Background Interleukin-2 (IL-2) induces regulatory T cells (Tregs) and reduces disease activity such as graft-versus-host disease and systemic lupus erythematosus. IL-2/anti-IL-2 monoclonal antibody immune complex (IL-2IC) increases the half-life of IL-2 in vivo and strongly induces Tregs. We previously demonstrated that administration of IL-2IC suppressed collagen-induced arthritis (CIA) in mice (EULAR 2015).
Objectives To clarify complex regulatory network of IL-2IC in autoimmune arthritis we examined the effects of IL-2IC on Th1, Th17, Treg inductions and microRNAs (miRNAs) expressions which regulate T cell inductions.
Methods Male DBA/1 mice were immunized by injection of 200μg of Type II collagen emulsified with an equal volume of Complete Freud Adjuvant intradermally at the base of the tail of mice (first immunization). Second immunization was given 21 days after first immunization. IL-2ICs were prepared by mixing 5μg of anti-IL-2 antibody (clone JES6–1D) with 1μg of mouse IL-2 for 15 minutes. The mice were injected with either PBS as a control or IL-2IC (5μg /mouse) intraperitoneally for 3 days. Mouse paws were scored for arthritis using a macroscopic scoring system ranging from 0 to 4 (0, no swelling or redness; 1, swelling/redness of paw or one joint; 2, two joints involved; 3, more than two joints involved; and 4, severe arthritis of the entire paw and joints). The arthritic score for each mouse is the sum of the scores of all four paws. Peripheral blood cells were stained with anti-CD25 (PC61), anti–CD4 (RM4–5), anti-Foxp3 (FJK-16s) and CD4+CD25+Foxp3+ Tregs were analyzed by flow cytometry. Th1 and Th17 cells infiltrating in the synovium were examine by immunohistochemistry stained with anti-IFN-g and IL-17 mAb. MiRNAs expressions in the serum from CIA mice were analyzed using panel real-time PCR analysis (miRCURY LNATM microRNA PCR). Statistical analysis was performed by paired t test.
Results To define the effect of IL-2IC on established CIA, IL-2IC was administered for 3 days from day 21 to day 23 after the first immunization (day 0 to day 2 after the second immunization) of CIA. To define the effect of IL-2IC on early stages of disease induction we administered IL-2IC from day 0 to day 2 after first immunization. We observed a significant decrease in both the incidence and severity of arthritis in these CIA mice. Injection of IL-2IC effectively elicited more than 2-fold expansion of CD4+CD25+Foxp3+ Tregs in peripheral blood cells than control mice. Th1 and Th17 cells infiltrations in the synovium were significantly inhibited by IL-2IC treatment. We identified 24 differentially expressed miRNAs (fold change >2.0) and 72 differentially expressed miRNAs (fold change <0.5) comparing untreated mice. Of note, we observed miR-17–5p and miR-19b-3p which inhibits iTreg induction was significantly augmented in CIA mice but decreased in CIA mice treated with IL-2IC. In addition, miR-326–3p which induces Th17 was significantly augmented in CIA mice but decreased in CIA mice treated with IL-2IC.
Conclusions These observations indicate that IL-2IC might play a complex regulatory role in autoimmune arthritis by regulating Th1, Th17, Tregs and miRNAs which regulate these T cell inductions.
Disclosure of Interest None declared
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