Authors
M. VAN HERCK (1), L. VONGHIA (2), P. MICHIELSEN (2), C. BRIDTS (3), D. EBO (3), J. DE MAN (4), B. DE WINTER (4), S. FRANCQUE (2) / [1] Universiteit Antwerpen / Antwerp University Hospital, WILRIJK (Antwerpen), Belgium, Laboratory of Experimental Medicine and Pediatrics, Division of Gastroenterology and Hepatology, [2] Universiteit Antwerpen / Antwerp University Hospital, WILRIJK (Antwerpen), Belgium, Gastroenterology and Hepatology, [3] Universiteit Antwerpen / Antwerp University Hospital, WILRIJK (Antwerpen), Belgium, Immunology - Allergology - Rheumatology, [4] University of Antwerp, Antwerp, Belgium, Laboratory of Experimental Medicine and Pediatrics, Division of Gastroenterology and Hepatology
Introduction
Non-alcoholic fatty liver disease (NAFLD) is a multisystem condition in which the liver, adipose tissue and the immune system are involved. T cells form a part of the adaptive immune system and can be subdivided in several subsets with distinct functions. We previously demonstrated that mice with severe NAFLD exhibit elevated hepatic T helper 17 (Th17) cells (CD4+ RORγt+), an abundance of visceral adipose tissue (VAT) CD8+ cytotoxic T (Tc) cells and a reduction of VAT regulatory T (Treg) cells (CD4+ CD25+ Foxp3+).
Aim
This study aimed at investigating the potential reversibility of these alterations upon diet reversal.
Methods
Male 8-week old C57BL/6J mice were fed a high-fat high-fructose diet (HFHFD) for 20 weeks. Subsequently, a diet reversal (DR) was performed by substituting the HFHFD with control diet (CD) and continuing the CD for 12 additional weeks. Three control groups were included: mice fed CD for 32 weeks, HFHFD for 20 weeks and HFHFD for 32 weeks. Liver tissue was assessed histologically and the NAFLD Activity Score (NAS) was calculated. T-cell subsets were characterised in liver and visceral tissue (VAT) via flow cytometry. Tc cells were expressed as a percentage of CD45+ CD3+ cells, Th17 and Treg cells as a percentage of CD3+ CD4+ cells. Data are represented as [median (IQR), p-value].
Results
Compared to CD mice, HFHFD-feeding for 20 and 32 weeks confirmed the previous findings of metabolic alterations, NAFLD development, and the associated hepatic and VAT T-cell alterations, specifically an increase in hepatic Th17 cells [1.6% (1.9) vs. 2.9% (2.8) and 5.7% (11.8) resp., p=0.031) and VAT Tc cells [22.3% (11.5) vs. 40.2% (11.5) and 37.7% (6.7) resp., p=0.004], as well as a reduction in VAT Treg cells [37.4% (28.2) vs. 12.3% (8.9) and 8.5% (9.5) resp., p=0.002]. Compared to mice fed HFHFD for 20 and 32 weeks, DR induced weight loss [resp. 47.3g (6.7) and 52.2g (6.6) vs. 39.4g (9.1), p<0.001], a decrease in cholesterol levels [resp. 140 mg/dL (37) and 170 mg/dL (33) vs. 62 mg/dL (16), p<0.001] and a decrease in NAS [resp. 4 (1) and 6 (1) vs. 1 (3), p<0.001]. No significant difference was observed in NAS between CD-fed mice and DR mice (p=0.160). Conversely, the alterations in hepatic and VAT T cell subsets were not affected by DR: hepatic Th17 cell and VAT Tc levels were significantly higher in DR mice compared to CD-fed mice [4.3% (2.2) vs. 1.5% (1.9), p=0.015 and 35.5% (6.5) vs. 22.3% (11.5), p=0.003 resp.], whereas no significant difference existed between the DR group and mice fed HFHFD for 20 and 32 weeks (p=0.594 and p=0.285 resp.). VAT Treg levels were significantly lower in DR mice compared to CD-fed mice [9.3% (7.1) vs. 37.4% (28.2), p=0.003], whereas no difference existed between the DR group and mice fed HFHFD for 20 and 32 wks (p=0.149).
Conclusions
Although diet reversal induced a metabolic and histological normalisation in HFHFD-fed mice, the HFHFD-induced alterations in hepatic Th17 cells, VAT Tc cells and VAT Treg cells were not reversed within a timeframe of 12 weeks. This finding challenges our current understanding of the reversibility of NAFLD-related inflammation upon life style modification.
