Oral N-acetylcysteine decreases IFN-γ production and ameliorates ischemia-reperfusion injury in steatotic livers

Abstract

Type 1 Natural Killer T-cells (NKT1 cells) play a critical role in mediating hepatic ischemia-reperfusion injury (IRI). Although hepatic steatosis is a major risk factor for preservation type injury, how NKT cells impact this is understudied. Given NKT1 cell activation by phospholipid ligands recognized presented by CD1d, we hypothesized that NKT1 cells are key modulators of hepatic IRI because of the increased frequency of activating ligands in the setting of hepatic steatosis. We first demonstrate that IRI is exacerbated by a high-fat diet (HFD) in experimental murine models of warm partial ischemia. This is evident in the evaluation of ALT levels and Phasor-Fluorescence Lifetime (Phasor-FLIM) Imaging for glycolytic stress. Polychromatic flow cytometry identified pronounced increases in CD45+CD3+NK1.1+NKT1 cells in HFD fed mice when compared to mice fed a normal diet (ND). This observation is further extended to IRI, measuring ex vivo cytokine expression in the HFD and ND. Much higher interferon-gamma (IFN-γ) expression is noted in the HFD mice after IRI. We further tested our hypothesis by performing a lipidomic analysis of hepatic tissue and compared this to Phasor-FLIM imaging using "long lifetime species", a byproduct of lipid oxidation. There are higher levels of triacylglycerols and phospholipids in HFD mice. Since N-acetylcysteine (NAC) is able to limit hepatic steatosis, we tested how oral NAC supplementation in HFD mice impacted IRI. Interestingly, oral NAC supplementation in HFD mice results in improved hepatic enhancement using contrast-enhanced magnetic resonance imaging (MRI) compared to HFD control mice and normalization of glycolysis demonstrated by Phasor-FLIM imaging. This correlated with improved biochemical serum levels and a decrease in IFN-γ expression at a tissue level and from CD45+CD3+CD1d+ cells. Lipidomic evaluation of tissue in the HFD+NAC mice demonstrated a drastic decrease in triacylglycerol, suggesting downregulation of the PPAR-γ pathway.

Keywords: IFN-gamma; N-acetylcysteine (NAC); NKT (natural killer T) cell; gadoxetate disodium; hepatic steatosis; ischemia-reperfusion injury (IRI); liver transplantation; phasor-FLIM.

Figure 1

Effects of HFD on liver function as assessed by DCE-MRI with the contrast agent Gadoxetate Disodium (Eovist^® Bayer). (A) Representative images of 26-30 week old male mice undergoing DCE-MRI, showing T1-weighted images performed before Eovist^® injection and at 4, 12 and 20 minutes post-injection (P-I). The yellow circle denotes a typical region of interest used to measure mean intensity. (B) Time course of relative liver enhanced T1 signal intensities after Eovist^® injection, comparing mice on ND (n=4), ND + NAC (n=5), HFD (n=5), and HFD + NAC (n=5). The differences in mean intensity between HFD and all the other groups (ND, ND + NAC and, HFD + NAC) were significant at 18- and 30-minutes post-injection. (C) Area under the curve (AUC) calculated from the data in (B). (D) The slope of T1 signal intensity increase for each group shown, corresponding to the rates of change within the first 20 min after Eovist^® injection. Statistical significance was established using a one-way ANOVA multiple comparisons test. “*” 0.05 to 0.01.

Figure 2

HFD Mice are prone to more severe IRI, which is significantly improved with NAC supplementation. (A) Serum ALT level following 24 hours of reperfusion. Number of mice per group is as follows: ND=11, ND (IRI)=13, ND + NAC= 5, ND + NAC (IRI)=10, HFD=5, HFD (IRI)=15, HFD + NAC, HFD + NAC (IRI)=8. (B) Representative images of baseline T2 mapping of mice on i) ND, iii), ND + NAC, v) HFD and vii) HFD + NAC. Representative images of T2 mapping twenty-four hours after IRI surgery of ii) ND, iv) ND + NAC, vi) HFD and viii) HFD + NAC. Blue represents liver parenchyma while green denotes edema. Graphical representation (xi) of the average of the T2 relaxation times of ROIs placed on the right and left areas of the corresponding livers (n=3-5 per group). “*” 0.05 to 0.01, “***” 0.001 to 0.0001, and “****” <0.0001.

Figure 3

IRI results in a higher degree of glycolytic stress in HFD mice. (A) Color mapped Phasor FLIM images of the ND and HFD liver samples in the absence (left) and presence (right) of NAC supplementation. The top row shows the sham samples, and the bottom row shows the IRI samples. More green, blue and red color represents more protein-bound NADH, free NADH and long lifetime species (LLS), respectively. (B) Phasor plot of autofluorescence FLIM in liver samples – the three colored circles represent the lifetime position of the three components: free and bound NADH, and LLS. (C) The concentration ratio of free to bound NADH was calculated from each mouse. The higher value represents more glycolytic stress. There is a higher level of glycolytic stress in HFD and HFD (IRI) samples. This is reduced in the HFD + NAC (IRI) samples, but not the HFD + NAC samples, respective to their HFD counterparts. (D) The fractional intensity of LLS calculated using three-component calculations to show the increasing LLS in high fat liver and how that increases with IRI (n=5-7 mice per group for both analyses). “*” 0.05 to 0.01.

Figure 4

NAC treatment significantly reduced hepatic steatosis in HFD mice. (A) The fluorescence intensity image, the LLS mapped image, the calculated size distribution maps, and the corresponding phasor plot. The red cursor was used to select the LLS lifetime signature originating from the intensity image and the LLS image was colored according to that phasor signature. The size map was created to show the distribution of the droplets. (B, C) The size map was created to show the distribution of the droplets. (B, C) The size distribution plots for Control (B) and NAC (C) treatment. The Y axis is shown in logscale to exemplify the increasing size as a function of diet and IRI. The distribution shows HFD and IRI induce the large lipid droplets and (especially above 10000 pixels) are lower in number in HFD + NAC, groups. (n=5-7 mice per group). (D) The average size distribution of lipid droplets from the livers of the animals in different feeding regimen. “*” 0.05 to 0.01, “**”0.01 to 0.001.

Figure 5

NAC treatment in HFD mice significantly altered the lipid profile within the liver. (A) Heat map demonstrating the alterations in lipid composition with the respective tissue. (B) Volcano plots demonstrating the differences in upregulated lipid species in HFD vs. ND and HFD vs. HFD + NAC mice, ultimately revealing changes TAG composition within these tissues. (C) Histogram representing quantitative differences in TAG composition between the treatment groups. “****” <0.0001.

Figure 6

IRI induced specific genetic upregulation in HFD mice. RT² profiler PCR array for cytokines and chemokines was utilized to examine differences in genetic regulation among treatment groups. Each group represents a single pooled sample from at least three separate liver-tissue specimens. (A) Heat map of cytokine and chemokine PCR analysis. (B) Venn Diagram demonstrating genes that are upregulated in HFD (IRI) and those shared amongst ND (IRI) and HFD (IRI).

Figure 7

IFN-γ producing Hepatic CD3+/NK1.1+ cells are contributors following IRI and successfully ameliorated with NAC supplementation. (A) Representative contour plots of the percentage of CD45+/CD3+/NK1.1+ NKT1 cells within Hepatic Mononuclear cells. Number of mice per group is as follows: ND=6, ND (IRI)=5, ND + NAC= 5, ND + NAC (IRI)=11, HFD=5, HFD (IRI)=5, HFD + NAC=5, HFD + NAC (IRI)=10. (B) Representative contour plots of the percentage of IFN-γ producing CD45+/CD3+/NK1.1+ NKT cells after IRI. Number of mice per group is as follows: ND (IRI)=5, ND + NAC (IRI)=5, HFD (IRI)=7, HFD + NAC (IRI)=10. (C) Histogram demonstrating the percentage of NKT1 cells illustrated in (A). (D) Histogram demonstrating the percentage of NKT1 cells illustrated in (B). “*” 0.05 to 0.01, “**”0.01 to 0.001, “***” 0.001 to 0.0001.

Figure 8

IFN-γ producing hepatic CD3+/CD1d+ cells are contributors following IRI and successfully ameliorated with NAC supplementation. (A) Representative contour plots of the percentage of IFN-γ producing CD45+/CD3+/CD1d+ NKT cells after IRI. Number of mice per group is as follows: ND (IRI)=5, ND + NAC (IRI)=5, HFD (IRI)=7, HFD + NAC (IRI)=10. (B) Histogram demonstrating the percentage of IFN-γ producing CD45+/CD3+/CD1d+ NKT cells illustrated in (A). “*” 0.05 to 0.01, “**”0.01 to 0.001.