Increased expression of TNF-α contributes to ethanol-induced liver injury (1
). Treatment of mice with adiponectin, a potent adipokine with anti-inflammatory properties, prevents ethanol-induced steatosis and TNF-α expression (10
). Kupffer cells isolated from rats exposed to chronic ethanol exhibit increased sensitivity to LPS-stimulated TNF-α expression and are used as a model system to understand the interaction between ethanol and LPS-mediated responses in macrophages (21
). Interestingly, the anti-inflammatory actions of adiponectin are enhanced in Kupffer cells isolated from rats chronically exposed to ethanol, compared to pair-fed controls (9
). Despite the efficacy of adiponectin in decreasing LPS-mediated responses, both in mouse models (10
) and primary cultures of Kupffer cells (9
), the development of adiponectin for therapeutic interventions in patients with alcoholic liver disease is likely of limited utility, due to the high concentration of adiponectin in the circulation, as well as the complex oligomeric structure of adiponectin. Therefore, here we made use of primary cultures of Kupffer cells to investigate the molecular mechanisms for the anti-inflammatory effects of adiponectin after chronic ethanol exposure. Understanding the mechanisms of adiponectin action, particularly in ethanol-treated macrophages, could illuminate molecular targets of adiponectin action that are more amenable to pharmacological intervention. Here we have identified an IL-10/STAT3/HO-1 dependent pathway that mediates the anti-inflammatory effects of adiponectin in Kupffer cells. The activity of this pathway is enhanced in Kupffer cells from ethanol-fed rats due to both an increased gAcrp-mediated expression of IL-10, as well as a greater IL-10 stimulated phosphorylation of STAT3 and expression of HO-1. Importantly, induction of HO-1 was also effective at normalizing LPS-stimulated TNF-α expression in an in vivo
model of chronic ethanol exposure.
Adiponectin has potent anti-inflammatory properties, both in vivo
and in cultured macrophages. Initially, treatment of macrophages with adiponectin increases the expression of inflammatory cytokines, such as TNF-α and IL-6 (11
). However, upon continued exposure to gAcrp, the expression of anti-inflammatory mediators, such as IL-10 and IL-1 receptor antagonist, is increased (11
). Increased expression of IL-10 is critical for the anti-inflammatory effects of adiponectin in macrophages; immunoneutralization of IL-10 prevents the suppression of LPS-stimulated TNF-α production by 1 μg/ml gAcrp in RAW 264.7 macrophages (11
). However, in one recent report from the Libby group, IL-10 was not critical in mediating the anti-inflammatory effects of 10 μg/ml full-length adiponectin in human macrophages (23
). Here we report that knock-down of IL-10 in primary cultures of Kupffer cells prevented gAcrp-mediated suppression of LPS-stimulated TNF-α mRNA accumulation, demonstrating that IL-10 is necessary and sufficient to mediate the anti-inflammatory effects of gAcrp in primary cultures of Kupffer cells. We also demonstrated that the induction of IL-10 by gAcrp in Kupffer cells was dependent on AdipoR1, but not AdipoR2, expression. The contribution of AdipoR1, which has a higher affinity for globular adiponectin compared to full-length adiponectin (24
), may explain the differences between our results indicating an essential role of IL-10 and that of the Libby group (23
), using higher concentrations of full-length adiponectin, that reported the induction of multiple anti-inflammatory mediators.
Kupffer cells isolated from ethanol-fed rats are more sensitive to the long-term anti-inflammatory effects of either gAcrp or full-length adiponectin, exhibiting decreased LPS-stimulated NFκB and MAPK activation, as well as decreased TNF-α expression relative to Kupffer cells from pair-fed controls (9
). Because IL-10 is essential to the anti-inflammatory role of gAcrp in Kupffer cells, we hypothesized that ethanol feeding increased the sensitivity to gAcrp via increased IL-10 expression and/or increased sensitivity to IL-10 mediated responses. Our data demonstrate that chronic ethanol feeding increased the sensitivity of Kupffer cells to gAcrp-stimulated IL-10 expression; expression of both IL-10 mRNA as well as the quantity of secreted IL-10 protein is increased in Kupffer cells from ethanol-fed rats compared to cells from control rats. Kupffer cells from ethanol-fed rats also exhibited enhanced IL-10-dependent signaling (), independent of any effect of chronic ethanol on the cell surface expression of IL-10RA, the ligand binding subunit of the IL-10 receptor complex (). Chronic ethanol accelerated and enhanced IL-10-stimulated phosphorylation of STAT3 () and increased expression of IL-10 dependent genes, including HO-1 and SOCS-3 mRNA ().
Very little is known about the impact of acute or chronic ethanol on IL-10 expression and signaling. After chronic ethanol exposure, plasma IL-10 concentrations are reduced in mice and IL-10 deficient mice exhibit an even greater sensitivity to LPS after ethanol feeding compared to wild type mice (4
). Short term/acute ethanol exposure increases IL-10 expression by monocytes in human subjects, as well as in mice in response to LPS. When human subjects consume a single dose of alcohol, the production of IL-10 by isolated monocytes in response to LPS is increased compared to controls (25
). This increase can be prevented by inhibiting HO-1 by pre-treatment with zinc protoporphyrin (26
). Taken together with the current data, it appears that while chronic ethanol exposure decreases circulating concentrations of IL-10 (4
), both short term/acute and chronic ethanol exposure contribute to an enhanced IL-10 expression in monocytes/macrophages in response to immunoregulatory signals, such as LPS or gAcrp.
IL-10 binds to a heterodimeric IL-10R, which undergoes transphosphorylation and then activates the Jak1/STAT3 pathway (27
). Activation of STAT3 is essential for IL-10-dependent signaling (2
). Chronic ethanol feeding increased IL-10 stimulated phosphorylation of JAK1 and STAT3 in Kupffer cells. Furthermore, inhibition of STAT3 signaling via chemical inhibitors or via siRNA knock-down ameliorated IL-10-dependent expression of HO-1 (). Reports in the literature suggest that the impact of chronic ethanol on the regulation of STAT3 is complex, and is likely to have ligand- and cell-type specific effects. Exposure of primary cultures of hepatocytes to ethanol suppresses IL-6-stimulated STAT3 activation (28
). Gao and colleagues have identified cell specific roles for STAT3 in hepatocytes compared to monocytes/macrophages in the liver (29
). Expression of STAT3 in hepatocytes had a negative impact on liver injury and promoted inflammation, while expression of STAT3 in monocytes/macrophages suppressed inflammation during ethanol exposure (29
). The anti-inflammatory role of STAT3 in monocytes/macrophages during chronic ethanol exposure is consistent with our identification of a critical contribution of STAT3 in Kupffer cells in mediating the anti-inflammatory effects of gAcrp.
Accumulating evidence suggests that HO-1 plays an important anti-inflammatory role in chronic inflammatory diseases and protects cells from oxidative insult (15
). Heme oxygenase catalyzes the initial and rate limiting step in oxidative degradation of heme, yielding equimolar amounts of biliverdin IXα, carbon monoxide, and free iron (30
). There are three isoforms of HO: HO-2 and HO-3 are constitutive forms, while HO-1 (also known as heat shock protein 32) is an inducible isozyme, with high expression levels in spleen and Kupffer cells (31
). HO-1 is a stress-responsive protein whose expression is upregulated by a broad spectrum of inducers, including heme, heavy metals, nephrotoxins, cytokines, endotoxins and oxidative stress. Interestingly, HO-1 expression was not increased by chronic ethanol exposure alone in either isolated Kupffer cells () or mouse liver (). However, literature suggests that HO-1 expression in response to ethanol may be dependent on the age of the animals studied (32
). In Kupffer cells, pharmacological inhibition of HO-1 or siRNA knock-down of HO-1 expression completely ameliorated the ability of gAcrp to inhibit LPS-stimulated TNF-α expression. Pharmacological induction of HO-1 in mice reduced LPS-stimulated TNF-α expression in the livers of ethanol-fed mice to that of pair-fed controls. Taken together, these data demonstrated a critical role for HO-1 in dampening the pro-inflammatory response to LPS both in Kupffer cells and in vivo
In summary, these data provide strong evidence for an essential role of IL-10/STAT3/HO-1 in mediating the anti-inflammatory function of gAcrp, demonstrating that gAcrp-dependent responses utilize two critical anti-inflammatory pathways. Importantly, after chronic ethanol exposure, Kupffer cells exhibit an increased sensitivity to the anti-inflammatory effects of both gAcrp and IL-10 and induction of HO-1 in vivo protects mice from the sensitizing effects of ethanol on LPS-stimulated TNF-α expression. The identification of HO-1 as a down-stream effector of gAcrp provides an exciting path for the design and development of novel therapeutic approaches for the resolution of chronic inflammation associated with alcoholic liver disease.