Retained NK Cell Phenotype and Functionality in Non-alcoholic Fatty Liver Disease

Abstract

Non-alcoholic fatty liver disease (NAFLD), and the progressive stage non-alcoholic steatohepatitis (NASH), is the predominant cause of chronic liver disease globally. As part of the complex pathogenesis, natural killer (NK) cells have been implicated in the development of liver inflammation in experimental murine models of NASH. However, there is a lack of knowledge on how NK cells are affected in humans with this disease. Here, we explored the presence of disease-specific changes within circulating and tissue-resident NK cell populations, as well as within other major immune cell subsets, in patients with liver biopsy-confirmed NAFLD. Using 18-color-flow cytometry, substantial changes were observed in certain myeloid populations in patients as compared to controls. NK cell numbers, on the other hand, were not altered. Furthermore, only minor differences in expression of activating and inhibitory NK cell receptors were noted, with the exception of an increased expression of NKG2D on NK cells from patients with NASH. NK cell differentiation remained constant, and NK cells from these patients retain their ability to respond adequately upon stimulation. Instead, considerable alterations were observed between liver, adipose tissue, and peripheral blood NK cells, independently of disease status. Taken together, these results increase our understanding of the importance of the local microenvironment in shaping the NK cell compartment and stress the need for further studies exploring how NASH affects intrahepatic NK cells in humans.

Keywords: NAFLD; adipose tissue immunology; liver immunology; natural killer cells; obesity.

Figure 1

Immunophenotyping of major peripheral blood immune cell subsets in NAFL and NASH. (A) Flow cytometry gating scheme used to identify the investigated immune cell subsets. Arrows indicate the sequence of gating. (B) Summary data for the frequency out of total leukocytes for the indicated immune cell populations in healthy controls (n = 10), NAFL (n = 4), and NASH (n = 11) patients. (C) Summary data of pDC frequency out of total leukocytes (left), absolute counts of pDCs (middle), and correlation between pDC frequency and ALT (right) in the indicated patient groups. Bars in (B,C) represent mean and error bars show SEM. ^**p < 0.01.

Figure 2

Phenotypic characterization of circulating NK cells from NAFLD patients. (A) Representative flow cytometry plots of NK cells from healthy, NAFL, and NASH patients. (B) Frequency of CD56^bright NK cells out of total NK cells in peripheral blood of healthy controls (n = 13), NAFL patients (n = 9), and NASH patients (n = 16). (C) Representative histograms for the indicated markers on CD56^bright and CD56^dim NK cells as well as internal negative control. The plots represent stainings from one healthy donor. (D) Heat map depicting the mean frequency of NK cells expressing CD16, CD44, CD57, KIRs, NKG2A, and NKG2C as well as the mean MFI of CD69, NKp46, CD161, Eomes, T-bet, and NKG2D on CD56^dim and CD56^bright NK cells for the indicated groups. (E) Representative histogram of NKG2D expression on NK cells from healthy control, NAFL, and NASH patients respectively. (F,G) Scatter plots of NKG2D MFI on CD56^dim and CD56^bright NK cells from the indicated groups. In (F), healthy controls (n = 11), NAFL (n = 6), and NASH (n = 13) patients, in (G) healthy controls (n = 11), obese individuals (n = 18). (H) Bar graph showing the frequency of CD56^dim NK cells that express different combination of NKG2A, KIRs, and CD57. Black circles indicate presence of the marker and white circles no expression. (I) Inverse Simpson Diversity index (SDI) analysis for healthy (n = 11), NAFL (n = 7), and NASH patients (n = 12). The Mann–Whitney U-test was used for comparison between groups. Bars in (B,F,G,I) show mean, error bars in H represent SEM. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 3

Retained functional capacity of circulating NK cells in NAFLD. (A) Representative concatenated flow cytometry plots showing CD56^dim NK cell responses following the indicated stimulations. (B) Heat map summarizing the mean frequency of responding NK cells for the measured functions and indicated stimulations in healthy controls (n = 15), NAFL patients (n = 10), and NASH patients (n = 16). (C) Multifunctional analysis of the combination of degranulation (CD107a) and cytokine production (IFN-γ, MIP-1β, and TNF) responses for IL-12 + IL-18 stimulation and/or co-culture with K562 target cells. (D) SNE plots of total cells from healthy donors subsequently divided in stimulated and un-stimulated cells. The residual plot highlights the decreased (blue) or more highly expressed (red) areas in the stimulated cells. Underneath, individual plots of the expression of 15 single markers are shown. (E) SNE plots depicting the difference between CD56^dim (left) and CD56^bright (right) NK cells from NAFL and NASH patients after IL-12 + IL-18 + K562 stimulation where the density plots highlight the specific changes in NASH patients.

Figure 4

Phenotype of NK cells in liver and adipose tissue. (A) Representative flow cytometry plots of NK cells derived from blood, liver, and adipose tissue. (B) Scatter plot of the frequency of total NK cells out of viable leukocytes and CD16⁻ NK cells out of total NK cells in peripheral blood, liver, and adipose tissue. PBMC (n = 22), adipose tissue (n = 19), and liver (n = 15). (C) Representative concatenated flow cytometry plots showing expression of differentiation markers NKG2A, KIRs, and CD57 on CD16⁻ or CD16⁺ NK cells in blood, liver, and adipose tissue. (D) Bar graphs showing frequency of cells expressing the indicated differentiation markers in CD16⁻ or CD16⁺ NK cells from blood, liver, and adipose tissue. PBMC (n = 22), adipose tissue (n = 19), and liver (n = 15). (E) SNE plot of total NK cells from adipose tissue (left) or liver (right) compared to peripheral blood. The residual plot highlights the decreased (blue) or more highly expressed (red) areas within tissue NK cells. (F,G) Representative flow cytometry plots showing CD69 and CD49a expression on total NK cells from the indicated tissues. (H) Expression of CD69 and CD49a on CD16⁻ and CD16⁺ NK cells in peripheral blood, liver, and adipose tissue. In H, CD69 on PBMC (n = 22), adipose tissue (n = 18), and liver (n = 15), CD49a on PBMC (n = 9), adipose tissue (n = 9), and liver (n = 7). Bars in (B,D,H) represent mean and error bars in (D,H) show SEM. ^**p < 0.01, ^***p < 0.001.

Figure 5

Tissue NK cells in relation to patient disease characteristics. (A) Representative hematoxylin and eosin stainings on normal, NAFL, and NASH liver sections. (B) Distribution of the patients in relation to age, BMI, and levels of alanine transferase (ALT) as a proxy for liver damage. (C) Heat map showing mean frequency of total, CD16⁺, and CD16⁻ NK cells for the indicated groups. (D) Heat map showing mean frequencies of NKG2A, CD57, KIRs, NKG2C, CD69, and CD49a expression on CD16⁻ and CD16⁺ NK cells for the indicated groups. ^*p < 0.05.