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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 the development of fibrosis induced by an MCD diet and expression of pro-fibrogenic genes in Balb/C miceWT or CCL2-KO mice were fed for 8 weeks on the control diet or the MCD diet. Groups are as in the legend to Figure 1. (A–D) At the end of the study protocol, animals were killed and liver sections stained with Sirius Red. (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) Sirius-Red-stained slides were subjected to image analysis, and the area occupied by fibrotic tissue was measured in WT and CCL2-KO mice fed on the control diet or the MCD diet. (F–I) RNA was isolated from liver tissue and the expression of TGF-β (F), type I procollagen (G), TIMP-1 (H) and α-smooth muscle actin (I) were measured by RT-PCR. *P<0.01 compared with WT animals fed on the control diet; **P<0.01 compared with WT animals fed on the MCD diet.
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Figure 4: Effects of CCL2 deficiency on the development of fibrosis induced by an MCD diet and expression of pro-fibrogenic genes in Balb/C miceWT or CCL2-KO mice were fed for 8 weeks on the control diet or the MCD diet. Groups are as in the legend to Figure 1. (A–D) At the end of the study protocol, animals were killed and liver sections stained with Sirius Red. (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) Sirius-Red-stained slides were subjected to image analysis, and the area occupied by fibrotic tissue was measured in WT and CCL2-KO mice fed on the control diet or the MCD diet. (F–I) RNA was isolated from liver tissue and the expression of TGF-β (F), type I procollagen (G), TIMP-1 (H) and α-smooth muscle actin (I) were measured by RT-PCR. *P<0.01 compared with WT animals fed on the control diet; **P<0.01 compared with WT animals fed on the MCD diet.

Mentions: As in other forms of chronic liver damage, the MCD diet triggers the initiation of a fibrogenic process that, in mice, leads to accumulation of fibrillar matrix after at least 8 weeks. To determine whether protection from necro-inflammatory damage is associated with changes in fibrogenesis, sections from mice fed on the control or MCD diets were stained with Sirius Red (Figures 4A–4D). As already described, administration of the MCD diet for 8 weeks induced the matrix accumulation predominantly in zone 3 of the hepatic acinus, with the appearance of a ‘chickenwire’ pattern. Fibrosis was also detectable in mice defective for CCL2, but in this case fibrotic bundles appeared to be less thick and more sparse compared with WT Balb/C mice. Computerized image analysis of the slides stained with Sirius Red demonstrated that in CCL2-KO mice accumulation of fibrillar collagen was significantly reduced compared with WT animals (Figure 4E).


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 the development of fibrosis induced by an MCD diet and expression of pro-fibrogenic genes in Balb/C miceWT or CCL2-KO mice were fed for 8 weeks on the control diet or the MCD diet. Groups are as in the legend to Figure 1. (A–D) At the end of the study protocol, animals were killed and liver sections stained with Sirius Red. (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) Sirius-Red-stained slides were subjected to image analysis, and the area occupied by fibrotic tissue was measured in WT and CCL2-KO mice fed on the control diet or the MCD diet. (F–I) RNA was isolated from liver tissue and the expression of TGF-β (F), type I procollagen (G), TIMP-1 (H) and α-smooth muscle actin (I) were measured by RT-PCR. *P<0.01 compared with WT animals fed on the control diet; **P<0.01 compared with WT animals fed on the MCD diet.
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Figure 4: Effects of CCL2 deficiency on the development of fibrosis induced by an MCD diet and expression of pro-fibrogenic genes in Balb/C miceWT or CCL2-KO mice were fed for 8 weeks on the control diet or the MCD diet. Groups are as in the legend to Figure 1. (A–D) At the end of the study protocol, animals were killed and liver sections stained with Sirius Red. (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) Sirius-Red-stained slides were subjected to image analysis, and the area occupied by fibrotic tissue was measured in WT and CCL2-KO mice fed on the control diet or the MCD diet. (F–I) RNA was isolated from liver tissue and the expression of TGF-β (F), type I procollagen (G), TIMP-1 (H) and α-smooth muscle actin (I) were measured by RT-PCR. *P<0.01 compared with WT animals fed on the control diet; **P<0.01 compared with WT animals fed on the MCD diet.
Mentions: As in other forms of chronic liver damage, the MCD diet triggers the initiation of a fibrogenic process that, in mice, leads to accumulation of fibrillar matrix after at least 8 weeks. To determine whether protection from necro-inflammatory damage is associated with changes in fibrogenesis, sections from mice fed on the control or MCD diets were stained with Sirius Red (Figures 4A–4D). As already described, administration of the MCD diet for 8 weeks induced the matrix accumulation predominantly in zone 3 of the hepatic acinus, with the appearance of a ‘chickenwire’ pattern. Fibrosis was also detectable in mice defective for CCL2, but in this case fibrotic bundles appeared to be less thick and more sparse compared with WT Balb/C mice. Computerized image analysis of the slides stained with Sirius Red demonstrated that in CCL2-KO mice accumulation of fibrillar collagen was significantly reduced compared with WT animals (Figure 4E).

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