Plasticity and regulatory mechanisms of human ILC2 functions

Highlights

• Human circulating ILC2 are modulated versus type 1 and type 3.

• Modulated ILC2 showed reduced abilty to induce IgE production by B cells.

• Treg cells reduced ILC2 proliferation and their CD154 expression.

Abstract

Human group 2 innate lymphoid cells (ILC2) represent the innate counterpart of Th2 cells and cooperate with them in helminths protection and in the pathogenesis of allergic diseases. Some reports described ILC2 plasticity and few studies investigated the cellular and molecular mechanisms regulating human ILC2 functions. The aim of this study is to define how immune deviation and immune regulation control human ILC2-mediated immune response. Human circulating ILC2 were expanded in vitro and then cultured in presence of IL-12 or IL-1β plus IL-23 or co-coltured in presence of circulating CD4+CD25highFoxp3+Treg. IL-12 induces IFN-γ production and upregulation of T-bet mRNA level on human circulating ILC2 whereas IL-1β and IL-23 mediate IL-22 production and upregulation of RORC mRNA level. In all these conditions, GATA-3 mRNA level is not reduced and the typical type 2 cytokines are only partially reduced. Moreover, “modulated” ILC2 have reduced ability to induce IgE producing by B cells. ILC2 proliferation, cytokines production and CD154 expression were inhibited by CD4+CD25highFoxp3+ Treg cells. TGF-β reduced CD154 expression on ILC2 stimulated with IL-25/IL-33. This study defines possible cellular and molecular mechanisms responsible for modulation and inhibition of human ILC2 activity. These results may be useful in the development of strategies aimed to dampen ILC2 function in type-2 mediated diseases.

Introduction

Innate lymphoid cells (ILCs) are an heterogeneous family of innate effectors cells involved in mucosal inflammation, including defence from pathogens, maintenance of epithelial barrier function, containment of commensal microbiota, tissue repair and regulation of metabolism [1,2].

The classification of ILCs into ILC1s, ILC2s, and ILC3s is similar to the one of CD4+ effector T cells, and is based on their expression of lineage-defining transcription factors, cytokines production, and functional attributes. In particular, group 1 ILCs (ILC1) depend on the T-box transcription factor T-bet and are characterized by the ability to produce the cytokines IFN-γ and TNF-α; group 2 ILCs (ILC2) depend on GATA-3 and produce IL-5 and IL-13, while group 3 ILCs (ILC3) express the transcription factor nuclear receptor RORγt and secrete IL-17 and/or IL-22 [3,4]. This initial simplifying classification has been reviewed in the last years based on recent findings that environmental milieu may modify ILCs in terms of transcription factors expression and cytokines production [[5], [6], [7]], and a plasticity feature of ILCs similar to the well-known plasticity described for CD4+ Th subsets, has been proposed [8]. For example, ILC3 cells can co-express T-bet and RORγt, produce IFN-γ and differentiate into ILC1 cells in response to a variety of inflammatory factors [[9], [10], [11], [12], [13], [14]]. This ability to switch is bi-directional, as also human ILC1 cells can convert into ILC3 cells [15]. Additionally, Toll-like-receptor-2 ligands can promote the production of IL-5 and IL-13 by ILC3 cells, suggesting that these cells might be able to differentiate into ILC2 cells [16]. Then ILC2s have been described to be capable of converting to an IFN-γ secreting phenotype in both humans and mice [[17], [18], [19]].

We recently demonstrated that human circulating ILC2 in response to IL-25/IL-33 stimulation or Toll-like receptors (TLRs) triggering produce IL-13 and IL-5 and express CD154 on their surface; more importantly stimulated ILC2 co-cultured with autologous B cells induced the production of all Ig (immunoglobulin) classes including IgE [20]. These data suggest that ILC2 contribute to type 2 immune response and of course play a key role in the pathogenesis of allergic diseases [21]. Taking into account these considerations, the aim of this study is to define the mechanisms of immune deviation of ILC2, as a possible tool for the modulation of type 2 response. First, we focused on ILC2 plasticity, confirming the possibility to shift them towards the ILC1 phenotype and demonstrating their ability to acquire an ILC3 similar phenotype. In particular, we found that human circulating ILC2 in presence of IL-12 acquired the ability to produce IFN-γ and upregulated the expression of T-bet without losing the capacity to produce type 2 cytokines (IL-13, IL-4 and IL-5) and to express GATA-3. As concern the plasticity versus the ILC3 subset, we found that the combination of IL-1β plus IL-23 mediated IL-22 production and upregulation of RORC expression. Also in this case ILC2 maintained the ability to produce type 2 cytokines and to express GATA-3. More importantly, we demonstrated that these modulated ILC2 have reduced ability to induce IgE production by autologous B cells.

Immune modulation represents one possible mechanism to interfere with a specific type of immune response and induce the shift of a functional phenotype towards a different one, but of course it is not the only one mechanism. In fact, T effector cells can be inhibited in their functional properties (mainly proliferation and cytokines production) by T regulatory cells [22,23]. Based on these considerations we tested the susceptibility of ILC2 to Treg cells activity, demonstrating that ILC2 proliferation, IL-13 and IL-4 production and CD154 expression are inhibited by Treg cells CD4+CD25highFoxp3+ . Moreover, we demonstrated that CD154 expression induced by IL-25/IL-33 stimulation is inhibited by TGF-β.