Ulmus davidiana var. japonica Nakai upregulates eosinophils and suppresses Th1 and Th17 cells in the small intestine

Abstract

The bark of Ulmus davidiana var. japonica Nakai (Ulmaceae) has been used in traditional Korean medicine for chronic inflammation in the gastrointestinal tract. Here we investigated the frequency and cytokine profile of the major immune cells in the small intestinal lamina propria (SI LP), spleen, and mesenteric lymph nodes (MLNs) of mice treated orally with Ulmus davidiana var. japonica Nakai bark water extract (UDE) to address the immunomodulatory role of this herb in intestinal homeostasis. B6 mice were given 5g/kg UDE once daily for 14 days. They were then sacrificed, and cells were isolated from the spleen, MLNs, and SI LP. The proportion of B versus T lymphocytes, CD4(+) versus CD8(+) T lymphocytes, Th1 and Th17 cells, and Foxp3(+) regulatory T cells in the spleen, MLNs, and SI LP were analyzed. The frequency of antigen-presenting cells (APCs), including dendritic cells, macrophages, and eosinophils in the SI LP and the expression of costimulatory molecules on APCs were also evaluated. The numbers and frequencies of Th1 and Th17 cells in the SI LP were significantly reduced in the UDE-treated mice compared with PBS controls. In addition, the proportion of IL-4-producing eosinophils in the SI LP was significantly elevated in the UDE-treated mice compared with controls. Taken together, these data indicate that UDE up-regulates the number and frequency of SI LP eosinophils, which can down-regulate the Th1 and Th17 responses via IL-4 secretion and contribute to intestinal homeostasis.

Figure 1. Decreased number of CD4⁺ T cells in the SI LP of UDE-administered mice.

(A) Cells isolated from spleen (SP), mesenteric lymph node (MLN), and small intestinal lamina propria (SI LP) were stained with TCRβ and CD19 and analyzed by flow cytometry. (B) Frequency of T and B cells in the SP, MLN, and SI LP of UDE- versus PBS-administered control mice. (C) T Cells from SP, MLN, and SI LP were stained with CD4 and CD8α and analyzed by flow cytometry. (D) Frequency of CD4 and CD8 cells in the SP, MLN, and SI LP of UDE- versus PBS-administered control mice. (E) Total number of T/B cells and CD4/CD8 cells in the SP, MLN, and SI LP of UDE- versus PBS administered control mice. *p < 0.05, **p < 0.01, NS=not significant.

Figure 2. Decreased number and frequency of Th1 and Th17 cells in the SI LP of UDE-administered mice.

(A) Intracellular cytokine staining for IL-17 and IFN-γ in CD4⁺ T cells in SP, MLN, SI LP from UDE- versus PBS-administered control mice. Cells were stimulated with PMA and Ionomycin for 4 hours and analyzed by flow cytometry. (B) Frequency and number of Th1 cells in the SP, MLN, and SI LP of UDE- versus PBS-administered control mice. (C) Frequency and number of Th17 cells in the SP, MLN, and SI LP of UDE- versus PBS-administered control mice. *p < 0.05, **p < 0.01, ***p < 0.001, NS= not significant.

Figure 3. No significant differences of Th2 cells in the SI LP and serum total IgE, UDE-specific IgE and IgG1 levels between UDE- and PBS-administered mice.

(A) Frequency and number of IL-4/GFP⁺ CD4⁺ T cells in the SI LP of UDE-versus PBS-administered control 4get mice. (B) Serum total IgE, UDE-specific IgE, and IgG1 secretion analyzed by ELISA. NS= not significant, OD₄₅₀=optical density at absorbance 450nm.

Figure 4. No significant increase of CD4⁺ Foxp3⁺ regulatory T cells and IL-10-producing CD4⁺ T cells in the SI LP of UDE-administered mice.

(A) Intracellular staining of Foxp3 in CD4⁺ T cells from UDE- versus PBS-administered control mice. (B) Frequency and number of CD4⁺ Foxp3⁺ regulatory T cells in the SP, MLN, and SI LP of UDE-versus PBS-administered control mice. (C) Intracellular cytokine staining for IL-10 in CD4⁺ T cells from UDE- and PBS-administered control mice. (D) Frequency and number of IL-10-producing T helper cells in the SP, MLN, and SI LP of UDE- versus PBS-administered control mice. *p < 0.05, **p < 0.01, NS=not significant.

Figure 5. Significant increase of IL-4-expressing eosinophils in the SI LP of UDE-administered mice.

(A) Cells isolated from SI LP were stained with CD11b, CD11c, and MHC class II and analyzed by flow cytometry. MHC class II ^lowCD11b^high CD11c^int cells (R1) are eosinophils. Among MHC class II^high cells, CD11b ^intCD11c^int cells (R4) and CD11c^high cells (R2 and R3) correspond to macrophages and dendritic cells, respectively. (B) Proportion and number of eosinophils, macrophages, and dendritic cells in the SP of UDE- versus PBS-administered control mice. (C) Antigen presenting cells isolated from SI LP were stained with CD40, CD80, and CD86 and analyzed by flow cytometry. Empty, solid line histograms represent cells from UDE-administered (red) versus PBS-administered control mice (black). Shaded histograms represent cells stained with isotype control of UDE-administered (dark gray) versus PBS-administered control mice (gray). (D) Mean fluorescence intensity of IL-4/GFP expressed by APCs from the SI LP of 4get mice were analyzed by flow cytometry. (E) Analysis of CD11b^highCD11c^int cells from LP of WT and ΔdblGATA mice for eosinophil markers CCR3 and Siglec F. *p<0.05, **p < 0.01, NS= not significant, LP: lamina propria, EO: eosionophil, DC: dendritic cell, Mϕ: macrophage, MFI: mean fluorescence intensity.