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Neuronal CCL2 is upregulated during hepatic encephalopathy and contributes to microglia activation and neurological decline.

McMillin M, Frampton G, Thompson M, Galindo C, Standeford H, Whittington E, Alpini G, DeMorrow S - J Neuroinflammation (2014)

Bottom Line: Systemic administration of INCB or C021 reduced liver damage as assessed by serum liver enzyme biochemistry.These findings suggest that CCL2 is elevated systemically following acute liver injury and that CCL2 is involved in both the microglia activation and neurological decline associated with hepatic encephalopathy.Methods used to modulate CCL2 levels and/or reduce CCR2/CCR4 activity may be potential therapeutic targets for the management of hepatic encephalopathy due to acute liver injury.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Internal Medicine, Texas A&M Health Science Center, College of Medicine, 1901 South 1st Street, Building 205, Temple, Texas, USA. demorrow@medicine.tamhsc.edu.

ABSTRACT

Background: Acute liver failure leads to systemic complications with one of the most dangerous being a decline in neurological function, termed hepatic encephalopathy. Neurological dysfunction is exacerbated by an increase of toxic metabolites in the brain that lead to neuroinflammation. Following various liver diseases, hepatic and circulating chemokines, such as chemokine ligand 2 (CCL2), are elevated, though their effects on the brain following acute liver injury and subsequent hepatic encephalopathy are unknown. CCL2 is known to activate microglia in other neuropathies, leading to a proinflammatory response. However, the effects of CCL2 on microglia activation and the pathogenesis of hepatic encephalopathy following acute liver injury remain to be determined.

Methods: Hepatic encephalopathy was induced in mice via injection of azoxymethane (AOM) in the presence or absence of INCB 3284 dimesylate (INCB), a chemokine receptor 2 inhibitor, or C 021 dihydrochloride (C021), a chemokine receptor 4 inhibitor. Mice were monitored for neurological decline and time to coma (loss of all reflexes) was recorded. Tissue was collected at coma and used for real-time PCR, immunoblots, ELISA, or immunostaining analyses to assess the activation of microglia and consequences on pro-inflammatory cytokine expression.

Results: Following AOM administration, microglia activation was significantly increased in AOM-treated mice compared to controls. Concentrations of CCL2 in the liver, serum, and cortex were significantly elevated in AOM-treated mice compared to controls. Systemic administration of INCB or C021 reduced liver damage as assessed by serum liver enzyme biochemistry. Administration of INCB or C021 significantly improved the neurological outcomes of AOM-treated mice, reduced microglia activation, reduced phosphorylation of ERK1/2, and alleviated AOM-induced cytokine upregulation.

Conclusions: These findings suggest that CCL2 is elevated systemically following acute liver injury and that CCL2 is involved in both the microglia activation and neurological decline associated with hepatic encephalopathy. Methods used to modulate CCL2 levels and/or reduce CCR2/CCR4 activity may be potential therapeutic targets for the management of hepatic encephalopathy due to acute liver injury.

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CCR2 and CCR4 signaling play a role in the progression of hepatic encephalopathy. (A) Representative immunoblots against CCR2 and CCR4 in vehicle- and AOM-treated cortex lysates. (B) Time to coma, in hours, of mice treated with AOM, AOM + INCB, or AOM + C021, n = 4. (C) Neurological score of mice treated with AOM, AOM + INCB, or AOM + C021, n = 4. Neurological score calculations are outlined in the methods section. (D) Serum ALT levels in mice treated with vehicle, AOM, AOM + INCB, or AOM + C021. (E) Bilirubin concentrations in serum for mice administered vehicle, AOM, AOM + INCB, or AOM + C021. (F) Hematoxylin and eosin stains in liver sections from mice treated with vehicle, AOM, AOM + INCB, or AOM + C021. For time to coma analyses, * P <0.05 compared to AOM-treated mice. For ALT and bilirubin assays, * P <0.05 compared to vehicle-treated mice, # P <0.05 compared to AOM-treated mice.
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Figure 3: CCR2 and CCR4 signaling play a role in the progression of hepatic encephalopathy. (A) Representative immunoblots against CCR2 and CCR4 in vehicle- and AOM-treated cortex lysates. (B) Time to coma, in hours, of mice treated with AOM, AOM + INCB, or AOM + C021, n = 4. (C) Neurological score of mice treated with AOM, AOM + INCB, or AOM + C021, n = 4. Neurological score calculations are outlined in the methods section. (D) Serum ALT levels in mice treated with vehicle, AOM, AOM + INCB, or AOM + C021. (E) Bilirubin concentrations in serum for mice administered vehicle, AOM, AOM + INCB, or AOM + C021. (F) Hematoxylin and eosin stains in liver sections from mice treated with vehicle, AOM, AOM + INCB, or AOM + C021. For time to coma analyses, * P <0.05 compared to AOM-treated mice. For ALT and bilirubin assays, * P <0.05 compared to vehicle-treated mice, # P <0.05 compared to AOM-treated mice.

Mentions: As CCL2 concentrations were systemically upregulated, the function of CCL2/CCR2 and CCL2/CCR4 receptor activity in this model was investigated. The protein expression of CCR2 and CCR4 was found to be unchanged in the cortex following AOM injection (Figure 3A). To assess the role of CCL2/CCR2 or CCL2/CCR4 signaling in the neurological complications of acute liver failure, antagonists against these receptors were injected prior to AOM administration. Pretreatment with either antagonist was found to significantly delay the neurological decline and increase the time taken to reach coma, indicating a neuroprotective effect of these treatments (Figure 3B,C). Analyses of liver enzymes after pretreatment with CCL2 receptor antagonists in AOM-treated mice demonstrated a significant reduction in the levels of ALT levels (Figure 3D) and bilirubin (Figure 3E) compared to AOM-treated mice alone, though levels were still.significantly elevated compared to controls indicating the presence of significant liver damage. This persistence of liver damage is evident in hematoxylin and eosin-stained liver sections, which indicate that significant necrosis and steatosis is still present in mice pretreated with either INCB or C021 (Figure 3F). Thus, it appears that even though significant liver damage was still present, pretreatment with INCB or C021 prior to AOM administration improved liver function.


Neuronal CCL2 is upregulated during hepatic encephalopathy and contributes to microglia activation and neurological decline.

McMillin M, Frampton G, Thompson M, Galindo C, Standeford H, Whittington E, Alpini G, DeMorrow S - J Neuroinflammation (2014)

CCR2 and CCR4 signaling play a role in the progression of hepatic encephalopathy. (A) Representative immunoblots against CCR2 and CCR4 in vehicle- and AOM-treated cortex lysates. (B) Time to coma, in hours, of mice treated with AOM, AOM + INCB, or AOM + C021, n = 4. (C) Neurological score of mice treated with AOM, AOM + INCB, or AOM + C021, n = 4. Neurological score calculations are outlined in the methods section. (D) Serum ALT levels in mice treated with vehicle, AOM, AOM + INCB, or AOM + C021. (E) Bilirubin concentrations in serum for mice administered vehicle, AOM, AOM + INCB, or AOM + C021. (F) Hematoxylin and eosin stains in liver sections from mice treated with vehicle, AOM, AOM + INCB, or AOM + C021. For time to coma analyses, * P <0.05 compared to AOM-treated mice. For ALT and bilirubin assays, * P <0.05 compared to vehicle-treated mice, # P <0.05 compared to AOM-treated mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4128607&req=5

Figure 3: CCR2 and CCR4 signaling play a role in the progression of hepatic encephalopathy. (A) Representative immunoblots against CCR2 and CCR4 in vehicle- and AOM-treated cortex lysates. (B) Time to coma, in hours, of mice treated with AOM, AOM + INCB, or AOM + C021, n = 4. (C) Neurological score of mice treated with AOM, AOM + INCB, or AOM + C021, n = 4. Neurological score calculations are outlined in the methods section. (D) Serum ALT levels in mice treated with vehicle, AOM, AOM + INCB, or AOM + C021. (E) Bilirubin concentrations in serum for mice administered vehicle, AOM, AOM + INCB, or AOM + C021. (F) Hematoxylin and eosin stains in liver sections from mice treated with vehicle, AOM, AOM + INCB, or AOM + C021. For time to coma analyses, * P <0.05 compared to AOM-treated mice. For ALT and bilirubin assays, * P <0.05 compared to vehicle-treated mice, # P <0.05 compared to AOM-treated mice.
Mentions: As CCL2 concentrations were systemically upregulated, the function of CCL2/CCR2 and CCL2/CCR4 receptor activity in this model was investigated. The protein expression of CCR2 and CCR4 was found to be unchanged in the cortex following AOM injection (Figure 3A). To assess the role of CCL2/CCR2 or CCL2/CCR4 signaling in the neurological complications of acute liver failure, antagonists against these receptors were injected prior to AOM administration. Pretreatment with either antagonist was found to significantly delay the neurological decline and increase the time taken to reach coma, indicating a neuroprotective effect of these treatments (Figure 3B,C). Analyses of liver enzymes after pretreatment with CCL2 receptor antagonists in AOM-treated mice demonstrated a significant reduction in the levels of ALT levels (Figure 3D) and bilirubin (Figure 3E) compared to AOM-treated mice alone, though levels were still.significantly elevated compared to controls indicating the presence of significant liver damage. This persistence of liver damage is evident in hematoxylin and eosin-stained liver sections, which indicate that significant necrosis and steatosis is still present in mice pretreated with either INCB or C021 (Figure 3F). Thus, it appears that even though significant liver damage was still present, pretreatment with INCB or C021 prior to AOM administration improved liver function.

Bottom Line: Systemic administration of INCB or C021 reduced liver damage as assessed by serum liver enzyme biochemistry.These findings suggest that CCL2 is elevated systemically following acute liver injury and that CCL2 is involved in both the microglia activation and neurological decline associated with hepatic encephalopathy.Methods used to modulate CCL2 levels and/or reduce CCR2/CCR4 activity may be potential therapeutic targets for the management of hepatic encephalopathy due to acute liver injury.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Internal Medicine, Texas A&M Health Science Center, College of Medicine, 1901 South 1st Street, Building 205, Temple, Texas, USA. demorrow@medicine.tamhsc.edu.

ABSTRACT

Background: Acute liver failure leads to systemic complications with one of the most dangerous being a decline in neurological function, termed hepatic encephalopathy. Neurological dysfunction is exacerbated by an increase of toxic metabolites in the brain that lead to neuroinflammation. Following various liver diseases, hepatic and circulating chemokines, such as chemokine ligand 2 (CCL2), are elevated, though their effects on the brain following acute liver injury and subsequent hepatic encephalopathy are unknown. CCL2 is known to activate microglia in other neuropathies, leading to a proinflammatory response. However, the effects of CCL2 on microglia activation and the pathogenesis of hepatic encephalopathy following acute liver injury remain to be determined.

Methods: Hepatic encephalopathy was induced in mice via injection of azoxymethane (AOM) in the presence or absence of INCB 3284 dimesylate (INCB), a chemokine receptor 2 inhibitor, or C 021 dihydrochloride (C021), a chemokine receptor 4 inhibitor. Mice were monitored for neurological decline and time to coma (loss of all reflexes) was recorded. Tissue was collected at coma and used for real-time PCR, immunoblots, ELISA, or immunostaining analyses to assess the activation of microglia and consequences on pro-inflammatory cytokine expression.

Results: Following AOM administration, microglia activation was significantly increased in AOM-treated mice compared to controls. Concentrations of CCL2 in the liver, serum, and cortex were significantly elevated in AOM-treated mice compared to controls. Systemic administration of INCB or C021 reduced liver damage as assessed by serum liver enzyme biochemistry. Administration of INCB or C021 significantly improved the neurological outcomes of AOM-treated mice, reduced microglia activation, reduced phosphorylation of ERK1/2, and alleviated AOM-induced cytokine upregulation.

Conclusions: These findings suggest that CCL2 is elevated systemically following acute liver injury and that CCL2 is involved in both the microglia activation and neurological decline associated with hepatic encephalopathy. Methods used to modulate CCL2 levels and/or reduce CCR2/CCR4 activity may be potential therapeutic targets for the management of hepatic encephalopathy due to acute liver injury.

Show MeSH
Related in: MedlinePlus