mNOX-E36 efficiently inhibits mouse monocyte migration in vitro

Recent studies using CCR2^−/− mice demonstrated that chemokine receptor CCR2 and its ligand MCP-1/CCL2 promote the infiltration of inflammatory Gr1⁺ (Ly6C⁺) monocytes into the liver.6 8 9 Subsequently, these monocytes were found to exert proinflammatory along with profibrogenic actions by the release of distinct cytokines and direct interactions with hepatic stellate cells. Thus, inhibition of hepatic monocyte infiltration via blocking MCP-1 could represent a promising therapeutic approach during fibrogenesis. The main mechanism of MCP-1 action is likely to promote exit of Gr1⁺ monocytes from the bone marrow into the blood.6 24 25 In order to elucidate the possibility of pharmacological inhibition of monocyte migration by the MCP-1 antagonist mNOX-E36, bone marrow cells from C57BL/6 WT mice were first investigated in a chemotaxis experiment. Simultaneous incubation of cells with mNOX-E36 efficiently and specifically inhibited the MCP-1-dependent migration of bone marrow monocytes in a transwell assay (figure 1). The inhibition of MCP-1 via mNOX-E36 restrained monocyte migration to a similar extent as in CCR2^−/− cells. Other leucocyte populations were not affected by mNOX-E36 (data not shown). This in vitro efficacy prompted us to assess whether mNOX-E36 is able to inhibit hepatic monocyte accumulation in liver injury models in vivo.

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Figure 1

mNOX-E36 inhibits monocyte chemoattractant protein-1 (MCP-1, CCL2)-dependent migration of monocytes in vitro. Monocytes were isolated from bone marrow of either wild-type (WT) or chemokine receptor C–C chemokine receptor 2-deficient (CCR2^−/−) mice. Migration of monocytes (defined as live CD11b⁺Ly6G⁻CD45⁺ cells) towards a gradient of 100 ng/ml recombinant murine CCL2 (MCP-1) was quantified after 4 h. Migration of unstimulated WT monocytes was set to 1. Data are expressed as mean±SD from three independent experiments. *p<0.05 (unpaired Student t test).

mNOX-E36 specifically inhibits hepatic macrophage infiltration in vivo upon acute liver injury

WT mice were challenged with a single dose of CCl₄ intraperitoneally to induce acute toxic hepatic damage. CCl₄ resulted in periportal necroses with maximal damage after 48 h (figure 2A). Correspondingly, highly elevated serum ALT activities (figure 2B) were measured. As assessed by FACS analysis (figure 2C) and immunohistochemistry (not shown) for the pan-leucocyte marker CD45 a severe accumulation of leucocytes in injured livers was also observed (figure 2C). Administration of mNOX-E36, however, efficiently reduced intrahepatic leucocyte infiltration, to a similar level to that seen in CCR2^−/− mice (figure 2C).

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Figure 2

mNOX-E36 specifically inhibits hepatic macrophage infiltration upon acute liver injury in vivo. Acute toxic liver injury was induced by 0.6 ml/kg carbon tetrachloride (CCl₄) intraperitoneally in C57bl/6 wild-type (WT) mice. mNOX-E36 at 20 mg/kg was administered subcutaneoulsy simultaneously. Mice were analysed 48 h thereafter. (A) Conventional H&E staining of liver paraffin sections showed necrotic patches and progressive mononuclear infiltrates in the periportal regions after acute liver injury. (B) Hepatic injury was also assessed by serum alanine aminotransferase (ALT) levels. (C) Infiltration of leucocytes into the liver was quantified by fluorescence-activated cell sorting (FACS; percentage of CD45⁺ cells from total liver cells), revealing decreased counts in chemokine receptor C–C chemokine receptor 2-deficient (CCR2^−/−) and mNOX-E36 treated mice. (D) FACS analysis of intrahepatic leucocytes, pregated on live (Hoechst dye⁻) CD45⁺Ly6G⁻ cells, showing reduced numbers of CD11b⁺F4/80⁺ intrahepatic macrophages after mNOX-E36 injection and in CCR2^−/− mice. Left, representative FACS plot; right, quantitative analysis. (E) FACS analysis of leucocytes from peripheral blood analysing circulating monocytes (defined as CD11b⁺Ly6G⁻ cells). (F) FACS analysis of leucocytes from bone marrow analysing monocytes (defined as CD11b⁺Ly6G⁻ cells). All data are expressed as the mean±SD from three independent experiments, comprising n=8–10 for animals in the treatment and n=5 animals in the control groups. *p<0.05, **p<0.005, ***p<0.001 (unpaired Student t test).

Previous studies revealed that in the early phase of liver injury macrophages are the predominantly infiltrating immune cells.6 20 Therefore, we purified leucocytes from liver tissues of WT animals and characterised the distinct cell types by FACS analysis. Intrahepatic macrophages were defined as living (Hoechst dye-negative) CD45⁺ cells that stain positive for the myeloid marker CD11b, positive for the macrophage marker F4/80 and negative for the neutrophil marker Ly6G.6 While WT mice showed massively increased hepatic macrophages after CCl₄ injection, these cells only mildly increased in livers of mNOX-E36-treated WT animals. Like CCR2^−/− mice, mNOX-E36-treated WT mice displayed significantly reduced macrophage infiltration following acute liver injury (figure 2D). Importantly, other leucocyte subpopulations such as T cells (CD8⁺, CD4⁺) or B cells (CD19⁺) were not affected by mNOX-E36 treatment (see supplementary figure 1 online).

Besides characterising the subsets of leucocytes in the liver, we also analysed corresponding monocyte populations in blood and bone marrow compartments. Of note, the hepatic CD11b⁺F4/80⁺ macrophages also stained positive for Gr1 (Ly6C), thus representing a vast majority of infiltrating monocytes from the circulation.6 Not only accumulation of these monocytes in injured liver, but also monocyte emigration out of the bone marrow was found to be inhibited by mNOX-E36. This effect was shown by an increased pooling of CD11b⁺Ly6G^– monocytes in blood and bone marrow compared with untreated mice (figure 2E,F). As described in previous studies,6 24 25 CCR2^−/− mice also displayed retention of monocytes in the bone marrow. The pharmacological effects on monocyte migration were specific for mNOX-E36 because the control Spiegelmer rev-mNOX-E36 did not show these effects on monocytes (data not shown). Furthermore, CCR2^−/− mice were not affected by mNOX-E36 with respect to liver injury, circulating or intrahepatic macrophages (see supplmentary figure 2 online), demonstrating that the effects of mNOX-E36 are mediated by specifically inhibiting MCP-1/CCR2-dependent mechanisms. The clear inhibition of hepatic macrophage accumulation upon injury by mNOX-E36 in an acute in vivo model built the basis to examine its application in a chronic setting.

mNOX-E36 specifically inhibits hepatic macrophage infiltration in vivo in two models of chronic liver injury

The infiltration of macrophages into the liver upon chronic injury has been convincingly linked to the progression of liver inflammation and fibrosis in mice and men.6 7 20 26 We therefore also investigated the therapeutic effects of mNOX-E36 in models of chronic liver damage. WT mice were challenged with CCl₄ intraperitoneally twice a week for 6 weeks and simultaneously received mNOX-E36 (20 mg/kg) subcutaneously three times a week. CCl₄-treated animals displayed severe toxic liver damage with increased serum ALT (figure 3A) and AST (data not shown), necrotic patches in histology and mononuclear infiltrates (figure 3B,C). The massive influx of leucocytes, especially macrophages, was visualised by immunohistological staining for CD45 and F4/80 (figure 3C). In line with the acute effects after single administration of CCl₄ and mNOX-E36, long-term treatment of mice with mNOX-E36 attenuated leucocyte influx in chronically damaged liver comparable with CCR2^−/− mice (figure 3B,C). Again, macrophages were the predominant leucocyte population in chronic injury, and (Ly6C⁺) macrophage accumulation was significantly reduced in mNOX-E36-treated and in CCR2^−/− mice, as demonstrated by immunohistochemistry (figure 3C) and FACS analyses (figure 3D). As expected and in line with the data of the acute model, the inactive Spiegelmer control had no effect on the amount of intrahepatic macrophages in this chronic setting (data not shown). Parallel to the intrahepatic macrophages we investigated other immune cells such as T cells (CD8⁺, CD4⁺) or natural killer (NK) cells (NK1.1⁺), which were affected neither in the CCR2-knockout nor by the mNOX-E36 treatment (see supplmentary figure 3 online). Besides liver tissue we again analysed blood and bone marrow by FACS. In blood we detected, concordant with the CCR2^−/− mice, lower levels of monocytes in the mNOX-E36-treated mice (figure 3E). CCR2^−/− mice, but not mNOX-E36-treated WT animals. retained monocytes in the bone marrow (figure 3F).

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Figure 3

mNOX-E36 specifically inhibits hepatic macrophage infiltration upon chronic carbon tetrachloride (CCl₄)-induced toxic injury in vivo. Chronic toxic liver injury was induced by 0.6 ml/kg CCl₄ intraperitoneally twice weekly in C57bl/6 wild-type (WT) mice. mNOX-E36 at 20 mg/kg was administered subcutaneously three times per week. Mice were analysed after 6 weeks of repetitive CCl₄ treatment, 48 h after the last injection. (A) Hepatic injury was assessed by serum alanine aminotransferase (ALT) levels. (B) Infiltration of leucocytes into the liver was quantified by fluorescence-activated cell sorting (FACS; percentage of CD45⁺ cells from total liver cells), revealing decreased intrahepatic leucocytes in chemokine receptor C–C chemokine receptor 2-deficient (CCR2^−/−) and mNOX-E36-treated mice. (C) Representative immunohistochemical stainings for total leucocytes (CD45) and macrophages (F4/80) on liver paraffin sections from different experimental conditions. (D) FACS analysis of intrahepatic leucocytes, pregated on live (Hoechst dye⁻) CD45⁺Ly6G⁻ cells, showing reduced numbers of CD11b⁺F4/80⁺ intrahepatic macrophages after mNOX-E36 injection and in CCR2^−/− mice. Left, representative FACS plot, right, quantitative analysis. (E) FACS analysis from peripheral blood analysing circulating monocytes (defined as CD11b⁺Ly6G⁻ cells). (F) FACS analysis of monocytes (defined as CD11b⁺Ly6G⁻ cells) from bone marrow. All data are expressed as the mean±SD from three independent experiments, comprising n=10–13 for animals in the treatment and n=5–7 animals in the control groups. *p<0.05, **p<0.005, ***p<0.001 (unpaired Student t test).

In order to exclude that these results had been confounded by model-specific effects, we employed a second model of liver injury. Mice were fed an MCD diet for 8 weeks, resulting in severe steatohepatitis at a macroscopic and microscopic level (figure 4A). Mice were severely ill with substantial weight loss (figure 4B) and elevated ALT (figure 4C) and AST (data not shown) levels in comparison with mice fed a control diet. Liver histology showed a diffuse infiltration with leucocytes, apoptotic areas and a massive fatty degeneration (figure 4A). By immunohistochemistry and FACS analysis, we again found decreased numbers of intrahepatic leucocytes as well as macrophages in CCR2^−/− and mNOX-E36-treated WT animals upon chronic metabolic injury (figure 4D). Other intrahepatic leucocyte populations were not affected in the MCD diet model either (see supplementary figure 4 online). Comparable with the toxic injury model, the total numbers of blood monocytes were decreased for CCR2^−/− and mNOX-E36-treated mice (figure 4E), whereas only in CCR2^−/− mice was the monocyte population retained in the bone marrow (figure 4F). Of note, lack of CCR2 or MCP-1 inhibition by mNOX-E36 also affected subpopulations of circulating blood monocytes, because Gr1^hi monocytes were reduced in comparison with their Gr1^low-expressing counterparts (see supplementary figure 4F online). Collectively, these data demonstrated that hepatic accumulation of macrophages upon chronic liver injury can be effectively attenuated by mNOX-E36 in vivo.

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Figure 4

mNOX-E36 specifically inhibits hepatic macrophage infiltration upon chronic methionine–choline-deficient (MCD) diet-induced metabolic injury in vivo. C57bl/6 wild-type (WT) mice were fed an MCD diet for 8 weeks. mNOX-E36 at 20 mg/kg was administered subcutaneously three times per week. (A) Representative macroscopic pictures of the livers (scale marks indicate 1 cm) and corresponding H&E staining of liver paraffin sections. (B) Change of body weight from baseline after 8 weeks of MCD diet in the different treatment groups. (C) Hepatic injury was assessed by serum alanine aminotransferase (ALT) levels. (D) Fluorescence-activated cell sorting (FACS) analysis of intrahepatic leucocytes, presented as either total leucocytes (defined as live CD45⁺ cells, left) or macrophages (defined as live CD45⁺CD11b⁺F4/80⁺Ly6G⁻ cells, right). (E) FACS analysis of leucocytes from peripheral blood analysing circulating monocytes (defined as CD11b⁺Ly6G⁻ cells). (F) FACS analysis of monocytes (defined as CD11b⁺Ly6G⁻ cells) from bone marrow. All data are expressed as the mean±SD from three independent experiments, comprising n=10–12 for animals in the treatment and n=5–7 animals in the control groups. *p<0.05, **p<0.005, ***p<0.001 (unpaired Student t test).

Reduction of liver-infiltrating macrophages via mNOX-E36 affects intrahepatic proinflammatory cytokines upon injury

We next investigated the consequences of blocking hepatic macrophage accumulation via mNOX-E36 in the liver. An important function of hepatic macrophages during disease progression is the secretion of proinflammatory cytokines.7 20 27 Therefore, cytokines were measured from total liver protein lysates by ELISA in both injury models. Animals that had received long-term treatment with mNOX-E36 had lower intrahepatic concentrations of TNFα, IL-6 and IFNγ upon CCl₄- (figure 5A–C) or MCD diet-induced chronic liver injury (figure 5D–F), which is in line with the reduced numbers of intrahepatic macrophages. Gene expression analysis by qPCR confirmed these observations (data not shown). As expected, mNOX-E36-treated animals showed much higher levels of MCP-1 in the liver by ELISA (data not shown), because MCP-1 that is functionally antagonised by mNOX-E36 is still detected by the ELISA.17 Altogether, the clear reduction of proinflammatory cytokines in livers of mNOX-E36-treated animals indicated that pharmacological inhibition of MCP-1 might be capable of limiting chronic liver injury in vivo.

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Figure 5

mNOX-E36 administration affects intrahepatic proinflammatory cytokines upon injury. C57bl/6 wild-type (WT) mice were subjected to 6 weeks of carbon tetrachloride (CCl₄) injury (A–C) or to 8 weeks of a methionine–choline-deficient (MCD) diet (D–F) and treated long term with mNOX-E36 (3× per week). Cytokines were measured from total liver protein extracts by ELISA. All data are expressed as the mean±SD from three independent experiments, comprising n=10–12 for animals in the treatment and n=5–7 animals in the control groups. *p<0.05, **p<0.005 (unpaired Student t test). IFNγ, interferon γ; IL-6, interleukin 6; TNFα, tumour necrosis factor α.

Steatosis development, but not fibrosis progression, is significantly attenuated in mNOX-E36-treated mice

We next analysed whether pharmacological inhibition of MCP-1 might represent a successful therapeutic approach to limit disease progression in chronic liver injury models. We thus assessed liver fibrosis progression in both models by quantifying collagen deposition in Sirius red staining as well as measuring the hepatic hydroxyproline content, an amino acid that is abundantly found in collagen fibres (figure 6). While CCR2^−/− mice showed an attenuated development of liver fibrosis in the chronic CCl₄ model (figure 6A–C), as anticipated from prior studies,6 8 9 and also after an MCD diet (figure 6D–F), treatment with mNOX-E36 did not significantly affect hepatic fibrogenesis in either experimental model.

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Figure 6

mNOX-E36 administration does not significantly alter hepatic fibrosis progression in two independent experimental models. C57bl/6 wild-type (WT) mice were subjected to 6 weeks of carbon tetrachloride (CCl₄) injury (A–C) or to 8 weeks of methionine–choline-deficient (MCD) diet (D–F) and treated long-term with mNOX-E36 (3× per week). Collagen deposition in the liver was visualised by Sirius red staining (A, D), showing bridging fibrosis after CCl₄ administration (A) and scattered collagen in steatotic livers after an MCD diet (D). Histological collagen deposition was quantified in a blinded, computer-based fashion (B, E). Hydroxyproline content of the livers was quantified as an additional measure of total hepatic collagen (C, F). Collectively, the data demonstrated attenuated fibrosis development in chemokine receptor C–C chemokine receptor 2-deficient (CCR2^−/−) mice, but no significant effect of mNOX-E36. All data are expressed as the mean±SD from three independent experiments, comprising n=10–13 for animals in the treatment and n=5–7 animals in the control groups. *p<0.05, **p<0.005, ***p<0.001 (unpaired Student t test).

Several recent observations have linked the progression of steatohepatitis to the stimulation of TNF-dependent signalling pathways in hepatocytes upon injury.28 29 Thus, we hypothesised that the reduction of intrahepatic macrophages and threir associated proinflammatory cytokines might reduce the development of steatosis in vivo. Indeed, fatty degeneration of the liver was attenuated in mice fed an MCD diet during mNOX-E36 treatment as demonstrated by reduced steatosis by Oil red O staining (figure 7A) as well as histopathological criteria scored by a blinded and experienced pathologist (figure 7B). Subsequently, hepatic triglyceride content was significantly decreased in mNOX-E36-treated animals in the MCD diet model (figure 7C). Similar findings were obtained in CCl₄-treated mice as well, although steatosis was much less prominent in this toxic model compared with the model with the MCD diet (data not shown). It has been reported previously that inflammatory cytokines from macrophages may induce the expression of important lipogenesis genes in injured liver.30 In line with the attenuated steatosis development, expression of the protective transcription factor PPARγ (peroxisome proliferator-activated receptor γ) was induced, whereas PEPCK (phosphoenolpyruvate carboxykinase), an important driver of hepatic gluconeogenesis, was reduced in mNOX-E36-treated animals in the MCD diet model (figure 7D,E). Thus, our results demonstrated that steatosis progression is closely associated with MCP-1-dependent macrophage recruitment and subsequent inflammation.

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Figure 7

mNOX-E36 protects from steatosis development in experimental liver injury. C57bl/6 wild-type (WT) mice were fed a methionine–choline-deficient (MCD) diet for 8 weeks. mNOX-E36 at 20 mg/kg was administered subcutaneously three times per week. (A) Oil red O staining of liver cryosections showed decreased levels of positive stained intracellular fat vacuoles in mNOX-E36-treated mice. (B) Pathological scoring of liver histology by a blinded pathologist showed attenuated fatty degeneration in mNOX-E36-injected mice. (C) Quantification of triglycerides from total liver lysates revealed reduced triglyceride content in murine liver after mNOX-E36 treatment. (D, E) Gene expression analysis from whole liver by quantitative real-time PCR showed induced hepatic peroxisome proliferator-activated receptor γ (PPARγ) and reduced phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels in mNOX-E36-injected mice. All data are expressed as the mean±SD from three independent experiments, comprising n=10–12 for animals in the treatment and n=5–7 animals in the control groups. *p<0.05, **p<0.005, ***p<0.001 (unpaired Student t test).

mNOX-E36 also ameliorates steatosis development upon therapeutic application

In order to test the therapeutic potential of mNOX-E36 to treat ongoing fatty liver degeneration, mice were kept on an MCD diet for 6 weeks without treatment, which induced severe steatohepatitis (not shown). While the MCD diet was continued, mNOX-E36 was administered during the last 2 weeks of injury. Clearly, mNOX-E36 treatment in this therapeutic setting reduced hepatic steatosis, as intrahepatic lipid vacuoles (figure 8A) and the histological fatty degeneration score (figure 8B) improved upon MCP-1 inhibition. Moreover, the hepatic triglyceride content showed a trend towards lower levels in the mNOX-36-treated animals (figure 8C). These data indicated that inhibition of MCP-1 might be a promising therapeutic strategy even in ongoing or established steatohepatitis.

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Figure 8

mNOX-E36 ameliorates hepatic steatosis in a therapeutic setting. C57bl/6 wild-type (WT) mice were fed a methionine–choline-deficient (MCD) diet for 8 weeks. mNOX-E36 at 20 mg/kg was administered subcutaneously three times per week only during the last 2 weeks of the diet. (A) Conventional H&E staining of liver paraffin sections showed fatty degeneration after feeding the MCD diet. Treatment with mNOX-E36 resulted in lower amounts of cells with large fat vacuoles. (B) Pathological scoring of liver histology by a blinded pathologist showed attenuated fatty degeneration in mNOX-E36-injected mice. (C) Quantification of triglycerides from total liver lysates revealed a trend of reduced triglyceride content in murine liver after mNOX-E36 treatment. All data are expressed as the mean±SD with n=4 animals per group. *p<0.05 (unpaired Student t test).