Unique and redundant functions of NKp46+ ILC3s in models of intestinal inflammation

Abstract

Group 3 ILCs (ILC3s) are innate sources of IL-22 and IL-17 and include lymphoid tissue-inducer (LTi)-like and NKp46(+) subsets. Both depend on RORγt and aryl hydrocarbon receptor, but NKp46(+)ILC3s also require Notch and T-bet for their development and are transcriptionally distinct. The extent to which these subsets have unique functions, especially in the context of T cell- and B cell-sufficient mice, remains largely unclear. To investigate the specific function of NKp46(+)ILC3s among other ILC3 subsets and T cells, we generated mice selectively lacking NKp46(+)ILC3s or all ILC3s and crossed them to T cell-deficient mice, thus maintaining B cells in all mice. In mice lacking T cells, NKp46(+)ILC3s were sufficient to promote inflammatory monocyte accumulation in the anti-CD40 innate colitis model through marked production of GM-CSF. In T cell-competent mice, lack of NKp46(+)ILCs had no impact on control of intestinal C. rodentium infection, whereas lack of all ILC3s partially impaired bacterial control. Thus, NKp46(+)ILC3s have a unique capacity to promote inflammation through GM-CSF-induced accumulation of inflammatory monocytes, but are superseded by LTi-like ILC3s and T cells in controlling intestinal bacterial infection.

Figure 1.

ILC3 deficiency protects mice from anti-CD40–induced colitis. ART and Tcrbd^−/− mice were injected with anti-CD40. (A) Mice were weighed daily, from day 0 to 7 p.i. (B) Colons were harvested and overall lengths measured 7 d p.i. Colon lengths were also assessed in Ahr^f/f x Tcrbd^−/− (flox control mice) and Rorγt-cre x Tcrbd^−/− (Cre control mice; not depicted). A and B represent five independent experiments with 4–10 mice/group per experiment. (C) Colons were stained with hematoxylin and eosin and scored for severity of colitis on day 7, where a higher score corresponds to increased pathology. Data represent two independent experiments with four to five mice/group. (D) mRNA expression of inflammatory mediators. Colons were collected from ART and Tcrbd^−/− mice at day 4 and 7 after anti-CD40 injection and the indicated transcripts were measured via quantitative RT-PCR. All gene expression values were normalized to Gapdh. (E) Protein expression of inflammatory mediators. Colonic lamina propria was processed at day 7 after anti-CD40 treatment; 10⁶ cells were isolated and cultured for 4 h. The indicated cytokines were measured by cytometric bead array of the culture supernatants. (D and E) Data represent two independent experiments with five mice/group. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, Student’s t test. Bars represent mean ± SEM.

Figure 2.

Evaluation of intestinal myeloid populations in ART and Tcrbd^−/− mice treated with anti-CD40. (A) Tcrbd^−/− mice were treated with anti-CD40 and sacrificed at days 0, 4, and 7. Cells were purified from colonic and small intestinal lamina propria. Ly6G⁺ neutrophils and Ly6G^–Ly6C^hi inflammatory monocytes were distinguished within the population of live CD45⁺CD19^–CD11b⁺ cells. Representative plots (top) and neutrophil and monocyte numbers among 10⁶ total leukocytes (bottom) are indicated. Data represent three independent experiments with two mice/time point. (B) Colonic lamina propria cells from Tcrbd^−/− and ART mice were collected at day 7 after anti-CD40 treatment. Live, CD45⁺CD19^– cells were analyzed for expression of Ly6G, Ly6C, CD11c, and CD11b. Data represent five independent experiments with two mice/group. Representative plots (top) and total numbers of neutrophils, monocytes, and DCs among 10⁶ total leukocytes (bottom) are indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001, student’s t test. Bars represent mean ± SEM.

Figure 3.

Adoptively transferred ILC3s cause severe colitis in NSG mice. 2 × 10⁴ small intestinal ILC3s (CD45⁺Lin^–Thy1⁺CD127⁺ cells) sort-purified from Rag1^−/− mice were intravenously injected into NSG mice. (A) After 8 wk, mice were treated with anti-CD40, and body weights were measured daily, from 0 to 7 d p.i. Data represent two independent experiments with four mice/group. (B) Cells were purified from the small intestinal lamina propria. ILC3s were identified as CD45⁺Lin⁻Thy1⁺RORγt⁺ cells. Data represent three independent experiments with two mice/group. (C) Ly6G⁺ neutrophils and Ly6G^–Ly6C^hi inflammatory monocytes were distinguished within the population of live CD45⁺CD19^–CD11b⁺ cells and (D) absolute numbers quantified. Data are representative of three experiments with two mice/group (C) or two independent experiments with five mice/group (D). *, P < 0.05, Student’s t test. Bars represent mean ± SEM.

Figure 4.

Impact of inflammatory monocytes and neutrophils depletion on anti-CD40–induced colitis. Tcrbd^−/− mice were treated with (A) anti-GR1 (RB6-8C5) or control IgG at days −1, 1, 3, and 5; (B) anti-CCR2 (MC-21) or control IgG at days −4, −1, 2, 3, and 4; (C) anti-Ly6G (1A8), or control IgG at days −1, 1, 3, and 5. Anti-CD40 was injected at day 0. (A–C) Numbers of intestinal neutrophils and inflammatory monocytes among 10⁶ total leukocytes present in the colon are indicated for each antibody treatment to indicate the efficacy of depletion. *, P < 0.05; ***, P < 0.001, Student’s t test. Data are representative of three experiments (A and C) or two experiments (B) with four to five mice/group. Bars represent mean ± SEM.

Figure 5.

Impact of GM-CSF and IL-22 neutralization on anti-CD40–induced colitis. (A) Representative plots show intracellular content of GM-CSF in LTi-like ILC3s (NKp46^–) and NKp46⁺ILC3s isolated from the small intestines of Tcrbd^−/− and ART mice, 6 d after treatment with anti-CD40. Cells were surface stained to identify NKp46⁺ and NKp46⁻ ILC3s; GM-CSF and TNF content were determined after 4-h culture in the presence of monensin. Data represent three independent experiments with two mice/group. (B and C) Tcrbd^−/− mice were treated with anti–GM-CSF (MP1-22E9), anti–IL-22 (8E11), or control IgG at days 0, 2, 4, and 6. ART mice received no neutralizing antibodies. Anti-CD40 was injected into all mice at day 0. (B) Mice were weighed daily, from 0 to 7 d p.i. Data represent three independent experiments with five mice/group. (C) Representative plots of intestinal inflammatory monocytes present in the colon of antibody-treated mice. (D) Quantification of inflammatory monocytes as represented in (C). C and D represent three independent experiments with two mice/group. *, P < 0.05; **, P < 0.01, Student’s t test. Bars represent mean ± SEM.

Figure 6.

Lack of NKp46⁺ ILC3s curbs inflammation during anti-CD40–induced colitis. RNT and Tcrbd^−/− mice were injected with anti-CD40. (A) Mice were weighed daily after antibody administration. Data represent five independent experiments with five mice/group. (B) Colons from Tcrbd^−/− and RNT mice were isolated 7 d after anti-CD40 injection and total lengths measured. Data represent three independent experiments with four to five mice/group. (C) Cells were purified from the small intestine LP. Ly6G⁺ neutrophils and Ly6G^–Ly6C^hi inflammatory monocytes were distinguished within the population of live CD45⁺CD19^–CD11b⁺ cells. Representative plots (left) and total neutrophil and monocyte numbers among 10⁶ total leukocytes (right) are indicated. Data represent three independent experiments with two mice/group. Small intestines (D) and colons (E) were collected at day 7 after anti-CD40 injection, RNA was extracted and cytokine content was measured by quantitative RT-PCR. Y axis demonstrates relative transcript abundance and x axis shows day p.i. Data in D and E represent three independent experiments with five mice/group (F) NKp46⁺ ILC3 (CD45⁺Thy1⁺CD127⁺NK1.1^–KLRG1^–NKp46⁺) and LTi-like ILC3 (CD45⁺Thy1⁺CD127⁺ NK1.1^–KLRG1^–NKp46^–) subsets from small intestine lamina propria were sort purified from Rag1^−/− mice and intravenously injected into NSG mice. After 8 wk, mice were treated with anti-CD40, and body weights were measured daily, from 0 to 7 d p.i. Data represent two independent experiments with five mice/group. *, P < 0.05; **, P < 0.01; ***, P < 0.001, Student’s t test. Bars represent mean ± SEM.

Figure 7.

Contribution of ILC3s to defense against C. rodentium infection. (A–D) C. rodentium (2 × 10⁹) was administrated by oral gavage to 8–12-wk-old B6 (A and B), AR (A), RN (B), Tcrbd^−/− (C and D), ART (C), and RNT (D) mice. Body weight and survival were monitored from 0 to 40 d p.i. (A–D) Data represent at least three independent experiments with 5–10 mice/group. (E) RNA was isolated from colons of Tcrbd^−/− and ART mice at day 7 after injection; Il22, Il17a, Il17f, Ifng, and Csf2 were measured by RT-PCR. All gene expression values were normalized to Gapdh. Data represent three independent experiments with five mice/group. (F) Neutrophil depletion in Tcrbd^−/− mice accelerates lethality to C. rodentium infection. Mice were infected and neutrophil depletion performed every 3 d by administration of 100 µg of anti-Ly6G (1A8) antibody. (E and F) Data are representative of three independent experiments with five mice/group. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, Student’s t test.