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

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.

Figure 1

Microglia activation occurs globally in the brain during hepatic encephalopathy. (A) Field IBA1 field fluorescence (red) in the cortex and cerebellum from vehicle and AOM-treated mice. (B) Quantification of IBA1 field fluorescence in the cortex and cerebellum from vehicle- and AOM-treated mice. (C) IBA1 immunofluorescence (red) in individual cells in vehicle cortex, AOM cortex, vehicle cerebellum, and AOM cerebellum. For immunofluorescence quantification analyses * P <0.05 compared to vehicle-treated mice, n = 3.

Figure 2

CCL2 levels are elevated following AOM-induced liver failure. (A) CCL2 concentrations in cortex lysates from vehicle and AOM-treated mice, n = 3. (B) CCL2 mRNA expression in the cortex of vehicle- and AOM-treated mice, n = 3. (C) Immunofluorescence for CCL2 (red) and NeuN (green) with DAPI (blue) used as a counterstain in vehicle- and AOM-treated mouse cortices. (D) Quantification of CCL2 immunofluorescence in vehicle- and AOM-treated mouse cortex, n = 3. (E) Serum levels of CCL2 in vehicle- and AOM-treated mice, n = 4. For ELISA, mRNA, and fluorescence quantification analyses * P <0.05 compared to vehicle-treated mice.

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.

Figure 4

CCR2 and CCR4 inhibition reduces microglia activation. (A) Cortical IBA1 field fluorescence (red) in vehicle-, AOM-, AOM + INCB-, and AOM + C021-treated mice. (B) Quantification of IBA1 field fluorescence in vehicle-, AOM-, AOM + INCB-, and AOM + C021-treated mice, n = 3. (C) IBA1 immunofluorescence (red) in individual cells in the cortex of vehicle-, AOM-, AOM + INCB-, and AOM + C021-treated mice. For quantitative immunofluorescence analyses * P <0.05 compared to vehicle-treated mice.

Figure 5

INCB or C021 pretreatment reduces G-protein signaling pathway activity and proinflammatory cytokine production. (A) Immunoblots and subsequent analyses for pERK1/2 and tERK1/2 in cortex lysates from mice administered AOM, AOM + INCB, and AOM + C021. (B) IL-1β mRNA expression in the cortex of vehicle-, AOM-, AOM + INCB-, and AOM + C021-treated mice, n = 3. (C) Cortical IL-6 mRNA expression in vehicle-, AOM-, AOM + INCB- and AOM + C021-treated mice, n = 3. For pERK1/2 immunoblotting analyses * P <0.05 compared to AOM-treated mice. For mRNA analyses * P <0.05 compared to vehicle-treated mice.