IL-1β and IL-2 convert human Treg into T_H17 cells

Abstract

Natural CD4⁺CD25⁺ regulatory T cells (Treg) and interleukin 17 (IL-17)-producing T helper cells (T_H17) carry out opposite functions, the former maintaining self-tolerance and the latter being involved in inflammation and autoimmunity. We report here that stimulation of human Natural Treg under T_H17 polarizing conditions in the presence of IL-2 converts them into T_H17 cells. Conversion of Tregs into T_H17 cells occurs both from natural naïve Tregs (NnTregs) and, to a higher extent, from memory Tregs (MTregs). Among antigen presenting cells, fresh monocytes activated by microbial stimuli were the most efficient inducers of T_H17 cells from Tregs. Conversion of Treg into T_H17 cells was induced by IL-1β and involved down-regulation of the Treg lineage transcription factor FOXP3 and suppressive functions. Our results indicate that, under inflammatory conditions, in the presence of IL-2, Treg can be converted into pro-inflammatory T_H17 cells and establish a functional link between inflammation and autoimmunity.

Introduction

Until recently, in both mice and humans, CD4⁺ T cell effectors have been classified in T_H1 and T_H2 type cells [1]. T_H1 cells mainly produce IFN-γ, are responsible for protection against intracellular pathogens and cancer and have been classically considered as the main subset involved in autoimmune diseases, whereas T_H2 cells produce IL-4, IL-5 and IL-13, are responsible for protection against extra-cellular parasites, and are believed to be mainly involved in the development of allergic disorders. Recently, however, the existence of additional CD4⁺ T cell lineages has been clearly documented. Among these, CD4⁺CD25⁺ natural regulatory T cells (Treg) have been described as a distinct lineage of anergic and suppressor cells arising in the thymus and characterized by the constitutive expression of the transcriptional suppressor FOXP3 [2], [3]. In both animal models and in humans, Treg have been shown to be in charge of controlling self-tolerance, constitutive absence or functional depletion of the subset resulting in autoimmunity [4], [5]. Among human circulating lymphocytes, natural Treg populations are readily distinguishable from CD25⁺ activated CD4⁺ T cells based on their ex-vivo CD127^lowFOXP3⁺ phenotype [6] and are present both at the naïve and memory stages [7]. In addition, lately, a distinct type of CD4⁺ helper T cells (T_H17 cells) producing IL-17, a pro-inflammatory mediator that stimulates the production of chemokines and anti-microbial peptides and leads to the recruitment of neutrophils, has received much attention [8]. Apart from their protective role towards extra-cellular microbial invasion, T_H17 cells have been clearly shown to be involved in autoimmune pathology, in studies using murine models of experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA), two experimental autoimmune diseases that had been previously attributed to unchecked T_H1 responses [8], [9], [10], [11]. Furthermore, recent findings of high numbers of IL-17 mRNA-expressing cells and high levels of secreted IL-17 in patients with several autoimmune diseases, are highly indicative of the involvement of T_H17 cells in human autoimmune disorders [12]. In healthy donors, a population of CD4⁺ T cells producing IL-17 ex-vivo has been recently identified in circulating lymphocytes from healthy individuals, among memory CD4⁺ T cells, mainly in the CCR6⁺ fraction, but not among naive CD4⁺ T cells [13], [14]. T_H17 cells with similar phenotypical and functional characteristics have also been found in the gut of Crohn's disease patients [14]. Both mouse and human T_H17 populations are characterized by high expression of the transcription factor RORγt [13], [14], [15].

Consistent with the opposite immune functions carried out by Treg and T_H17 cells, several studies have reported the existence of reciprocal developmental pathways for these two subsets. Thus, at least in mice, TGF-β1 alone promotes the differentiation of Treg, whereas the association of IL-6 and TGF-β1 has been reported to inhibit the differentiation of Treg and to promote the differentiation of T_H17 cells [16], [17]. In humans, T_H17 differentiation from naïve CD4⁺CD25⁻ T cells has been initially suggested to be induced by IL-1β and IL-6 but suppressed by TGF-β [18], [19]. However, recent reports have shown that TGF-β1 in combination with IL-21 or with IL-1β and IL-23 induces differentiation of T_H17 cells from human naïve CD4⁺ T cells [20], [21], [22]. IL-2, that is indispensable for the development of Treg [23], has been initially reported to inhibit the differentiation of T_H17 cells both [18], [24]. However, this effect is abolished in the presence of IL-1β [25] and, under these conditions, IL-2 has instead a positive effect on IL-17 production by human CD4⁺ T cells [20].

Together, the results of these studies have suggested the existence of not only a functional antagonism between the Treg and T_H17 subsets, but also of a dichotomy, mutually exclusive, in their generation. The relationship between the two subsets, however, is complex and has not been completely elucidated. In particular, the analysis of the interplay between the two subsets during chronic inflammation, that often leads to the development of autoimmune pathology, is of great interest.

Whereas under physiologic conditions natural Treg are anergic and suppressor, different factors, including cytokines produced under inflammatory conditions, TLR stimulation, or interaction with activated APC, have been shown to be able to abrogate their suppressive effects, resulting in autoimmune reactions [26], [27], [28], [29]. Because many of the factors that influence the development of natural Treg are also involved in the differentiation of T_H17 cells, we addressed if stimulation of Treg under T_H17 polarizing conditions, in the presence of factors that break their anergy, could convert them into T_H17 cells. In the present study, we report that stimulation of human Treg in the presence of activated monocytes and IL-2 induced their conversion into T_H17 cells. The conversion was mediated by IL-1β, was dependent on the presence of IL-2, and involved down-regulation of FOXP3 and suppressor functions. It occurred from both natural naïves and, to a larger extent, from memory Treg. Together, our results indicate that, under inflammatory conditions, in the presence of IL-2, natural Treg can be converted into T_H17 cells by IL-1β-producing APC, lose their suppressor function, and may contribute instead, both directly and/or indirectly, to the autoimmune pathology.