Type 1 Innate Lymphoid Cells Protect Mice from Acute Liver Injury via Interferon-γ Secretion for Upregulating Bcl-xL Expression in Hepatocytes

Abstract

Although type 1 innate lymphoid cells (ILC1s) have been originally found as liver-resident ILCs, their pathophysiological role in the liver remains poorly investigated. Here, we demonstrated that carbon tetrachloride (CCl₄) injection into mice activated ILC1s, but not natural killer (NK) cells, in the liver. Activated ILC1s produced interferon-γ (IFN-γ) and protected mice from CCl₄-induced acute liver injury. IFN-γ released from activated ILC1s promoted the survival of hepatocytes through upregulation of Bcl-xL. An activating NK receptor, DNAM-1, was required for the optimal activation and IFN-γ production of liver ILC1s. Extracellular adenosine triphosphate accelerated interleukin-12-driven IFN-γ production by liver ILC1s. These findings suggest that ILC1s are critical for tissue protection during acute liver injury.

Keywords: ATP; Bcl-xL; CCl(4); DNAM-1; IFN-γ; IL-12; IL-7; ILC1; acute liver injury; hepatocyte.

Figure 1.. Liver ILC1 but not NK cells are activated and produce IFN-γ after CCl₄ injection

(A) Gating strategy and expression of CD200R for NK cells and ILC1 from the liver of mice before (naïve) and 18 h after CCl₄ injection (n = 2–5 for gating strategy and n = 3–4 for CD200R). (B) The percentages and the number of NK cells and ILC1 in the liver before (naïve) and 18 h after CCl₄ injection. Data were pooled from 6 experiments (n = 5–14). (C) Expression of CD69 on NK cells and ILC1 in the liver before and 18 h after CCl₄ injection (n = 2–5). (D) Mean fluorescence intensity (MFI) of CD69 on NK cells and ILC1 in the liver before and 18 h after CCl₄ injection. Data were pooled from 4 experiments (n = 4–14). (E) Expression of CD25 on NK cells and ILC1 in the liver before and 18 h after CCl₄ injection (n = 2–5). (F) MFI of CD25 on NK cells and ILC1 in the liver before and 18 h after CCl₄ injection. Data were pooled from 2 experiments (n = 4). (G) Expression of CD69 on ILC1 in the liver of mice before CCl₄ injection and on CD25⁻ ILC1 and CD25⁺ ILC1 in the liver 15 h after CCl₄ injection (n = 2–5). (H) MFI of CD69 on NK cells and ILC1 in the liver of mice before CCl₄ injection and on NK cells, CD25⁻ ILC1, and CD25⁺ ILC1 in the liver 15 h after CCl₄ injection. Data were pooled from 3 experiments (n = 9–13). (I) Kinetics of the percentages of IFN-γ⁺ cells in NK cells, CD25⁻ ILC1, and CD25⁺ ILC1 in the liver after CCl₄ injection (n = 4). *p<0.05 vs. NK cells and CD25⁻ ILC1. (J) The percentages of IFN-γ⁺ cells in ILC1 in the liver before CCl₄ injection and those in CD25⁻ ILC1 and CD25⁺ ILC1 in the liver 15 h after CCl₄ injection (n = 2–5). (K) The percentages of IFN-γ⁺ cells in NK cells and ILC1 in the liver before CCl₄ injection and those in NK cells, CD25⁻ ILC1, and CD25⁺ ILC1 in the liver 15 h after CCl₄ injection. Data were pooled from 3 experiments (n = 9–13). Data are representative of more than 20 (A, C, and E), 3 (G and J), and 2 (I, and A (CD200R)) independent experiments. **p<0.005. Error bars show s.d. See also Figures S1 and S2.

Figure 2.. IFN-γ released from liver ILC1 has a protective role in CCl₄-induced acute liver injury

(A) The percentages and number of CD25⁺ cells in the liver of Rag1^−/− mice before (naïve) and 18 h after CCl₄ injection. Data were pooled from 2 experiments (n = 4–6). (B) The percentages of CD25⁺ (7D4⁺) ILC1 (upper left) (n = 3) and number of each lymphocyte subset in the liver (lower left) and plasma concentrations of IFN-γ (upper right) and ALT (lower right) of Rag1^−/− mice that had been injected with a depletion anti-CD25 mAb (PC61) or isotype-matched Ig (Isotype Ig) 18 h after CCl₄ injection. Data were pooled from 2 (lower panels) (n = 6) and 3 (upper right) (n = 6–10) experiments. (C) Plasma concentrations of ALT of WT and Zfp683^−/− mice 18 h after CCl₄ injection. Data were pooled from 2 experiments (n = 4–8). (D) Plasma concentrations of ALT of Eomes^fl/fl and Ncr1^creEomes^fl/fl mice 18 h after CCl₄ injection. Data were pooled from 2 experiments (n = 7). (E) Expression of CD25 on ILC1 in the liver of WT and Ifng^−/− mice before (naïve) and 18 h after CCl₄ injection (n = 2–4). (F) MFI of CD25 on NK cells and ILC1 in the liver of WT and Ifng^−/− mice before and 18 h after CCl₄ injection. Data were pooled from 2 experiments (n = 3–7). (G) Expression of CD69 on ILC1 in the liver of WT and Ifng^−/− mice before and 18 h after CCl₄ injection (n = 2–4). (H) MFI of CD69 on NK cells, CD25⁻ ILC1, and CD25⁺ ILC1 in the liver of WT and Ifng^−/− mice before and 18 h after CCl₄ injection. Data were pooled from 2 experiments (n = 3–7). (I) Plasma concentrations of ALT before and 18 h after CCl₄ injection. Data were pooled from 3 experiments (n = 4–10). (J) Histology of the liver (hematoxylin and eosin staining) before and 18 h after CCl₄ injection (n = 2–4). Scale bars represent 500 μm. (K) Quantified damaged areas around the central veins and the portal veins of mice before and 18 h after CCl₄ injection. Data were pooled from 2 experiments (n = 3–7). (L) Plasma concentrations of IFN-γ (upper) and ALT (lower) of mice (that had been injected with a neutralizing anti-IFN-γ mAb or isotype Ig 6 h before and 6 h after CCl₄ injection) 18 h after CCl₄ injection. Data were pooled from 2 experiments (n = 8). Data are representative of 2 independent experiments (B (upper left), E, G, and J). *p<0.05, **p<0.005. Error bars show s.d. See also Figure S3.

Figure 3.. DNAM-1 and IL-7 are required for optimal activation of liver ILC1 after CCl₄ injection

(A) Expression of DNAM-1 on NK cells and ILC1 in the liver (n = 2–5). (B) Expression of Pvr mRNA in the liver of mice before (naïve) and 18 h after CCl₄ injection (n = 3–4). (C) Expression of CD69 on ILC1 in the liver of WT and Cd226^−/− mice before and 18 h after CCl₄ injection. (D) MFI of CD69 on NK cells and ILC1 in the liver of WT and Cd226^−/− mice before and 18 h after CCl₄ injection (n = 2–5). (E) Expression of CD25 on ILC1 in the liver of WT and Cd226^−/− mice before and 18 h after CCl₄ injection. (F) MFI of CD25 on NK cells and ILC1 in the liver of WT and Cd226^−/− mice before and 18 h after CCl₄ injection (n = 2–5). (G) Expression of CD25 on liver ILC1 (left) (n = 2–4) and MFI of CD25 on liver NK cells and liver ILC1 (right) after crosslinking with isotype Ig or anti-DNAM-1 mAb. Data were pooled from 3 experiments (right) (n = 8). (H) Expression of Il7 mRNA in the liver of mice before and 18 h after CCl₄ injection (n = 3–4). (I) Expression of CD25 on ILC1 (left) (n = 3) and MFI of CD25 on NK cells and ILC1 (right) in the liver of mice (that had been injected with a neutralizing anti-IL-7Rα mAb or isotype Ig 6 h before and 6 h after CCl₄ injection) before and 18 h after CCl₄ injection. Data were pooled from 2 experiments (right) (n = 3–6). (J) Plasma concentrations of ALT of mice (that had been injected with a neutralizing anti-IL-7Rα mAb or isotype Ig 6 h before and 6 h after CCl₄ injection) before and 18 h after CCl₄ injection. Data were pooled from 2 experiments (n = 3–6). (K) Expression of CD25 on liver ILC1 (left) (n = 2) and MFI of CD25 on liver NK cells and liver ILC1 (right) in the absence or presence of IL-7. Data were pooled from 3 experiments (right) (n = 4). Data are representative of 3 (A, G (left), and K (left)), 2 (B, H, and I (left)), and 4 (C-F) independent experiments. *p<0.05, **p<0.01, ***p<0.005. Error bars show s.d.

Figure 4.. DNAM-1 is critical for IFN-γ production by liver ILC1 after CCl₄ injection

(A) The percentages of IFN-γ⁺ cells in ILC1, CD25⁻ ILC1, and CD25⁺ ILC1 in the liver of in WT and Cd226^−/− mice before (naïve) and 15 h after CCl₄ injection (n = 2–5). (B) The percentages of IFN-γ⁺ cells in NK cells, CD25⁻ ILC1, and CD25⁺ ILC1 in the liver of in WT and Cd226^−/− mice before and 15 h after CCl₄ injection. Data were pooled from 2 experiments (n = 5–9). (C) Plasma concentrations of IFN-γ (left) and ALT (middle) of WT and Cd226^−/− mice or Rag1^−/− and Rag1^−/−Cd226^−/− mice (right) before and 18 h after CCl₄ injection. Data were pooled from 4 (left) (n = 4–15), 5 (middle) (n = 5–17), and 3 (right) (n = 3–10) experiments. (D) Plasma concentrations of ALT of Rag1^−/− and Rag1^−/−Cd226^−/− mice (that had been injected with a depletion anti-CD25 mAb or isotype Ig) 18 h after CCl₄ injection (n = 3). (E) The percentages of IFN-γ⁺ cells in liver ILC1 (that had been primed with IL-2 and IL-7 in vitro) (left) (n = 2–5) and those in naïve or primed liver NK cells and liver ILC1 (right) (n = 5–7) after crosslinking with anti-DNAM-1 mAb or Isotype Ig. Data were pooled from 2 experiments (right). Data are representative of 2 (A and D) and 3 (E (left)) independent experiments. *p<0.05, **p<0.005. Error bars show s.d. See also Figure S4.

Figure 5.. ATP accelerates IL-12-driven IFN-γ production by liver ILC1

(A) Expression of P2RX7 on NK cells and ILC1 in the liver (n = 3–5). (B) Plasma concentration of extracellular ATP from the proximal inferior vena cava before (naïve) and 15 h after CCl₄ injection. Data were pooled from 2 experiments (n = 6–10). (C) Expression of CD25 on NK cells and ILC1 in the liver of mice (that had been injected or not with BBG) before and 18 h after CCl₄ injection (n = 2–5). (D) Expression of CD69 on NK cells, CD25⁻ ILC1, and CD25⁺ ILC1 in the liver of mice (that had been injected or not with BBG) before and 18 h after CCl₄ injection (n = 2–5). (E) The percentages of IFN-γ⁺ cells in ILC1, CD25⁻ ILC1, and CD25⁺ ILC1 in the liver of mice (that had been injected or not with BBG) before and 15 h after CCl₄ injection (n = 2–5). (F) The percentages of IFN-γ⁺ cells in NK cells, CD25⁻ ILC1, and CD25⁺ ILC1 in the liver of mice (that had been injected or not with BBG) before and 15 h after CCl₄ injection. Data were pooled from 2 experiments (n = 5–10). (G) Plasma concentration of ALT in mice (that had been injected or not with BBG) before and 18 h after CCl₄ injection. Data were pooled from 2 experiments (n = 4–8). (H) Expression of Il12a and Il12b mRNA in the liver before and 18 h after CCl₄ injection (n = 3–4). (I) Expression of Il12a and Il12b mRNA in myeloid cell subsets (CD11b⁻ DC, CD11b⁺ DC, Kupffer cells (KC), and macrophages (Mϕ)) in the liver before and 18 h after CCl₄ injection. Data were pooled from 3 experiments (n = 4–5). (J) The percentages of IFN-γ⁺ cells in liver NK cells and liver ILC1 after stimulation with or without IL-12, ATP or both. Data were pooled from 2 experiments (n = 6). (K) The percentages of IFN-γ⁺ cells in liver NK cells, liver CD25⁻ ILC1, and liver CD25⁺ ILC1 (isolated from mice before (naïve) and 18 h after CCl₄ injection) after stimulation with or without IL-12 and ATP. Data were pooled from 3 experiments (n = 10–16). Data are representative of 7 (A) and 2 (C, D, E, and H) independent experiments. *p<0.05, **p<0.005. Error bars show s.d. See also Figure S5.

Figure 6.. IFN-γ contributes to the survival of hepatocytes through upregulation of Bcl-xL

(A) Expression of IFN-γR1 on hepatocytes (n = 2). (B) Expression of intracellular Bcl-2, Bcl-xL, and Mcl-1 in hepatocytes of WT and Ifng^−/− mice before (naïve) and 18 h after CCl₄ injection (n = 2–3). (C) MFI of intracellular Bcl-2, Bcl-xL, and Mcl-1 in hepatocytes of WT and Ifng^−/− mice before and 18 h after CCl₄ injection. Data were pooled from 2 experiments (n = 2–6). (D) Expression of Bcl-xL in hepatocytes cultured in the presence or absence of CCl_4, IFN-γ, or both (n = 3). (E) MFI of Bcl-xL in hepatocytes cultured in the presence or absence of CCl_4, IFN-γ, or both (n = 3). (F) Survival of hepatocytes cultured in the presence or absence of CCl_4, IFN-γ, or both (n = 4). The percentages of living hepatocytes are shown. Data are representative of 3 (A, D, E, and F) and 2 (B) independent experiments. *p<0.05. **p<0.01, ***p<0.005. Error bars show s.d. See also Figure S6.

Figure 7.. Liver ILC1-derived IFN-γ is sufficient to ameliorate CCl₄-induced acute liver injury

(A) Experimental design of ILC1 transfer into Rag2^−/−Il2rg^−/− mice. (B) Phenotypical characterization of donor-derived ILC1 in the liver of recipient Rag2^−/−Il2rg^−/− mice that had been transferred with purified donor ILC1 together with or without IL-15 (n = 2–4). (C) The number of donor-derived ILC1 in the liver of recipient Rag2^−/−Il2rg^−/− mice that had been transferred with purified donor ILC1 together with or without IL-15. Data were pooled from 4 experiments (n = 4). (D) Expression of CD25 on donor-derived ILC1 in the liver of Rag2^−/−Il2rg^−/− mice (that had been transferred with purified donor ILC1 together with IL-15) before (naïve) and 18 h after CCl₄ injection on day 7 post-transfer (n = 2–3). (E) MFI of CD25 on donor-derived ILC1 in the liver of Rag2^−/−Il2rg^−/− mice (that had been transferred with purified donor ILC1 together with IL-15) before and 18 h after CCl₄ injection on day 7 post-transfer. Data were pooled from 2 experiments (n = 4). (F) Expression of CD69 on donor-derived ILC1, CD25⁻ ILC1, and CD25⁺ ILC1 in the liver of Rag2^−/−Il2rg^−/− mice (that had been transferred with purified donor ILC1 together with IL-15) before and 18 h after CCl₄ injection on day 7 post-transfer (n = 2–3). (G) MFI of CD69 on donor-derived CD25⁻ ILC1 and CD25⁺ ILC1 in the liver of Rag2^−/−Il2rg^−/− mice (that had been transferred with purified donor ILC1 together with IL-15) before and 18 h after CCl₄ injection on day 7 post-transfer. Data were pooled from 2 experiments (n = 4). (H) Kinetics of IFN-γ⁺ cells in donor-derived CD25⁻ ILC1 and CD25⁺ ILC1 in the liver of Rag2^−/−Il2rg^−/− mice (that had been transferred with purified donor ILC1 together with IL-15) after CCl₄ injection. Data were pooled from 4 experiments (n = 5–15). *p<0.005 vs. CD25⁻ ILC1. (I) The percentages of IFN-γ⁺ cells in donor-derived ILC1, CD25⁻ ILC1, and CD25⁺ ILC1 in the liver of Rag2^−/−Il2rg^−/− mice (that had been transferred with purified donor ILC1 together with IL-15) before and 15 h after CCl₄ injection (n = 4–6). (J) The percentages of IFN-γ⁺ cells in donor-derived CD25⁻ ILC1 and CD25⁺ ILC1 in the liver of Rag2^−/−Il2rg^−/− mice (that had been transferred with purified donor ILC1 together with IL-15) before and 15 h after CCl₄ injection (n = 4–6). (K) Plasma concentrations of ALT in Rag2^−/−Il2rg^−/− mice (that had been transferred or not with purified donor ILC1 together with IL-15) before and 18 h after CCl₄ injection on day 7 post-transfer, which were also injected with a depletion anti-CD25 mAb (left), a neutralizing anti-IFN-γ mAb (right), or isotype Ig 6 h before and 6 h after CCl₄ injection. Data were pooled from 2 (left) (n = 4–5) and 3 (right) (n = 6–7) experiments. Data are representative of 4 (B) and 3 (D, F, I, and J) independent experiments. *p<0.05, **p<0.01, ***p<0.005. Error bars show s.d. See also Figure S7.