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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Objective

From an immunologic standpoint, the mechanisms by which treatment with tocilizumab (TCZ), a humanized anti–interleukin-6 (anti–IL-6) receptor antibody, results in improvement in rheumatoid arthritis (RA) patients are still not fully understood. In vitro studies and studies in mouse models have demonstrated the critical role of IL-6 in Th17 cell differentiation. Th17 lymphocytes have been shown to be strongly involved in RA pathogenesis, and the purpose of this study was to investigate the effect of IL-6 blockade on the balance between Th17 cells and Treg cells in patients with active RA.

Methods

Patients with active RA for whom TCZ had been prescribed by a rheumatologist were enrolled in this study. Phenotypic analyses of T cell populations were performed, and the Disease Activity Score in 28 joints (DAS28) was assessed. Serum cytokine levels and other parameters of inflammation were measured before the first infusion and after the third infusion of TCZ (8 mg/kg).

Results

Compared to controls, levels of Th17 cells (CD4+IL-17+) were increased and Treg cells (CD4+CD25highFoxP3+) were decreased in the peripheral blood of patients with active RA. The suppressive function of circulating Treg cells was not impaired in patients with active RA. TCZ treatment induced a significant decrease in the DAS28 associated with a significant decrease in the percentage of Th17 cells (from a median of 0.9% to 0.45%; P = 0.009) and an increase in the percentage of Treg cells (from a median of 3.05% to 3.94%; P = 0.0039) in all patients.

Conclusion

This study demonstrates for the first time that inhibition of IL-6 function by TCZ corrects the imbalance between Th17 cells and Treg cells in patients with RA.

The balance between Th17 cells and Treg cells is of major importance in autoimmunity. In general, Th17 cells promote autoimmunity, whereas Treg cells protect against the occurrence of autoimmune diseases (1). In rheumatoid arthritis (RA), Th17 cells have been shown to play a central role by secreting interleukin-17 (IL-17), which activates numerous cell types involved in the pathogenesis of RA, including synovial fibroblasts, monocytes, macrophages, chondrocytes, and osteoblasts (2, 3). In addition, RA is associated with the production of proinflammatory cytokines, such as IL-6, tumor necrosis factor α (TNFα), and IL-1β. Inhibition of IL-6 signaling by blocking the gp130 pathway or by knocking out the IL-6 gene significantly improves autoimmune arthritis in experimental animal models (4, 5). Moreover, tocilizumab (TCZ), a humanized anti–IL-6 receptor antibody, has been shown to be an effective treatment for RA (6, 7). However, why IL-6 blockade improves RA is still unclear since the cytokine may play a dual role. While IL-6 may trigger the hepatic acute-phase response and directly activate different cells such as B and T lymphocytes, macrophages, and osteoclasts, it may also act at an earlier stage in RA pathogenesis.

IL-6 has been shown to be of particular importance in Th17 cell differentiation in mice. The addition of IL-6 to murine CD4+ T cells cultured in the presence of transforming growth factor β (TGFβ) skews their differentiation toward Th17 instead of immunosuppressive FoxP3+ Treg cells (1). In humans, the mechanisms of Th17 cell differentiation are still incompletely characterized. While IL-6 has also been shown to promote Th17 cell polarization in vitro, previous reports have demonstrated that other cytokines, such as IL-1β and IL-23, are involved in this process as well (8). Unlike the mechanisms described in mice (1), TGFβ plays an indirect role in humans by inhibiting Th1 cell generation, and thereby enhancing polarization of Th17 cells from precursor cells defined by a CD161+CD4+ phenotype (9, 10).

In mice, it has been shown that blockade of the IL-6 pathway results in a decrease in the Th17 immune response (5). In patients with RA, TCZ has been demonstrated to induce clinical improvement and to reduce levels of various parameters of inflammation. However, whether the efficacy of TCZ may be related to its ability to negatively affect Th17 cell differentiation in vivo has not been investigated (6, 7). In the current study, we used the ability of TCZ to inhibit IL-6 function in order to decipher the role of IL-6 on the immune balance between Th17 cells and Treg cells in patients with active RA.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Study population.

Fifteen patients (11 women and 4 men) fulfilling the American College of Rheumatology/European League Against Rheumatism 2010 criteria for RA (11), with a median Disease Activity Score in 28 joints (DAS28) (12) of 5.53 (interquartile range [IQR] 4.32–6.31), for whom TCZ was prescribed by a rheumatologist, were enrolled in this study. Informed consent was obtained from all patients. The study was approved by the local ethics committee (2010–018696-21). All patients had seropositive RA, with a median duration of 11 years (IQR 4–13 years). They had all previously received methotrexate (MTX), and 6 of them (40%) were still receiving MTX when TCZ was started. Patients had received a median of 2 courses of therapy with biologic agents (IQR 1–3) before they began treatment with TCZ, including infliximab (n = 5), etanercept (n = 12), adalimumab (n = 7), certolizumab (n = 1), rituximab (n = 5), anakinra (n = 1), and abatacept (n = 3). Because of persistently active disease, 11 patients (73.33%) were also treated with steroids at a median daily dose of 20 mg of prednisone (IQR 10–40). TCZ was prescribed monthly at a dose of 8 mg/kg for all patients. Blood samples were collected just before the first and fourth infusions of TCZ. TCZ treatment was stopped in 2 patients before trial completion because of urticaria and in 1 patient because of diarrhea with fever. Paired data from 6 women and 3 men with a median age of 56 years (IQR 37–63 years), a median disease duration of 10 years (IQR 3.5–12.5 years), and a median baseline DAS28 score of 5.53 (IQR 4.37–6.16) were analyzed before and after TCZ treatment.

The control group consisted of 17 healthy volunteers. The controls had no evidence of an inflammatory syndrome (serum levels of C-reactive protein <0.5 mg/dl), no history of cancer, no recent acute or chronic infectious diseases, and no autoimmune or autoinflammatory diseases, and had not recently been treated with steroids or immunosuppressive drugs.

Cell preparation, culture, and flow cytometry.

Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll gradient centrifugation. CD4+ T cells were then purified by magnetic-activated cell sorting (Miltenyi Biotec) and stimulated with 0.1 μg/ml of phorbol myristate acetate (PMA) and 1 μg/ml of ionomycin (Sigma-Aldrich) for 8 hours; 1 μl/ml of brefeldin A (Golgi Plug; BD Biosciences) was added for the last 4 hours. The cells were stained with phycoerythrin (PE)–conjugated anti–IL-17A and allophycocyanin (APC)–conjugated anti–interferon-y (anti-IFNγ) monoclonal antibodies (eBioscience). CD4+ lymphocytes were isolated before cytokine staining since stimulation with PMA triggers the internalization and degradation of CD4, which disturbs the identification of Th1 (CD4+IFNγ+) and Th17 (CD4+IL-17+) cells (13). Treg cells (CD4+CD25highFoxP3+) were stained with PE–Cy5.5–conjugated anti-CD4, PE-conjugated anti-CD25, and Alexa Fluor 488–conjugated anti-FoxP3 (Human Treg Flow Kit) according to the instructions of the manufacturer (BioLegend Ozyme). Flow cytometric staining was performed and the results analyzed using the cytometry platform at the Institut Fédératif de Recherche 100, using an LSRII flow cytometer (BD Biosciences) and FlowJo software.

Proliferation assays.

CD4+CD25high (Treg) and CD4+CD25− (T effector) cells were isolated from PBMCs using a human Treg cell isolation kit according to the instructions of the manufacturer (Miltenyi Biotec). T effector cells were stained with a cell trace violet cell proliferation kit (Invitrogen) and cultured with or without anti-CD2/CD3/CD28 microbeads (Treg Cell Suppression Inspector; Miltenyi Biotec) and Treg cells, as indicated. After 4 days of culture, cell trace dilution was analyzed by flow cytometry, and the proliferation index was calculated using ModFit LT 3.0 software.

Cytokine assays.

Serum levels of IL-17A, IL-1β, and IL-6 were quantified by enzyme-linked immunosorbent assay (eBioscience).

Statistical analysis.

Wilcoxon's signed rank test was used to compare parameters in individual patients before and after treatment. The Mann-Whitney U test was used to compare patients before treatment with the control group. P values less than 0.05 (2-tailed) were considered significant. Data are presented as the median (IQR). Analyses were performed using GraphPad Prism software.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Increased levels of Th17 cells and decreased levels of Treg cells in patients with active RA.

The percentage of circulating Th17 cells (CD4+IL-17+) was significantly increased in RA patients (median 0.88%; n = 15) compared to controls (0.36%; n = 17) (P = 0.0008) (Figures 1A and B). Analyses confirmed that these cells exhibit a phenotype consistent with that of Th17 lymphocytes (CCR6+CD161+CD45RA−) in both groups (data not shown). In contrast, no difference in the percentage of Th1 cells (CD4+IL-17−IFNγ+) was observed between RA patients (12.30%) and healthy subjects (12.42%) (P = 0.8799) (Figures 1A and 1B). The percentage of Treg cells (CD4+CD25highFoxP3+) was reduced in patients with active RA (3.05%) as compared to controls (4.31%) (P = 0.0396) (Figure 1B). However, the immunosuppressive function of Treg cells (CD4+CD25high) was not different between RA patients and healthy volunteers (Figure 1C). The median ratio of Th17 cells to Treg cells was increased in patients with RA (0.25) as compared to controls (0.09) (P < 0.0001) (Figure 1D). IL-6 was increased in the serum of some but not all patients with active RA, whereas it was not detected in the serum of healthy controls (P = 0.006) (data not shown). The serum levels of IL-17 and IL-1β were very low and not different between RA patients and healthy controls (data not shown).

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Figure 1. Increased circulating Th17 cells and decreased, but functional, Treg cells in patients with rheumatoid arthritis (RA). A and B, Flow cytometric analysis of Th1 (CD4+IL-17−IFNγ+), Th17 (CD4+IL-17+), and Treg (CD4+CD25highFoxp3+) cells in RA patients (n = 15) and healthy subjects (n = 17) was performed (A), and the results were quantified (B). C, Functional analysis of Treg cells (CD4+CD25high) was performed, and the proliferation index was measured using ModFit LT 3.0 software. The percentage of inhibition was calculated using the proliferation index of stimulated T effector cells without Treg cells as the reference. The percentage of inhibition at a 1:2 ratio and a 1:1 ratio of Treg cells to T effector cells was compared in patients with active RA (n = 6), patients with inactive RA after treatment with tocilizumab (n = 3), and healthy controls (n = 6). D, The ratio of Th17 cells to Treg cells in patients with RA (n = 15) and healthy subjects (n = 17) was determined. Data in B are shown as box plots. Each box represents the interquartile range (IQR). Lines inside the boxes represent the median. Whiskers represent the highest and lowest values. Histograms in C and D represent the median and IQR. P values were determined by Mann-Whitney U test (B and D) or Kruskal-Wallis test (C). IL-17 = interleukin 17; IFNγ = interferon-γ; NS = not significant.

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TCZ-induced reduction in the ratio of Th17 cells to Treg cells.

In all 9 patients, RA responded to TCZ treatment, as demonstrated by a significant decrease in the DAS28 (Table 1 and Figure 2B). No significant difference was seen in the evolution of neutrophils or CD4 and CD8 T lymphocytes after TCZ treatment (Table 1). Blockade of IL-6 with TCZ did not significantly modify the levels of Th1 lymphocytes (Figure 2A). Interestingly, a significant decrease in the percentage of Th17 cells was detected after TCZ treatment, from a median of 0.9% to 0.45% of total CD4+ cells (P = 0.009) (Figure 2A). In contrast, TCZ administration resulted in an increase in the percentage of Treg cells, from a median of 3.05% to 3.94% of total CD4+ cells (P = 0.0039) (Figure 2A). Importantly, Treg cells from TCZ-treated patients were still capable of suppressing the proliferation of autologous T effector cells (Figure 1C). Thus, TCZ induced a clinical improvement associated with a correction of the ratio of Th17 cells to Treg cells, from a median of 0.3 to 0.11 (P = 0.009) (Figure 2C). The serum level of IL-6 was not significantly modified by TCZ (from a median of 13.14 pg/ml before treatment to 8.43 pg/ml after treatment; P = 0.6406) (data not shown). Interestingly, we observed that Th17 cells began to decrease and Treg cells to increase before the complete remission of the disease (DAS28 score <2.6) (Figure 2D).

Table 1. Characteristics of the patients with rheumatoid arthritis before and after treatment with TCZ*
 Before treatment, median (IQR)After treatment, median (IQR)P
  • *

    Four infusions of tocilizumab (TCZ; 8 mg/kg) were administered 1 month apart. IQR = interquartile range; DAS28 = Disease Activity Score in 28 joints; ESR = erythrocyte sedimentation rate; VAS = visual analog scale.

Leukocytes, gm/liter10,400 (7,800–11,055)8,000 (6,150–10,380)0.4409
Neutrophils, gm/liter4,990 (4,040–7,677)3,580 (2,372–6,660)0.4258
Lymphocytes, gm/liter2,461 (1,300–3,793)2,930 (2,440–3,352)0.3594
 T cells, gm/liter1,936 (877–2,715)2,250 (2,085–2,780)0.4258
 CD4+ T cells, gm/liter1,477 (577–1,842)1,536 (1,080–1,823)0.25
 CD8+ T cells, gm/liter605 (277–873)576 (501–922)0.6523
 B cells, gm/liter194 (92–375)248 (104–363)0.7344
DAS285.61 (4.37–6.16)2.00 (1.34–2.73)0.0039
 Tender joint count4 (2.5–12)2 (0–3.5)0.0249
 Swollen joint count8 (2–19)1 (0–3.5)0.0156
 ESR, mm/hour28 (13.8–60.8)2 (2–4.5)0.0078
 Patient global assessment, 1–100–mm VAS70 (53–73)20 (20–38)0.009
C-reactive protein, mg/liter13 (4–70.4)2 (0.5–2.5)0.0078
Prednisone, mg/day10 (4.5–50)9 (0–17.5)0.125
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Figure 2. Link between clinical response to tocilizumab (TCZ) and correction of the ratio of Th17 cells to Treg cells in patients with rheumatoid arthritis. A, Percentages of Th1, Th17, and Treg cells in 9 patients before and after TCZ treatment. B, Disease Activity Score in 28 joints (DAS28) before and after treatment. C, Ratio of Th17 cells to Treg cells before and after treatment. D, Evolution of the DAS28 and the percentages of Treg cells and Th17 cells during TCZ treatment in 1 patient. Values are the median and interquartile range. P values were determined by Wilcoxon's signed rank test. NS = not significant.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

This study confirmed the major imbalance between Th17 cells and Treg cells in patients with active RA, as previously reported in the literature (14, 15). Furthermore, we have demonstrated that the Th17/Treg cell imbalance occurring in patients with RA can be corrected by TCZ-mediated blockade of IL-6 signals, which is associated with improved clinical outcome. These results emphasize the benefit of therapeutic approaches based on inhibition of Th17 cells and/or promotion of Treg cells in RA. By blocking the IL-6 pathway, TCZ may decrease IL-6–induced inflammation and/or affect Treg cell and Th17 cell differentiation.

Interestingly, levels of IL-6 were not elevated in the serum of all RA patients. Furthermore, TCZ treatment did not induce a significant decrease in serum IL-6 concentrations, whereas clinical improvement associated with a decrease in the ratio of Th17 cells to Treg cells was observed in all of these patients. These results therefore suggest that reduction of IL-6–induced inflammation may not be the primary mode of action of TCZ. Since TCZ corrects the Th17/Treg cell imbalance in RA patients before complete clinical remission has occurred, the modulation of these 2 antagonistic lymphocyte subsets may represent a likely mechanism of action of TCZ underlying its clinical efficacy. Evidence has been provided that T helper lymphocytes can be redirected to other lineages depending on the cytokine environment (15). In fact, that environment can differentially activate master regulator genes (such as T-bet, GATA-3, and retinoic acid receptor–related orphan nuclear receptor γt), which, in association with proteins such as STAT-3, STAT-4, STAT-5, STAT-6, or suppressor of cytokine signaling 3, change T helper cell polarization (16).

Although additional studies are needed to identify the molecular bases controlling TCZ-mediated Treg cell increase and Th17 cell reduction, the present study demonstrates for the first time that in vivo inhibition of IL-6 function with TCZ can restore a physiologic Th17/Treg cell balance in patients with RA. This study opens a potential new avenue for therapy, or for monitoring of current anticytokine therapies, in RA and other autoimmune diseases in which IL-6, Treg cells, and/or Th17 cells may be involved.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Bonnotte had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Samson, Maillefert, Bonnotte.

Acquisition of data. Samson, Audia, Ciudad, Trad, Fraszczak, Ornetti, Maillefert, Bonnotte.

Analysis and interpretation of data. Samson, Janikashvili, Miossec, Bonnotte.

ROLE OF THE STUDY SPONSOR

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Roche Chugai Laboratories had no role in the study design or in the collection, analysis, or interpretation of the data, the writing of the manuscript, or the decision to submit the manuscript for publication. Publication of this article was not contingent upon approval by Roche Chugai Laboratories.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

We acknowledge Corinne Chevalier for her help with the collection of clinical data and Serge Aho-Glele for conducting the statistical analyses.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES