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- MATERIALS AND METHODS
Hepatic ischemia/reperfusion injury (IRI), an exogenous, antigen-independent, local inflammation response, occurs in multiple clinical settings, including liver transplantation, hepatic resection, trauma, and shock. The nervous system maintains extensive crosstalk with the immune system through neuropeptide and peptide hormone networks. This study examined the function and therapeutic potential of the vasoactive intestinal peptide (VIP) neuropeptide in a murine model of liver warm ischemia (90 minutes) followed by reperfusion. Liver ischemia/reperfusion (IR) triggered an induction of gene expression of intrinsic VIP; this peaked at 24 hours of reperfusion and coincided with a hepatic self-healing phase. Treatment with the VIP neuropeptide protected livers from IRI; this was evidenced by diminished serum alanine aminotransferase levels and well-preserved tissue architecture and was associated with elevated intracellular cyclic adenosine monophosphate (cAMP)–protein kinase A (PKA) signaling. The hepatocellular protection rendered by VIP was accompanied by diminished neutrophil/macrophage infiltration and activation, reduced hepatocyte necrosis/apoptosis, and increased hepatic interleukin-10 (IL-10) expression. Strikingly, PKA inhibition restored liver damage in otherwise IR-resistant VIP-treated mice. In vitro, VIP not only diminished macrophage tumor necrosis factor α/IL-6/IL-12 expression in a PKA-dependent manner but also prevented necrosis/apoptosis in primary mouse hepatocyte cultures. In conclusion, our findings document the importance of VIP neuropeptide–mediated cAMP-PKA signaling in hepatic homeostasis and cytoprotection in vivo. Because the enhancement of neural modulation differentially regulates local inflammation and prevents hepatocyte death, these results provide the rationale for novel approaches to managing liver IRI in transplant patients. Liver Transpl 19:945–956, 2013. © 2013 AASLD.
Hepatic ischemia/reperfusion injury (IRI) remains the major challenge in clinical liver transplantation, hepatic resection, trauma, and shock. Two phases of ischemia/reperfusion (IR)-triggered hypoxic cellular stress and inflammation-mediated hepatocellular injury can be distinguished. First, endogenous reactive oxygen species (ROS)–inflicted damage initiates local circulatory disturbances and inflammation, and this leads ultimately to hepatocyte death. Second, the activation of the host innate immune system exacerbates local liver injury. Our group was among the first to document that the activation of sentinel toll-like receptor 4 (TLR4) signaling is required for the mechanism of liver IRI and that IR-triggered TLR4, primarily on Kupffer cells/macrophages, facilitates downstream signature proinflammatory cytokine programs [ie, tumor necrosis factor α (TNF-α), interferon β (IFN-β), and chemokine (C-X-C motif) ligand (CXCL10)].[3, 4]
The immune system and the nervous system maintain active communication, and they mount integrated responses to danger signals through intricate chemical messengers, neuropeptides, and peptide hormones. The innate immune system acts as the first defensive firewall against invading pathogens through the recognition of pathogen-associated molecular patterns, phagocytosis, and the release of proinflammatory mediators. These immune components convey the peripheral message to the brainstem and pre-optic area of the anterior hypothalamus. Then, the regional neural-hormonal-stress response may amplify the local inflammation immune cascade to eliminate pathogens,[6-9] restore host homeostasis, and return to a resting state.
Vasoactive intestinal peptide (VIP), which has 28 amino acids, was first isolated from the gastrointestinal tract as a vasodilator. Later, VIP was recognized as a widely distributed neuroregulator in the central and peripheral nervous system. VIP has shown similarities to other gastrointestinal hormones such as secretin, glucagon, and pituitary adenylate cyclase activating polypeptide (PACAP; 68% identity). VIP conveys its immunoregulatory function almost exclusively through 2 G protein–coupled receptors: VPAC1, which is constitutively expressed by lymphocytes/macrophages, and VPAC2, which is expressed selectively by activated lymphocytes/macrophages. PACAP acts on these same receptors with high affinity, but it also interacts with the highly selective PAC1 receptor, which is also expressed in macrophages. Hepatocytes, on the other hand, constitutively express all 3 receptors: VPAC1, VPAC2, and PAC1.
The widespread distribution of VIP correlates with its involvement in various biological processes. Indeed, VIP peptides inhibit phagocytic activity, the production of free radicals, and the adherence/migration of macrophages and neutrophils. VIP peptides inhibit the production of proinflammatory cytokines/chemokines; down-regulate the expression of inducible nitric oxide synthase and the subsequent release of nitric oxide; and enhance anti-inflammatory interleukin-10 (IL-10), which is produced by activated macrophages, microglia, and monocytes.[17, 18] We recently showed that PACAP-mediated cyclic adenosine monophosphate (cAMP)–protein kinase A (PKA) activation provided protection in a murine model of liver IRI,[19, 20] whereas the ability of VIP to act in this manner is unknown. Experiments for determining this are important for 2 reasons: (1) cytoprotective actions of PACAP are thought to be mediated primarily by the PAC1 receptor, which is highly selective (1000-fold higher affinity) for PACAP, and (2) VIP neuropeptides and long-acting VIP analogues are currently being developed clinically for the treatment of chronic inflammatory lung disorders in sarcoidosis, bronchial asthma/chronic obstructive pulmonary disease, and pulmonary arterial hypertension[24, 25] as well as neuroblastoma and Alzheimer's disease. Although recent studies have implied beneficial effects of the VIP neuropeptide on liver IRI,[27, 28] the underlying mechanisms remain to be determined.
This study examined the putative therapeutic function and mechanisms by which VIP may affect IR hepatocellular insult and contribute to liver homeostasis. Because stress triggers proinflammatory and anti-inflammatory neuropeptides, we first determined endogenous VIP expression in hepatic IRI. We then determined whether exogenous VIP could diminish the proinflammatory response and promote hepatocyte survival in an IR-stressed liver, and we studied its actions in cultured macrophages and hepatocytes. Finally, we examined the dependence of these in vivo and in vitro actions on the cAMP-PKA signaling pathway.
- Top of page
- MATERIALS AND METHODS
Although VIP neuropeptides regulate macrophage activation and stimulate glucose-induced insulin secretion,[17, 18, 30] their role in innate immunity-driven liver inflammation and IRI remains ill defined. Here we show that (1) VIP was induced in a mouse model of liver warm IR damage, (2) exogenous VIP protected livers against IRI by inhibiting TLR4 activation and improving hepatocyte survival, and (3) VIP-mediated cytoprotection was cAMP-PKA–dependent. These results are consistent with our findings for PACAP neuropeptides in this model.
In the present study, we first found local VIP expression in IR-stressed livers, the levels of which were elevated between 12 and 24 hours of reperfusion. This may imply a regulatory role for intrinsic VIP in liver self-repair. We then asked whether the administration of exogenous VIP would attenuate liver IRI. Because IR-induced liver damage peaks at 6 hours of reperfusion, we focused on this time point to demonstrate the modulatory role of VIP neuropeptides. Strikingly, VIP treatment diminished hepatocellular damage, as evidenced by decreased sALT levels and an amelioration of cardinal features of liver IRI (ie, edema, vacuolization, and necrosis). These findings are consistent with the ability of VIP to prevent transient ischemic brain damage in a rat model of focal cerebral ischemia.
We found increased infiltration by CD68+ macrophages, which was consistent with a preferential proinflammatory chemokine gene expression profile in IR-stressed livers.[1-4] Because VIP therapy suppresses macrophage function,[13, 17, 18] others have suggested that VIP may act as an essential neural immunomodulator in autoimmune diseases. We observed reduced macrophage migration and decreased activation/function along with diminished expression of IRI signature genes [ie, TNF-α, IL-1β, IL-6, CXCL10, and CCL2 (monocyte chemoattractant protein 1]. Indeed, CXCL10, one of the key mediators in the type I IFN pathway downstream of TLR4,[3, 4] may be directly regulated by VIP. In agreement with our in vivo findings, VIP nearly abolished TLR4-mediated proinflammatory cytokine programs in BMM cultures.
The PKA pathway in VIP regulation[17, 18] may modulate multiple intracellular events. We have identified cAMP-PKA activation as a regulator that halts pathological cell recruitment, prevents destructive immune reactions, and promotes hepatocyte survival. This implies that PKA activation may raise defensive thresholds against the IR inflammatory response. Indeed, VIP treatment enhanced hepatic cAMP levels and augmented PKA activity, whereas PKA inhibition restored a proinflammatory profile in VIP-treated BMM cultures. Strikingly, in vivo PKA antagonism restored liver IRI pathology in otherwise IR-resistant VIP-treated hosts.
TLR4-mediated innate immune activation progresses through myeloid differentiation factor 88–dependent and/or Toll-interleukin 1 receptor domain-containing adapter inducing interferon-beta (TRIF)-dependent pathways. Our previous studies have indicated that signaling via TRIF/IFN regulatory factor 3 rather than myeloid differentiation factor 88 is instrumental for downstream NF-κB activation, IR inflammation, and hepatocellular damage.[2, 4] We have shown that cAMP-PKA activation may directly inhibit NF-κB by modulating p65 phosphorylation, stabilizing/elevating IκB, and regulating the transactivation/stability of NF-κB complexes. The cAMP-PKA signaling cascade may also indirectly enhance the phosphorylation of cyclic adenosine monophosphate response element binding (CREB), which has a higher affinity for CREB-binding protein, and result in competitive sequestration of p65/CREB-binding protein complexes in IR livers. Here we show that VIP-induced cAMP-PKA activation decreased the phosphorylation/proteolytic degradation of the IκB subunit, suppressed the phosphorylation of NF-κB p65 and downstream proinflammatory programs, yet augmented IL-10, and all of these things enhanced hepatocyte survival. In agreement with the in vivo data, we found that PKA activation diminished the proinflammatory cytokine profile in LPS-activated BMM cultures.
Activated neutrophils (polymorphonuclear neutrophils (PMNs)) generate ROS to promote tissue damage in the second phase of liver IRI. In contrast to the increased neutrophil infiltration/MPO activity in controls, livers in VIP-conditioned mice showed decreased Ly-6G+ neutrophil infiltration and MPO activity and depressed levels of CXCL1 (Kupffer cells (KC)), the key neutrophil chemoattractant. Because neutrophil activity can be enhanced by macrophage-produced cytokines, VIP can also exert its regulatory function during liver IRI through proinflammatory cytokine/chemokine networks.
Both necrosis and apoptosis are responsible for IR hepatocyte damage. Death receptor activation, mitochondrial Ca2+ loading, and ROS promote the mitochondrial permeability transition and lead to hepatocellular swelling, rupture of the plasma membrane, and release of cytochrome C, which ultimately result in adenosine triphosphate depletion–dependent oncotic necrosis and caspase-dependent apoptosis. Hepatocyte oncotic necrosis and apoptosis, which proceed via DNA degradation, can be detected with a TUNEL assay. Interestingly, VIP treatment inhibited necrosis/apoptosis, as evidenced by the decreased frequency of TUNEL-positive cells and caspase-3 activity in IR livers. VIP enhanced the hepatic expression of Bcl2/Bcl-xL, and this suggested PKA activation–mediated cytoprotection by antinecrotic/apoptotic proteins. It is plausible that neural immunomodulation prevents hepatocellular damage by modifying the proapoptotic/antiapoptotic ratio, maintaining mitochondrial integrity, or promoting adenosine triphosphate generation. To distinguish between necrosis and apoptosis in hepatocyte cultures, we employed H2O2 to mimic in vivo ROS-triggered necrosis or TNF-α to induce apoptosis. VIP supplementation diminished hepatocyte death, reduced caspase-3 activity, and ameliorated ALT/LDH release in both culture systems. These results, in agreement with our in vivo data, reinforce the immunomodulatory role of VIP in depressing NF-κB in nonparenchymal and parenchymal liver compartments with resultant improvements in hepatocellular function. Moreover, PKA inhibition exacerbated hepatocyte death, and this confirms that neural regulation at the hepatocyte level is cAMP-PKA–dependent.
In conclusion, this study is the first to reveal the mechanisms of the exogenous VIP neuropeptide for attenuating liver IRI by depressing macrophage function and improving hepatocyte survival in a cAMP-PKA–dependent manner. Harnessing immunoregulatory and cytoprotective mechanisms via VIP may be essential to the maintenance of hepatic homeostasis in vivo through the minimization of local organ damage and the promotion of IL-10 cytoprotection. Because VIP is being developed into a therapeutic principle for humans,[22-26] this very important peptide should also be considered as a novel therapy for targeting IR-triggered hepatic inflammation and damage in liver transplant patients.