Patrick S. Asmawidjaja

Th17 cells, but not Th1 cells, from patients with early rheumatoid arthritis are potent inducers of matrix metalloproteinases and proinflammatory cytokines upon synovial fibroblast interaction, including autocrine interleukin‐17A production

OBJECTIVE Both Th1 cells and Th17 cells have been recognized in rheumatoid arthritis (RA); however, it remains unclear whether Th1 cells and/or Th17 cells are involved in driving disease chronicity and destructiveness. The aim of this study was to identify and characterize the functional role of Th17 cells in early RA. METHODS Flow cytometry analysis was performed on peripheral blood mononuclear cells (PBMCs) from treatment-naive patients with early RA and age-matched healthy volunteers. PBMCs from these patients, naive T cells, and primary CCR6- Th1 cells and CCR6+ Th17 cells were sorted and cultured in the absence or presence of synovial fibroblasts from patients with early RA (RASFs), and cytokine expression and gene transcription were analyzed. In addition, tumor necrosis factor α (TNFα)- and interleukin-17A (IL-17A)-blocking experiments were performed. RESULTS In the PBMCs of treatment-naive patients with early RA, an increased fraction of IL-17A-and TNFα-producing CCR6+ Th17 cells was observed. When cocultured with RASFs, these primary Th17 cells were potent inducers of IL-6 and IL-8 and the tissue-destructive enzymes matrix metalloproteinase 1 (MMP-1) and MMP-3, whereas primary Th1 cells or naive T cells were not. Importantly, specific up-regulation of IL-17A but not TNFα or interferon-γ was observed in RASF/Th17 cell cocultures. In addition to TNFα blocking, IL-17A neutralization was required to further down-regulate Th17 activity in RASF/Th17 cell cocultures. CONCLUSION Th17 cells, but not Th1 cells, cooperated with RASFs in a proinflammatory feedback loop, revealing a potential mechanism by which human Th17 cells drive chronic destructive disease in patients with RA. Furthermore, the neutralization of IL-17A activity is essential in current anti-TNF therapies to suppress Th17 cell activity in patients with early RA and potentially other Th17 cell-mediated disorders.

1,25-Dihydroxyvitamin D3 modulates Th17 polarization and interleukin-22 expression by memory T cells from patients with early rheumatoid arthritis

OBJECTIVE To examine the immunologic mechanism by which 1,25-dihydroxyvitamin D(3) (1,25[OH](2)D(3)) may prevent corticosteroid-induced osteoporosis in patients with early rheumatoid arthritis (RA), with a focus on T cell biology. METHODS Peripheral blood mononuclear cells (PBMCs) and CD4+CD45RO+ (memory) and CD4+CD45RO- (non-memory) T cells separated by fluorescence-activated cell sorting (FACS) from treatment-naive patients with early RA were stimulated with anti-CD3/anti-CD28 in the absence or presence of various concentrations of 1,25(OH)(2)D(3), dexamethasone (DEX), and 1,25(OH)(2)D(3) and DEX combined. Levels of T cell cytokines were determined by enzyme-linked immunosorbent assay and flow cytometry. RESULTS The presence of 1,25(OH)(2)D(3) reduced interleukin-17A (IL-17A) and interferon-gamma levels and increased IL-4 levels in stimulated PBMCs from treatment-naive patients with early RA. In addition, 1,25(OH)(2)D(3) had favorable effects on tumor necrosis factor alpha (TNFalpha):IL-4 and IL-17A:IL-4 ratios and prevented the unfavorable effects of DEX on these ratios. Enhanced percentages of IL-17A- and IL-22-expressing CD4+ T cells and IL-17A-expressing memory T cells were observed in PBMCs from treatment-naive patients with early RA as compared with healthy controls. Of note, we found no difference in the percentage of CD45RO+ and CD45RO- cells between these 2 groups. Interestingly, 1,25(OH)(2)D(3), in contrast to DEX, directly modulated human Th17 polarization, accompanied by suppression of IL-17A, IL-17F, TNFalpha, and IL-22 production by memory T cells sorted by FACS from patients with early RA. CONCLUSION These data indicate that 1,25(OH)(2)D(3) may contribute its bone-sparing effects in RA patients taking corticosteroids by the modulation of Th17 polarization, inhibition of Th17 cytokines, and stimulation of IL-4.

GATA-3 protects against severe joint inflammation and bone erosion and reduces differentiation of Th17 cells during experimental arthritis

OBJECTIVE Rheumatoid arthritis is associated with the infiltration of T helper cells into the joints. It is unclear whether interferon-gamma (IFNgamma)-producing Th1 cells or the novel T helper subset, interleukin-17 (IL-17)-producing Th17 cells, are the pathogenic mediators of joint inflammation in chronic nonautoimmune arthritis. Therefore, this study was aimed at examining whether the Th2-specific transcription factor GATA-3 can regulate arthritis, in an experimental murine model, by modulating Th1 and/or Th17 cell polarization. METHODS Arthritis was induced with methylated bovine serum albumin (mBSA) in both wild-type and CD2 T cell-specific GATA-3 (CD2-GATA-3)-transgenic mice. At days 1 and 7 after the induction of arthritis, knee joints were scored macroscopically for arthritis severity and for histologic changes. Single-cell suspensions were generated from the spleens, lymph nodes, and inflamed knee joints. Cytokine expression by CD4+ T cells was determined using flow cytometry, and IL-17 expression in the inflamed knee joints was determined by enzyme-linked immunosorbent assay. Analyses of gene expression were performed for Th17-associated factors. RESULTS Wild-type mice developed severe joint inflammation, including massive inflammatory cell infiltration and bone erosion that increased significantly over time, reaching maximal arthritis scores at day 7. In contrast, only mild joint inflammation was observed in CD2-GATA-3-transgenic mice. This mild effect was further accompanied by systemic and local reductions in the numbers of IL-17+IFNgamma- and IL-17+IFNgamma+, but not IL-17-IFNgamma+, CD4+ T cells, and by induction of Th2 cytokine expression. Moreover, GATA-3 overexpression resulted in reduced gene expression of the Th17-associated transcription factor retinoic acid-related orphan receptor gammat. CONCLUSION These results indicate that enforced GATA-3 expression protects against severe joint inflammation and bone erosion in mice, accompanied by reduced differentiation of Th17 cells, but not Th1 cells, during mBSA-induced arthritis.

Interleukin-23 promotes Th17 differentiation by inhibiting T-bet and FoxP3 and is required for elevation of interleukin-22, but not interleukin-21, in autoimmune experimental arthritis.

OBJECTIVE To examine the role of interleukin-23 (IL-23) in subgroup polarization of IL-17A-positive and/or interferon-gamma (IFNgamma)-positive T cells in autoimmune disease-prone DBA/1 mice with and without collagen-induced arthritis. METHODS A magnetic-activated cell sorting system was used to isolate CD4+ T cells from the spleen of naive and type II collagen (CII)-immunized DBA/1 mice. These CD4+ T cells were stimulated in vitro under Th0, Th1, or different Th17 culture conditions. Intracellular staining for IL-17A and IFNgamma was evaluated by flow cytometry. In addition, Th17 cytokines and T helper-specific transcription factors were analyzed by enzyme-linked immunosorbent assay and/or quantitative polymerase chain reaction. RESULTS In CD4+ T cells from naive DBA/1 mice, IL-23 alone hardly induced retinoic acid-related orphan receptor gammat (RORgammat), Th17 polarization, and Th17 cytokines, but it inhibited T-bet expression. In contrast, transforming growth factor beta1 (TGFbeta1)/IL-6 was a potent inducer of RORgammat, RORalpha, IL-17A, IL-17F, IL-21, and FoxP3 in these cells. In contrast to TGFbeta1/IL-6, IL-23 was critical for the induction of IL-22 in CD4+ T cells from both naive and CII-immunized DBA/1 mice. Consistent with these findings, IL-23 showed a more pronounced induction of the IL-17A+IFNgamma- subset in CD4+ T cells from CII-immunized mice. However, in CD4+ T cells from naive mice, IL-23 significantly increased the TGFbeta1/IL-6-induced Th17 polarization, including elevated levels of IL-17A and IL-17F and decreased expression of T-bet and FoxP3. Of note, the IL-23-induced increase in IL-17A and IL-17F levels was prevented in T-bet-deficient mice. CONCLUSION IL-23 promotes Th17 differentiation by inhibiting T-bet and FoxP3 and is required for elevation of IL-22, but not IL-21, levels in autoimmune arthritis. These data indicate different mechanisms for IL-23 and TGFbeta1/IL-6 at the transcription factor level during Th17 differentiation in autoimmune experimental arthritis.

TNF blockade requires 1,25(OH)2D3 to control human Th17-mediated synovial inflammation

Objectives T helper 17 (Th17) cells from patients with early rheumatoid arthritis (RA) induce a proinflammatory feedback loop upon RA synovial fibroblast (RASF) interaction, including autocrine interleukin (IL)-17A production. A major challenge in medicine is how to control the pathogenic Th17 cell activity in human inflammatory autoimmune diseases. The objective of this study was to examine whether tumour necrosis factor (TNF) blockade and/or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) controls Th17-mediated synovial inflammation. Methods Peripheral CD4+CD45RO+CCR6+ Th17 cells of patients with early RA, Th17–RASF cocultures and synovial biopsy specimens were cultured with or without 1,25(OH)2D3 and/or TNFα blockade. Intracellular cytokine expression was detected by flow cytometry. Cytokine and matrix metalloprotease (MMP) production was determined by ELISA. Results The authors show that the 1,25(OH)2D3, but not TNFα blockade, significantly suppressed autocrine IL-17A production in Th17–RASF and synovial biopsy cultures. Combining 1,25(OH)2D3 and TNFα blockade had a significant additive effect compared with single treatment in controlling synovial inflammation, indicated by a further reduction in IL-6, IL-8, MMP-1 and MMP-3 in Th17–RASF cocultures and IL-6 and IL-8 expression in cultures of RA synovial tissue. Conclusions These data show that TNF blockade does not suppress IL-17A and IL-22, which can be overcome by 1,25(OH)2D3. The combination of neutralising TNF activity and 1,25(OH)2D3 controls human Th17 activity and additively inhibits synovial inflammation. This indicates more valuable therapeutic potential of activation of Vitamin D receptorsignalling over current TNF neutralisation strategies in patients with RA and potentially other Th17-mediated inflammatory diseases.

The Varicellovirus-Encoded TAP Inhibitor UL49.5 Regulates the Presentation of CTL Epitopes by Qa-1b1

Impairment of MHC class I Ag processing is a commonly observed mechanism that allows viruses and tumors to escape immune destruction by CTL. The peptide transporter TAP that is responsible for the delivery of MHC class I-binding peptides into the endoplasmic reticulum is a pivotal target of viral-immune evasion molecules, and expression of this transporter is frequently lost in advanced cancers. We recently described a novel population of CTL that intriguingly exhibits reactivity against such tumor-immune escape variants and that recognizes self-peptides emerging at the cell surface due to defects in the processing machinery. Investigations of this new type of CTL epitopes are hampered by the lack of an efficient inhibitor for peptide transport in mouse cells. In this article, we demonstrate that the varicellovirus protein UL49.5, in contrast to ICP47 and US6, strongly impairs the activity of the mouse transporter and mediates degradation of mouse TAP1 and TAP2. Inhibition of TAP was witnessed by a strong reduction of surface MHC class I display and a decrease in recognition of conventional tumor-specific CTL. Analysis of CTL reactivity through the nonclassical molecule Qa-1b revealed that the presentation of the predominant leader peptide was inhibited. Interestingly, expression of UL49.5 in processing competent tumor cells induced the presentation of the new category of peptides. Our data show that the varicellovirus UL49.5 protein is a universal TAP inhibitor that can be exploited for preclinical studies on CTL-based immune intervention.

Interleukin-23 is critical for full-blown expression of a non-autoimmune destructive arthritis and regulates interleukin-17A and RORγt in γδ T cells

IntroductionInterleukin (IL)-23 is essential for the development of various experimental autoimmune models. However, the role of IL-23 in non-autoimmune experimental arthritis remains unclear. Here, we examined the role of IL-23 in the non-autoimmune antigen-induced arthritis (AIA) model. In addition, the regulatory potential of IL-23 in IL-17A and retinoic acid-related orphan receptor gamma t (RORγt) expression in CD4+ and TCRγδ+ T cells was evaluated systemically as well as at the site of inflammation.MethodsAntigen-induced arthritis was induced in wild-type, IL-23p19-deficient and IL-17 Receptor A - knockout mice. At different time points, synovial cytokine and chemokine expression was measured. At days 1 and 7 of AIA, splenocytes and joint-infiltrating cells were isolated and analyzed for intracellular IL-17A and interferon (IFN)-γ ex-vivo by flow cytometry. In splenic CD4+ and TCRγδ+ T cells gene expression was quantified by flow cytometry and quantitative PCR.ResultsIL-23 was critical for full-blown AIA. Lack of IL-23 did not prevent the onset of joint inflammation but stopped the progression to a destructive synovitis. IL-23 regulated IL-17A expression in CD4+ T cells in the spleen. Of note, IL-17A and IFN-γ expression was reduced in CD4+ T cells in the inflamed joints of IL-23p19-deficient mice. Interestingly, IL-23 was also critical for the induction of IL-17A and RORγt but not IFN-γ in TCRγδ+ T cells in the inflamed joints. The importance of the IL-23/IL-17 axis was further confirmed using IL-17 Receptor A knockout mice showing significantly milder AIA compared to control mice, with a disease course comparable to that of IL-23p19-deficient mice.ConclusionsThese data show that IL-23 is critical for full-blown expression of a non-autoimmune destructive arthritis and regulates the proportion of IL-17A and IFN-γ-positive CD4+ T cells at the site of inflammation. Furthermore, IL-23 regulates IL-17A and RORγt expression in TCRγδ T cells in arthritis. These findings indicate that regulating the IL-23 pathway may have therapeutic potential in non-autoimmune arthritis.

Absence of Interleukin-17 Receptor A Signaling Prevents Autoimmune Inflammation of the Joint and Leads to a Th2-like Phenotype in Collagen-Induced Arthritis

Interleukin‐17A (IL‐17A) signals through the IL‐17 receptor (IL‐17R) A/C heterodimer. IL‐17RA serves as a common receptor subunit for several IL‐17 cytokine family members. Lack of IL‐17RA signaling may therefore have additional effects beyond those of lack of IL‐17A alone. The present study was undertaken to determine the role of IL‐17RA signaling in autoimmune arthritis.

IL-23 dependent and independent stages of experimental arthritis: no clinical effect of therapeutic IL-23p19 inhibition in collagen-induced arthritis.

IL-23p19 deficient mice have revealed a critical role of IL-23 in the development of experimental autoimmune diseases, such as collagen-induced arthritis (CIA). Neutralizing IL-23 after onset of CIA in rats has been shown to reduce paw volume, but the effect on synovial inflammation and the immunological autoimmune response is not clear. In this study, we examined the role of IL-23 at different stages of CIA and during T cell memory mediated flare-up arthritis with focus on changes in B cell activity and Th1/Th17 modulation. Anti-IL-23p19 antibody (anti-IL23p19) treatment, starting 15 days after the type II collagen (CII)-immunization but before clinical signs of disease onset, significantly suppressed the severity of CIA. This was accompanied with significantly lower CII-specific IgG1 levels and lower IgG2a levels in the anti-IL-23p19 treated mice compared to the control group. Importantly, neutralizing IL-23 after the first signs of CIA did not ameliorate the disease. This was in contrast to arthritic mice that underwent an arthritis flare-up since a significantly lower disease score was observed in the IL-23p19 treated mice compared to the control group, accompanied by lower synovial IL-17A and IL-22 expression in the knee joints of these mice. These data show IL-23-dependent and IL-23-independent stages during autoimmune CIA. Furthermore, the memory T cell mediated flare-up arthritis is IL-23-mediated. These data suggest that specific neutralization of IL-23p19 after onset of autoimmune arthritis may not be beneficial as a therapeutic therapy for patients with rheumatoid arthritis (RA). However, T cell mediated arthritis relapses in patients with RA might be controlled by anti-IL-23p19 treatment.

Enhanced Expression of Bruton's Tyrosine Kinase in B Cells Drives Systemic Autoimmunity by Disrupting T Cell Homeostasis.

Upon BCR stimulation, naive B cells increase protein levels of the key downstream signaling molecule Bruton’s tyrosine kinase (BTK). Transgenic CD19-hBtk mice with B cell–specific BTK overexpression show spontaneous germinal center formation, anti-nuclear autoantibodies, and systemic autoimmunity resembling lupus and Sjögren syndrome. However, it remains unknown how T cells are engaged in this pathology. In this study, we found that CD19-hBtk B cells were high in IL-6 and IL-10 and disrupted T cell homeostasis in vivo. CD19-hBtk B cells promoted IFN-γ production by T cells and expression of the immune-checkpoint protein ICOS on T cells and induced follicular Th cell differentiation. Crosses with CD40L-deficient mice revealed that increased IL-6 production and autoimmune pathology in CD19-hBtk mice was dependent on B–T cell interaction, whereas IL-10 production and IgM autoantibody formation were CD40L independent. Surprisingly, in Btk-overexpressing mice, naive B cells manifested increased CD86 expression, which was dependent on CD40L, suggesting that T cells interact with B cells in a very early stage of immune pathology. These findings indicate that increased BTK-mediated signaling in B cells involves a positive-feedback loop that establishes T cell–propagated autoimmune pathology, making BTK an attractive therapeutic target in autoimmune disease.