J Rheum Dis.  2013 Apr;20(2):94-102. 10.4078/jrd.2013.20.2.94.

Reciprocal Regulation of TH17 and Regulatory T Cells by Methotrexate and Its Therapeutic Effects in Collagen-induced Arthritis (CIA)

• Park EM
• Park MK
• Lee DG
• Baek SY
• Woo JW
• Kwok SK
• Cho ML
• Park SH

• KMID: 2223033
• DOI: http://doi.org/10.4078/jrd.2013.20.2.94

Abstract

OBJECTIVE
Methotrexate is the first-line drug in treatment of rheumatoid arthritis (RA) exhibiting higher efficacy and better tolerability than most other DMARDs. To have a better understanding of the anti-arthritic mechanism of methotrexate, we investigated the effect of methotrexate on suppressing the autoimmune inflammatory and destructive arthritis in collagen-induced arthritis (CIA) mice.
METHODS
The effects of methotrexate on joint inflammation were assessed by clinical scoring and histologic analysis. Levels of cytokines and autoreactive antibodies were analyzed by immunohistochemistry and ELISA. The population of TH17 and Foxp3+ regulatory T (Treg) cells and phosphorylation of their critical transcription activators, STAT3 and STAT5, were examined by fluorescence microscopy and flow cytometry, respectively.
RESULTS
Treatment with methotrexate significantly alleviated joint inflammation and cartilage destruction in CIA. Serum levels of total immunoglobulins G, G1, G2a specific to type II collagen were also reduced considerably in methotrexate-treated mice. The drug inhibited the expression of proinflammatory cytokines such as IL-1beta, TNF-alpha, IL-6 and IL-17 in arthritic joints ex vivo as well as by splenocytes in vitro. Moreover, methotrexate treatment resulted in reciprocal modulation of TH17 cells and Foxp3+ regulatory T (Treg) cells in spleen tissues, in which TH17 cells were decreased and Treg cells in number were increased. Subsequent analysis of CD4+T cells showed that phosphorylation of STAT3 was decreased whereas phosphorylation of STAT5 was increased in methotrexate-treated mice.
CONCLUSION
Methotrexate treatment effectively suppressed autoimmune arthritis and restored homeostasis of the immune system by reciprocal regulation of TH17 and Treg cells in a mouse model of collagen-induced arthritis.

Keyword

Methotrexate; Collagen-induced arthritis; Cytokines; TH17 cells; Regulatory T cells

MeSH Terms

Animals
Antibodies
Antirheumatic Agents
Arthritis
Arthritis, Experimental
Arthritis, Rheumatoid
Cartilage
Collagen Type II
Cytokines
Enzyme-Linked Immunosorbent Assay
Flow Cytometry
Homeostasis
Immune System
Immunoglobulins
Immunohistochemistry
Inflammation
Interleukin-17
Interleukin-6
Joints
Methotrexate
Mice
Microscopy, Fluorescence
Phosphorylation
Spleen
T-Lymphocytes, Regulatory
Th17 Cells
Tumor Necrosis Factor-alpha
Antibodies
Antirheumatic Agents
Collagen Type II
Cytokines
Immunoglobulins
Interleukin-17
Interleukin-6
Methotrexate
Tumor Necrosis Factor-alpha

Figure

• 

  Figure 1. Suppression of arthritis development in MTX-treated CIA mice. Arthritis was induced by immunization with CII in Freund's complete adjuvant on day 0. On day 7, mice also received PBS or MTX (1 mg/kg or 7.5 mg/kg) three times per week for 7 weeks. (A) Severity of arthritis was determined as described in Materials and Methods. (B) Representative histological analysis of knee joints and paws was assessed by H & E, toluidine blue, and safranin o staining. Original magnification: ×40 and ×200 for H&E staining and ×200 for toluidine blue and safranin o staining. Values of histological scores of inflammation and cartilage damage were shown in the right panel. (C) Anti-collagen antibody levels in CIA mice. The levels of IgG anti-collagen antibodies were measured by ELISA. Values are expressed as the optical density (O.D.) ∗p<0.01, † p<0.001 compared to the vehicle control mice.

• 

  Figure 2. Immunohistologies of joints tissues from MTX treated CIA mice. Tissue sections from mice joints of each group were stained with anti-TNF-α, anti-IL-1β, anti-IL-6, anti-IL-17 antibodies or isotype antibodies, respectively. Cells stained with each antibody are shown in brown. Original magnification: ×400.

• 

  Figure 3. Reciprocal regulation of TH17 and Foxp3+ regulatory T cells in MTX-treated CIA mice. Spleen from vehicle and MTX-treated CIA mice were stained by anti-CD4 (green), anti-CD25 (blue), and anti-IL-17 (red, upper), anti-Foxp3 (red, lower) antibodies. Populations of CD4+ CD25+ Foxp3+ T cells and CD4+ IL-17+ T cells were analyzed using laser confocal mi-croscopy. Original magnification: ×400. The graphs represent the number of positive cells. ∗p<0.05 compared to the vehicle control mice.

• 

  Figure 4. STATs-dependent regulation of TH17 and Foxp3+ regulatory T cells in MTX-treated CIA mice. Spleens from mice in each group were stained with antibodies against anti-CD4 (green, upper; white, lower), anti-Foxp3 (red), phos-pho-STAT3 (red) or phospho-STAT5 (blue). Original magnification: ×400. The graphs represent the number of positive cells. Data are expressed as the mean± SD. ∗p<0.05 compared to the vehicle control mice.

• 

  Figure 5. Suppressive effect of MTX on production of inflammatory cytokines. Splenocytes of DBA/1J mice were cultured with LPS (100 ng/mL) (A) or anti-CD3 (1 μ g/mL)/CD28 (2 μ g/mL) (B) in the presence or absence of MTX (0.5 μ g/mL) for 24 hours in vitro. TNF-α, IL-6 and IL-17 in the cell supernatant was determined by ELISA. Data are expressed as the mean± SD. ∗p<0.01, † p <0.001 compared to the LPS or CD3/CD28-treated cells.

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