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. 2022 Nov;237(11):4215-4225.
doi: 10.1002/jcp.30877. Epub 2022 Sep 13.

Differential recruitment of monocyte-derived macrophages in control and stellate cell-depleted mice during recurrent carbon tetrachloride-induced acute liver injury

Akanksha Sharma  1   2 , Ramesh Kudira  1 , Jiang Wang  3 , Alexander Miethke  1 , Chandrashekhar R Gandhi  1   2   4
Affiliations

Differential recruitment of monocyte-derived macrophages in control and stellate cell-depleted mice during recurrent carbon tetrachloride-induced acute liver injury

Akanksha Sharma et al. J Cell Physiol. 2022 Nov.

Abstract

Liver depleted of hepatic stellate cells (HSCs) is resistant to ischemia/reperfusion-, concanavalin A-, and acetaminophen-induced acute injury. Whether HSCs regulate carbon tetrachloride (CCl4 )-induced acute liver injury is not known. CCl4 treatment damages pericentral hepatocytes that express CCl4 -metabolizing Cyp2E1 and activates HSCs. We investigated whether HSC-depletion in mice transgenic for thymidine kinase under the glial fibrillary acidic protein promoter (GFAP-TK-Tg) confers resistance to injury and inflammation due to CCl4 rechallenge. GFAP-TK-Tg or wild type (WT) mice were administered 0.16 ml/kg CCl4 (3× at 3 days intervals), then 40 μg/g/day ganciclovir for 10 days. The treatment depletes ~70%-75% HSCs from GFAP-TK-Tg but not WT mice while the liver recovers from earlier CCl4 -induced injury. Mice were then administered CCl4 , and liver injury and inflammation were determined at 24 h. HSC-depleted and HSC-sufficient mice showed similar CCl4 -induced hepatocyte necrosis and oxidative stress. However, increase in F4/80+ macrophages, but not CD68+ cells, was greater in CCl4 rechallenged HSC-depleted compared to HSC-sufficient mice. Expression of tumor necrosis factor-α (TNF-α), CCL2, and CXCL1 increased similarly, whereas increase in interleukin-6 (IL6), IL1β, and IL10 expression was higher in CCl4 rechallenged HSC-depleted compared to HSC-sufficient mice. CCl4 rechallenge of HSC-sufficient mice rapidly activated HSCs causing significant fibrosis with increased expression of Col1a1, transforming growth factor β1 (TGFβ1), tissue inhibitors of metalloproteinases 1 (TIMP1); increase in TIPM1 was much lower and metalloproteinases 13 (MMP13) greater in CCl4 rechallenged HSC-depleted mice. Interestingly, hepatic recruitment of both profibrogenic (Ly6Chi ) and antifibrogenic restorative (Ly6Clo ) macrophages, and neutrophils was significantly greater in CCl4 rechallenged HSC-depleted mice. These data suggest that CCl4 directly damages hepatocytes but HSCs regulate inflammation. Rapid fibrogenesis in CCl4 rechallenged HSC-sufficient mice recovered from earlier injury indicates that even transiently activated HSCs that had reverted to the quiescent phenotype remain primed to become reactivated.

Keywords: acute liver injury; fibrosis; inflammation; macrophages; stellate cells.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

FIGURE 1
FIGURE 1
CCl4-induced liver injury, recovery and rechallenge. WT-B6 mice were subjected to 3-CCl4 administrations and euthanized 24 h after the last CCl4 administration (3-CCl4) or allowed to recover for 10 days (recovered). The recovered mice were administered vehicle (oil) or CCl4 (1-CCl4) and euthanized at 24 h. Naïve mice were euthanized 24 h after single-CCl4 administration. (a) H/E-stained liver sections show necrotic damage and inflammation (scale bar = 50 μm). (b) Inflammation scores. (c) Percent necrotic area. (d) Serum ALT levels. (e) 4-HNE staining demonstrates oxidative stress (scale bar = 100 μm). Liver sections were stained for (f) F4/80 or (g) CD68 (scale bar = 20 μm). (h) Expression of indicated cytokines by qRT-PCR. (i) Hepatic concentrations of indicated cytokines. ALT, alanine transaminase; CCl4, carbon tetrachloride; HNE, 4-hydroxy-2-nonenal; qRT-PCR, quantitative reverse transcription polymerase chain reaction; WT, wild type. *p < 0.05; **p < 0.01; ***p < 0.001 versus control.
FIGURE 2
FIGURE 2
CCl4-induced fibrosis, recovery, and recurrence. WT-B6 mice were subjected to 3-CCl4 administrations and euthanized 24 h after the last CCl4 administration (3-CCl4) or allowed to recover for 10 days (recovered). The recovered mice were administered vehicle (oil) or CCl4 (1-CCl4) and euthanized at 24 h. Naïve mice were euthanized 24 h after single CCl4 administration. Liver sections stained (a) for αSMA (scale bar = 50 μm) or (b) with Sirius Red (scale bar = 100 μm). Bar graphs show quantification of (c) αSMA-stained areas or (d) Sirius Red-stained areas. (e–j) Hepatic mRNA expression of indicated proteins by qRT-PCR. αSMA, smooth muscle α-actin; CCl4, carbon tetrachloride; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; WT, wild type. *p < 0.05; **p < 0.01; ***p < 0.001 versus control or as indicated.
FIGURE 3
FIGURE 3
Hepatic injury, oxidative stress, and macrophages and cytokines in CCl4-challenged HSC-depleted mice. HSC-sufficient or HSC-depleted mice were challenged with single CCl4 administration and euthanized at 24 h. (a) H/E-stained liver sections show hepatic necrosis and inflammation (scale bar = 100 μm). (b) Serum ALT levels. (c) Oxidative stress as determined by 4HNE staining (scale bar = 100 μm) and its quantification (bar graph). (d) Liver sections stained for F4/80 or CD68 (scale bar = 20 μm); bar graph shows quantification. (e) Expression of indicated cytokines as measured by qRT-PCR. (f) Hepatic concentrations of indicated cytokines. ALT, alanine transaminase; CCl4, carbon tetrachloride; HSC, hepatic stellate cells; IL6, interleukin-6; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; TNF-α, tumor necrosis factor-α. *p < 0.05; **p < 0.01; ***p < 0.001 versus control or as indicated.
FIGURE 4
FIGURE 4
Hepatic fibrosis in CCl4-challenged HSC-depleted mice. HSC-sufficient or HSC-depleted mice were challenged with a single CCl4 injection and euthanized at 24 h. Liver sections were stained (a) for αSMA (scale bar = 100 μm) or (b) with Sirius Red (scale bar = 100 μm). (c–e) Hepatic mRNA expression of indicated proteins as measured by qRT-PCR. αSMA, smooth muscle α-actin; CCl4, carbon tetrachloride; HSC, hepatic stellate cells; MMP, matrix metalloproteinases; mRNA, messenger RNA; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; TGFβ1, transforming growth factor β1; TIMP1, tissue inhibitors of metalloproteinase 1. *p < 0.05; **p < 0.01; ***p < 0.001 versus control or as indicated.
FIGURE 5
FIGURE 5
Macrophage populations in CCl4 rechallenged HSC-sufficient and HSC-depleted mice. Representative images show hepatic mononuclear cells in untreated control mice, and CCl4-challenged HSC-sufficient or HSC-depleted mice as analyzed by flow cytometry. (a) FACS plots show Kupffer cells (KC) (F4/80+CD11blow/−Ly6G−Lin−CD45+eflour506−), monocyte-derived macrophages (MM) (F4/80+CD11b+Ly6G−Lin−CD45+eflour506−), and monocytes (M) (CD11b+F4/80−Ly6G−Lin−CD45+eflour506−). (b) CD11b+F4/80−Ly6G−Lin−CD45+eflour506− cells, further characterized for proinflammatory monocytes (Ly6C+CD11b+F4/80−Ly6G−Lin−CD45+eflour506−) and anti-inflammatory monocytes (CD11b+F4/80−Ly6G−Lin−CD45+eflour506−). (c) Flow cytometry analysis of neutrophils (Ly6G+CD11b+Lin−CD45+eflour506−). Numbers in the FACS plots indicate percent CD45+ individual mononuclear cell populations. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparison test. ANOVA, analysis of variance; CCl4, carbon tetrachloride; FACS, fluorescence-activated single cell sorting; HSC, hepatic stellate cells. *p < 0.05, **p < 0.01, ***p < 0.001; ****p < 0.0001. Gating strategy is also described below each plot.
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