ILC2s are the predominant source of intestinal ILC-derived IL-10

Abstract

Although innate lymphoid cells (ILCs) functionally analogous to T helper type 1 (Th1), Th2, and Th17 cells are well characterized, an ILC subset strictly equivalent to IL-10-secreting regulatory T cells has only recently been proposed. Here, we report the absence of an intestinal regulatory ILC population distinct from group 1 ILCs (ILC1s), ILC2s, and ILC3s in (1) mice bred in our animal facility; (2) mice from The Jackson Laboratory, Taconic Biosciences, and Charles River Laboratories; and (3) mice subjected to intestinal inflammation. Instead, a low percentage of intestinal ILC2s produced IL-10 at steady state. A screen for putative IL-10 elicitors revealed that IL-2, IL-4, IL-27, IL-10, and neuromedin U (NMU) increased IL-10 production in activated intestinal ILC2s, while TL1A suppressed IL-10 production. Secreted IL-10 further induced IL-10 production in ILC2s through a positive feedback loop. In summary, ILC2s provide an inducible source of IL-10 in the gastrointestinal tract, whereas ILCregs are not a generalizable immune cell population in mice.

Figure 1.

No evidence for IL-10–producing Lin⁻CD127⁺Thy1⁺ cells in naive small and large intestine. (A and B) Representative FACS plots showing IL-10–eGFP expression in small intestine (SI) and large intestine (LI) lamina propria (LP) CD45⁺ lymphocyte-sized Lin⁻CD45⁺CD127⁺ cells (A) and CD4⁺ T cells (B). (C) Frequencies of eGFP⁺ cells among lymphocyte-sized Lin⁻CD45⁺CD127⁺Thy1⁺ cells and CD4⁺ T cells in small intestine and large intestine lamina propria in multiple mice (n = 4). (D) Modified gating scheme to account for all cell lineages that expressed eGFP within the lymphocyte-based FSC/SSC gate. (E) Modified gating scheme to include cells with an expanded FSC/SSC profile. Below, the FSC/SSC profile of CD3e⁺ cells is shown as a reference for where lymphocytes lie in the plot. Bars indicate means ± SD. Data are representative of two independent experiments.

Figure 2.

No evidence for lineage-negative ILCs distinct from ILC2s and ILC3s in small intestines of mice bred at WUSM and mice purchased from commercial vendors. (A) Left: Gating strategy to identify lineage-negative events that lacked markers for ILC2s and ILC3s. Right: Frequencies and total numbers of Lin⁻CD45⁺CD127⁺ cells that were GATA3^hi (ILC2s) or RORγt⁺ (ILC3s) or that lacked markers for both ILC2s and ILC3s (G6) in small intestine lamina propria of WT mice bred at WUSM (n = 8). (B–D) Frequencies of Lin⁻CD45⁺CD127⁺ cells that lacked ILC2 and ILC3 markers in mice purchased from JAX (n = 8; B), Taconic Biosciences (n = 8; C), and Charles River Laboratories (n = 8; D). (E) Representative flow cytometry plots showing backgated G6. ILC2s are shown as a reference for lineage-negative cells. Right, quantified gMFI of lineage staining in G6 and ILC2s (n = 4). (F) T-bet staining in G6 (n = 4). (G) Frequencies of G6, ILC2s, and ILC3s using a modified lineage staining panel (Modified Lin) in which anti-F4/80 antibodies were added while anti-B220 antibodies were excluded (n = 4). (H) gMFI of Modified Lin staining in G6 and ILC2s (n = 4). (I) Frequencies of T-bet⁺ cells within G6 obtained using Modified Lin staining (n = 4). Bars indicate means ± SD. Data were pooled from two independent experiments (A–D) or are representative of two independent experiments (D–I). ****, P < 0.0001.

Figure 3.

No evidence for ILCreg induction during intestinal inflammation. (A and B) Representative flow cytometry plots showing IL-10–eGFP expression in Lin⁻CD45⁺CD127⁺ cells (A) and CD4⁺ T cells (B) on day 8 of DSS treatment. (C) Frequencies of Lin⁻CD45⁺CD127⁺ cells and CD4⁺ T cells expressing eGFP in small intestine lamina propria in multiple mice (n = 8). (D) Body weight loss as a percentage of initial weight at day 8 of DSS treatment (n = 6). D0, day 0; D8, day 8. (E and F) Frequencies of Lin⁻CD45⁺CD127⁺ cells that lacked markers for both ILC2s and ILC3s in small intestine lamina propria of mice treated with DSS in drinking water for 8 d (n = 5; E) or in mice infected for 8 d with C. rodentium (n = 5; F). Frequencies of ILC2s and ILC3s are shown as controls. Bars indicate means ± SD. Data were pooled from two independent experiments (C) or are representative of two independent experiments (D–F).

Figure 4.

IL-10 production by intestinal ILC2s is induced by IL-2, IL-4, IL-10, IL-27, and NMU, and suppressed by TL1A. (A) Representative flow cytometry plots showing IL-25R, KLRG1, and GATA3 staining in Lin⁻CD45⁺CD127⁺Thy1⁻ cells. (B) Representative flow cytometry plots showing expression of eGFP in Lin⁻CD45⁺CD127⁺KLRG1⁺ ILC2s that are Thy1⁺ and Thy1⁻. (C) Heatmap depicting frequencies of eGFP⁺ cells obtained by flow cytometric analysis of ILC2s cultured 2 d with IL-33, IL-25, and either IL-7 or TSLP, in combination with the indicated molecules. (D and E) Validation of results in C. Cells were cultured for 3 d with IL-33, IL-25, IL-7, and the indicated conditions (n = 5–6). (F) ILC2s cultured as in C using pre-activated ILC2s. (G) IL-4- or IL-2-elicited eGFP expression in ILC2s treated with TL1A (n = 4). (H and I) Isolated ILC2s were treated with IL-25, IL-33, IL-7, and the indicated molecules in the presence or absence of IL-10–blocking antibodies (n = 5–6; H) or in the presence or absence of IL-2- and IL-4–blocking antibodies (n = 3; I). Bars indicate means ± SD. Data from D, E, and H were analyzed from the same experiments. Data were pooled from two independent experiments (E and H) or are representative of two independent experiments (A, B, G, and I). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure S1.

IL-10 production in NK cells, ILC1s, and ILC3s. (A) Expression of IL-10–eGFP in NK1.1⁺ cells (pooled ILC1s and NK cells) and ILC3s isolated from naive small intestine lamina propria. (B) Screen for IL-10 elicitors using sorted small intestine ILC3s cultured for 2 d with IL-23, IL-1β, IL-7, and a candidate mediator. (C) Expression of IL-10–eGFP in small intestine lamina propria ILC3s sorted from biological replicates and cultured for 2 d with IL-23, IL-1β, IL-7, and molecules that were found to induce IL-10 in ILC2s (n = 4). (D) Expression of IL-10–eGFP in NK1.1⁺ cells (pooled ILC1s and NK cells), ILC3s, and CD4⁺ T cells isolated from small intestine lamina propria of mice 4 d after inoculation with 10⁸ CFU of C. difficile VPI 10463 (n = 3). Bars indicate means ± SD. Data from A, C, and D are representative of two independent experiments.