Saireito (114) Increases IC50 and Changes T-Cell Phenotype When Used in Combination with Prednisolone Therapy in Human Peripheral Blood Mononuclear Cells

Abstract

Prednisolone (PSL), a type of corticosteroid used to treat autoimmune diseases, can increase the risk of infection and osteoporosis. Saireito (114), a Kampo medicine, has an immunosuppressive effect; with its use, the dose of steroids can be reduced. However, its mechanism when used with PSL is still unclear. We used peripheral blood mononuclear cells (PBMCs) from healthy adults to examine the effect of 114 and PSL treatment on PBMC proliferation, T-cell subsets, and cytokine production. PBMCs were cotreated with concanavalin A and 300 μM 114 (either Tsumura & Co. (TJ) or Kracie Holdings (KR)) and 0.0001-1.0 μM PSL for 96 h to create the T-cell mitogen. We then measured the PBMC proliferation; ratio of CD4⁺ T cells, CD8⁺ T cells, and T-follicular helper (Tfh) cells; and concentration of cytokines (TNF, IFN-γ, IL-6, IL-10, IL-17A, and IL-21). The proliferation of PBMCs was dose dependently suppressed in both the PSL and PSL + 114 groups (p < 0.05). Combination therapy increased the IC₅₀ in the PSL group (0.0947 μM) by 2.02 and 1.64-fold in the PSL + TJ114 and PSL + KR114 groups, respectively. Both the PSL + 114 groups had an increased ratio of CD4⁺ T cells compared to the PSL group, with no effect on the ratio of CD8⁺ T and Tfh cells. Furthermore, the PSL + 114 groups showed increased IL-6 and IL-10 compared to the PSL monotherapy group, although the difference was not significant. There was no significant difference in the TNF, IFN-γ, IL-17A, and IL-21 concentrations between the PSL and PSL + 114 groups. The elevated IC₅₀ with 114 cotreatment suggests diminished immunosuppressive action. Moreover, increased cytokine production by Th2 with 114 cotreatment suggests a restoration of T-cell balance in Th1-mediated autoimmune diseases. However, increased IL-6 suggests potential exacerbation of IL-6-mediated diseases, such as rheumatoid arthritis. Therefore, it is necessary to monitor these clinical parameters when using 114 in combination with PSL.

Figure 1

Inhibition of PBMC proliferation with PSL and 114 treatments. Proliferation of PBMCs stimulated by ConA after addition of PSL and 114 cultures. Each treatment was compared to its respective blank using Dunnett's multiple comparison test (^∗p < 0.05; ^∗∗p < 0.01). The PSL monotherapy group and the PSL + 114 combination therapy groups were compared using the Tukey-Kramer multiple comparison test (mean ± SD, n = 6). ConA: concanavalin A, PBMCs: peripheral blood mononuclear cells, DMSO: dimethyl sulfoxide, PSL: prednisolone, and 114: saireito.

Figure 2

Typical dose curves of PSL, 114, and PSL + 114 for the proliferation of ConA-activated PBMCs from healthy subjects (n = 6). ConA: concanavalin A, PBMCs: peripheral blood mononuclear cells, PSL: prednisolone, and 114: saireito.

Figure 3

Dot plot and cell positive rate of CD4⁺ T cells in the PBMCs after PSL and 114 treatments. (a) Dot plot of CD4⁺ T cells in the PBMCs stimulated by ConA after PSL and 114 treatments (indicated in the square). Vertical axis: PerCP-Cy5-5A; horizontal axis: FSC-A. The inside of the square frame was CD4⁺ T cells, and the ratio was calculated by the number of CD4 stained cells / lymphocyte count ∗ 100. (b) Ratio of CD4⁺ T cells in PBMCs after PSL and 114 treatments. Each treatment was compared to its respective blank using Dunnett's multiple comparisons test (^∗p < 0.05; ^∗∗p < 0.01). The PSL monotherapy group and the PSL + 114 combination therapy groups were compared using the Tukey–Kramer multiple comparison test (mean + SD, n = 6). ConA: concanavalin A, PBMCs: peripheral blood mononuclear cells, PSL: prednisolone, and 114: saireito.

Figure 4

Dot plot and cell positive rate of CD8⁺ T cells in the PBMCs after PSL and 114 treatments. (a) Dot plot upon gating for CD8⁺ T cells in the PBMCs stimulated by ConA after PSL and 114 treatments (indicated in the square). Gating was conducted using APC-Cy7-A on the vertical axis and FSC-A on the horizontal. The inside of the square frame was CD8⁺ T cells, and the ratio was calculated by the number of CD8-stained cells / lymphocyte count ∗ 100. (b) Ratio of CD8⁺ T cells in PBMCs after PSL and 114 treatments. Each treatment was compared to its respective blank using Dunnett's multiple comparisons test (^∗p < 0.05; ^∗∗p < 0.01), and the PSL monotherapy group and the PSL + 114 combination therapy groups were compared using the Tukey–Kramer multiple comparison test (mean + SD, n = 6). ConA: concanavalin A, PBMCs: peripheral blood mononuclear cells, PSL: prednisolone, and 114: saireito.

Figure 5

Dot plot and cell positive rate of Tfh cells in the PBMCs after PSL and 114 treatments. (a) Dot plot upon gating CXCR5⁺PD-1⁺ cells (Tfh cells) in the subcellular fractions of CD4⁺ T cells of PBMCs stimulated by ConA after PSL and 114 treatments. After gating in a PerCP-Cy5-5A-FSC-A dot plot, FITC-A in the subcellular fractions was gated on the vertical axis, and Alexa Fluor 647-A was gated on the horizontal axis. The first quadrant of the dot plot was Tfh cells, and the ratio was calculated by the number of CXCR5 and PD-1-stained cells / the number of CD4-stained cells ∗ 100. (b) Ratio of Tfh cells in PBMCs stimulated by ConA after PSL and 114 treatments. Each treatment was compared to its respective blank using Dunnett's multiple comparisons test (^∗p < 0.05; ^∗∗p < 0.01). The PSL monotherapy group and the PSL + 114 combination therapy groups were compared using the Tukey–Kramer multiple comparison test (mean + SD, n = 6). ConA: concanavalin A, PBMCs: peripheral blood mononuclear cells, PSL: prednisolone, and 114: saireito.

Figure 6

Concentration of cytokines in the PBMCs after PSL and 114 treatments. ConA, PSL, and 114 were added to PBMCs, and then the solution was incubated for 96 h. For flow cytometry, beads were used to analyze the concentration of TNF, IFN-γ, IL-6, IL-10, IL-17A, and IL-21 in the culture supernatant. Each treatment was compared to its respective blank using Dunnett's multiple comparisons test (^∗p < 0.05; ^∗∗p < 0.01). The PSL monotherapy group and the PSL + 114 combination therapy groups were compared using the Tukey–Kramer multiple comparison test (median + SD, n = 6). ConA: concanavalin A, PBMCs: peripheral blood mononuclear cells, PSL: prednisolone, and 114: saireito.