RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells

Abstract

The mucosal immune system of the intestine is separated from a vast array of microbes by a single layer of epithelial cells. Cues from the commensal microflora are needed to maintain epithelial homeostasis, but the molecular and cellular identities of these cues are unclear. Here we provide evidence that signals from the commensal microflora contribute to the differentiation of a lymphocyte population coexpressing stimulatory natural killer cell receptors and the transcription factor RORgammat that produced interleukin 22 (IL-22). The emergence of these IL-22-producing RORgammathiNKp46+NK1.1(int) cells depended on RORgammat expression, which indicated that these cells may have been derived from lymphoid tissue-inducer cells. IL-22 released by these cells promoted the production of antimicrobial molecules important in the maintenance of mucosal homeostasis.

Figure 1

Lamina propria NKp46⁺ cells have an immature phenotype. (a,b) Flow cytometry of lamina propria (LP) lymphocytes from the small intestine and spleen cells stained with anti-CD3 and anti-NKp46. (a) Numbers adjacent to outlined areas indicate percent NKp46⁺CD3⁻ cells (mean ± s.e.m.). (b) Gating on NKp46⁺CD3⁻ cells: filled histograms, specific staining (antibodies to markers below plots); open histograms, isotype-matched control antibody. Data are representative of at least eight independent experiments. (c) Flow cytometry of splenocytes and lamina propria cells stained with anti-NKp46, anti-CD3, anti-CD11b and anti-CD27, gated on NKp46⁺CD3⁻ cells. Numbers in quadrants indicate percent cells in each. Data are representative of three independent experiments. (d) Flow cytometry of lamina propria cells and splenocytes (mouse strains, above plots) stained with anti-NKp46 and anti-CD3. Numbers above outlined areas indicate percent NKp46⁺CD3⁻ cells. Data are representative of three independent experiments. (e,f) Flow cytometry of B6 and Il15^−/− lamina propria cells stained with anti-CD3 and anti-NKp46 (e) or anti-NK1.1 (f). Numbers adjacent to outlined areas indicate percent cells in gate. Data are representative of four independent experiments. (g,h) Absolute numbers of various cell populations (above graphs) in the spleen (g) or intestinal lamina propria (h). LPL, lamina propria lymphocytes. Data are representative of three independent experiments (error bars, s.d.; n = 6 mice).

Figure 2

Mucosal NKp46⁺ cells localize in cryptopatches. (a–c) Immunofluorescence staining of sections from the small intestines and spleens of mice (strains, above images). White arrowheads indicate scattered NKp46⁺ cells outside cryptopatches in B6 mice (a) and Il15^−/− mice (c); yellow and red arrowheads indicate splenic NKp46⁺Ly49G2⁺ and NKp46⁺Ly49G2⁻ NK cells, respectively (b). Markers (and staining color), right margins. Original magnification, ×40. Data are representative of five independent experiments.

Figure 3

NKp46⁺ cells of the lamina propria express RORγt. (a) Sections from the small intestines of Rorc(γt)^GFP/+ mice, stained with anti-GFP (green; RORγt) or anti-NKp46 (red). Original magnification, ×40. Data are representative of four independent experiments. (b–d) Flow cytometry of Rorc(γt)^GFP/+ and Rorc(γt)^GFP/GFP lamina propria lymphocytes stained with anti-CD3, anti-NKp46 and anti-NK1.1 and gated on CD3⁻ cells. (b,d) Numbers adjacent to outlined areas indicate percent cells in gate. (c) NK1.1 expression by various cell populations (right margin). MFI, mean fluorescence intensity. Data are representative of five independent experiments. (e) Flow cytometry of B6 and Il15^−/− lamina propria lymphocytes stained with anti-CD3 anti-NK1.1 and a monoclonal antibody specific for RORγt, or with isotype-matched control antibody and gated on CD3⁻ cells. Numbers adjacent to outlined areas indicate percent cells in gate. Data are representative of three independent experiments. (f) Absolute numbers of various lamina propria cell populations (above graphs). Data are representative of three independent experiments (error bars, s.d.; n = 4 mice).

Figure 4

Mucosal NKp46⁺ cells have only weak NK cell effector functions. (a) Cytotoxicity of lamina propria lymphocytes or splenocytes from B6 mice injected twice with anti-NK1.1 or isotype-matched control antibody and, 1 d later, with PBS, lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (Poly(I:C)), assessed 24 h later as killing of YAC-1 mouse lymphoma target cells at effector/target ratios of 6:1, 2:1, 0.6:1 and 0.1:1. (b) Cytotoxicity of sorted lamina propria lymphocyte populations (RORγt^neg–intNKp46⁺NK1.1^hi and RORγt^hiNKp46⁺NK1.1^int) or splenocyte cells (RORγt⁻NKp46⁺NK1.1⁺) from Rorc(γt)^GFP/+ mice injected with polyinosinic-polycytidylic acid, assessed 1 d later as killing of YAC-1 target cells. (c) IFN-γ-producing cells among Rorc(γt)^GFP/+ lamina propria lymphocyte and splenocyte populations (key) incubated for 6 h in the presence of brefeldin A with various stimuli (above graphs) and then stained with anti-IFN-γ (top row), and IFN-γ-producing cells among lamina propria and spleen cells from Rorc(γt)^GFP/+ mice injected with various Toll-like receptor ligands (above graphs) and, 1 d later, incubated for 6 h with YAC-1 cells in the presence of brefeldin A and then stained with anti-IFN-γ (bottom row). (d,e) Quantitative RT-PCR analysis of the expression of IL-17A mRNA (Il17a; d) and IL-17F mRNA (Il17f; e) in sorted lamina propria CD3⁺ and NKp46⁺CD3⁻ (NKp46⁺) cells. (f) Flow cytometry of Rorc(γt)^GFP/+ lamina propria cells stained with anti-CD3, anti-NK1.1 or anti-IL-17 or isotype-matched control antibody, gated on subsets above plots. Numbers above lines indicate percent IL-17⁺ cells. Data are representative of four (a), three (b–e) or five (f) independent experiments.

Figure 5

Lamina propria RORγt^hiNKp46⁺NK1.1^int cells produce IL-22. (a) Quantitative RT-PCR analysis of IL-22 mRNA expression (Il22) in splenic and lamina propria NKp46⁺CD3⁻ cells sorted from B6 mice. Data are representative of three experiments. (b,c) Quantitative RT-PCR analysis of IL-22 mRNA expression (b) and ELISA of IL-22 protein production (c) by sorted splenic NKp46⁺CD3⁻ cells (Spleen) and of RORγt^hiNKp46⁺NK1.1^int (RORγt^hi), RORγt^neg–intNKp46⁺NK1.1^int (RORγt^neg–int) and CD4⁺RORγt^int (LP T_H-17) lamina propria cells sorted from Rorc(γt)^GFP/+ mice. Data are representative of three experiments. (d) Flow cytometry of B6 lamina propria cells stained with anti-NK1.1 and anti-RORγt (far left), or with anti-IL-22 or an isotype-matched control antibody and gated on subsets above plots (right). Numbers above lines indicate percent IL-22⁺ cells. Data are representative of four experiments. (e,f) Quantitative RT-PCR analysis of IL-22 mRNA expression (b) and ELISA of IL-22 protein production (c) by RORγt^hiNKp46⁺NK1.1^int cells sorted from Rorc(γt)^GFP/+ mice and RORγt^intNKp46⁺NK1.1^hi cells from Rorc(γt)^GFP/GFP mice; for ELISA, cultures were stimulated for 72 h with IL-23. ND, not detectable. Data are representative of three independent experiments.

Figure 6

Germ-free mice lack IL-22-producing RORγt^hiNKp46⁺NK1.1^int cells. (a) Flow cytometry of lamina propria lymphocytes from conventional and germ-free B6 mice, stained with anti-NK1.1 and anti-RORγt. Numbers adjacent to outlined areas indicate percent cells in each gate. Data are representative of three independent experiments with a total of 20 conventional mice and 24 germ-free mice. (b) Absolute numbers of lamina propria cell populations. Data are representative of three independent experiments (error bars, s.d.; n = 4 mice). (c) NK1.1^int cells among all CD3⁻RORγt^hi cells in the lamina propria of conventional B6 mice, of germ-free B6 mice and of germ-free mice recolonized with normal microflora (time, horizontal axis). Data are representative of three independent experiments (error bars, s.d.; n = 3 mice). (d) IL-22-producing RORγt^hiNK1.1^int cells among lamina propria cells from conventional or germ-free B6 mice, incubated for 6 h with IL-23 in the presence of brefeldin A, then stained with anti-NK1.1, anti-RORγt and anti-IL-22. Data are representative of two independent experiments with ten mice per group. (e) NK1.1^int cells among all CD3⁻RORγt^hi cells in the lamina propria of conventional B6 mice, germ-free B6 mice and Il6^−/− mice. Data are representative of three independent experiments (error bars, s.d.; n = 3 mice). (f) Quantitative RT-PCR analysis of RegIIIβ (Reg3b) and RegIIIγ (Reg3g) mRNA in epithelial cells purified from B6 mice (top row) or Rag2^−/− mice (middle row) injected three times every other day with 200 µg anti-IL-22 (α-IL-22; B6) or 200 µg anti-NK1.1 (α-NK1.1; Rag2^−/−) or with isotype-matched control antibody (Ctrl Ig), analyzed 2 d after the final injection, or from Rorc(γt)^GFP/+ and Rorc(γt)^GFP/+ mice (bottom row). (g) Quantitative RT-PCR analysis as in f of epithelial cells from conventional B6 mice (B6) and germ-free mice recolonized with normal microflora (time, horizontal axis). Data are representative of three experiments (f,g).