Polymerized-type I collagen induces upregulation of Foxp3-expressing CD4 regulatory T cells and downregulation of IL-17-producing CD4⁺ T cells (Th17) cells in collagen-induced arthritis

Abstract

Previous studies showed that polymerized-type I collagen (polymerized collagen) exhibits potent immunoregulatory properties. This work evaluated the effect of intramuscular administration of polymerized collagen in early and established collagen-induced arthritis (CIA) in mice and analyzed changes in Th subsets following therapy. Incidence of CIA was of 100% in mice challenged with type II collagen. Clinimorphometric analysis showed a downregulation of inflammation after administration of all treatments (P < 0.05). Histological analysis showed that the CIA-mice group had extensive bone erosion, pannus and severe focal inflammatory infiltrates. In contrast, there was a remarkable reduction in the severity of arthritis in mice under polymerized collagen, methotrexate or methotrexate/polymerized collagen treatment. Polymerized Collagen but not methotrexate induced tissue joint regeneration. Polymerized Collagen and methotrexate/polymerized collagen but not methotrexate alone induces downregulation of CD4(+)/IL17A(+) T cells and upregulation of Tregs and CD4(+)/IFN-γ(+) T cells. Thus, Polymerized Collagen could be an effective therapeutic agent in early and established rheumatoid arthritis by exerting downregulation of autoimmune inflammation.

Figures

Figure 1

Preventive and therapeutic effects of polymerized-type I collagen on mouse CIA. (a) Paw thickness in early arthritis model. One hundred microliters of Polymerized Collagen, 100 μL of Polymerized Collagen, and 100 μL of methotrexate (2.5 mg/kg) or 100 μL of methotrexate (2.5 mg/kg)/Polymerized Collagen were administered intramuscular once a week during 6 weeks at the same time of that of the booster. Citric/citrate buffer was injected as a vehicle control. Data represent mean ± SEM (each group, n = 6). (b) Clinical arthritis score in early arthritis model (n = 6). (c) Paw thickness in established arthritis model. One hundred microliters of Polymerized Collagen, 100 μL of Polymerized Collagen, and 100 μL of methotrexate (2.5 mg/kg) or 100 μL of methotrexate (2.5 mg/kg)/Polymerized Collagen were administered intramuscularly once a week during 6 weeks. Treatments were started on 14 days after the booster. Citric/citrate buffer was injected as a vehicle control. Data represent mean ± SEM (each group, n = 6). (d) Clinical arthritis score in established arthritis model (n = 6). *P < 0.05.

Figure 2

Preventive effect of polymerized-type I collagen on the histological damage in CIA mice. (a) Representative section of joint histopathology is shown. Upper panel: hematoxylin and eosin stained and lower panel: PAS stained; magnification: 10x. Arrow heads point out nodules. (b) Pathology scores of each group were calculated and expressed as mean ± SEM (n = 6). *P < 0.05.

Figure 3

Representative flow plots of IL-17A- and Foxp3-expressing CD4+ T cells.

Figure 4

Effect of polymerized-type I collagen on the ex vivo intracellular cytokine production and on CD4+ T cell subsets regulation in splenocytes. (a) Spleen cells obtained immediately ex vivo in early arthritis model on day 35 after booster immunization. (b) Splenocytes obtained immediately ex vivo in early arthritis model on day 98 after booster immunization. (c) Spleen cells obtained immediately ex vivo in established arthritis model during first sacrifice. (d) Splenocytes obtained from established arthritis model during second sacrifice. Intracellular production of IL-17A, IL-4, IFN-γ, and Foxp3 by CD4+ T cells was detected by flow cytometry. Results are representative of 6 mice analyzed in each group. Horizontal dotted line represents mean normal values, obtained from mice (n = 3) without CIA. Data represent mean ± SEM. *P < 0.05.

Figure 5

Effect of polymerized-type I collagen on the ex vivo NF-κB and IκB-α in splenocytes. (a) Spleen cells obtained immediately ex vivo in early arthritis model on day 35 after booster immunization. (b) Splenocytes obtained immediately ex vivo in early arthritis model on day 98 after booster immunization. (c) Spleen cells obtained immediately ex vivo in established arthritis model during first sacrifice. (d) Splenocytes obtained from established arthritis model during second sacrifice. Intracellular levels of NF-κBp65 and IκBα cells were detected by flow cytometry. Results are representative of 6 mice analyzed in each group. Horizontal dotted line represent mean normal values, obtained from mice (n = 3) without CIA. Data represent mean ± SEM. *P < 0.05.