Eosinophils from Murine Lamina Propria Induce Differentiation of Naïve T Cells into Regulatory T Cells via TGF-β1 and Retinoic Acid

Abstract

Treg cells play a crucial role in immune tolerance, but mechanisms that induce Treg cells are poorly understood. We here have described eosinophils in lamina propria (LP) that displayed high aldehyde dehydrogenase (ALDH) activity, a rate-limiting step during all-trans retinoic acid (ATRA) synthesis, and expressed TGF-β1 mRNA and high levels of ATRA. Co-incubation assay confirmed that LP eosinophils induced the differentiation of naïve T cells into Treg cells. Differentiation promoted by LP eosinophils were inhibited by blocked either TGF-β1 or ATRA. Peripheral blood (PB) eosinophils did not produce ATRA and could not induce Treg differentiation. These data identifies LP eosinophils as effective inducers of Treg cell differentiation through a mechanism dependent on TGF-β1 and ATRA.

Fig 1. High ALDH expression on CD11c^int CD11b^hi LP eosinophils.

(A) Cells from LP were stained with anti-mouse CD11c, CD11b and Siglec-F Abs, and then CD11c⁺ CD11b⁻ (P1), CD11c⁺ CD11b⁺ (P2), CD11c⁻ CD11b⁺ side scatter (SSC)^lo Siglec-F⁻ (P3) and CD11c⁻ CD11b⁺ SSC^hi Siglec-F⁺ cells (P4) were gated by flow cytometry. (B) Cells from LP were stained with a panel of Abs and the indicated surface molecules of four populations were analyzed by flow cytometry. Filled histograms are the isotype control.(C) Four subsets were sorted and ALDH activity of four subsets were determined by Aldefluor assay. (D) Statistical summary of ALDH activity of four subsets. Data from experiments such as that shown in C. Bars show the means ± SD of three independent experiments. (E) CD11b⁺ cells were isolated from the LP and stained with anti-mouse Siglec-F and CCR3 Abs, the percentage of Siglec-F⁺CCR3⁺ eosinophils were analyzed by flow cytometry.(F) May-Grunwald-Giemsa staining of sorted CD11b⁺Siglec-F⁺CCR3⁺ cells from LP. Scale bar, 20 μm.

Fig 2. LP eosinophils produce ATRA and TGF-β1.

(A) Structures of ATRA and acitretin. (B) LP eosinophils or CD11c⁺MHC-II⁺CD103⁺ LP DC were sorted and extracted. ATRA in LP eosinophils or CD11c⁺MHC-II⁺CD103⁺ LP DC were quantified by LC/MS/MS. The acitretin was used as the internal standard for ATRA quantification. (C) ATRA produced by LP eosinophils and CD11c⁺MHC-II⁺CD103⁺ LP DC. Bars show the means ± SD of three independent experiments. (D) RNA was isolated from purified LP eosinophils or CD11c⁺MHC-II⁺CD103⁺ LP DC, and expression of tgfb1 were analyzed by quantitative real-time PCR. Values are expressed relative to Gapdh. Bars show the means ± SD of three independent experiments.

Fig 3. Induction of Foxp3⁺ Treg cells by LP eosinophils but not by PB eosinophils.

(A) CD11b⁺ cells were isolated from the PB and stained with anti-mouse Siglec-F and CCR3 Abs, the percentage of Siglec-F⁺CCR3⁺ eosinophils were analyzed by flow cytometry. (B) Cells from BP were stained with a panel of Abs and the indicated surface molecules of the cells were analyzed by flow cytometry. Filled histograms are the isotype control. (C) MACS-sorted CD4⁺CD62L⁺ T cell from the OT-II mouse were cultured under activating coditions with LP or PB eosinophils, and Foxp3 expression by CD4⁺ T cells was analyzed by flow cytometry after 4 d of culture. (D) CFSE-labeled CD4⁺ T cells were cultured with sorted LP eosinophil-induced Treg at the indicated ratios (Teff:Treg×10⁵) under activating conditions for 3 d. The proliferation of CD4⁺ T cells was analyzed by flow cytometry.

Fig 4. TGF-β1 enhances the conversion of Treg cells in the presence of Lp eosinophils.

(A) MACS-sorted CD4⁺CD62L⁺ T cell from the OT-II mouse were cultured under activating coditions with LP or PB eosinophils as described in Fig 3 with IgG1 isotype control antibody (isotype) or TGF-β neutralizing antibody (α-TGF-β). The cells were stain for Foxp3 and CD4 and analyzed by flow cytometry. (B) RNA was isolated from purified LP or PB eosinophils, and expression of tgfb1 were analyzed by quantitative real-time PCR. Values are expressed relative to Gapdh. Bars show the means ± SD of three independent experiments. (C) MACS-sorted CD4⁺CD62L⁺ T cell from the OT-II mouse were cultured under activating coditions with LP or PB eosinophils as described in Fig 3 with the indicated concentrations of TGF-β1. The cells were stain for Foxp3 and CD4 and analyzed by flow cytometry.

Fig 5. ATRA produced by Lp eosinophils is responsible for conversion of Treg cells.

(A) BP eosinophils were sorted and extracted. ATRA in BP eosinophils were quantified by LC/MS/MS. The acitretin was used as the internal standard for ATRA quantification. (B) MACS-sorted CD4⁺CD62L⁺ T cell from the OT-II mouse were cultured under activating coditions with LP or PB eosinophils as described in Fig 3 in the absence (–) or presence of 1ng/ml TGF-β1, or the RA receptor antagonist LE135 and LE540 (RAi). CD4⁺Foxp3⁺ Treg cells differentiation was analyzed by intracellular staining and flow cytometry.