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

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.


Introduction
The immune system needs to carefully balance both immune responses and immune tolerance [1]. Immune responses eliminate harmful antigens such as pathogens and dead or aberrant host cells [1,2]. At the same time, immune tolerance is needed in order to avoid damaging normal host tissues and to allow the presence of harmless antigens such as commensal bacteria and food antigens in the intestinal tract [3].
Regulatory T (Treg) cells play a crucial role in generation and maintenance of immune tolerance [4]. It has been shown that transforming growth factor-beta (TGF-β) stimulates naïve CD4 + CD25 − T cells to differentiate into either CD4 + CD25 + Foxp3 + Treg cells or Th17 cells [5,6], while all-trans retinoic acid (ATRA) from intestinal dendritic cells (DC) induces the differentiation of naïve T cells into Treg cells in presence of TGF-β1 but suppress the differentiation of Th1, Th2 and Th17 cells [7][8][9]. The activation of aldehyde dehydrogenase (ALDH), a unique rate-limiting enzyme during ATRA synthesis, has been considered as the signal for cells to produce ATRA [10]. The mucosal immune system rather than systemic immune system acts as the main sensor and effector in responses to exogenous antigens [11]. Gut-associated lymphoid tissue (GALT), the largest lymphoid organ in the mucosal immune system, is comprised of Peyer's patches, interdigitating lymphocytes, plasma cells and lymphocytes in the LP, and mesenteric lymph nodes, in which LP is the loci for the highest frequency of Treg cells expansion [12]. In LP, CD103 + DC and CD11b + F4/80 + CD11c − macrophages can induce the generation of Treg cells, while CD11c + CD11b + CD103 − DC induce the differentiation of Th17 cells [13,14]. However, except for DC and macrophages, no other immune cells have been reported to induce the differentiation of naïve CD4 + CD25 − T cells.
While investigating the function of DC and macrophages from murine LP, we identified eosinophils that displayed a high activity of ALDH as well as producing high levels of ATRA and expressing TGF-β1 mRNA. In the present study, we provided evidence that this subset of eosinophils (LP eosinophils) represents a novel inducer of the differentiation of naïve T cells into Treg cells.

Materials and Methods Mice
Female wild-type or OT-II transgenic C57BL/6 mice of 6-10 weeks of age were kindly provided by Joint Venture SIPPR-BK Experimental Animal Company (Shanghai, China) or Dr. Jian-Li Wang (Department of Immunology, Zhejiang University School of Medicine, China).
All mice were maintained in a specific pathogen-free animal facility with a standardized light (12 h light/dark cycle), temperature (22±1°C) and humidity (55±15%). Animals were fed food and water freely. Cages were changed weekly. At this study, mice were sacrificed by cervical dislocation. All of animal experimental protocols were approved by the Ethics Committee for Animal Experiment of Zhejiang University.

Detection of ALDH activity of LP cells
The aldehyde dehydrogenase (ALDH) activity of the LP cells was detected using an ALDE-FLUOR staining kit (StemCell Technologies, Canada). Briefly, the LP cells in ALDEFLUOR buffer containing ALDH substrate were incubated at 37°C for 45 min in the dark. After washing with the buffer and centrifugation, the cell pellet was suspended in ice-cold ALDEFLUOR buffer, add and incubate with biotin-conjugated monoclonal anti-CD11c, anti-CD11b and anti-Siglec-F, and then detected by flow cytometry [15]. The ALDH activity was measured by the difference values of mean fluorescence intensity (MFI) of the LP cells before or after treatment with 300 μM ALDH inhibitor DEAB (StemCell Technologies).

Morphological examination of LP eosinophils
May-Grunwald-Giemsa dye was used to stain LP eosinophils and observed under a light microscope as previously described [16].

Determination of ATRA produced by LP and PB eosinophils
A LC(liquid chromatography)/MS/MS-based method was established for quantitative detection of ATRA in cell cultures referring to a previous report [17]. Briefly, 10 5 CD11b + Siglec-F + CCR3 + LP or PB eosinophils or CD11c + MHC-II + CD103 + LP DC was mixed with 1 ml 0.025 M KOH-ethanol, and then added internal standard. The mixture was added with 10 ml hexane for vortex, followed by a short centrifugation for stratification. The lower aqueous phase was collected to mix with 60 μl of 4 M HCl and then added with hexane as above. After centrifugation, the hexane phase was collected to remove the solvent with a gentle nitrogen stream. The residue was dissolved in 100 μl acetonitrile for measurement of ATRA using a triple-quadrupole mass spectrometer (Agilent Technologies, USA). In the detection, acitretin (Sigma), an analogue of ATRA, was used as the internal reference [18].

Real-time PCR
Total RNA was extracted from purified LP eosinophils, CD11c + MHC-II + CD103 + LP DC or PB eosinophils using RNeasy Mini Kit (Qiagen, Germany). cDNA was generated using the Omniscript RT kit (if starting material was >50 ng) or Sensiscript RT kit (if starting material was <50 ng) (Qiagen). cDNA was used as a template for quantitative real-time PCR using SYBR Green Master Mix (Applied Biosystems, USA), and the following gene-specific primers: tgfb1 primers ACCATGCCAACTTCTGTCTG, CGGGTTGTGTTGGTTGTAGA; and Gapdh primers TGGCAAAGTGGAGATTGTTGCC, AAGATGGTGATGGGCTTCCCG. PCR was performed using an ABI 7500 Real-time PCR System (Applied Biosystems). Gene expression was calculated relative to Gapdh.

Detection of LP and PB eosinophils-induced Treg response
Naïve T cells from splenocytes of OT-II transgenic C57BL/6 mice were isolated using a CD4 +-CD62L + T cell isolation kit-II (Miltenyi Biotec). Eosinophils were cultured with 5 ng/mL recombinant murine GM-CSF (Peprotech, USA) to sustain their viability. The naïve T cells (10 5 cells) were co-cultured with the LP eosinophils (10 5 cells) isolated from wild-type C57BL/ 6 mice in 10% FCS RPMI-1640 medium containing 1.5 μg OVA peptide (ISQVHAAHAEI-NEAGR) and 10 4 irradiated murine spleen CD11c + cells at 37°C in an atmosphere of 5% CO 2 for 4 d [14,19]. In some experiments, the following were included in the coculture conditions: (a) Anti-human TGF-β1 (Peprotech) or rat IgG1 isotype control antibody (eBioscience) was added to cultures at a final concentration of 1 μg/ml. (b) Recombinant human TGF-β1 (Peprotech) at varying concentrations; and (c) RA receptor inhibitors LE540 (Wako Chemicals, Japan) and LE135 (Tocris Bioscience, UK), each at 1μM. The differentiated Treg cells in the 4 d co-cultures were identified by flow cytometry using FITC-conjugated anti-CD4 and APC-conjugated anti-Foxp3 in a Treg cell identification kit (eBioscience). At the same time, the ability of the PB eosinophils to induce a Treg response during co-cultivation was also determined as above.

In vitro suppressor assay
To examine the suppressive capacity of LP eosinophil-induced Treg cells. LP eosinophilinduced Treg cells were generated in vitro and sorted to >99% purity. 10 5 CFSE-labeled murine splenic CD4 + T cells were stimulated with 1 μl anti-CD3/CD28 coated beads (Invitrogen, USA), and the numbers of LP eosinophil-induced Treg cells indicated in the figures for 3 d.

Quantitative measurement of ATRA by LC/MS/MS
ATRA is a crucial factor during differentiation of naïve T cells into Treg cells [7]. In previous studies, CD11c + MHC-II + CD103 + LP DC was found to promote de novo generation of Foxp3 Treg cells via ATRA [13]. Therefore, to quantify ATRA produced by the CD11b + Siglec-F + CCR3 + LP eosinophils (the CD11c − CD11b + SSC hi Siglec-F + subset from the P4 in Fig 1A) or CD11c + MHC-II + CD103 + LP DC (P1), a LC/MS/MS-based method was established for quantitative detection of ATRA, in which acitretin, an analogue of ATRA, was used as the internal standard (Fig 2A) [17,18]. The results showed that the LC/MS/MS method could accurately quantify the ATRA in LP eosinophils and CD11c + MHC-II + CD103 + LP DC (Fig 2B).

Treg differentiation induced by LP eosinophils
We next examined whether Lp eosinophils could induce Treg cells conversion. To this end, eosinophils from the LP or PB were isolated (Figs 1E and 3A). Compared to the LP eosinophils (Fig 1B), the PB eosinophils also expressed F4/80 but did not express CD11c and CD80 ( Fig  3B). However, neither the LP eosinophils nor the PB eosinophils expressed MHC II, DEC-205, CD86, CD40 and CD103. Purified OT-II CD4+ T cells, which express a transgenic T cell receptor specific for ovalbumin (OVA) peptide presented by major histocompatibility complex class II molecules, cultured together with LP or PB eosinophils in the presence of OVA peptide and irradiated DC [28]. After 4 d of co-incubation, we compared the ability of LP and PB eosinophils to induce expression of Foxp3 in CD4 + T cells, it was found that only LP eosinophils was able to induce expression of Foxp3 (Fig 3C). Next, we tested the suppressive capacity of LP eosinophil-induced Treg cells. The data show that LP eosinophil-induced Treg cells could suppress proliferation of T effector cells in vitro (Fig 3D).

TGF-β1 enhances the conversion of Treg cells in the presence of Lp eosinophils
As we have shown that LP eosinophils express TGF-β1 mRNA, we analyzed whether TGF-β1 played any role in the development of Treg cells. Thus, naive T cells were again co-cultured with LP or PB eosinophils, with or without antibody neutralization of TGF-β1. Neutralization of TGF-β1 lead to a significant decrease in the frequency of Treg cells (6.58% vs 0.38%) ( Fig  4A). Next, we asked whether the functional difference between LP and PB eosinophils was related to the ability of producing TGF-β1. LP eosinophils expressed higher levels of tgfb1 ( Fig  4B). To test whether TGF-β1 was able to exert an enhancing effect on Treg cells conversion, we added increasing doses of recombinant TGF-β1 to cultures. At low TGF-β1 concentrations (0.1 ng/ml), Treg cells conversion increased by 120% in LP eosinophils co-cultures (from 6.52 to 14.4%), and inclusion of 1 ng/ml TGF-β1 resulted in the expression of Foxp3 by~40% of T cells (Fig 5C). Although PB eosinophils induce minor Foxp3 expression in the absence of exogenous TGF-β1, they remained much lower than LP eosinophils at inducing T reg cells (Fig 4C). The data suggested that the LP eosinophils have the ability to induce differentiation of naïve T cells into Treg cells via TGF-β1-dependent mechanisms.

ATRA produced by Lp eosinophils is responsible for conversion of Treg cells
Differentiation of Treg cells is enhanced by the ATRA [7]. We have previously observed that the LP eosinophils could produce a high level of ATRA. And we now show that PB eosinophils cannot produce ATRA (Fig 5A). As a result, we investigated the function of the ATRA in this  process. To assess whether ATRA was involved in the differentiation of TGF-β1-dependent Foxp3 + Treg cells induced by LP eosinophils, we added the synthetic ATRA receptor antagonist LE540 and LE135 (RAi) to co-cultures. The addition of RAi reduced the frequency of Foxp3 + Treg cells generated by LP eosinophils from 6.18% to 1.17%, and neutralization of ATRA inhibited Treg cells conversion by 74% and 64% in LP and PB eosinophils co-cultures in presence of TGF-β1 ( Fig 5B). Therefore, the mechanisms by which LP eosinophils induced the generation of Treg cells involved ATRA.

Discussion
Immune tolerance in the intestinal tract is required for the presence of the normal gut microbiota and the uptake of macromolecular nutrients from food [1,3]. Although the mechanisms of immune tolerance are complicated, Treg cells are considered as the crucial inducers of immune tolerance [4,5,16]. It has been reported that CD4 + CD25 + Foxp3 + Treg cells could be induced by the CD103 + DC from mouse mesenteric lymph nodes (MLN) or intestinal LP via a TGF-β1-and ATRA-dependent mechanism [13,19]. Denning et al confirmed that the CD11b +-F4/80 + CD11Cmacrophages from the murine intestinal LP also have the ability to induce Treg cells during co-incubation of the macrophages with naïve T cells [14]. However, except for DC and macrophages, no other immune cells have been shown to possess the ability to induce Treg differentiation. Uematsu et al. described an eosinophils subset expressing the F4/80 macrophage marker in murine colon LP (LP eosinophils) [16], but the function of LP eosinophils had not been characterized yet. In the present study, we observed LP eosinophils of C57BL/6 mice consistent with the eosinophils subset previously described [16]. While investigating the function of the LP eosinophils, we unexpectedly found that the LP eosinophils could produce ATRA and express TGF-β1 mRNA. In particular, both the ALDH activity and ATRA levels produced by the LP eosinophils were significantly higher than for the CD103 + DC isolated from murine LP, strongly implying that the LP eosinophils could have the potential to induce differentiation of naïve T cells into Treg cells.
Studies have demonstrated that a specific reduction in Treg cells was observed in the LP of eosinophil-deficient mice [29]. We confirmed these findings in vitro co-culture experiments that LP eosinophils could induce the differentiation of Foxp3 + Treg cells.
Compared to the LP eosinophils (CD11c + CD80 + ), the eosinophils from mouse peripheral blood expressed a different phenotype (CD11c − CD80 − ) but also lacked the ability to produce ATRA and express TGF-β1 mRNA to induce Treg response.
Eosinophils are produced in the bone marrow from pluripotent stem cells, and then into a separate eosinophil lineage like LP and PB eosinophils. We speculate that the need for the immune system to tolerate the gut microbiota and food in the intestines could account for this functional difference between LP and PB eosinophils. Taken together, this study reveals that LP eosinophils, unlike PB eosinophils, can induce Treg differentiation through a mechanism dependent on ATRA and TGF-β1.