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Lack of CC chemokine ligand 2 differentially affects inflammation and fibrosis according to the genetic background in a murine model of steatohepatitis.

Galastri S, Zamara E, Milani S, Novo E, Provenzano A, Delogu W, Vizzutti F, Sutti S, Locatelli I, Navari N, Vivoli E, Caligiuri A, Pinzani M, Albano E, Parola M, Marra F - Clin. Sci. (2012)

Bottom Line: Sirius Red staining demonstrated pericellular fibrosis in zone 3, and image analysis showed a significantly lower matrix accumulation in CCL2-KO mice.This was associated with reduced hepatic expression of TGF-β (transforming growth factor-β), type I procollagen, TIMP-1 (tissue inhibitor of metalloproteinases-1) and α-smooth muscle actin.We conclude that, in experimental murine steatohepatitis, the effects of CCL2 deficiency are markedly dependent on the genetic background.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Medicina Interna, University of Florence, Florence, Italy.

ABSTRACT
Expression of CCL2 (CC chemokine ligand 2) (or monocyte chemoattractant protein-1) regulates inflammatory cell infiltration in the liver and adipose tissue, favouring steatosis. However, its role in the pathogenesis of steatohepatitis is still uncertain. In the present study, we investigated the development of non-alcoholic steatohepatitis induced by an MCD diet (methionine/choline-deficient diet) in mice lacking the CCL2 gene on two different genetic backgrounds, namely Balb/C and C57/Bl6J. WT (wild-type) and CCL2-KO (knockout) mice were fed on a lipid-enriched MCD diet or a control diet for 8 weeks. In Balb/C mice fed on the MCD diet, a lack of CCL2 was associated with lower ALT (alanine transaminase) levels and reduced infiltration of inflammatory cells, together with a lower generation of oxidative-stress-related products. Sirius Red staining demonstrated pericellular fibrosis in zone 3, and image analysis showed a significantly lower matrix accumulation in CCL2-KO mice. This was associated with reduced hepatic expression of TGF-β (transforming growth factor-β), type I procollagen, TIMP-1 (tissue inhibitor of metalloproteinases-1) and α-smooth muscle actin. In contrast, in mice on a C57Bl/6 background, neither ALT levels nor inflammation or fibrosis were significantly different comparing WT and CCL2-KO animals fed on an MCD diet. In agreement, genes related to fibrogenesis were expressed to comparable levels in the two groups of animals. Comparison of the expression of several genes involved in inflammation and repair demonstrated that IL (interleukin)-4 and the M2 marker MGL-1 (macrophage galactose-type C-type lectin 1) were differentially expressed in Balb/C and C57Bl/6 mice. No significant differences in the degree of steatosis were observed in all groups of mice fed on the MCD diet. We conclude that, in experimental murine steatohepatitis, the effects of CCL2 deficiency are markedly dependent on the genetic background.

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Effects of CCL2 deficiency on liver histology and inflammation following administration of an MCD diet in Balb/C miceWT or CCL2-KO mice were fed for 8 weeks on the control diet or the MCD diet. (A–D) At the end of the study protocol, animals were killed and liver sections were stained with haematoxylin and eosin. (A) WT animals fed on the control diet (WT-Cnt); (B) CCL2-KO animals fed on the control diet (KO Cnt); (C) WT animals fed on the MCD diet (WT MCD); (D) CCL2-KO animals fed on the MCD diet (KO MCD). (E) Individual necro-inflammatory scores in the four groups of mice are indicated. (F) Cells positive for the monocyte/macrophage marker MoMa2 were counted in slides from each group of mice indicated as above. *P<0.01 compared with WT animals fed on the control diet; **P<0.01 compared with WT animals fed on the MCD diet. (G,H) Representative immunofluorescence staining for MoMa2-positive cells (green) in WT animals fed on the MCD diet (G) and CCL2-KO animals fed on the MCD diet (H).
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Figure 2: Effects of CCL2 deficiency on liver histology and inflammation following administration of an MCD diet in Balb/C miceWT or CCL2-KO mice were fed for 8 weeks on the control diet or the MCD diet. (A–D) At the end of the study protocol, animals were killed and liver sections were stained with haematoxylin and eosin. (A) WT animals fed on the control diet (WT-Cnt); (B) CCL2-KO animals fed on the control diet (KO Cnt); (C) WT animals fed on the MCD diet (WT MCD); (D) CCL2-KO animals fed on the MCD diet (KO MCD). (E) Individual necro-inflammatory scores in the four groups of mice are indicated. (F) Cells positive for the monocyte/macrophage marker MoMa2 were counted in slides from each group of mice indicated as above. *P<0.01 compared with WT animals fed on the control diet; **P<0.01 compared with WT animals fed on the MCD diet. (G,H) Representative immunofluorescence staining for MoMa2-positive cells (green) in WT animals fed on the MCD diet (G) and CCL2-KO animals fed on the MCD diet (H).

Mentions: Feeding with an MCD diet is associated with steatosis, hepatocellular injury and inflammation, which recapitulate the histological picture observed in patients with NASH. In MCD-diet-fed WT mice, the appearance of macrovescicular steatosis was accompanied by infiltration with inflammatory cells and degenerative changes in hepatocytes, including lobular necrosis (Figures 2A–2D). In addition, aggregates of inflammatory cells surrounding fat-laden hepatocytes were observed, forming lipogranulomas. In CCL2-KO animals, no differences in steatosis were evident, whereas inflammatory changes were less marked, with a lower number of inflammatory cell aggregates and less dense infiltrates (Figures 2A–2D). When fed on a control diet, the livers of CCL2-KO mice had a histological appearance indistinguishable from that of WT mice. Blinded evaluation of necro-inflammatory changes observed in the different genotypes demonstrated an increased score in WT mice fed on the MCD diet (Figure 2E). Necro-inflammation was less severe in CCL2-KO mice fed on an MCD diet, with an overall score significantly lower than that of WT animals (Figure 2E). Scoring of steatosis confirmed that no significant differences were present comparing CCL2-KO and WT mice (results not shown).


Lack of CC chemokine ligand 2 differentially affects inflammation and fibrosis according to the genetic background in a murine model of steatohepatitis.

Galastri S, Zamara E, Milani S, Novo E, Provenzano A, Delogu W, Vizzutti F, Sutti S, Locatelli I, Navari N, Vivoli E, Caligiuri A, Pinzani M, Albano E, Parola M, Marra F - Clin. Sci. (2012)

Effects of CCL2 deficiency on liver histology and inflammation following administration of an MCD diet in Balb/C miceWT or CCL2-KO mice were fed for 8 weeks on the control diet or the MCD diet. (A–D) At the end of the study protocol, animals were killed and liver sections were stained with haematoxylin and eosin. (A) WT animals fed on the control diet (WT-Cnt); (B) CCL2-KO animals fed on the control diet (KO Cnt); (C) WT animals fed on the MCD diet (WT MCD); (D) CCL2-KO animals fed on the MCD diet (KO MCD). (E) Individual necro-inflammatory scores in the four groups of mice are indicated. (F) Cells positive for the monocyte/macrophage marker MoMa2 were counted in slides from each group of mice indicated as above. *P<0.01 compared with WT animals fed on the control diet; **P<0.01 compared with WT animals fed on the MCD diet. (G,H) Representative immunofluorescence staining for MoMa2-positive cells (green) in WT animals fed on the MCD diet (G) and CCL2-KO animals fed on the MCD diet (H).
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Figure 2: Effects of CCL2 deficiency on liver histology and inflammation following administration of an MCD diet in Balb/C miceWT or CCL2-KO mice were fed for 8 weeks on the control diet or the MCD diet. (A–D) At the end of the study protocol, animals were killed and liver sections were stained with haematoxylin and eosin. (A) WT animals fed on the control diet (WT-Cnt); (B) CCL2-KO animals fed on the control diet (KO Cnt); (C) WT animals fed on the MCD diet (WT MCD); (D) CCL2-KO animals fed on the MCD diet (KO MCD). (E) Individual necro-inflammatory scores in the four groups of mice are indicated. (F) Cells positive for the monocyte/macrophage marker MoMa2 were counted in slides from each group of mice indicated as above. *P<0.01 compared with WT animals fed on the control diet; **P<0.01 compared with WT animals fed on the MCD diet. (G,H) Representative immunofluorescence staining for MoMa2-positive cells (green) in WT animals fed on the MCD diet (G) and CCL2-KO animals fed on the MCD diet (H).
Mentions: Feeding with an MCD diet is associated with steatosis, hepatocellular injury and inflammation, which recapitulate the histological picture observed in patients with NASH. In MCD-diet-fed WT mice, the appearance of macrovescicular steatosis was accompanied by infiltration with inflammatory cells and degenerative changes in hepatocytes, including lobular necrosis (Figures 2A–2D). In addition, aggregates of inflammatory cells surrounding fat-laden hepatocytes were observed, forming lipogranulomas. In CCL2-KO animals, no differences in steatosis were evident, whereas inflammatory changes were less marked, with a lower number of inflammatory cell aggregates and less dense infiltrates (Figures 2A–2D). When fed on a control diet, the livers of CCL2-KO mice had a histological appearance indistinguishable from that of WT mice. Blinded evaluation of necro-inflammatory changes observed in the different genotypes demonstrated an increased score in WT mice fed on the MCD diet (Figure 2E). Necro-inflammation was less severe in CCL2-KO mice fed on an MCD diet, with an overall score significantly lower than that of WT animals (Figure 2E). Scoring of steatosis confirmed that no significant differences were present comparing CCL2-KO and WT mice (results not shown).

Bottom Line: Sirius Red staining demonstrated pericellular fibrosis in zone 3, and image analysis showed a significantly lower matrix accumulation in CCL2-KO mice.This was associated with reduced hepatic expression of TGF-β (transforming growth factor-β), type I procollagen, TIMP-1 (tissue inhibitor of metalloproteinases-1) and α-smooth muscle actin.We conclude that, in experimental murine steatohepatitis, the effects of CCL2 deficiency are markedly dependent on the genetic background.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Medicina Interna, University of Florence, Florence, Italy.

ABSTRACT
Expression of CCL2 (CC chemokine ligand 2) (or monocyte chemoattractant protein-1) regulates inflammatory cell infiltration in the liver and adipose tissue, favouring steatosis. However, its role in the pathogenesis of steatohepatitis is still uncertain. In the present study, we investigated the development of non-alcoholic steatohepatitis induced by an MCD diet (methionine/choline-deficient diet) in mice lacking the CCL2 gene on two different genetic backgrounds, namely Balb/C and C57/Bl6J. WT (wild-type) and CCL2-KO (knockout) mice were fed on a lipid-enriched MCD diet or a control diet for 8 weeks. In Balb/C mice fed on the MCD diet, a lack of CCL2 was associated with lower ALT (alanine transaminase) levels and reduced infiltration of inflammatory cells, together with a lower generation of oxidative-stress-related products. Sirius Red staining demonstrated pericellular fibrosis in zone 3, and image analysis showed a significantly lower matrix accumulation in CCL2-KO mice. This was associated with reduced hepatic expression of TGF-β (transforming growth factor-β), type I procollagen, TIMP-1 (tissue inhibitor of metalloproteinases-1) and α-smooth muscle actin. In contrast, in mice on a C57Bl/6 background, neither ALT levels nor inflammation or fibrosis were significantly different comparing WT and CCL2-KO animals fed on an MCD diet. In agreement, genes related to fibrogenesis were expressed to comparable levels in the two groups of animals. Comparison of the expression of several genes involved in inflammation and repair demonstrated that IL (interleukin)-4 and the M2 marker MGL-1 (macrophage galactose-type C-type lectin 1) were differentially expressed in Balb/C and C57Bl/6 mice. No significant differences in the degree of steatosis were observed in all groups of mice fed on the MCD diet. We conclude that, in experimental murine steatohepatitis, the effects of CCL2 deficiency are markedly dependent on the genetic background.

Show MeSH
Related in: MedlinePlus