In inflammatory diseases occurring outside the CNS, communication between the periphery and the brain via humoral and/or neural routes results in central neural changes and associated behavioral alterations. We have recently identified another immune-to-CNS communication pathway in the setting of organ-centered peripheral inflammation: namely, the entrance of immune cells into the brain. In our current study, using a mouse model of inflammatory liver injury, we have confirmed the significant infiltration of activated monocytes into the brain in mice with hepatic inflammation and have defined the mechanism that mediates this trafficking of monocytes. Specifically, we show that in the presence of hepatic inflammation, mice demonstrate elevated cerebral monocyte chemoattractant protein (MCP)-1 levels, as well as increased numbers of circulating CCR2-expressing monocytes. Cerebral recruitment of monocytes was abolished in inflamed mice that lacked MCP-1/CCL2 or CCR2. Furthermore, in mice with hepatic inflammation, microglia were activated and produced MCP-1/CCL2 before cerebral monocyte infiltration. Moreover, peripheral tumor necrosis factor (TNF)␣ signaling was required to stimulate microglia to produce MCP-1/CCL2. TNF␣ signaling via TNF receptor 1 (TNFR1) is required for these observed effects since in TNFR1 deficient mice with hepatic inflammation, microglial expression of MCP-1/CCL2 and cerebral monocyte recruitment were both markedly inhibited, whereas there was no inhibition in TNFR2 deficient mice. Our results identify the existence of a novel immune-to-CNS communication pathway occurring in the setting of peripheral organ-centered inflammation which may have specific implications for the development of alterations in cerebral neurotransmission commonly encountered in numerous inflammatory diseases occurring outside the CNS.

Fulminant liver failure (FLF) consists of a cascade of events beginning with a presumed uncontrolled systemic activation of the immune system. The etiology of FLF remains undefined. In this study, we demonstrate that CCR5 deficiency promotes the development of acute FLF in mice following Con A administration by preventing activated hepatic CD1d-restricted NKT cells (but not conventional T cells) from dying from activation-induced apoptosis. The resistance of CCR5-deficient NKT cells from activation-induced apoptosis following Con A administration is not due to a defective Fas-driven death pathway. Moreover, FLF in CCR5-deficient mice also correlated with hepatic CCR5-deficient NKT cells, producing more IL-4, but not IFN-γ, relative to wild-type NKT cells. Furthermore, FLF in these mice was abolished by IL-4 mAb or NK1.1 mAb treatment. We propose that CCR5 deficiency may predispose individuals to the development of FLF by preventing hepatic NKT cell apoptosis and by regulating NKT cell function, establishing a novel role for CCR5 in the development of this catastrophic liver disease that is independent of leukocyte recruitment.

Signaling occurs between the liver and brain in cholestatic liver disease, giving rise to sickness behaviors such as fatigue. However, the signaling pathways involved are poorly defined. Circulating inflammatory mediator levels are increased in cholestasis, leading to speculation that they may be capable of activating circulating immune cells that subsequently could gain access to the brain. Indeed, we have identified that at day 10 after bile duct resection-induced cholestasis, there is activation of circulating monocytes that express tumor necrosis factor ␣ (TNF-␣) in conjunction with increased expression of adhesion molecules by cerebral endothelium. Moreover, using intravital microscopy, we have identified markedly enhanced leukocytes rolling along cerebral endothelial cells, mediated by P-selectin, in bile duct-resected (BDR) but not control mice. In addition, we have identified increased infiltration of monocytes (but not lymphocytes) into the brains of BDR mice and found that these infiltrating monocytes produce TNF-␣. Furthermore, infiltration of TNF-␣-secreting monocytes into the brains of cholestatic mice is associated with a broad activation of resident brain macrophages to produce TNF-␣. In conclusion, cholestasis is associated with an activation of cerebral endothelium that recruits TNF-␣-producing monocytes into the brain. We hypothesize that enhanced TNF-␣ release within the brain may contribute to the development of cholestasis-associated sickness behaviors, including fatigue. (HEPATOLOGY 2006;43:154-162.)

T cell‐mediated hepatitis is associated with significant morbidity and mortality worldwide. Levels of C‐C chemokine ligand 3/macrophage inflammatory protein‐1α (CCL3/MIP‐1α) are elevated in the serum of patients with T cell‐mediated liver diseases, but its role is not fully understood. Con A‐induced hepatitis is a murine liver‐specific inflammation mediated by activated T cells and is driven by an up‐regulation of the hepatic expression of IFN‐γ. In this study, we have used CCL3/MIP‐1α gene‐deficient mice to examine the role of CCL3/MIP‐1α in the pathogenesis of Con A‐induced hepatitis. We demonstrate a novel pro‐inflammatory role for CCL3/MIP‐1α since CCL3/MIP‐1α deficiency significantly attenuated hepatic injury, both biochemically and histologically. Moreover, the recruitment of CCR1‐expressing CD4+ T cells to the liver after Con A treatment was strikingly attenuated by CCL3/MIP‐1α deficiency. Correspondingly, hepatic IFN‐γ produced by the recruited CD4+ T cells was significantly reduced by CCL3/MIP‐1α deficiency during Con A‐induced hepatitis. Furthermore, treatment of mice with a dual CCR1/CCR5 peptide antagonist, methionylated RANTES, also markedly reduced hepatic injury and decreased the numbers of CD4+ T cells within the liver producing IFN‐γ during Con A‐induced hepatitis. These findings demonstrate that blockade of the CCL3/MIP‐1α‐CCR1 pathway may represent a novel therapeutic target for treating T cell‐mediated liver diseases.

Natural killer T (NKT) cells and regulatory T cells (Tregs)are both found within the liver and are known to exhibit immune regulatory functions. Hepatic NKT cells are activated early during inflammatory responses and release cytokines, including interferon gamma (IFN-␥), which we speculated could regulate Treg recruitment to the liver. To examine this, we treated C57BL/6 mice with a specific NKT cell activating ligand ␣ galactosyl-C18-ceramide (␣Gal-C18-Cer) and examined the hepatic recruitment of Tregs. We found a time-dependant increase in the hepatic recruitment of Tregs after NKT cell activation, which was absent in NKT cell-deficient mice. Most recruited Tregs expressed interleukin (IL) 10, and to a lesser extent transforming growth factor beta (TGF-␤). Because IFN-␥ induces the production of chemokine (C-X-C motif) ligand 10 (CXCL10), and Tregs can express the cognate receptor for CXCL10 (that is, CXCR3), we considered that CXCL10 might mediate the hepatic recruitment of Tregs after NKT cell activation. Hepatic CXCL10 levels were markedly increased after ␣Gal-C18-Cer administration in wild-type but not in NKT celldeficient mice. Moreover, approximately 50% of Tregs recruited to the liver after ␣Gal-C18-Cer administration expressed CXCR3 and CXCR3؉ Treg recruitment into the liver was significantly inhibited in IFN-␥ KO mice, and after CXCL10 neutralization. In addition, prevention of CXCR3؉ Treg recruitment into the liver enhanced inflammatory effector cell recruitment into the liver after ␣Gal-C18-Cer treatment. Conclusion: These results show that activated NKT cells can induce the hepatic recruitment of Tregs through a cytokine-tochemokine pathway, which could be relevant in the development of chemokine blocking or NKT cell activating strategies to treat liver diseases.