyrin repeats that are likely to be important in protein-protein interactions.

Formation of a regulatory factor X/X2 box-binding protein/nuclear factor-Y multiprotein complex on the conserved regulatory regions of HLA class II genes.

Coordinate regulation of MHC class II genes occurs in a tissue-specific and cytokine-inducible manner. While the upstream regulatory sequences are conserved among all MHC class II genes, multiple base pair changes are found, even within the essential X box region. Analysis of all class II X boxes reveals differential binding between two transcription factors known to interact with the X box region, regulatory factor X and X2 box-binding protein (RFX and X2BP) of the HLA-DRA gene. These data presented a paradox with regard to the coordinate regulation of the class II genes if the factors though to regulate the HLA-DRA gene do not bind to the homologous sequence of all class II genes. Previous results suggested that cooperative interactions between the DNA binding proteins may be the key to understanding this paradox. Here RFX/X2BP/DNA complexes were formed on all class II isotypes regardless of the ability of the X box region to bind either factor individually. To further determine the role of the interactions between the X and Y factors, multiprotein/DNA complexes containing RFX, X2BP, NF-Y, and X-Y box DNA of the DRA and DRB genes were formed. This quaternary complex was extremely stable to competitor DNA, with a half-life > 4 h. These results suggest that the conserved X and Y boxes of class II genes function similarly and define a single multiprotein regulatory complex for class II expression in B cells.

HLA-DMA and HLA-DMB gene expression functions through the conserved S-X-Y region.

The MHC class II homologous proteins HLA-DMA and HLA-DMB function in the loading of peptides onto class II molecules. Like the class II genes, the HLA-DM genes contain upstream regulatory sequences similar to the S-X-Y regulatory region as well as additional putative regulatory sites. To determine whether the DM genes are regulated in a similar manner as class II genes, a series of in vivo and in vitro analyses was performed. Deletion analysis showed that expression from the DM promoters is dependent on the conserved S-X-Y region. The class II-specific transcription factors RFX and CIITA are also required for expression, as cell lines deficient in these factors failed to allow transcription from the DM promoters. In addition, in vivo footprint analysis showed the putative X and Y boxes to be occupied by transcription factors in wild-type B cells, but not in RFX-deficient B cells. In astrocytes, IFN-gamma treatment induced increased occupancy of these sites. None of the other putative regulatory sites was occupied in vivo, indicating that they may not be functional. Finally, gel shift analysis showed synergistic complex formation between proteins that bind to the putative X boxes of the DM genes, as is found for the DRA gene. Therefore, the DM genes share a common mechanism of regulation with the class II genes.

Qualitative and quantitative analysis of HLA-DRB gene expression.

Determination of the molecular mechanisms influencing HLA-DRB gene expression would allow better understanding of the regulation of the immune response in normal as well as in pathological conditions. We started an extensive sequencing analysis of the proximal promoter regions of the DRB genes and observed a nucleotide polymorphism involving the classical regulatory regions of the DRB promoters. These nucleotide substitutions were observed to induce variations of the promoter activities as assessed by chloramphenicol acetyl transferase assays and the quantification of DRB transcripts. Similar analysis performed in patients with rheumatoid arthritis strongly suggests dysregulation of HLA-DRB gene expression.