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. 2021 Dec;20(12):e13512.
doi: 10.1111/acel.13512. Epub 2021 Nov 11.

Necroptosis contributes to chronic inflammation and fibrosis in aging liver

Sabira Mohammed  1 , Nidheesh Thadathil  2 , Ramasamy Selvarani  2 , Evan H Nicklas  2 , Dawei Wang  2 , Benjamin F Miller  2   3   4 , Arlan Richardson  1   2   3   5 , Sathyaseelan S Deepa  1   2   3
Affiliations

Necroptosis contributes to chronic inflammation and fibrosis in aging liver

Sabira Mohammed et al. Aging Cell. 2021 Dec.

Abstract

Inflammaging, characterized by an increase in low-grade chronic inflammation with age, is a hallmark of aging and is strongly associated with various age-related diseases, including chronic liver disease (CLD) and hepatocellular carcinoma (HCC). Because necroptosis is a cell death pathway that induces inflammation through the release of DAMPs, we tested the hypothesis that age-associated increase in necroptosis contributes to chronic inflammation in aging liver. Phosphorylation of MLKL and MLKL oligomers, markers of necroptosis, as well as phosphorylation of RIPK3 and RIPK1 were significantly upregulated in the livers of old mice relative to young mice and this increase occurred in the later half of life (i.e., after 18 months of age). Markers of M1 macrophages, expression of pro-inflammatory cytokines (TNFα, IL6 and IL1β), and markers of fibrosis were all significantly upregulated in the liver with age and the change in necroptosis paralleled the changes in inflammation and fibrosis. Hepatocytes and liver macrophages isolated from old mice showed elevated levels of necroptosis markers as well as increased expression of pro-inflammatory cytokines relative to young mice. Short-term treatment with the necroptosis inhibitor, necrostatin-1s (Nec-1s), reduced necroptosis, markers of M1 macrophages, fibrosis, and cell senescence as well as reducing the expression of pro-inflammatory cytokines in the livers of old mice. Thus, our data show for the first time that liver aging is associated with increased necroptosis and necroptosis contributes to chronic inflammation in the liver, which in turn appears to contribute to liver fibrosis and possibly CLD.

Keywords: aging; fibrosis; inflammation; liver; necroptosis; necrostatin-1s.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

FIGURE 1
FIGURE 1
Necroptosis markers increase with age in the livers of mice. (a) Left: Annexin VFITC/PI staining of liver cells from 7, 12, 18, and 22 to 24‐month‐old mice. The upper right quadrant (FITC+/PI+) represents late apoptotic/necroptotic population, the lower right quadrant (FITC+/PI−) represents early apoptotic population and the upper left quadrant (FITC−/PI+) represents the necrotic population. Right: Graphical representation of the early apoptotic, late apoptotic/necroptotic and necrotic populations. (b) Left: Immunoblots of liver extracts prepared from 7 (white bars), 12 (gray bars), 18 (blue bars), and 22 to 24‐month‐old (red bars) mice for PMLKL, MLKL and β‐tubulin. Right: Graphical representation of quantified blots normalized to β‐tubulin. (c) Left: Immunoblots of liver extracts for P‐RIPK3, RIPK3, P‐RIPK1, RIPK1 and β‐tubulin. Right: Graphical representation of quantified blots normalized to β‐tubulin. (d) Left: Immunostaining of liver sections from young (white bar), old (red bar) and Mlkl knockout (yellow bar) mice for P‐MLKL. Scale bar: 50 µM. Right: Graphical representation of the percentage of red fluorescent area. (e) Top: Immunoblots probed using anti‐MLKL antibody for oligomers (>250 kDa). β‐tubulin was used as loading control. Bottom: Graphical representation of quantified oligomer normalized to β‐tubulin. (f) Left: Immunoblots of plasma samples for HMGB1. Coomassie stained gel is used as loading control. Right: Graphical representation of quantified blot normalized to Coomassie stained gel. Data represented as mean ± SEM, *p < 0.05, **p < 0.005, ***p < 0.0005, n = 5–7 per group
FIGURE 2
FIGURE 2
Increased expression of necroptosis markers in hepatocytes isolated from old mice. (a) Left: Representative image of Annexin V/PI staining of hepatocytes isolated from young (white bars) and old mice (red bars). The upper right quadrant (FITC+/PI+) represents late apoptotic/necroptotic population, the lower right quadrant (FITC+/PI−) represents early apoptotic population and the upper left quadrant represents necrotic population. Right: Graphical representation of the early apoptotic, late apoptotic/necroptotic and necrotic populations. (b) Left: Immunoblots of hepatocytes isolated from young and old mice for P‐MLKL, MLKL and GAPDH. Right: Graphical representation of quantified blot normalized to GAPDH. (c) Immunostaining for the albumin (green) and P‐MLKL (red) in the livers from old mice. Arrows indicate co‐localization (yellow). Scale bar: 50 µM. (d) Left: Immunoblots of isolated hepatocytes from young and old mice for MLKL oligomer. Right: Graphical representation of quantified oligomer band normalized to GAPDH. (e) Top: Immunoblots of isolated hepatocytes from young and old mice for P‐RIPK3, RIPK3, P‐RIPK1, RIPK1 and GAPDH. Bottom: Graphical representation of quantified blots normalized to GAPDH. (f) Immunostaining for F4/80 (green) and P‐MLKL (red) in livers from old mice. Arrows indicate co‐localization (yellow). Scale bar: 50 µM. Data represented as mean ± SEM, *p < 0.05, **p < 0.005, ***p < 0.0005, n = 5–7/group
FIGURE 3
FIGURE 3
Markers of inflammation increase with age in the livers of mice. (a) Left: Representative flow cytometric analysis of CD45+ cells. Right: Graphical representation of the percentage population of CD45+ cells in the liver of young (white bar) and old mice (red bar), gated on live cell population. (b) Left: Representative flow cytometric analysis of F4/80+ cells. Right: Graphical representation of the percentage population of F4/80+ cells in the livers of young and old mice, gated on live, CD45+ cells. (c) Left: Representative flow cytometric analysis of M1 macrophages (CD80+ F4/80+ cells). Right: Graphical representation of the percentage population of M1 macrophages (CD80+ F4/80+ cells) in the livers of young and old mice, gated on live, CD45+ CD11b+ Ly6G− cells. (d) Left: Representative flow cytometric analysis of M2 macrophages (CD206+ F4/80+ cells). Right panel: Graphical representation of the percentage population of M1 macrophages (CD206+ F4/80+ cells) in the livers of young and old mice, gated on live, CD45+ CD11b+ Ly6G− cells. (e) Transcript levels of TNFα, IL6, IL‐1β in the livers of 7 (white bars), 12 (gray bars), 18 (blue bars), and 22 to 24‐month‐old (red bars) mice normalized to β‐ microglobulin and expressed as fold change. (f) Left: Immunoblots of liver extracts for TNFα, cleaved IL‐1β and β‐tubulin. Right: Graphical representation of quantified blots normalized to β‐tubulin. Transcript levels of TNFα, IL6, IL‐1β and MCP‐1 in isolated F4/80+ cells (g) hepatocytes (h) from young and old mice normalized to β‐microglobulin and expressed as fold change. Data represented as mean ± SEM, *p < 0.05, **p < 0.005, ***p < 0.0005, n = 5–7/group
FIGURE 4
FIGURE 4
Markers of fibrosis increase with age in the livers of mice. (a) Transcript levels of TGFβ in liver of 7 (white bars), 12 (gray bars), 18 (blue bars), and 22 to 24‐month‐old mice (red bars) normalized to β‐microglobulin and expressed as fold change. (b) Left: Immunoblots of liver extracts for desmin and β‐tubulin. Right: Graphical representation of quantified blots normalized to β‐tubulin. (c) Transcript levels of Col1α1, Col3α1 in livers normalized to β‐microglobulin and expressed as fold change. (d) Levels of hydroxyproline in the livers, expressed as microgram of hydroxyproline/g of liver tissue. (e) Left: Picrosirius red staining in young and old mice. Scale bar: 200µm. Right: Quantification of fibrotic area in young (white bar) and old (red bar) mice. Data represented as mean ± SEM, *p < 0.05, **p < 0.005, ***p < 0.0005, n = 7–10/group
FIGURE 5
FIGURE 5
Nec‐1s treatment reduces markers of necroptosis, inflammation, fibrosis, and hepatic damage in the livers of old mice. (a) Left: Immunoblots of liver extracts from young (7‐month, white bars), old (24‐month, red bars), and old mice treated with Nec‐1s (24 months, old‐Nec‐1s, green bars) for P‐MLKL, MLKL and β‐tubulin. Right: Graphical representation of quantified blots normalized to β‐tubulin. (b) Left: Immunoblots of non‐reduced samples for MLKL oligomer and β‐tubulin. Right: Graphical representation of quantified oligomer normalized to β‐tubulin. (c) Transcript levels of p16 and p21 normalized to β‐microglobulin and expressed as fold change. (d) Left: Immunostaining for F4/80, CD68 and CD206 (green). Nucleus counterstained with DAPI (blue). Scale bar: 75 µM. Right: Graphical representation of the percentage area of green fluorescence. (e) Left: Immunoblots of liver extracts for TNFα and β‐tubulin. Right: Graphical representation of quantified blots normalized to β‐tubulin. (f) ALT activity measured in the serum expressed as IU/L. (g) Levels of OHP expressed as microgram of hydroxyproline/g of liver tissue. (h) Left: Picrosirius red staining. Scale bar: 200 µm. Right: Quantification of staining. Data represented as mean ± SEM, *p < 0.05, **p < 0.005, ***p < 0.0005, n = 5–7/group
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