CXC Chemokines Function as a Rheostat for Hepatocyte Proliferation and Liver Regeneration

<div><p>Background</p><p>Our previous in vitro studies have demonstrated dose-dependent effects of CXCR2 ligands on hepatocyte cell death and proliferation. In the current study, we sought to determine if CXCR2 ligand concentration is responsible for the divergent effects of these mediators on liver regeneration after ischemia/reperfusion injury and partial hepatectomy.</p><p>Methods</p><p>Murine models of partial ischemia/reperfusion injury and hepatectomy were used to study the effect of CXCR2 ligands on liver regeneration.</p><p>Results</p><p>We found that hepatic expression of the CXCR2 ligands, macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine (KC), was significantly increased after both I/R injury and partial hepatectomy. However, expression of these ligands after I/R injury was 30-100-fold greater than after hepatectomy. Interestingly, the same pattern of expression was found in ischemic versus non-ischemic liver lobes following I/R injury with expression significantly greater in the ischemic liver lobes. In both systems, lower ligand expression was associated with increased hepatocyte proliferation and liver regeneration in a CXCR2-dependent fashion. To confirm that these effects were related to ligand concentration, we administered exogenous MIP-2 and KC to mice undergoing partial hepatectomy. Mice received a “high” dose that replicated serum levels found after I/R injury and a “low” dose that was similar to that found after hepatectomy. Mice receiving the “high” dose had reduced levels of hepatocyte proliferation and regeneration whereas the “low” dose promoted hepatocyte proliferation and regeneration.</p><p>Conclusions</p><p>Together, these data demonstrate that concentrations of CXC chemokines regulate the hepatic proliferative response and subsequent liver regeneration.</p></div

Differential hepatic expression of CXC chemokines in I/R injury and hepatectomy.

<p>Expression of MIP-2 and KC in the remnant liver after partial hepatectomy or ischemic and non-ischemic lobes after I/R injury. Data are mean ± SEM with n = 3–6 per group. *<i>P</i><0.05 compared to sham group. **<i>P</i><0.05 compared to ischemic liver.</p

Rab27 proteins are not regulated by CXCR1 or CXCR2.

<p>Protein expression of Rab27a and Rab27b in wild-type and CXCR1-deficient (A) or CXCR2-deficient (B) hepatocytes was determined by Western blot. β-actin was used as a control protein. Results shown are representative of three independent experiments.</p

Chemokine Receptors, CXCR1 and CXCR2, Differentially Regulate Exosome Release in Hepatocytes

<div><p>Exosomes are small membrane vesicles released by different cell types, including hepatocytes, that play important roles in intercellular communication. We have previously demonstrated that hepatocyte-derived exosomes contain the synthetic machinery to form sphingosine-1-phosphate (S1P) in target hepatocytes resulting in proliferation and liver regeneration after ischemia/reperfusion (I/R) injury. We also demonstrated that the chemokine receptors, CXCR1 and CXCR2, regulate liver recovery and regeneration after I/R injury. In the current study, we sought to determine if the regulatory effects of CXCR1 and CXCR2 on liver recovery and regeneration might occur via altered release of hepatocyte exosomes. We found that hepatocyte release of exosomes was dependent upon CXCR1 and CXCR2. CXCR1-deficient hepatocytes produced fewer exosomes, whereas CXCR2-deficient hepatocytes produced more exosomes compared to their wild-type controls. In CXCR2-deficient hepatocytes, there was increased activity of neutral sphingomyelinase (Nsm) and intracellular ceramide. CXCR1-deficient hepatocytes had no alterations in Nsm activity or ceramide production. Interestingly, exosomes from CXCR1-deficient hepatocytes had no effect on hepatocyte proliferation, due to a lack of neutral ceramidase and sphingosine kinase. The data demonstrate that CXCR1 and CXCR2 regulate hepatocyte exosome release. The mechanism utilized by CXCR1 remains elusive, but CXCR2 appears to modulate Nsm activity and resultant production of ceramide to control exosome release. CXCR1 is required for packaging of enzymes into exosomes that mediate their hepatocyte proliferative effect.</p></div

Exosomes from CXCR1-deficient hepatocytes lack neutral ceramidase and sphingosine kinase activity.

<p>Ceramide concentration (A), neutral ceramidase activity (B), and sphingosine kinase activity (C) were measured in exosomes from wild-type and CXCR1- and CXCR2-deficient hepatocytes. Data are mean ± SEM with n = 3 per group. *P<0.05 compared to wild-type exosomes.</p

CXCR1 and CXCR2 regulate exosome release during hepatic I/R injury.

<p>Knockout of CXCR1 (A) reduced, while knockout of CXCR2 (B) increased the number of exosomes in the serum during ischemia/reperfusion (I/R) injury. After 24 and 96 hours of reperfusion, serum was taken for analysis. Serum exosomes were determined via CD81 antigen ELISA. Data are mean ± SEM with n = 4–11 per group. *P<0.05 compared to wild-type mice. Liver histology in CXCR1-knockout mice was similar to wild-types (C), whereas CXCR2-knockout mice showed improve liver architecture after I/R injury (D). Similarly, serum ALT values in CXCR1-knockout mice were similar to wild-type controls (E), while CXCR2-knockout mice had significantly less ALT than their wild-type controls (F). Data are mean ± SEM with n = 3–4 per group. *P<0.05 compared to wild-type mice.</p

Inhibition of Nsm reduces exosome release in CXCR2-deficient hepatocytes.

<p>Exosomes released by wild-type and CXCR2-deficient hepatocytes treated with the Nsm inhibitor, GW4869, were measured by CD81 antigen ELISA. Data are mean ± SEM with n = 4–8 per group. *P<0.05 compared to control group (0 μM GW4869).</p

CXCR1 and CXCR2 regulate exosome release in hepatocytes independent of ligand.

<p>Exosomes released by wild-type and CXCR1-deficient (A) or CXCR2-deficient (B) hepatocytes (HEP), Kupffer cells (KC), and liver sinusoidal endothelial cells (SEC) were measured by CD81 antigen ELISA. Data are mean ± SEM with n = 3–6 per group. *P<0.05 compared to wild-type cells. (C) Wild-type hepatocytes were treated with 0, 100, or 5000 ng/ml MIP-2 and exosomes in the culture media were determined via CD81 ELISA. Data are mean ± SEM with n = 7–13 per group.</p

CXCR2, but not CXCR1, modulates Nsm and ceramide to regulate exosome release.

<p>Accumulated ceramide in wild-type and CXCR1-deficient or CXCR2-deficient hepatocytes was detected fluorescence microscopy with Cy-3-labeled antibody (A). Intracellular hepatocyte ceramide concentrations (B, D) and Nsm activity (C, E) were determined. Data are mean ± SEM with n = 4 per group. *P<0.05 compared to wild-type mice. *P<0.05 compared to wild-type mice.</p

Knockout of CXCR1 abrogates the proliferative effects of hepatocyte exosomes.

<p>Primary hepatocytes from wild-type mice were treated with exosomes from wild-type and CXCR1-deficient (A) or CXCR2-deficient (B) hepatocytes for 24 hours. Cell proliferation was determined by BrdU incorporation assays. Data are mean ± SEM with n = 8 per group. *P<0.05 compared to control group (0 μg/ml exosomes).</p