Our results show that an underlying condition of obesity exacerbates steatohepatitic lesions when mice are subjected to metabolic oxidative stress arising from carbon tetrachloride exposure. Obese mice have significantly higher lipid peroxidation, ALT levels and ATP release than their lean counterparts. Our electron microscopy studies highlight the increased degeneration of hepatocytes and membrane leakage following such metabolic oxidative stress induced by CCl4
metabolism. These studies are in agreement with studies by Donthamsetty et al. and Ikejima et al. describing the increased sensitivity to carbon tetrachloride in diet-induced obesity (43
). In these studies, the roles of mitochondria together with higher leptin levels were shown as likely causes of the increased susceptibility to liver injury in obese rodents. However, there had previously been no detailed mechanistic studies documenting (a) the crosstalk of hepatocytes and Kupffer cells, (b) molecular mediators of free radical generation and (c) Kupffer cell activation through damage-associated molecular patterns. Our studies, for the first time, show the importance of hepatocyte-Kupffer cell crosstalk in potentiating the inflammatory events through the P2X7 receptor-NADPH oxidase axis.
The primary target of CYP2E1 metabolism of hepatotoxins such as carbon tetrachloride is the hepatocyte (45
). The free radical metabolites within the hepatocytes and (to a lesser extent) other non-parenchymal cells are responsible for lipid peroxidation and membrane damage (46
). We thus investigated the possible underlying causes that would explain the susceptibility of DIO hepatocytes to increased cellular damage and inflammation. We hypothesized that diet-induced obesity was an underlying cause of exacerbated liver injury and membrane fragility through its microenvironment of continuous low-level inflammation.
In testing this hypothesis, we found increased ATP release in DIO hepatocytes at 24 hours compared to that in lean controls (, lower panel). This result suggested that the underlying low inflammatory trigger indicated by higher than normal levels of TNF-α (data not shown) and reported in the literature in DIO mice (47
) can cause membrane damage in hepatocytes from these mice. Furthermore, low doses of CCl4
might have exacerbated the already fragile membrane integrity of these hepatocytes and released even higher concentrations of ATP into the extracellular matrix as seen by us (). Binding of extracellularly released ATP to different purinergic receptors on monocytes and microglia has been noted and is concentration-dependent (48
). We found that higher levels of ATP are released extracellularly from DIO mouse hepatocytes, which might cause ATP to bind to P2X7 receptors either in Kupffer cells (which are macrophages) or other non-parenchymal cells in a paracrine fashion and exacerbate liver injury. As ATP can act as a damage-associated molecular signal in the event of tissue injury and can stimulate sterile inflammation through binding with purinergic receptors (18
), this result (exacerbation of liver inflammation) is not surprising.
After ATP binds to purinergic receptors, the production of reactive oxygen species and reactive nitrogen species can increase in resident macrophages, namely the Kupffer cells. NADPH oxidase-induced superoxide formation and subsequent peroxynitrite formation in immune cells have been shown to follow P2X7 receptor stimulation by extracellular ATP (22
). To explain the possible paracrine effects of ATP release in Kupffer cells, especially on protein oxidation and release of inflammatory mediators, we examined the effect of free radical formation, nitrotyrosine formation in proteins, and release of cytokines and chemokines in a model of metabolic oxidative stress-induced steatohepatitis. We found that in CCl4
-treated DIO mice, Kupffer cell protein radical formation and post-translational tyrosine nitration increased both in vivo and in vitro and were NADPH oxidase- and P2X7 receptor-dependent (). Kupffer cell TNF-α and MCP-2 release increased in CCl4
-treated DIO mice and was blocked by both NADPH oxidase and P2X7 receptor antagonists and in gene-deficient mice, suggesting that the ATP-mediated P2X7 receptor-NADPH oxidase axis plays an important role in exacerbating liver injury ().
One of the known mechanisms of antigen presentation by macrophages, especially through MHC class II molecules on activated macrophages, proceeds through formation of reactive oxygen species. Furthermore, post-translational oxidative modification on proteins is also believed to trigger increased presentation of modified proteins on MHC class I and MHC class II molecules (52
). We therefore examined MHC class II protein expression on the surface of isolated Kupffer cells from CCl4
-treated DIO mice and NADPH oxidase and P2X7 receptor gene-deficient mice. Since F4/80 molecules are exclusive for resident macrophages like Kupffer cells, lung bronchoalveolar regions and splenic red pulp, we used F4/80 as a marker for Kupffer cells. For flow cytometry, equal number F480 cells were gated (based on 5000 events) and cell numbers that were positive for both F480 and MHC Class II or CD80 were calculated. We observed a significant increase in MHC Class II protein/ F4/80 positive cells on CCl4
-treated DIO mouse cells as compared to those from DIO mice or p47 phox and P2X7 receptor gene-deficient mice, suggesting that free radical-mediated oxidative stress through NADPH oxidase and the P2X7 receptor-NADPH oxidase axis might be a prime mechanism for increased F/480-MHC class II positive cells (). This result is significant because increased MHC Class II containing cells mark an activated state of this important resident macrophage and can bridge innate immunity to TH1-type adaptive immune responses along with heightened cytokines coming primarily from Type 1 T helper cells.
Thus, our data seem to suggest that in DIO mouse hepatocytes treated with CCl4, increased ATP release and its resultant downstream events exacerbate liver injury. ATP might stimulate the P2X7 receptor on Kupffer cells and form protein radicals and post-translational tyrosine nitration through NADPH oxidase activity. Furthermore, the heightened leukocyte infiltration observed in livers from CCl4-treated DIO mice might be explained by the increased free radical cascade and P2X7 receptor-NADPH oxidase-primed release of TNF-α, the increase in monocyte chemoattractant protein-2, and the MHC class II protein expression in Kupffer cells ( and ). Our data thus links ATP-induced P2X7 receptor stimulation, NADPH oxidase activation, and the proinflammatory status of the liver microenvironment in CCl4 - induced metabolic oxidative stress coupled with obesity.
Though much of our data suggested a strong participation of the P2X7 receptor and NADPH oxidase in the oxidative stress-dependent increase in inflammatory events in the obese liver, at this point it remained unclear whether the P2X7 receptor and NADPH oxidase were working in concert. It also remained to be seen whether P2X7 receptor stimulation was upstream to NADPH oxidase activation and subsequent protein radical formation and tyrosine nitration. Our observed increases in free radical-mediated oxidation and cytokine release were more pronounced at 24 h than at earlier time points, suggesting that P2X7 receptor stimulation might be upstream to NADPH oxidase activation. Our quantitative RT-PCR studies indicated that there was no change in P2X7 receptor expression in high fat-fed NADPH oxidase-deficient mice (data not shown). However, P47 phox expression, but not gp91 phox, was significantly downregulated in P2X7 receptor-deficient mice, suggesting that the P2X7 receptor plays a significant role in p47 phox expression ().
To further examine these questions, we studied the functional activation of NADPH oxidase that led to association of P47 phox to its membrane subunit gp91 phox in P2X7 receptor-deficient mice (). We found that the presence of the P2X7 receptor is required for p47 phox association with gp91 phox, and that there exists a dual mechanism of downstream mediators of P2X7 receptor stimulation. It not only exerts its effect on the expression of the NADPH oxidase subunit, but also exerts a direct effect on the membrane association of its subunits. Parvathenani et al. showed that P2X7 receptor stimulation influenced NADPH oxidase-induced oxidant generation in rat microglia (54
). The same work also demonstrated that P2X7 receptor stimulation triggered the translocation of p67 phox, an event similar to the present study where we show NADPH oxidase activation through p47 phox binding to gp91. However, this present report is the first to indicate strong evidence of P2X7 receptor stimulation and p47 phox expression in Kupffer cells of DIO mice treated with CCl4
Histopathological examination and serum ALT of mouse liver from P2X7 receptor knockout mice showed that there was significantly less tissue injury and leucocyte infiltration in P2X7 receptor knockout mice treated with CCl4, thus strengthening the argument that the P2X7 receptor mediates the proinflammatory events in CCl4-treated obese mice and causes an exacerbation of steatohepatitic lesions (). However, it is important to note that obesity-induced liver disease is a culmination of multiple factors, and P2X7 receptor stimulation-NADPH oxidase activation may be only one of the many factors that determine the proinflammatory makeup of the disease. Further studies will be needed to identify the downstream mediators of P2X7 receptor stimulation and subsequent synergistic factors in obesity that link NADPH oxidase activation; such studies would be significant in explaining the proinflammatory events in metabolic oxidative stress-induced exacerbation of steatohepatitic lesions in obese mice. Further studies will also be needed to show whether NADPH oxidase-induced signaling mechanisms alone or subsequent free radical chemistry or both are responsible for the shift towards increased inflammatory status of the liver.
In summary, we have identified P2X7 receptor-mediated NADPH oxidase activation as a primary event in exacerbation of steatohepatitic lesions in obese mice. We also report that in Kupffer cells in obese mice, P2X7 receptor stimulation is involved in the formation of protein radicals and post-translational nitration of proteins, which have a role in the exacerbation of inflammatory responses in the liver. Here we have also identified the use of P2X7 receptor antagonists as a possible therapeutic approach for treatment of the early steatohepatitic lesions in obesity.