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, 174 (5), 1766-75

Dual Role of CCR2 in the Constitution and the Resolution of Liver Fibrosis in Mice

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Dual Role of CCR2 in the Constitution and the Resolution of Liver Fibrosis in Mice

Claudia Mitchell et al. Am J Pathol.

Abstract

Inflammation has been shown to induce the progression of fibrosis in response to liver injury. Among inflammatory cells, macrophages and lymphocytes play major roles in both the constitution and resolution of liver fibrosis. The chemokine receptor CCR2 is involved in the recruitment of monocytes to injury sites, and it is known to be induced during the progression of fibrosis in humans. However, its specific role during this process has not yet been unveiled. We first demonstrated that, compared with wild-type mice, CCR2 knockout animals presented a delay in liver injury after acute CCl(4) injection, accompanied by a reduction in infiltrating macrophage populations. We then induced fibrosis using repeated injections of CCl(4) and observed a significantly lower level of fibrotic scars at the peak of fibrosis in mutant animals compared with control mice. This diminished fibrosis was associated with a reduction in F4/80(+)CD11b(+) and CD11c(+) populations at the sites of injury. Subsequent analysis of the kinetics of the resolution of fibrosis showed that fibrosis rapidly regressed in wild-type, but not in CCR2(-/-) mice. The persistence of hepatic injury in mutant animals was correlated with sustained tissue inhibitor of metalloproteinase-1 mRNA expression levels and a reduction in matrix metalloproteinase-2 and matrix metalloproteinase-13 expression levels. In conclusion, these findings underline the role of the CCR2 signaling pathway in both the constitution and resolution of liver fibrotic scars.

Figures

Figure 1
Figure 1
Analysis of inflammation markers and liver injury in oil- or CCl4-injected wild-type (white bars) or CCR2−/− mice (black bars), at 12, 18, 24, or 48 hours after injection. A: Real-time RT-PCR analysis of the hepatic expression of various chemokines, chemokine receptors, and cytokines during acute liver injury after CCl4 injection. B: Liver injury was assessed by measuring serum transaminases (alanine aminotransferases). Excipient (oil)-injected animals were sacrificed 12 hours after injury. *P < 0.05, **P < 0.01.
Figure 2
Figure 2
Fibrotic markers at peak fibrosis (day 2) after 6 weeks of bi-weekly injections of CCl4 in wild-type (WT) or CCR2−/− mice. A and B: Sirius Red staining of liver sections showing less collagen deposit in CCR2−/− mutant mice. C: Quantification of Sirius Red staining in oil- or CCl4-injected wild-type or CCR2−/− animals, showing a significant reduction in fibrosis in CCR2−/− mutant mice compared with wild-type mice. D and E: α-SMA staining of liver sections with less staining in mutant mice. F: Quantification of α-SMA-positive area in oil- or CCl4-injected animals. *P < 0.05, **P < 0.01. Original magnifications, ×100.
Figure 3
Figure 3
Histological analysis of wild-type (WT) livers or CCR2−/− livers at peak fibrosis (day 2) after 6 weeks of CCl4 injury. A and B: H&E-stained sections show a greater inflammatory reaction and injury in wild-type mice. CD68 (C and D) and F4/80 (E and F) immunostainings showing positive cells underlining the bridging fibrosis that is less extensive in CCR2−/− mutant mice.
Figure 4
Figure 4
Fluorescence-activated cell sorting analysis showing increased percentages of macrophages and dendritic cells in the livers of wild-type (WT) compared with CCR2−/− mice after acute and chronic CCl4 injury. Nonparenchymal liver cells were collected after excipient (oil) injection, at 12 hours after acute CCl4 injection, at 2 days (day 2) after the end of chronic treatment (peak fibrosis), or at 7 days (day 7) after the end of chronic treatment (recovery phase). A: Representative data of three animals per time point showing double-positive CD11b and F4/80 cell populations in wild-type and CCR2−/− mice, with the corresponding mean percentages ± SE. B: Representative data of three animals per time point showing CD11c+ populations in wild-type and CCR2−/− mice, with the corresponding mean percentages ± SE. Excipient (oil)-injected animals were analyzed 12 hours after acute injury. *P < 0.05, **P < 0.01.
Figure 5
Figure 5
A--F: Sirius Red staining during the resolving phase after 2 (A and B), 7 (C and D), and 14 (E and F) days of spontaneous recovery after the last CCl4 injection in wild-type (WT) and CCR2−/− mutant mice. G: Persistence of the total area of fibrosis detected by Sirius Red staining after 7 and 14 days of recovery in CCR2−/− mice compared with control mice. *P < 0.05, **P < 0.01. Original magnifications, ×100.
Figure 6
Figure 6
Analysis of calcium deposit in necrotic areas. The same necrotic area was stained by H&E (A) and Von Kossa (B) staining. C: Quantification of Von Kossa-positive areas expressed in percent of total area in liver sections analyzed. Necrotic areas decreased significantly in wild-type (WT) animals between 2 and 7 days, but not in CCR2−/− mice. *P < 0.05. D–I: Von Kossa staining in liver sections of wild-type mice and CCR2−/− mice 2 (D and E), 7 (F and G), and 14 (H and I) days after the last CCl4 injection.
Figure 7
Figure 7
Analysis of liver expression of MMP-2, MMP-13, and TIMP-1 during the recovery phase after CCl4 treatment. A diminished expression of MMP-2 was seen in CCR2−/− mice (black bars) by real-time RT-PCR (A) and Western blot (B), during the initial phase of recovery. C: MMP-13 expression was also diminished at 7 days but increased at 14 days after the last CCl4 injection, whereas TIMP-1 (D) showed the inversed pattern of expression. *P < 0.05.

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