KN-93, an inhibitor of calcium/calmodulin-dependent protein kinase IV, promotes generation and function of Foxp3⁺ regulatory T cells in MRL/lpr mice

Abstract

Objective: Foxp3(+) regulatory T cells (Treg) are pivotal for the maintenance of peripheral tolerance and prevent development of autoimmune diseases. We have reported that calcium/calmodulin-dependent protein kinase IV (CaMK4) deficient MRL/lpr mice display less disease activity by promoting IL-2 production and increasing the activity of Treg cells. To further define the mechanism of CaMK4 on Treg cells in systemic lupus erythematosus (SLE), we used the Foxp3-GFP reporter mice and treated them with KN-93, an inhibitor of CaMK4.

Methods: We generated MRL/lpr Foxp3-GFP mice to record Treg cells; stimulated naïve CD4(+) T cells from MRL/lpr Foxp3-GFP mice under Treg polarizing conditions in the absence or presence of KN-93; evaluated the number of GFP positive cells in lymphoid organs and examined skin and kidney pathology at 16 weeks of age. We also examined the infiltration of cells and recruitment of Treg cells in the kidney.

Results: We show that culture of MRL/lpr Foxp3-GFP T cells in the presence of KN-93 promotes Treg differentiation in a dose-dependent manner. Treatment of MRL/lpr Foxp3-GFP mice with KN-93 results in a significant induction of Treg cells in the spleen, peripheral lymph nodes and peripheral blood and this is accompanied by decreased skin and kidney damage. Notably, KN-93 clearly diminishes the accumulation of inflammatory cells along with reciprocally increased Treg cells in target organ.

Conclusion: Our results indicate that KN-93 treatment enhances the generation of Treg cells in vitro and in vivo highlighting its potential therapeutic use for the treatment of human autoimmune diseases.

Keywords: Calcium/calmodulin-dependent protein kinase IV; KN-93; organ damage; regulatory T cells; systemic lupus erythematosus.

Figure 1

CaMK4 inhibition enhanced the percentage of T_reg cells in the peripheral blood from MRL/lpr Foxp3-GFP mice. T_reg (GFP positive) cells in the peripheral blood were measured by flow cytometry after treatment. KN-93 was initiated from 6 weeks of age (A) or 12 weeks of age (B) (*p < 0.05; mean ± SEM; cumulative results of three independent experiments with 3–5 mice per group).

Figure 2

CaMK4 inhibition increased the percentage of T_reg cells in the lymphoid organs from MRL/lpr Foxp3-GFP mice. (A) Representative flow cytometric staining profile for GPF positive cells in in vivo experiments at 16 weeks of age. KN-93 was initiated from 6 weeks of age. (B) Percentage of GFP positive cells in spleen, thymus, peripheral LN, mesenteric LN and Peyer’s patch (n = 4–6). (C) Immunofluorescence staining of spleen sections for GFP (green) and DAPI (blue) expression. Scale bar = 50 µm. (Color online.)

Figure 3

CaMK4 inhibition enhanced T_reg cells differentiation from naïve CD4 T cells of MRL/lpr Foxp3-GFP mice. Representative flow cytometric staining profile for T_reg cells in in vitro experiments. Naïve T cells from MRL/lpr Foxp3-GFP mice were differentiated in T_reg condition in the absence or presence (4 or 10 µM). A profile representative of six mice per group is shown (*p < 0.05, **p < 0.01; mean ± SEM).

Figure 4

KN-93 treatment ameliorates organ damage in MRL/lpr Foxp3-GFP mice. Deposition of C3 in the skin (A) or glomeruli (B) from MRL/lpr Foxp3-GFP mice at 16 weeks of age treated with PBS or KN-93 was detected by immunofluorescence with anti-C3 staining. Results are representative of 4–6 mice/group. Bar, 100 µm. (C) The graph represents the C3 and the IgG deposition scores in the glomeruli of the indicated mice as detected by immunofluorescence (n = 4–6/group). Data are representative of two independent experiments (*p < 0.05; mean ± SEM). (D) Facial skin lesion of MRL/lpr Foxp3-GFP mice treated with PBS or KN-93 starting at 6 weeks of age. (E) Deposition of IgG in the glomeruli from MRL/lpr Foxp3-GFP mice at 16 weeks of age treated with PBS or KN-93 was detected by immunofluorescence with anti-IgG staining. Results are representative of 4–6 mice/group. Bar, 100 µm.

Figure 5

Loss of CaMK4 prevents recruitment of inflammatory cells into kidneys of MRL/lpr Foxp3-GFP mice. (A) Representative staining profiles of cells isolated from kidneys of MRL/lpr Foxp3-GFP mice treated with PBS or KN-93. Data are representative of 6–8 mice per group. (B) Flow cytometric analysis of cells indicated as in (A), showing number of CD3⁺, CD45⁺, CD3⁺CD4⁻CD8⁻ (DN-T), CD3⁺CD4⁺ and CD3⁺CD8⁺ cells (*p < 0.05; mean ± SEM; n = 6–8). (C) Representative flow cytometric staining profile for GFP positive cells in the kidneys (*p < 0.05; mean ± SEM; n = 6–8). Data are cumulative results of two independent experiments with 3–4 mice per group.