The Obese Liver Environment Mediates Conversion of NK Cells to a Less Cytotoxic ILC1-Like Phenotype - PubMed (original) (raw)
The Obese Liver Environment Mediates Conversion of NK Cells to a Less Cytotoxic ILC1-Like Phenotype
Antonia O Cuff et al. Front Immunol. 2019.
Abstract
The liver contains both NK cells and their less cytotoxic relatives, ILC1. Here, we investigate the role of NK cells and ILC1 in the obesity-associated condition, non-alcoholic fatty liver disease (NAFLD). In the livers of mice suffering from NAFLD, NK cells are less able to degranulate, express lower levels of perforin and are less able to kill cancerous target cells than those from healthy animals. This is associated with a decreased ability to kill cancer cells in vivo. On the other hand, we find that perforin-deficient mice suffer from less severe NAFLD, suggesting that this reduction in NK cell cytotoxicity may be protective in the obese liver, albeit at the cost of increased susceptibility to cancer. The decrease in cytotoxicity is associated with a shift toward a transcriptional profile characteristic of ILC1, increased expression of the ILC1-associated proteins CD200R1 and CD49a, and an altered metabolic profile mimicking that of ILC1. We show that the conversion of NK cells to this less cytotoxic phenotype is at least partially mediated by TGFβ, which is expressed at high levels in the obese liver. Finally, we show that reduced cytotoxicity is also a feature of NK cells in the livers of human NAFLD patients.
Keywords: ILC1; NAFLD; NK cells; TGFβ; immunometabolism; obesity.
Copyright © 2019 Cuff, Sillito, Dertschnig, Hall, Luong, Chakraverty and Male.
Figures
Figure 1
NK cells in the livers of obese mice are less cytotoxic than those in lean mice. (A) Immune cells were isolated from mouse livers. NK cells were identified by scatter, and as live CD45+ Lineage-negative NK1.1+ CD49b+ cells. ILC1 were identified as live CD45+ Lineage-negative NK1.1+ CD49a+ cells. The frequency of NK cells and ILC1 as a percentage of live CD45+ cells in the spleens and livers of lean and obese mice is shown. n = 12 mice per group, medians and IQRs are shown. (B) Intrahepatic leukocytes were cultured for 4 h in the presence of anti-CD107a and Brefeldin A. Representative CD107a staining of NK cells from a lean (left, blue) and an obese (right, red) mouse and summary data are shown. (C) Representative perforin staining in liver NK from a lean (blue trace) and an obese (red trace) mouse. MFI of perforin in splenic NK, liver NK, and liver ILC1 from lean and obese mice are shown. (D) Representative granzyme B staining in liver NK from a lean (blue trace) and an obese (red trace) mouse. Gray traces represent internal negative controls. MFI of granzyme B in splenic NK, liver NK, and liver ILC1 from lean and obese mice are shown. (E) NK cells were sorted from the spleens and livers of lean and obese mice and cultured with the cancerous NK cell target line YAC-1. YAC-1 death at 24 h is shown. (F) CTV-labeled RMA/S (NK cell targets) CTB-labeled RMA (recovery control) cells were mixed in a 1:1 ratio and intravenously injected into lean or obese mice. Representative input cells (leftmost, black) and recovered cells from lean (center, blue) and obese (rightmost, red) mice are shown. The RMA/S:RMA ratio in recovered splenocytes, normalized to input ratio, is shown. For panels (B–F), n = 6 mice per group; significance was determined using Mann Whitney _U_-Tests; medians and IQRs are shown.
Figure 2
Perforin-mediated killing promotes fibrosis in NAFLD. (A) Growth curves for wild type and perforin knockout mice on the obesogenic diet. (B) Liver weights for wild type and perforin knockout mice after 24 weeks on the obesogenic diet. (C) Plasma glucose levels. (D) Representative H&E and Picrosirius red staining in a wild type and a perforin knockout mouse. (E) Histological score in wild type and perforin knockout mouse livers, after 24 weeks on the obesogenic diet. (F) Plasma ALT levels. (G) Picrosirius red positive area in representative histological fields. (H,I) Acta2 and Col1a1 mRNA in livers of wild type and perforin knockout mice, after 24 weeks on the obesogenic diet. n = 8 mice per group; significance was determined using Mann Whitney _U_-Tests; medians and IQRs are shown.
Figure 3
NK cells in the livers of obese mice acquire an ILC1-like transcriptional profile. (A) NK cells and ILC1 were sorted from the livers of lean and obese mice and examined by RNASeq. Genes that were differentially expressed by >2-fold, with padj <0.05, in NK cells of lean vs. obese mice are shown. Genes that are also differentially expressed in ILC1 compared to NK (>2-fold, with padj <0.05) are marked with an asterisk. (B,C) Protein expression of the inhibitory receptors CD200R1 (B) and LAG3 (C) in NK cells and ILC1 of lean (blue) compared to obese (red) mice. Gray traces represent FMO controls. (D) Expression of Eomes in NK cells and ILC1 of lean (blue) compared to obese (red) mice. Gray traces represent internal negative controls. (E) Expression of CD49a in Lineage-negative NK1.1+ CD49b+ cells (NK) and Lineage-negative NK1.1+ CD49b− cells (ILC1) of lean (blue) compared to obese (red) mice. Gray traces represent FMO controls. n = 6 mice per group; significance was determined using Mann Whitney _U_-Tests; medians and IQRs are shown.
Figure 4
NK cells in the livers of obese mice are metabolically reprogrammed. Forward and side scatter (A), pS6 staining (B), and Glut1 staining (C) of freshly isolated NK cells and ILC1 from lean (blue) and obese (red) mice. n = 12 mice per group (A), six mice per group (B) and seven mice per group (C); significance was determined using Mann Whitney _U_-Tests; medians and IQRs are shown.
Figure 5
TGFβ in obese mouse livers limits NK cell degranulation and alters their metabolic profile. (A) Tgfb1 mRNA in the livers of lean and obese mice, normalized so that mean Tgfb1 transcript expression in the livers of lean mice = 1. (B,C) TGFβ1 in lean and obese liver conditioned medium (B) and lean and obese mouse plasma (C) measured by ELISA. n = 8 mice per group (A,C) and six conditioned media per group (B); significance was determined using Mann Whitney _U_-Tests; medians and IQRs are shown. (D) Splenic NK cells were cultured for 24 h in medium alone, lean or obese liver conditioned medium, or 10 ng/ml TGFβ1. For the last 4 h of culture, CD107a antibody and Brefeldin A were added to media. n = 6 culture conditions per group; significance was determined using Mann Whitney _U_-Tests with Holm's correction for multiple comparisons; medians and IQRs are shown. (E) Splenic NK cells were cultured as described in (D), with the addition of 10 μg/ml anti-TGFβ antibody or isotype control. n = 6 culture conditions per group; significance was determined using a Wilcoxon signed ranks test, where each pair is obese liver conditioned medium from the same mouse, with anti-TGFβ antibody or isotype control. Degranulation in NK cells cultured in lean liver conditioned medium with isotype control are shown for comparison, but were not statistically tested. (F) Splenic NK cells were cultured for 24 h in medium alone, lean or obese liver conditioned medium, or 10 ng/ml TGFβ1, before staining for pS6. n = 6 culture conditions per group; significance was determined using Mann Whitney _U_-Tests with Holm's correction for multiple comparisons; medians and IQRs are shown. (G,H) Splenic NK cells were cultured for 5 d in 10 ng/ml TGFβ1. CD49a (G) and CD200R (H) staining before and after culture are shown. For this small dataset, no statistical testing was carried out.
Figure 6
NK cells in the livers of NAFLD patients are less cytotoxic than those from healthy controls. (A) Immune cells were isolated from human livers. NK cells were identified by scatter, and as live CD45+ CD56+ CD3− Tbethi Eomeslo cells. Liver-resident NK cells (lrNK) were identified as live CD45+ CD56+ CD3− Tbetlo Eomeshi cells. (B,C) Intrahepatic leukocytes were cultured for 4 h in the presence of anti-CD107a and Brefeldin A. Representative CD107a staining of NK cells from a healthy (B) and a NAFLD (C) liver is shown. (D) Inverse correlation between NK cell degranulation and histological score in human livers. H, healthy; SS, simple steatosis; NAS, NASH Activity Score; C, cirrhosis. Significance was determined using Spearman's Rank Correlation. (E) CD56+ CD3− CD16+ CXCR6− NK cells were sorted from healthy (n = 4) and NAFLD (n = 3) livers and cultured for 4 h with the human NK target cell, K562. At the end of culture, target cell death was assessed using AnnexinV staining. (F) Inverse correlation between liver-resident NK cell degranulation and histological score in human livers. Significance was determined using Spearman's Rank Correlation. (G) CD56+ CD3− CXCR6+ liver-resident NK cells were sorted from healthy (n = 4) and NAFLD (n = 3) livers and cultured for 4 h with the human NK target cell, K562. At the end of culture, target cell death was assessed using AnnexinV staining. For (E,G), medians are shown. For these small datasets, no statistical testing was carried out.
References
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical
Molecular Biology Databases