Kupffer Cells Undergo Fundamental Changes during the Development of Experimental NASH and Are Critical in Initiating Liver Damage and Inflammation - PubMed (original) (raw)

Kupffer Cells Undergo Fundamental Changes during the Development of Experimental NASH and Are Critical in Initiating Liver Damage and Inflammation

D T Reid et al. PLoS One. 2016.

Abstract

Non-alcoholic fatty liver disease has become the leading liver disease in North America and is associated with the progressive inflammatory liver disease non-alcoholic steatohepatitis (NASH). Considerable effort has been made to understand the role of resident and recruited macrophage populations in NASH however numerous questions remain. Our goal was to characterize the dynamic changes in liver macrophages during the initiation of NASH in a murine model. Using the methionine-choline deficient diet we found that liver-resident macrophages, Kupffer cells were lost early in disease onset followed by a robust infiltration of Ly-6C+ monocyte-derived macrophages that retained a dynamic phenotype. Genetic profiling revealed distinct patterns of inflammatory gene expression between macrophage subsets. Only early depletion of liver macrophages using liposomal clodronate prevented the development of NASH in mice suggesting that Kupffer cells are critical for the orchestration of inflammation during experimental NASH. Increased understanding of these dynamics may allow us to target potentially harmful populations whilst promoting anti-inflammatory or restorative populations to ultimately guide the development of effective treatment strategies.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1

MCD diet feeding results in liver injury and inflammation (A) Timeline of the development of steatosis, NASH and fibrosis in mice fed MCD diet. Schematic represents timeline of data collection (red arrows) during MCD diet feeding treatment (n = 7-9/group). (B) ALT levels are significantly elevated after 7 (p<0.05), 14 (p<0.01) and 21 days (p<0.001) of feeding compared to D0. (C) Mice fed MCD diet for 7, 14 and 21 days had a significantly greater NAS score compared to mice at D0 (p<0.0001). Mice fed MCD diet for 14 and 21 days developed steatohepatitis compared to mice fed MCD diet for 7 days (p<0.0001). (D) Representative images of H&E stained liver sections following 0, 7, 14 or 21 days of MCD diet feeding. White arrow indicates lipid accumulation and black arrow indicates inflammatory foci. 20x magnification, PT: portal tract, CV: central vein.

Fig 2

Fig 2. Changes in resident and recruited macrophage populations.

(A) Effect of MCD diet feeding over 21 days on resident macrophages and recruited populations of monocyte-derived macrophages (gated on F4/80+). (B) MCD diet feeding resulted in significantly more cellular infiltration after 21 days compared to D7 (p<0.05). (C) One week of MCD diet feeding resulted in significantly reduced numbers of Ly-6Clo liver-resident macrophages compared to D21 (p<0.05). (D) Numbers of Ly-6Cint monocyte-derived macrophages was significantly elevated after 21 days of MCD diet feeding (p<0.05). (E) Recruitment of Ly-6Chi monocyte-derived macrophages was significantly elevated between D7 and D14 (p<0.05) and after 21 days of MCD diet feeding compared to D0 and D7 (p<0.0001) and D14 (p<0.05).

Fig 3

Fig 3

Transcriptomics analysis of resident and recruited macrophages in the liver during MCD diet treatment (A) Fluorescence-activated cell sorting gating strategy for liver macrophages. Sorting purity exceeded 90% for all populations. (B) Gene expression profile of inflammation genes associated with steatohepatitis reveals differences between circulating monocytes, resident KC and recruited Ly-6C+ macrophage populations. (C) Genes highlighted by yellow boxes demonstrate changes in expression levels, either increased or decreased, in the known cytokine-cytokine receptor interaction pathway between sorted cell populations depicted using the Kyoto Encyclopedia of Genes and Genomes pathway mapper mmu04060.

Fig 4

Fig 4

Cell surface expression of chemokines during steatohepatitis (A) Recruitment of Ly-6C+ monocyte-derived macrophages can be characterized by CCR2 and CX3CR1 expression. (B) Infiltration of Ly-6Chi monocyte-derived macrophages is partially dependent on CCR2 and CX3CR1. (C) Ly-6Cint monocyte-derived macrophages are recruited in part to the liver via CX3CR1 and CCR2. (D) Kupffer cells do not express CCR2 or CX3CR1. (E) Ly-6Clo macrophages are predominately CCR2+ appearing only after 14 days of MCD diet feeding.

Fig 5

Fig 5

Leukocyte recruitment is a later event (A) Emergence of leukocytes is a late event following MCD diet treatment. (B-C) CD4+ and CD8+ T lymphocytes are observed after 14 days of MCD diet feeding. (D) NK cells and NKT cells are more prominent on D21 compared to D14.

Fig 6

Fig 6

CLL treatment protects mice from steatohepatitis (A) Regime of clodronate-loaded liposome (CLL) treatment or PBS initiated at D0, D7 or D14 (green arrows) and repeated on a weekly basis until termination of the study (red arrow). All groups of mice (n = 8/group) were fed MCD diet for the same three-week duration. (B) A significant reduction in serum ALT was observed when CLL was initiated at D0 (p<0.05) and D7 (p<0.01) compared to PBS control whereas no difference was detected when CLL was started at D14. (C) Mice are protected from developing steatohepatitis when CLL is initiated on D0 (p<0.001) and D7 (p<0.0001) compared to PBS control but not when CLL begins at D14 of MCD diet feeding. (D) Representative images of H&E stained liver sections after 21 days of MCD diet feeding following CLL treatment at D0, D7 or D14. 20x magnification, PT: portal tract, CV: central vein.

Fig 7

Fig 7

Recruitment of Ly-6C+ monocytes is reduced with CLL treatment (A) Clodronate-loaded liposome (CLL) treatment or PBS initiated at onset of MCD diet feeding leads to significant changes in Ly-6C+ cell populations. (B) A significant reduction in Ly-6Chi (p<0.05), Ly-6Cint (p<0.05) and Ly-6Clo (p<0.01) populations can be detected after 21 days of MCD diet feeding when CLL treatment is initiated at onset of MCD diet feeding. (C) Ly-6Chi monocyte-derived macrophages are significantly reduced when CLL treatment is initiated at D7 of MCD diet feeding compared to PBS control (p<0.05). (D) No difference between cell populations is detected when CLL treatment is initiated 14 days after MCD diet feeding is started.

Fig 8

Fig 8. Reduction of leukocytes following CLL treatment.

(A-B) Clodronate-loaded liposome (CLL) treatment or PBS initiated at onset of MCD diet feeding leads to significant changes in lymphoid cell populations (CD4+: blue CD8+: red). (C) A significant reduction in CD4+ (p<0.05) and CD8+ (p<0.05) T cell populations can be detected after 21 days of MCD diet feeding when CLL treatment is initiated at onset of MCD diet feeding. (D-E) No difference is detected in CD4+ and CD8+ lymphocytes when CLL is initiated at D7 or D14 following MCD diet treatment. (F-G) A significant reduction in NK cells (p<0.01) and NKT cells (p<0.01) can be detected after 21 days of MCD diet feeding when CLL treatment is initiated at onset of MCD diet feeding.

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Grants and funding

DTR – Natural Sciences and Engineering Research Council of Canada Postgraduate Doctoral Scholarship, Alberta Innovates Technology Futures Graduate Scholarship, Cumming School of Medicine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. BE—Canadian Institutes of Health Research Signature Initiative Team Grant in Health Challenges in Chronic Inflammation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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