Neuropeptide PACAP in mouse liver ischemia and reperfusion injury: Immunomodulation by the cAMP-PKA pathway

Authors

  • Haofeng Ji,

    1. Dumont-UCLA Transplant Center, Division of Liver and Pancreas Transplantation, Department of Surgery, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA
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  • Yu Zhang,

    1. Dumont-UCLA Transplant Center, Division of Liver and Pancreas Transplantation, Department of Surgery, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA
    2. Department of Surgery, Division of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
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  • Xiu-da Shen,

    1. Dumont-UCLA Transplant Center, Division of Liver and Pancreas Transplantation, Department of Surgery, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA
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  • Feng Gao,

    1. Dumont-UCLA Transplant Center, Division of Liver and Pancreas Transplantation, Department of Surgery, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA
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  • Cynthia Y. Huang,

    1. Dumont-UCLA Transplant Center, Division of Liver and Pancreas Transplantation, Department of Surgery, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA
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  • Catalina Abad,

    1. Semel Institute for Neuroscience and Human Behavior, Department of Psychiatry, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA
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  • Ronald W. Busuttil,

    1. Dumont-UCLA Transplant Center, Division of Liver and Pancreas Transplantation, Department of Surgery, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA
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  • James A. Waschek,

    1. Semel Institute for Neuroscience and Human Behavior, Department of Psychiatry, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA
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  • Jerzy W. Kupiec-Weglinski

    Corresponding author
    1. Dumont-UCLA Transplant Center, Division of Liver and Pancreas Transplantation, Department of Surgery, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA
    • M.D., Ph.D., Dumont-UCLA Transplant Center, 77-120 CHS, 10833 Le Conte Avenue, Los Angeles, CA 90095
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    • fax: 310-267-2358


  • Potential conflict of interest: Nothing to report.

  • See Editorial on Page 878

Abstract

Hepatic ischemia and reperfusion injury (IRI), an exogenous antigen-independent local inflammation response, occurs in multiple clinical settings, including liver transplantation, hepatic resection, trauma, and shock. The immune system and the nervous system maintain extensive communication and mount a variety of integrated responses to danger signals through intricate chemical messengers. This study examined the function and potential therapeutic potential of neuropeptide pituitary adenylate cyclase-activating polypeptides (PACAP) in a murine model of partial liver “warm” ischemia (90 minutes) followed by reperfusion. Liver IRI readily triggered the expression of intrinsic PACAP and its receptors, whereas the hepatocellular damage was exacerbated in PACAP-deficient mice. Conversely, PACAP27, or PACAP38 peptide monotherapy, which elevates intracellular cyclic adenosine monophosphate/protein kinase A (cAMP-PKA) signaling, protected livers from IRI, as evidenced by diminished serum alanine aminotransferase levels and well-preserved tissue architecture. The liver protection rendered by PACAP peptides was accompanied by diminished neutrophil/macrophage infiltration and activation, reduced hepatocyte necrosis/apoptosis, and selectively augmented hepatic interleukin (IL)-10 expression. Strikingly, PKA inhibition readily restored liver damage in otherwise IR-resistant, PACAP-conditioned mice. In vitro, PACAP treatment not only diminished macrophage tumor necrosis factor alpha/IL-6/IL-12 levels in a PKA-dependent manner, but also prevented necrosis and apoptosis in primary mouse hepatocyte cultures. Conclusion: Our novel findings document the importance of PACAP-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 manage liver inflammation and IRI in transplant patients. (HEPATOLOGY 2013)

Hepatic ischemia and reperfusion injury (IRI), an exogenous antigen-independent inflammation response, occurs in multiple clinical settings, including liver transplantation, hepatic resection, trauma, and shock.1 Liver ischemia and reperfusion (IR)-mediated local tissue damage combines two phases of ischemia-trigged hypoxic cellular stress and inflammation-mediated reperfusion injury. Endogenous reactive oxygen species (ROS)-inflicted tissue damage initiates circulatory disturbances and cascade of inflammation responses, leading to the ultimate hepatocyte death. Our group was among the first to document that activation of sentinel Toll-like receptor 4 (TLR4) signaling is required in the mechanism of liver IRI.2 We then provided evidence that IR-triggered TLR4, primarily on Kupffer cells/macrophages, activates downstream “signature” proinflammatory programs, such as tumor necrosis factor alpha (TNF-α), interferon-beta (IFN-β), and C-X-C motif chemokine (CXCL)10.3, 4

The immune system and the nervous system maintain extensive communication and mount a variety of integrated responses to danger signals through intricate chemical messengers. The innate immune system provides the first defense line against invading pathogens through recognition of pathogen-associated molecular patterns and releasing proinflammatory mediators.5 These immune components convey the peripheral message to the brainstem and preoptic area of the anterior hypothalamus, the activate systemic neuroendocrine hypothalamus, and regional neural-hormonal–stress response, which amplify local inflammation to eliminate pathogens.6-9 This interplay constitutes an important feedback loop that optimizes, monitors, and adjusts innate inflammation by stimulation of efferent vagus nerve activity.6, 7 The neural modulation of local inflammation eventually restores host homestasis and the return to a resting status.10

The mammalian nervous system, equipped with neuropeptides and peptide hormones with pro- and anti-inflammatory properties, may directly defend the host from microbial assault.9 Pituitary adenylate cyclase-activating polypeptides (PACAP), a 38-amino-acid neuropeptide (PACAP38), and a C-terminally truncated 27-amino-acid form (PACAP27), originally isolated from ovine hypothalamus,11 belong to the secretin/glucagon/vasoactive intestinal peptide (VIP) family. The PACAP sequence shows a 68% homology with VIP and was identified as a hypothalamic hormone that stimulates adenylate cyclase in pituitary cells.12 PACAP is expressed throughout the nervous system, adrenal gland, gastrointestinal tract, pancreas, and liver.12 Interestingly, PACAP storage/gene expression is found in central (e.g., thymus) and peripheral (e.g., spleen and lymph nodes) lymphoid organs and some lymphoid cells.13 PACAP exerts its function through three G-protein-coupled receptors.12 These include vasoactive receptors with high affinity for VIP and PACAP (i.e., VIP/PACAP receptor [VPAC]1, constitutively expressed in lymphocytes/macrophages, and VPAC2, expressed selectively in stimulated lymphocytes/macrophages).12 The third receptor, PAC1, favors PACAP (300- to 1,000-fold higher binding affinity than VIP) and macrophages.12 Hepatocytes express all three PACAP receptors.14

The highly conserved sequence, with wide expression and storage locations, suggest that PACAP may affect different physiological functions.15 Indeed, PACAP peptides inhibit LPS-induced TNF-α by increasing the activity of the cyclic adenosine monophosphate/protein kinase A (cAMP-PKA) axis and cAMP response element-binding protein (CREB)16 and by modulating nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) activity.17 We have shown that by differentially regulating local inflammation, the activation of cAMP-PKA prevented hepatocyte death,18 whereas others reported that PACAP deficiency resulted in higher susceptibility to retinal ischemic injury.19 These data suggesting therapeutic potential of PACAP neuropeptide warrant confirmation in animal inflammatory disease models.

This study was designed to examine putative therapeutic effects and mechanisms by which PACAP may contribute to liver homeostasis in IR-mediated hepatocellular insult. Because stress triggers pro- and anti-inflammatory response by neuropeptides/peptide hormones, we first determined the function of endogenous PACAP in the pathophysiology of liver IRI. The question then arose of whether exogenous PACAP could diminish proinflammatory response and promote hepatocyte survival. Finally, a key issue as to whether PACAP-induced cAMP-PKA activation is essential for liver homeostasis warrants critical evaluation while considering neural immunomodulation as a novel therapeutic concept in the management of liver inflammation.

Abbreviations

7-AAD, 7-aminoactinomycin D; ActD, actinomycin D; ALT, alanine aminotransferase; ATP, adenosine triphosphate; Bcl-2, B-cell lymphoma 2; Bcl-xl, B-cell lymphoma extra large; BMMs, bone-marrow–derived macrophages; cAMP, cyclic adenosine monophosphate; CCL, C-C motif ligand; CREB, cAMP response element-binding protein; CXCL, C-X-C motif chemokine; DMSO, dimethyl sulfoxide; FACS, fluorescence-activated cell sorting; H&E, hematoxylin and eosin; HPF, high-power fields; HPRT, hypoxanthine-guanine phosphoribosyl transferase; IFN-β, inteferon-beta; IκBα, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha; IL, interleukin; IR, ischemia and reperfusion IRI, ischemia and reperfusion injury; IV, intravenous; KO, knockout; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; mAb, monoclonal antibody; MPO, myeloperoxidase; MyD88, myeloid differentiation primary response gene 88; NF-κB, nuclear factor kappa light-chain enhancer of activated B cells; PACAP, pituitary adenylate cyclase-activating polypeptides; PBS, phosphate-buffered saline; PD-1, programmed death-1; p-IκBα, phosphorylated IκBα; PKA, protein kinase A; p-NF-κB, phosphorylated NF-κB; qRT-PCR, guantitative reverse-transcription polymerase chain reaction; ROS, reactive oxygen species; sALT, serum alanine aminotransferase; TLR4, Toll-like receptor 4; TNF-α, tumor nexrosis factor alpha; TRIF, TIR-domain–containing adapter-inducing IFN-β; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; VIP, vasoactive intestinal peptide; VPAC 1/2, VIP/PACAP receptor 1/2; WT, wide type.

Materials and Methods

Animals.

Male (8-12 weeks old) wild-type (WT) (Jackson Laboratory, Bar Harbor, ME) and PACAP-deficient mice20 on a C57BL/6 background (back-crossed for at least 12 generations) were used. Animals were housed in the University of California Los Angeles animal facility under specific pathogen-free conditions and received humane care according to the criteria outlined in Guide for the Care and Use of Laboratory Animals (prepared by the National Academy of Sciences; National Institutes of Health publication 86-23, revised 1985).

Mouse Warm Liver IRI Model.

We have used a mouse model of partial “warm” hepatic IRI.2 In brief, animals were anesthetized, injected with heparin (100 U/kg, intraperitoneally), and the arterial/portal venous blood supply to the cephalad lobes was interrupted by an atraumatic clip for 90 minutes. Sham-operated mice underwent the same procedure, but without vascular occlusion. In the treatment groups, animals were infused 1 hour before the onset of liver ischemia with a single dose of PACAP27 or PACAP38 neuropeptide (50 nmol/mouse, intravenously [IV]; Phoenix Pharmaceuticals, Burlingame, CA) dissolved in phosphate-buffered saline (PBS). Some recipients were given H-89 (cAMP-PKA inhibitor; 20 nmol/mouse, IV; Sigma-Aldrich, St. Louis, MO) dissolved in dimethyl sulfoxide (DMSO). Mice were sacrificed at various time points of reperfusion; liver and serum samples were collected for analysis.

The Hepatocellular Damage.

Serum alanine aminotransferase (sALT) levels were measured by the IDEXX Laboratory (Westbrook, ME). Culture medium alanine aminotransferase (ALT) levels were measured by an ALT kit (Stanbio, Boerne, TX). Untreated hepatocyte lysates were used to determine total ALT level. Cell death was expressed as ALT released from treated cells (percentage of the total ALT).

Histopathology.

Liver specimens (4 μm), stained with hematoxylin and eosin (H&E), were analyzed blindly by modified Suzuki's criteria.21 Primary monoclonal antibody (mAb) against mouse neutrophils (Ly-6G) (1A8; BD Biosciences, San Jose, CA) and macrophages (CD68) (FA-11; AbD Serotec, Raleigh, NC) were used.21 Liver sections were evaluated blindly by counting labeled cells in 10 high-power fields (HPF).

Myeloperoxidase Activity Assay.

The presence of myeloperoxidase (MPO) was used as an index of neutrophil accumulation in the liver.21 One absorbance unit of MPO activity was defined as the quantity of enzyme degrading 1 mol of peroxide/min at 25°C/g of tissue.

Quantitative Reverse-Transcription Polymerase Chain Reaction.

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed with a platinum SYBR green quantitative PCR kit (Invitrogen, Carlsbad, CA) by the Chromo 4 detector (MJ Research, Waltham, MA). Primers to amplify specific gene fragments were published.21 The sequence of PACAP and PACAP receptor primers is shown in Supporting Table 1. Target gene expressions were calculated by their ratios to the housekeeping gene, hypoxanthine-guanine phosphoribosyl transferase (HPRT).

Western Blottings.

Western blottings were performed with liver proteins (30 μg/sample) and rabbit antimouse B-cell lymphoma 2 (Bcl-2), B-cell lymphoma extra large (Bcl-xl), phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (p-IκBα), phosphorylated NF-κB (p-NF-κB) p65, and β-actin mAbs (Cell Signaling Technology, Danvers, MA).21 Relative quantities of protein were determined by densitometer and are expressed in AU.

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling Assay.

DNA fragments in liver sections, resulting from oncotic necrosis and apoptosis, were detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method (Klenow-FragEL DNA Fragmentation Detection Kit; Calbiochem, La Jolla, CA).21 TUNEL-positive cells were counted in 10 HPF/section under light microscopy (x400).

Caspase-3 Activity Assay.

Caspase-3 activity was performed using the Caspase-3 Cellular Activity Assay Kit (Calbiochem). Liver tissue sample and cell lysis were used according to the manufacturer's instruction.

The cAMP/PKA Kinase Activity Assays.

The cAMP levels and PKA activity in tissue samples were measured by the cAMP Enzyme Immunoassay and PKA kinase activity kits, respectively (Enzo Life Sciences, Farmingdale, NY).

Cell Cultures.

Bone-marrow–derived macrophages (BMMs), separated from femurs/tibias of C57BL/6 mice, were cultured (5 × 106/well) with 10% L929 conditioned medium for 6 days. The cell purity was 94%-99% CD11b+. BMMs were activated by lipopolysaccharide (LPS) (10 ng/mL; Sigma-Aldrich) in the presence of PACAP27, PACAP38 (10 nM), or PBS control and incubated for 24 hours. H-89 (10 μM) pretreatment at 1 hour before LPS stress was used to block the cAMP-PKA pathway. Cell-free supernatants were assayed for cytokine levels by enzyme-linked immunosorbent assay (eBioscience, San Diego, CA).

Mouse hepatocytes were isolated by in situ two-stage collagenase perfusion method, cultured with complete L-15 medium plus 6.25 μg/mL of insulin, 1 μM of dexamethasone, and 10% fetal bovine serum for 24 hours before experiments. Hepatocyte viability after isolation was 95%-99%. After pretreatment with PACAP27, PACAP38 (10 nM), with H-89 (10 μM) or DMSO control for 1 hour, hepatocyte death was induced by hydrogen peroxide (5 mM; Sigma-Aldrich), or TNF-α (10 ng/mL; R&D Systems, Minneapolis, MN) in combination with actinomycin D (ActD; 0.4 μg/mL; Sigma-Aldrich) during a 5-hour incubation period. Cells were processed for flow cytometry/caspase-3 activity, whereas supernatants were assessed for ALT/lactate dehydrogenase (LDH) levels.

LDH Release Assay.

Culture medium LDH activity was measured by LDH kit (Stanbio Laboratory, Boerne, TX). Untreated hepatocyte lysates were used to determine total LDH activity. Cell death was expressed as LDH activity released from the treated cells as a percentage of the total LDH activity.

Flow Cytometry.

Hepatocytes stained with fluorescein isothiocyanate/Annexin V and 7-aminoactinomycin D (7-AAD) (BD Biosciences, Mountain View, CA) were analyzed on a FACSCalibur cytometer (BD Biosciences). Dead cells were identified as Annexin V+7-AAD+.

Statistical Analysis.

All values are expressed as the mean ± standard deviation. Data were analyzed with an unpaired, two-tailed Student t test. P < 0.05 was considered to be statistically significant.

Results

Local PACAP Expression Profile in Liver IRI.

First, we determined whether IR triggers the expression of endogenous PACAP and PACAP receptor genes in mouse livers subjected to 90 minutes of warm ischemia, followed by reperfusion. Compared with sham controls, PACAP messenger RNA levels transiently dropped after the ischemia insult (0 hours) and increased progressively thereafter, peaking by 12-24 hours of reperfusion (Fig. 1A). The hepatic expression of PACAP receptors (VPAC1 and VPAC2) increased sharply at the beginning of reperfusion. Although the expression of VPAC1 reduced gradually and VPAC2 dropped rapidly during the first 6 hours of reperfusion, both increased steadily thereafter. The expression of the PAC1 receptor increased gradually from the onset of ischemia throughout the 24-hour reperfusion period (Fig. 1A).

Figure 1.

(A) Liver IRI triggers PACAP and PACAP receptor (VPAC1, VPAC2, and PAC1) gene expression. Liver samples were harvested from B6 mice that were either sham operated or subjected to 90 minutes of partial warm ischemia, followed by various lengths of reperfusion. Livers in groups of WT PACAP KO mice and WT mice pretreated with PACAP27, PACAP38, or PBS were subjected to ischemia (90 minutes). At 6 hours of reperfusion, hepatocellular function was analyzed by (B and D) sALT levels and (C and E) liver histology (representative H&E staining; magnification: ×100 and ×400) (*P < 0.001; n = 10-12/group).

PACAP Deficiency Sensitizes Liver to IRI.

To address whether the expression of PACAP neuropeptide is essential in liver homeostasis, we assessed the effect of PACAP deficiency in our model of 90-minute ischemia, followed by 6 hours of reperfusion. Indeed, PACAP knockout (KO) mice showed increased susceptibility to hepatic IRI, evidenced by higher sALT levels (Fig. 1B: 31,172 ± 6,994 versus 4,680 ± 554 U/L; P < 0.001) and liver histology, with more severe lobular edema, widespread hemorrhage, and congestion/hepatocellular necrosis, compared to WT controls (Fig. 1C).

Treatment With PACAP Neuropeptide Ameliorates Liver IRI.

To directly test the functional significance of PACAP, separate groups of WT mice were pretreated with PACAP neuropeptide. Unlike controls given PBS, mice conditioned with PACAP27/PACAP38 were resistant against IRI, evidenced by reduced sALT levels (Fig. 1D: 831 ± 76/984 ± 165 versus 5,225 ± 630 U/L; P < 0.001), well-preserved hepatic architecture (Fig. 1E: minimal sinusoidal congestion, no edema, vacuolization, or necrosis), and decreased Suzuki score (P < 0.001; Supporting Fig. 1).

PACAP Depresses Neutrophil/Macrophage Sequestration in IR Livers.

MPO-based liver neutrophil activity (U/g) was depressed in mice pretreated with PACAP27/PACAP38, compared to controls (Fig. 2A: 0.46 ± 0.22/0.67 ± 0.06 versus 1.56 ± 0.34; P < 0.01). These correlated with the frequency of neutrophils sequestered in the livers. Their accumulation in PACAP27/PACAP38-treated mice was decreased, compared to controls (Fig. 2B: 2.3 ± 1.3/3.3 ± 1.3 versus 27.8 ± 6.8; P < 0.001). The parallel macrophage recruitment was also ameliorated in PACAP27/PACAP38-treated ischemic livers (Fig. 2C: 3.5 ± 1.3/3.8 ± 1.0 versus 62.8 ± 3.8; P < 0.001).

Figure 2.

Accumulation of neutrophils and macrophages in IR livers after administration of neuropeptide PACAP (6 hours of reperfusion after 90 minutes of ischemia). (A) MPO levels, (B) Ly-6G+ neutrophils, and (C) CD68+ macrophages in IR liver lobes (magnification: ×400) (**P < 0.01; n = 10-12/group). qRT-PCR-assisted detection of cytokines/chemokines in mouse livers (6 hours of reperfusion after 90 minutes of ischemia: (D) CXCL1, CCL2, and CXCL10; (E) IL-10; and (F) TNF-α, IL-1β, IL-6, and IFN-β. Data normalized to HPRT gene expression (***P < 0.05; **P < 0.01; *P < 0.001; n = 4-6/group).

PACAP Differentially Regulates IR-Induced Liver Cytokine/Chemokine Programs.

To assess the immunoregulatory function of PACAP neuropeptide, we next analyzed hepatic chemokine/cytokine expression patterns. The neutrophil/monocyte-derived proinflammatory chemokine (CXCL1, C-C motif ligand [CCL]2, and CXCL10) and cytokine (TNF-α, IL-1β, IL-6, and IFN-β) programs were markedly and uniformly suppressed in PACAP treatment groups, compared to controls (Fig. 2D,F; P < 0.001; P < 0.01; and P < 0.05). However, elevated IL-10 levels were noted selectively after PACAP treatment (Fig. 2E; P < 0.001).

PACAP Inhibits IR-Mediated Liver Necrosis/Apoptosis.

We next screened for IR-induced hepatic oncotic necrosis and apoptosis. PACAP27/PACAP38 treatment diminished otherwise abundant hepatocellular necrosis/apoptosis, evidenced by reduced frequency of TUNEL+ cells (Fig. 3A,B: 2.8 ± 1.0/3.0 ± 1.4 versus 30.6 ± 4.9 [PBS]; P < 0.001) and decreased caspase-3 activity (Fig. 3C: 5.8 ± 0.8/4.6 ± 0.9 versus 22.2 ± 1.0 [PBS]; P < 0.01). In addition, western blotting analysis revealed selectively increased expression of Bcl-2/Bcl-xl, yet suppressed phosphorylation of IκBα/NF-κB p-65 proteins after PACAP treatment, compared to the PBS group (Fig. 3D).

Figure 3.

Necrosis/apoptosis in IR livers (6 hours of reperfusion after 90 minutes of ischemia). (A and B) TUNEL-assisted detection of hepatic necrosis/apoptosis (dark arrows) in ischemic liver lobes (magnification: ×400), (C) caspase-3 activity, and (d) western blotting–assisted detection of Bcl-2/Bcl-xl, p-IκBα, and p-NF-κB p65 (**P < 0.01; *P < 0.001; n = 4-6/group).

The cAMP-PKA Pathway Is Critical in PACAP Regulation of Liver IRI.

Having shown that PACAP suppressed macrophage function by cAMP-PKA,17 we then asked whether PACAP may trigger cAMP-PKA signaling in our model. Indeed, we have recently found that IR itself may trigger cAMP expression.18 Interestingly, administration of PACAP27/PACAP38 neuropeptide increased cAMP levels (Fig. 4A: 1,025 ± 224/1,085 ± 233 versus 510 ± 88; umol/g; P < 0.01) and PKA activity (Fig. 4B: 9.9 ± 0.2/10.4 ± 1.5 versus 5.0 ± 0.2; ng/g; P < 0.01), compared to controls.

Figure 4.

Functional significance of cAMP-PKA in PACAP neural regulation. Administration of PACAP peptides increased liver cAMP levels (A) and elevated PKA activity (B). Adjunctive use of H-89, a PKA inhibitor, restored liver injury in PACAP-pretreated groups, evidenced by (C) sALT levels (*P < 0.001), (D) liver histology (representative H&E; magnification: ×100 and ×400), and (E) CXCL-10, TNF-α, and IL-1β expression (***P < 0.05; *P < 0.001).

Next, we used H-89, a specific PKA inhibitor, to study whether cAMP-PKA activation is essential for PACAP-mediated neural immunomodulation. Strikingly, adjunctive inhibition of PKA activity not only restored, but even exacerbated liver IRI in PACAP-pretreated mice, evidenced by increased sALT levels (Fig. 4C: 6,115 ± 2,141 [PACAP27+H-89] versus 1,165 ± 496 [PACAP27]; 6,911 ± 1,668 [PACAP38+H-89] versus 2,371 ± 680 [PACAP38] U/L; P < 0.001) and hepatic histology. Livers after combined PACAP and PKA inhibition therapy were characterized by extended zonal/panlobular parenchyma necrosis, with widespread sinusoidal congestion and severe edema (Fig. 4D), comparable to PBS controls (Supporting Fig. 1). Intrahepatic expression of proinflammatory CXCL10, TNF-α, and IL-1β was uniformly heightened, whereas IL-10 levels concomitantly diminished after PACAP plus PKA antagonist treatment (Fig. 4E).

PACAP Directly Regulates Macrophage TLR4 Response.

TLR4 activation represents the pivotal triggering step in IR-mediated liver inflammation.2 Because macrophages express three PACAP receptors,14 TLR4 regulation may contribute to the beneficial effect of PACAP in our model. First, we asked whether and how PACAP-triggered cAMP-PKA may affect macrophage TLR4 responses. BMM cultures were stimulated with LPS in the absence or presence of PACAP; with H-89 (PKA inhibitor) or DMSO (control). Figure 5A-C shows that PACAP28/PACAP38 supplement depressed (P < 0.01 and P < 0.001) otherwise enhanced LPS-induced expression (pg/mL) of TNF-α (353.8 ± 14.8/481.2 ± 39.8 versus 959.6 ± 52.5), IL-6 (301.2 ± 59.8/565.6 ± 120.0 versus 2,188.0 ± 142.5), and IL-12p40 (2,145.9 ± 99.0/2,382.9 ± 117.7 versus 5,225.5 ± 80.9), but increased anti-inflammatory IL-10 (Fig. 5D: 3,823.1 ± 188.2/3,031.5 ± 93.9 versus 1,161.3 ± 23.1; P < 0.001). In contrast, H-89-facilitated PKA inhibition resulted in enhanced (P < 0.01 and P < 0.001) TNF-α, IL-6, and IL-12p40 levels (pg/mL) in PACAP27/PACAP38 groups (Fig. 5A-C: TNF-α: 1,074.1 ± 33.8/1,117.8 ± 22.6; IL-6: 1,690.9 ± 174.9/1,986.4 ± 97.6; and IL-12p40: 4,805.1 ± 271.0/5,347.1 ± 168.1), compared with PACAP cultures only. Moreover, IL-10 levels decreased (P < 0.001) in BMM cultures supplemented with PACAP plus H-89 (Fig. 5D: 833.2 ± 124.9/981.1 ± 126.8), compared with PACAP alone.

Figure 5.

Effects of PACAP upon macrophage TLR4 activation in vitro. BMMs were stimulated with LPS in the absence or presence of PACAP peptides with H-89 (PKA inhibitor), or DMSO (control). The production of (A) TNF-α, (B) IL-6, (C) IL-12p40, and (D) IL-10 in culture supernatants (**P < 0.01; *P < 0.001). Representative of n = 3/group.

PACAP Prevents Hepatocyte Death.

To analyze the immunomodulatory function of cAMP-PKA signaling in hepatocytes, we designed primary hepatocyte culture systems to mimic liver IR-mediated hepatocellular damage in vivo. Because necrosis and apoptosis are essential in the mechanism of liver IRI, we used H2O2 to mimic in vivo ROS-triggered hepatocyte necrosis or TNF-α/ActD to induce apoptosis. Native mouse hepatocytes were cultured in the presence of PACAP, with H-89 (PKA antagonist) or DMSO (control). The addition of PACAP27/PACAP38 consistently suppressed hepatocyte death, assessed by fluorescence-activated cell-sorting (FACS)-assisted frequency (%) of Annexin V+7-AAD+ cells (Fig. 6A: H2O2: 3.3 ± 2.6/3.4 ± 2.8 versus 13.8 ± 3.6; TNF-α+ActD: 4.8 ± 2.3/3.1 ± 2.5 versus 15.6 ± 2.5; P < 0.001), diminished caspase-3 activity (pmol/min/5 × 10 E4 cells) (Fig. 6B: H2O2: 0.09 ± 0.07/0.09 ± 0.07 versus 0.29 ± 0.17; TNF-α+ActD: 0.58 ± 0.13/0.58 ± 0.13 versus 1.91 ± 0.32; P < 0.001), LDH release (%) (Fig. 6C: H2O2: 10.39 ± 2.29/10.36 ± 2.28 versus 19.19 ± 5.26; TNF-α+ActD: 15.58 ± 4.23/15.54 ± 4.22 versus 37.62 ± 9.58; P < 0.01), and ALT release (%) (Fig. 6D: H2O: 10.98 ± 2.06/11.06 ± 2.03 versus 22.58 ± 4.58; TNF-α+ActD: 13.97 ± 3.80/14.10 ± 3.75 versus 36.36 ± 8.58; P < 0.01), as compared to controls. In contrast, PKA inhibition enhanced hepatocyte death (Fig. 6A: H2O2: 10.1 ± 3.1/11.2 ± 3.2; TNF-α+ActD: 13.4 ± 2.7/13.3 ± 2.8) and caspase-3 activity (Fig. 6B: H2O2: 0.27 ± 0.17/0.26 ± 0.16; TNF-α+ActD: 1.85 ± 0.31/1.74 ± 0.30). In addition, PKA inhibition increased LDH (Fig. 6C: H2O2: 18.63 ± 5.03/18.45 ± 5.03; TNF-α+ActD: 36.22 ± 9.24/35.88 ± 9.22), and ALT (Fig. 6D: H2O2: 21.97 ± 4.63/22.20 ± 4.57; TNF-α+ActD: 35.15 ± 8.49/35.52 ± 8.39) release in hepatocyte cultures.

Figure 6.

Cytoprotective effects of PACAP peptides upon hepatocytes in vitro. H2O2 or TNF-α+ActD were used to induce primary murine hepatocyte necrosis/apoptosis in the absence or presence of PACAP peptides with H-89 (PKA inhibitor) or DMSO (control). (A) FACS-assisted detection of Annexin V+7-AAD+ dead cells, (B) hepatocellular caspase-3 activity, (C) LDH release, and (D) ALT levels in supernatants (***P < 0.05; **P < 0.01; *P < 0.001). Representative of n = 3/group.

Discussion

Although PACAP neuropeptide regulates macrophage cytokine programs and stimulates hepatocyte glucose production,22 its role in innate immunity-driven liver inflammation and IR hepatocellular injury have not been explored. Here, we show that (1) PACAP and its intrinsic receptors were induced in mouse livers subjected to warm IR, (2) PACAP deficiency exacerbated liver damage, implying that PACAP is essential for liver homeostasis, (3) exogenous PACAP protected livers against IRI by inhibiting macrophage function and improving hepatocyte survival, and (4) PACAP-mediated regulatory/cytoprotective function was cAMP-PKA dependent.

PACAP neuropeptide may affect a diverse range of physiological functions. Indeed, PACAP-deficient mice display increased cold stress,23 decreased reproductive function,24 and altered metabolism.25 It was also shown that PACAP ablation results in higher susceptibility to renal IRI,26, 27 consistent with PACAP-facilitated cytoprotection against oxidative stress in an in vitro primary kidney cell culture.28 However, PACAP failed to salvage hepatocellular carcinoma cell lines, perhaps because of uncertain expression of PACAP receptors on tumorized cells.28 We first found that warm IR did trigger local PACAP and all three receptor expressions in the stressed liver, the levels of which were elevated between 12 and 24 hours of reperfusion (self-repair phase). This may imply the importance of PACAP neural regulation in the liver's self-healing as a result of IRI. Then, we used PACAP KO mice to study the requirement for PACAP innervations/regulation in hepatic homeostasis. Strikingly, mice lacking PACAP neuropeptide experienced heightened liver damage, evidenced by sALT levels and histological Suzuki's grading of liver injury. We reported similarly exacerbated IRI in livers deficient of programmed death-1 (PD-1)21 and T-cell immunoglobulin mucin domain-conatining molecule 329 negative T-cell costimulation signaling. In analogy with cytoprotection rendered by stimulating the PD-1/B7-H1 pathway,21 we then asked whether the administration of PACAP neuropeptide may affect liver function. Strikingly, both PACAP27 and PACAP38 diminished IR hepatocellular damage, evidenced by decreased sALT levels and amelioration of cardinal features of liver injury (i.e., edema, vacuolization, and necrosis).

In the initial IR-mediated inflammation phase, we found increased activation/recruitment of CD68+ macrophages, consistent with preferential proinflammatory chemotactic gene expression in IR-stressed livers.2-4 Because PACAP therapy suppressed macrophage function,16 others have suggested that PACAP may act as an essential neural immunomodulator in autoimmune diseases.30 We observed decreased CD68+ macrophage infiltration and diminished activation/function, evidenced by immunohistology and decreased expression of IRI signature markers, including TNF-α, IL-1β, IL-6, CXCL10, and CCL2 (monocyte chemoattractant protein-1). Indeed, CXCL-10, one of the key mediators in the type I IFN pathway downstream of TLR4 in liver IRI,3, 4 may be directly regulated by PACAP. In agreement with our in vivo findings, PACAP supplement diminished TLR4-mediated proinflammatory cytokine programs in the BMM culture system.

cAMP-PKA intracellular signaling is involved in neural regulation by PACAP17, 31 and may modulate multiple intracellular events.32 We have identified cAMP-PKA activation as a regulator of the liver IRI cascade, which halts pathological cell sequestration, prevents destructive immune reactions, and ultimately promotes parenchymal cell survival.18 It is plausible that PKA activation raises the defensive threshold to inflammatory response in IR livers. Indeed, the administration of PACAP27/PACAP38 augmented cAMP levels and enhanced PKA activity in IR livers. Furthermore, inhibition of PKA recreated proinflammatory cytokine profiles in PACAP-treated BMM cultures, confirming the altered liver inflammation phenotype to be responsible for local cytoprotection. Strikingly, in vivo PKA antagonism not only rendered otherwise IR-resistant PACAP-treated hosts susceptible to the panoply of hepatic proinflammatory events, but also readily restored liver IRI pathology.

TLR4 activation promotes innate responses through the myeloid differentiation primary response gene 88 (MyD88)- or TIR-domain–containing adapter-inducing IFN-β (TRIF)-dependent pathway.33 Our previous studies indicated that signaling by TRIF-IRF3, rather than MyD88, is instrumental for downstream NF-κB activation, local 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, as well as regulating transactivation/stability of NF-κB complexes.18 cAMP-PKA may also indirectly enhance CREB phosphorylation, which has higher affinity for CREB-binding protein, resulting in the sequestration of p65/CBP complexes in IR livers.18 Here, PACAP-induced cAMP-PKA activation decreased the phosphorylation/proteolytic degradation of the IκB subunit and suppressed the phosphorylation of NF-κB p65 (Fig. 7). Furthermore, our qRT-PCR showed that PACAP inhibited downstream TLR4-NF-κB proinflammatory programs, abolished TNF receptor/IL-1 receptor de novo activation, yet augmented IL-10, all of which enhance hepatocyte survival. In agreement with in vivo data, we found that PKA activation diminished the proinflammatory cytokine profile in LPS-activated BMM cultures.

Figure 7.

Scheme of molecular mechanisms of cAMP-PKA-dependent PACAP-mediated inhibition of the TLR-4/NF-κB axis. PACAP binding to its receptors on macrophages (A) triggers the cAMP-PKA pathway, which (B) directly prevents NF-κB translocation and (C) stabilizes the IκB inhibitor. This transcriptional machinery suppresses proinflammatory programs, but promotes IL-10.

Activated neutrophils generate ROS to dominate tissue damage in the second phase of liver IRI.1 Indeed, unlike in sham controls, Ly-6G+ neutrophil infiltration and MPO activity increased in PBS-treated IRI. In contrast, livers in PACAP-conditioned mice were characterized by decreased neutrophil infiltration/MPO activity and depressed CXCL1 (KC) levels, the key neutrophil chemoattractant. Because neutrophil activation and target tissue sequestration can be enhanced by macrophage-derived inflammatory cytokines, PACAP can exert its regulatory function during liver IRI through cytokine/chemokine networks.

Both necrosis and apoptosis are responsible for hepatocyte damage in liver IRI.34 Death-receptor activation, mitochondrial Ca2+ loading, and ROS promote mitochondrial permeability transition, leading to hepatocellular swelling, rupture of the plasma membrane, and release of cytochrome C, ultimately resulting in adenosine triphosphate (ATP) depletion-dependent oncotic necrosis and caspase-dependent apoptosis.1 Hepatocyte oncotic necrosis and apoptosis, which render parenchymal cytodestruction, proceed through DNA degradation detected by TUNEL assay.34 Consistent with the essential role of PACAP in hepatic homeostasis, PACAP deficiency exacerbated hepatodestruction, increased frequency of TUNEL+ cells, and augmented caspase-3 activity (data not shown). Conversely, PACAP treatment inhibited necrosis/apoptosis, evidenced by decreased frequency of TUNEL+ cells and caspase-3 activity in IR livers. Interestingly, PACAP enhanced the hepatic expression of Bcl-2/Bcl-xl, suggesting PKA activation-mediated cytoprotection by antinecrotic/apoptotic proteins. It is plausible that neural immunomodulation prevents hepatocellular damage by modifying pro-/antiapoptotic ratio, decreasing the release of apoptogenic factors (e.g., cytochrome c) from mitochondria into the cytosol, maintaining mitochondria integrity, or promoting ATP generation.35

To distinguish between necrosis and apoptosis in our in vitro hepatocyte cultures, we employed H2O2 to mimic in vivo ROS-triggered necrosis and TNF-α to induce apoptosis. Interestingly, PACAP supplement diminished hepatocyte death, reduced capase-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 PACAP to depress NF-κB not only in nonparenchymal, but also in parenchyma cells, with resultant improvement of liver function. Furthermore, PKA inhibition exacerbated hepatocyte death, confirming that this neural regulation at the hepatocyte level is cAMP-PKA dependent.

In conclusion, this study is the first to document the (1) essential role of intrinsic PACAP neuropeptide to maintain hepatic homeostasis in liver IR inflammation/damage and (2) efficacy of exogenous PACAP to ameliorate liver IRI by depressing macrophage function in a cAMP-PKA-dependent manner and to improve hepatocyte survival. Harnessing immune-regulatory and cytoprotective mechanisms by neuropeptide PACAP may be essential in the maintenance of hepatic homeostasis in vivo by minimizing local organ damage and promoting IL-10-dependent cytoprotection. Several clinical trials suggest that PACAP38, at picomolar concentrations, is safe for clinical use and has no direct effect on the circulation or regional cerebral blood flow.36, 37 As neuropeptides are currently being developed into a new therapeutic principle for chronic inflammatory lung disorders in sarcoidosis patients,38 they should also be considered as a novel therapeutic means to manage liver inflammation and IRI in humans.

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