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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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Lack of CCL2 reduces the expression of proinflammatory genes and limits generation of oxidative stress induced by an MCD diet 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,B) At the end of the study protocol, RNA was isolated from liver tissue and expression of CD11b (A) or of TNFα (B) was measured by RT-PCR. (C,D) A sample of fresh liver tissue was harvested, and levels of ROS (C) and of HNE (D) were assayed as described in the Materials and methods sections. *P<0.01 compared with WT animals fed on the control diet; **P<0.01 compared with WT animals fed on the MCD diet. UF, units of fluorescence.
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Figure 3: Lack of CCL2 reduces the expression of proinflammatory genes and limits generation of oxidative stress induced by an MCD diet 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,B) At the end of the study protocol, RNA was isolated from liver tissue and expression of CD11b (A) or of TNFα (B) was measured by RT-PCR. (C,D) A sample of fresh liver tissue was harvested, and levels of ROS (C) and of HNE (D) were assayed as described in the Materials and methods sections. *P<0.01 compared with WT animals fed on the control diet; **P<0.01 compared with WT animals fed on the MCD diet. UF, units of fluorescence.

Mentions: To provide further evidence for the protective effects of CCL2 deficiency on inflammation, we measured the gene expression of CD11b, a marker of inflammatory cells. A 5-fold significant increase in CD11b expression was observed in WT mice fed on the MCD diet in comparison with those fed on the control diet (Figure 3A). Expression of CD11b was significantly reduced in MCD-diet-fed CCL2-KO mice, confirming reduced inflammation and in agreement with the results of the histological examination. TNFα (tumour necrosis factor α) is a major inflammatory cytokine and has been implicated in the pathogenesis of NASH-associated hepatocyte damage [3]. Transcripts encoding the TNFα gene were markedly more abundant after feeding WT mice with the MCD diet (Figure 3B), and also in this case CCL2 deficiency reduced the expression of this cytokine.


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)

Lack of CCL2 reduces the expression of proinflammatory genes and limits generation of oxidative stress induced by an MCD diet 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,B) At the end of the study protocol, RNA was isolated from liver tissue and expression of CD11b (A) or of TNFα (B) was measured by RT-PCR. (C,D) A sample of fresh liver tissue was harvested, and levels of ROS (C) and of HNE (D) were assayed as described in the Materials and methods sections. *P<0.01 compared with WT animals fed on the control diet; **P<0.01 compared with WT animals fed on the MCD diet. UF, units of fluorescence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369401&req=5

Figure 3: Lack of CCL2 reduces the expression of proinflammatory genes and limits generation of oxidative stress induced by an MCD diet 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,B) At the end of the study protocol, RNA was isolated from liver tissue and expression of CD11b (A) or of TNFα (B) was measured by RT-PCR. (C,D) A sample of fresh liver tissue was harvested, and levels of ROS (C) and of HNE (D) were assayed as described in the Materials and methods sections. *P<0.01 compared with WT animals fed on the control diet; **P<0.01 compared with WT animals fed on the MCD diet. UF, units of fluorescence.
Mentions: To provide further evidence for the protective effects of CCL2 deficiency on inflammation, we measured the gene expression of CD11b, a marker of inflammatory cells. A 5-fold significant increase in CD11b expression was observed in WT mice fed on the MCD diet in comparison with those fed on the control diet (Figure 3A). Expression of CD11b was significantly reduced in MCD-diet-fed CCL2-KO mice, confirming reduced inflammation and in agreement with the results of the histological examination. TNFα (tumour necrosis factor α) is a major inflammatory cytokine and has been implicated in the pathogenesis of NASH-associated hepatocyte damage [3]. Transcripts encoding the TNFα gene were markedly more abundant after feeding WT mice with the MCD diet (Figure 3B), and also in this case CCL2 deficiency reduced the expression of this cytokine.

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