This information is current as of April 3, 2014.

NLRP3 Regulates Neutrophil Functions and Contributes to Hepatic Ischemia− Reperfusion Injury Independently of Inflammasomes

J Immunol published online 2 April 2014 http://www.jimmunol.org/content/early/2014/04/01/jimmun ol.1302039

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http://www.jimmunol.org/content/suppl/2014/04/02/jimmunol.130203 9.DCSupplemental.html

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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Yoshiyuki Inoue, Koumei Shirasuna, Hiroaki Kimura, Fumitake Usui, Akira Kawashima, Tadayoshi Karasawa, Kenji Tago, Katsuya Dezaki, Satoshi Nishimura, Junji Sagara, Tetsuo Noda, Yoichiro Iwakura, Hiroko Tsutsui, Shun'ichiro Taniguchi, Ken Yanagisawa, Toshihiko Yada, Yoshikazu Yasuda and Masafumi Takahashi

Published April 2, 2014, doi:10.4049/jimmunol.1302039 The Journal of Immunology

NLRP3 Regulates Neutrophil Functions and Contributes to Hepatic Ischemia–Reperfusion Injury Independently of Inflammasomes

Inflammation plays a key role in the pathophysiology of hepatic ischemia–reperfusion (I/R) injury. However, the mechanism by which hepatic I/R induces inflammatory responses remains unclear. Recent evidence indicates that a sterile inflammatory response triggered by I/R is mediated through a multiple-protein complex called the inflammasome. Therefore, we investigated the role of the inflammasome in hepatic I/R injury and found that hepatic I/R stimuli upregulated the inflammasome-component molecule, nucleotide-binding oligomerization domain–like receptor (NLR) family pyrin domain–containing 3 (NLRP3), but not apoptosisassociated speck–like protein containing a caspase recruitment domain (ASC). NLRP32/2 mice, but not ASC2/2 and caspase-12/2 mice, had significantly less liver injury after hepatic I/R. NLRP32/2 mice showed reduced inflammatory responses, reactive oxygen species production, and apoptosis in I/R liver. Notably, infiltration of neutrophils, but not macrophages, was markedly inhibited in the I/R liver of NLRP32/2 mice. Bone marrow transplantation experiments showed that NLRP3 not only in bone marrow–derived cells, but also in non-bone marrow–derived cells contributed to liver injury after I/R. In vitro experiments revealed that keratinocyte-derived chemokine–induced activation of heterotrimeric G proteins was markedly diminished. Furthermore, NLRP32/2 neutrophils decreased keratinocyte-derived chemokine–induced concentrations of intracellular calcium elevation, Rac activation, and actin assembly formation, thereby resulting in impaired migration activity. Taken together, NLRP3 regulates chemokine-mediated functions and recruitment of neutrophils, and thereby contributes to hepatic I/R injury independently of inflammasomes. These findings identify a novel role of NLRP3 in the pathophysiology of hepatic I/R injury. The Journal of Immunology, 2014, 192: 000–000. epatic ischemia–reperfusion (I/R) injury is a major cause of liver dysfunction and serious complications in hepatic surgery and liver transplantation. Systemic low-flow ischemia and hypoxia, such as trauma, hemorrhagic shock, sepsis, congestive heart failure, and respiratory failure, may also lead to hepatic I/R injury. One prominent feature of hepatic I/R injury is excessive inflammatory responses characterized by the release of inflammatory cytokines and chemokines that recruit circulating leukocytes, mainly neutrophils, into the ischemic tissues (1). The recruited neutrophils in the ischemic tissues can then lead to dramatic hepatic I/R injury. Indeed, experimental studies have shown that the inhibition of inflammatory responses by neutrophil

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depletion or by neutralizing Abs against chemokines for neutrophils can reduce hepatic I/R injury (2). However, the mechanism by which I/R stimuli trigger inflammatory responses after I/R in the liver is still unknown. An accumulating body of evidence indicates that inflammation in the absence of pathogens, which is referred to as sterile inflammation, is mediated through the inflammasome, a large multipleprotein complex in the cytosol that regulates proinflammatory cytokine IL-1b production (3, 4). The best characterized inflammasome is the nucleotide-binding oligomerization domain–like receptor (NLR) family pyrin domain–containing 3 (NLRP3; also known as NALP3 and cryopyrin) inflammasome, which is known

*Division of Inflammation Research, Center for Molecular Medicine, Jichi Medical University, Tochigi 329-0498, Japan; †Department of Surgery, Jichi Medical University, Tochigi 329-0498, Japan; ‡Department of Biochemistry, Jichi Medical University, Tochigi 329-0498, Japan; xDepartment of Physiology, Jichi Medical University, Tochigi 329-0498, Japan; {Department of Translational Systems Biology and Medicine Initiative, The University of Tokyo, Tokyo 113-0033, Japan; ‖Department of Molecular Oncology, Shinshu University Graduate School of Medicine, Nagano 3908621, Japan; #Department of Cell Biology, Japanese Foundation for Cancer Research, Cancer Institute, Tokyo 135-8550, Japan; **Research Institute for Biomedical Science, Tokyo University of Science, Chiba 278-8510, Japan; and ††Department of Microbiology, Hyogo College of Medicine, Nishinomiya 663-8501, Japan

Address correspondence and reprint requests to Prof. Masafumi Takahashi, Division of Inflammation Research, Center for Molecular Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. E-mail address: masafumi2@ jichi.ac.jp

Received for publication August 2, 2013. Accepted for publication February 19, 2014. This work was supported by the Japan Society for the Promotion of Science through the funding program for Next Generation World-Leading Researchers (NEXT) initiated by the Council for Science and Technology Policy (M.T.) and a grant-in-aid for research activity start-up in Jichi Medical University (Y.I.). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302039

The online version of this article contains supplemental material. Abbreviations used in this article: ALT, alanine aminotransferase; ASC, apoptosisassociated speck–like protein containing a caspase recruitment domain; AST, aspartate aminotransferase; BMT, bone marrow transplantation; [Ca2+]i, concentrations of intracellular calcium; 4-HNE, 4-hydroxy-2-nonenal; I/R, ischemia–reperfusion; KC, keratinocyte-derived chemokine; NLR, nucleotide-binding oligomerization domain– like receptor; NLRP3, nucleotide-binding oligomerization domain–like receptor family pyrin domain–containing 3; NPC, nonparenchymal cell; 8-OHdG, 8-hydroxy-29deoxyguanosine; ROS, reactive oxygen species; WT, wild-type. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

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Yoshiyuki Inoue,*,† Koumei Shirasuna,* Hiroaki Kimura,* Fumitake Usui,* Akira Kawashima,* Tadayoshi Karasawa,* Kenji Tago,‡ Katsuya Dezaki,x Satoshi Nishimura,{ Junji Sagara,‖ Tetsuo Noda,# Yoichiro Iwakura,** Hiroko Tsutsui,†† Shun’ichiro Taniguchi,‖ Ken Yanagisawa,‡ Toshihiko Yada,x Yoshikazu Yasuda,† and Masafumi Takahashi*

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Materials and Methods Animals All experiments in this study were performed in accordance with the Jichi Medical University Guide for Laboratory Animals. ASC-deficient (ASC2/2), caspase-12/2, and IL-1b2/2 mice were generated as previously described (8–11) and backcrossed for at least 12 generations onto the C57BL/6J background. NLRP32/2 mice were kindly provided by Dr. Vishva M. Dixit (Genentech, South San Francisco, CA) (12). C57BL/6J (wild-type [WT]) were purchased from SLC Japan (Shizuoka, Japan). All mice used in this study had a C57BL/6J genetic background (male, 8–12 wk old).

Hepatic I/R injury The mice underwent hepatic I/R surgery or sham operations. Partial hepatic ischemia was induced as previously described (13). In brief, mice were anesthetized with isoflurane. A midline laparotomy was performed and an atraumatic clip (Fine Science Tools, Foster City, CA) was placed across the portal vein, hepatic artery, and bile duct to interrupt blood supply to the left lateral and median lobes (∼70%) of the liver. After 60 min of partial hepatic ischemia, the clip was removed to initiate reperfusion. Sham control mice underwent the same protocol without vascular occlusion. Mice were sacrificed at the indicated periods of reperfusion, and samples of blood and ischemic lobes were collected for analyses.

Measurement of serum parameters Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, blood urea nitrogen, and creatinine were measured using chemical analyzer Fuji-drychem (Fuji Film, Tokyo, Japan) according to the manufacturer’s instructions.

Histology and immunohistochemistry The I/R livers, perfused with saline, were fixed with 10% (w/v) formalin in PBS and embedded in paraffin. The paraffin-embedded samples were cut into 4-mm-thick sections and stained with H&E, sirius red, and Masson’s trichrome. Quantification of injured area using H&E sections was conducted in a double-blind manner by involving at least two independent researchers. Immunohistochemical analysis was performed with Abs against the oxidative stress markers 4-hydroxy-2-nonenal (4-HNE; clone HNEJ-2; Japan Institute for the Control of Aging, Nikken SEIL; Shizuoka, Japan), 8-hydroxy-29-deoxyguanosine (8-OHdG; clone N45.1; Nikken SEIL), and the smooth muscle cell marker a-smooth muscle actin (clone 1A4; Sigma, St. Louis, MO). These were followed by incubation with biotin-conjugated secondary Abs. The sections were washed and treated with avidin-peroxidase (M.O.M. Immunodetection Kit; Vector Laboratories, Burlingame, CA). The reaction was developed using a DAB

substrate kit (Vector Laboratories). The sections were then counterstained with hematoxylin. No signals were detected when an irrelevant IgG (Vector Laboratories) was used instead of the primary Ab as a negative control. Liver neutrophils were stained using a naphthol AS-D chloroacetate esterase staining kit (Muto Pure Chemical, Tokyo, Japan), which identifies specific leukocyte esterases. The staining sections were digitalized and analyzed using a microscope (FSX-100; Olympus, Tokyo, Japan).

Detection of apoptosis Apoptotic cells were identified using an In situ Apoptosis Detection Kit (Takara Bio, Shiga, Japan) using a TUNEL method.

Real-time RT-PCR Total RNA was prepared using ISOGEN (Nippon Gene, Toyama, Japan) according to the manufacturer’s instructions. Real-time RT-PCR analysis was performed using the Thermal Cycler Dice Real Time System II (Takara Bio, Shiga, Japan) to detect the mRNA expression of Nlrp3, Asc, Il1b, Il6, Tnfa, Ifng, Ccl2, and Actb. The primers are listed in Supplemental Table I. The expression levels of each target gene were normalized by subtracting the corresponding b-actin threshold cycle (CT) value; normalization was carried out using the DD CT comparative method.

Flow cytometry Cells were analyzed using flow cytometry. The cells were double-labeled with the following Abs: FITC-conjugated anti-CD45R (eBioscience), PE-conjugated anti–Gr-1 (Miltenyi Biotec), FITC-conjugated anti-F4/80 (eBioscience), PE-conjugated anti-CD11b (eBioscience), FITC-conjugated anti-CXCR1 (Biorbyt), PerCP/Cy5.5-conjugated anti-CXCR2 (Biolegend), and FITC- and PE-conjugated anti-Ly6G (BD Bioscience). The cells were examined by flow cytometry (FACSCalibur; Becton Dickinson, Franklin Lakes, NJ) and analyzed using CellQuest software version 3.3 (Becton Dickinson). Isotype control Abs were used as negative controls to exclude nonspecific background staining.

Bone marrow transplantation Whole bone marrow cells were harvested by flushing femurs and tibias with PBS. RBCs were lysed in hypotonic buffer. The cells were washed twice with PBS and resuspended. Six-week-old recipient mice were lethally irradiated with a total dose of 9 Gy and were injected with bone marrow cells (2 3 106) via a cervical vein. To verify the reconstitution of bone marrow after transplantation by this protocol, we used GFP mice as donors. Flow cytometry analysis showed that 8 wk after bone marrow transplantation (BMT), peripheral blood cells consisted of .90% GFP+ cells (5, 6). Using this protocol, we produced six types of BMTWT to WT, BMTWT to NLRP32/2, BMTNLRP32/2 to WT, BMTNLRP32/2 to NLRP32/2, BMTWT to ASC2/2, and BMTASC2/2 to WT mice.

Cell culture and in vitro experiments Hepatocytes were isolated from mice using two-step collagenase perfusion. In brief, mice were anesthetized and livers were perfused with 0.5 mM EGTA and 25 mM HEPES in Ca2+- and Mg2+-free HBSS (Invitrogen) for 5 min, followed by perfusion with 0.05% collagenase (Wako Chemicals), 0.005% trypsin inhibitor (Wako Chemicals), and 15 mM HEPES in DMEM (Sigma) for 10 min at 37˚C. The suspension was centrifuged at 30 3 g for 2 min and washed three times with DMEM at room temperature. The cells were resuspended in DMEM and the percentage of intact cells was determined by trypan blue staining. Hepatocytes were plated on collagen-coated dishes in DMEM supplemented with 10% FCS, 1 mM dexamethasone, and 1 mM insulin. Nonparenchymal cells (NPCs) were also isolated using collagenase perfusion. The suspension was digested in a beaker with stirring at 37˚C for another 15 min. After hepatocytes were removed by low-speed centrifugation, NPCs were purified via density gradient centrifugation using 28% Nycodenz (Axis-Shield, Oslo, Norway) at 4˚C at 1400 3 g for 20 min, and cultured in DMEM supplemented with 10% FCS.

In vitro hypoxia experiments For hypoxia experiments, cells were placed in Aneropac (Mitsubishi Gas Chemical, Tokyo, Japan) (14). To verify the hypoxic conditions, we measured the pO2 of the culture media under hypoxic conditions using StatProfile Critical Care Xpress (Nova Biomedical, Waltham, MA). The levels of pO2 under normoxic and hypoxic conditions were 173.2 6 2.0 and 42.5 6 7.3 mm Hg, respectively (n = 4, p , 0.01).

Neutrophil isolation For isolation of neutrophils, mice received two i.p. injections with 1 ml of 9% casein at 0 h and after 12 h, and mice were sacrificed 3 h later. The

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to participate in the pathophysiology of sterile inflammation. The NLRP3 inflammasome contains NLRP3 associated with apoptosisassociated specklike protein containing a caspase recruitment domain (ASC), which recruits caspase-1 and induces its activation. Because caspase-1 is known as an IL-1b–converting enzyme, it processes pro–IL-1b into its mature form and induces IL-1b release, thereby causing inflammation and tissue damage. We have recently shown the importance of the inflammasome in the pathogenesis of several disease conditions (5–7). In particular, using mice deficient in ASC and caspase-1, we demonstrated that inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes, is crucially involved in the initial inflammatory response after myocardial I/R injury (5). Therefore, we hypothesized that the NLRP3 inflammasome is involved in inflammatory responses and subsequent injury after I/R in the liver. To test this hypothesis, we used mice deficient in NLRP3, ASC, and caspase-1, and unexpectedly found that the deficiency of NLRP3, but not ASC and caspase-1, resulted in less inflammation and injury after hepatic I/R, indicating the inflammasome-independent role of NLRP3. Furthermore, we identified that NLRP3 regulates chemokine-mediated signaling pathway and neutrophil functions. These findings demonstrate a novel role for NLRP3 in the pathophysiology of hepatic I/R independent of inflammasomes, and provide new insight into the mechanisms underlying the postI/R inflammatory responses in the liver.

NLRP3 IN HEPATIC I/R INJURY

The Journal of Immunology peritoneal cavity was lavaged with PBS, and RBCs were lysed with hypotonic buffer. The cells were washed and resuspended in RPMI 1640 (Invitrogen) supplemented with 0.1% BSA. The isolated neutrophils were incubated for 1 h at 37˚C and then stimulated with keratinocyte-derived chemokine (KC; Peprotech, Rocky Hill, NJ).

Neutrophil migration assay To analyze neutrophil migration, we performed a transwell migration assay using 24-well tissue culture plates with 3-mm pore polycarbonate membrane (BD Biosciences). Cells (1 3 106) were placed in the upper chamber and incubated for 1 h at 37˚C. Cells migrated to the lower chamber were collected and analyzed by flow cytometry and Giemsa staining.

Western blotting

Preparation of membrane fractions Isolated PMNs were washed with PBS and suspended in hypotonic buffer containing 20 mM Tris-HC1 (pH 7.5), 1 mM MgCl2, 1 mM DTT, and protease inhibitor mixture. Then the cells were passed through a 21G needle five times using a 10-ml syringe. The disrupted cell suspension was centrifuged at 450 3 g for 5 min at 4˚C to remove the nuclei. The supernatant containing the membrane fraction was transferred to a fresh tube. A crude plasma membrane fraction was obtained by centrifuging the supernatant at 100,000 3 g for 40 min at 4˚C. The pellet was washed with hypotonic buffer and suspended in buffer containing Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 1 mM DTT and protease inhibitor, using 27G needle.

[35S]GTPgS binding assay [35S]GTPgS binding to the membrane fractions was assayed as described previously (15) with a few modifications. In brief, the membrane fractions (10 mg) were stimulated with 10 ng/ml KC (dissolved in distilled water) or distilled water as a control for 30 s. Then the binding reaction was started by adding 5 mM [35S]GTPgS in 100 ml reaction buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2, and 1 mM EDTA at 25˚C for 5 min. At the end of the incubation period, reaction mixtures were filtered through GF/B Glass Microfiber Filters (GE Healthcare Life Sciences), followed by several washes with the wash buffer (20 mM Tris-HCl [pH 7.5], 100 mM NaCl, 25 mM MgCl2). The radioactivity captured on Glass filters was measured by scintillation counter.

Measurement of Rac1 activity Rac1 activity was measured using a Rac1 Activation Assay Biochem Kit (Cytoskeleton, Denver, CO) according to the manufacturer’s instructions. In brief, cell lysates were prepared and incubated with PAK-PBD beads for 1 h at 4˚C. The immunocomplex beads were collected and washed with cold wash buffer. Proteins were eluted from beads by boiling in Laemmli sample buffer and analyzed by Western blotting. Blots were probed with Rac1 Abs, followed by a secondary Ab conjugated with HRP (Invitrogen).

Measurement of concentrations of intracellular calcium Concentrations of intracellular calcium ([Ca2+]i) in neutrophils were measured by dual-wavelength Fura-2 microfluorometry with excitation at 340/380 nm and emission at 510 nm using a cooled charge-coupled device camera (16). The ratio image was produced on an Aquacosmos system (Hamamatsu Photonics, Hamamatsu, Japan). Maximum value and peak time of [Ca2+]i after KC stimulation were analyzed.

F-actin formation assay Cells were fixed and permeabilized using a FoxP3 Staining Buffer Set (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. For F-actin staining, the cells were incubated in

Acti-stain 488 fluorescent phalloidin (Cytoskeleton), and fluorescence was analyzed using flow cytometry (FACSCalibur; Becton Dickinson) or confocal laser-scanning microscopy (FV-10i; Olympus, Tokyo, Japan).

Statistical analysis Data are expressed as mean 6 SEM. An unpaired t test was used for comparisons between two groups. For comparisons between multiple groups, the significance of differences in between-group means was determined by one-way ANOVA combined with a post hoc test. A p value ,0.05 was considered to be statistically significant.

Results Role of NLRP3 and ASC in hepatic I/R injury To investigate whether the inflammasome is involved in the process of hepatic I/R injury, we measured expression levels of NLRP3 and ASC in liver tissues after I/R. Real-time RT-PCR analysis showed that mRNA expression of Nlrp3, but not Asc, was increased in response to hepatic I/R (Fig. 1A), suggesting that the NLRP3 inflammasome plays a role in hepatic I/R development. To further investigate the role of the inflammasome, we subjected NLRP32/2, ASC2/2, and caspase-12/2 mice to 60 min of partial hepatic ischemia (∼70%), followed by reperfusion. Normal liver architecture was observed in NLRP32/2, ASC2/2, and caspase-12/2 mice at baseline condition (data not shown). After 6 h of reperfusion, histological analysis showed a large area of the liver injury in WT mice (Fig. 1B). Unexpectedly, however, the liver injury was drastically attenuated in NLRP32/2 mice, but not in ASC2/2 and caspase-12/2 mice, compared with WT mice. NLRP32/2 mice also showed less liver injury after 3 and 6 h of reperfusion than the WT, ASC2/2, and caspase-12/2 mice, as determined by serum ALT levels (Fig. 1C). Although serum AST levels tended to decrease in NLRP32/2 mice, there were no significant differences in other biological parameters such as serum total bilirubin, blood urea nitrogen, or creatinine among these mice (Supplemental Fig. 1A). We also assessed IL-1b levels in the I/R liver using ELISA. The IL-1b levels were significantly higher in the I/R liver compared with those in the liver of sham-operated mice, and this IL1b elevation was significantly lower in the I/R liver of NLRP32/2 mice, but not of ASC2/2 and caspase-12/2 mice (Fig. 1D). Interestingly, IL-1b2/2 mice had less serum ALT levels and liver injury after hepatic I/R (Supplemental Fig. 1B), suggesting the role of inflammasome-independent IL-1b–driven inflammatory responses in hepatic I/R injury. Inflammatory cytokines, reactive oxygen species, apoptosis, and fibrosis in hepatic I/R injury We next performed real-time RT-PCR to detect the expression of inflammatory cytokines. The expression of Il1b, Il6, Ccl2, and Tnfa was increased at 3 h after reperfusion in the liver of WT mice, and the increased expression of Il1b, Il6, and Ccl2, but not Tnfa, was decreased in NLRP32/2 mice (Fig. 2). Because I/R initiates reactive oxygen species (ROS) generation, leading to apoptotic and necrotic cell death in the liver (1, 17), we performed immunohistochemical analysis with Abs against 4-HNE and 8OHdG and TUNEL staining to assess ROS generation and apoptosis, respectively. A number of cells positive for 4-HNE and 8-OHdG were clearly visualized in the I/R liver of WT mice (Fig. 3A). In addition, the number of these positive cells was markedly lower in the I/R liver of NLRP32/2 mice than in WT mice. TUNEL+ cells were also detected in the injured area and in border areas, but not in the noninjured area of the I/R liver, and they were significantly decreased in the injured area of NLRP32/2 mice (Fig. 3B, 3C). However, there were no differences among the sham-operated, WT, and NLRP32/2 mice in liver fibrosis, as

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Expression of NLRP3 and ASC was analyzed by Western blotting. Cells lysates were prepared with 1% Nonidet P-40 buffer and subjected to SDSPAGE under reducing conditions, and the protein bands were then transferred to a PVDF membrane. The membrane was blocked for 1 h at room temperature with 5% skim milk and then incubated for 1 h at room temperature with the primary Abs, followed by incubation for 1 h with the secondary Ab, conjugated HRP. The primary Abs against NLRP3, ASC (Enzo Life Sciences), and b-actin (Sigma) were used. Immunoreactive bands were visualized by Western BLoT Quant HRP Substrate (Takara Bio, Shiga, Japan). The expression levels of b-actin served as an internal control for protein loading.

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FIGURE 1. Role of NLRP3 and ASC in hepatic I/R injury. Liver samples were obtained from nonoperated, sham-operated (Sham), WT, NLRP32/2, ASC2/2, and caspase-12/2 mice at 3 and 6 h after hepatic I/R injury. (A) Hepatic mRNA levels of Nlrp3 and Asc were assessed at 3 h after I/R in WT mice by using real-time RT-PCR analysis (n = 4). (B) Liver sections were stained with H&E at 6 h after I/R, and quantitative analysis of the injured area was performed (n = 5). (C) Serum levels of ALT (3 and 6 h after I/R) were assessed (n = 7–10). (D) The levels of IL-1b in the I/R liver were assessed (n = 7–10). Data are expressed as means 6 SEM. *p , 0.05.

determined by sirius red and Masson’s trichrome staining, or in myofibroblasts, as determined by a-smooth muscle actin immunostaining (Supplemental Fig. 2A). Infiltration of inflammatory cells To investigate the infiltration of inflammatory cells, we performed flow cytometry for neutrophils (CD45+/Gr1+/CD45R2) and macrophages (CD45+/CD11b+/F4/80+) in the liver. The number of infiltrated neutrophils was significantly increased in the liver of WT

mice at 3 h after I/R, and this increased neutrophil infiltration was significantly inhibited in the liver of NLRP32/2 mice (Fig. 4A, 4B). Because Gr-1 includes both Ly6G and Ly6C, we used Ly6G as a specific maker for neutrophils. The number of Ly6G+ cells was significantly increased in the I/R liver of WT mice, and this increased number of Ly6G+ cells was significantly suppressed in the I/R liver of NLRP32/2 mice (Supplemental Fig. 2B, 2C). Further, Gr1+ population was almost identical to Ly6G+ population in the I/R liver (Supplemental Fig. 2D). In contrast, there

The Journal of Immunology

was no difference in the levels of infiltrated macrophages between WT and NLRP32/2 mice (Fig. 4C, 4D). Prevention of neutrophil infiltration in the I/R liver of NLRP32/2 mice was confirmed by naphthol AS-D chloroacetate esterase staining (Supplemental Fig. 2E). Contribution of bone marrow–derived cells To explore the mechanism of preventing hepatic I/R injury in NLRP32/2 mice, we determined the expression of NLRP3 and ASC in primary hepatocytes and NPCs by Western blotting. The presence of NLRP3 and ASC was confirmed in primary NPCs, whereas the same was not detected in primary hepatocytes (Fig. 5A). No expression of NLRP3 and ASC was confirmed in primary hepatocytes and Hepa1-6 cells under hypoxic conditions (Supplemental Fig. 3). J774 macrophages served as a positive control. This finding indicates a limited role of hepatocytes in NLRP3 inflammasome activation. Therefore, we produced six types of BMT mice (BMTWT to WT, BMTWT to NLRP32/2, BMTNLRP32/2 to WT, BMTNLRP32/2 to NLRP32/2, BMTWT to ASC2/2, and BMTASC2/2 to WT mice) (5). The protocol used in this study could not achieve full reconstitution of some hematopoietic cells such as mast cells and Kupffer cells because these cells are relatively irradiation resistant (18, 19). The liver injury after I/R, as determined by histological examination and serum ALT levels, was significantly reduced in BMTNLRP32/2 to NLRP32/2 and BMTNLRP32/2 to WT mice, compared with that in BMTWT to WT mice (Fig. 5B, 5C). In addition, the I/R injury was also significantly reduced in BMTWT to NLRP32/2 mice. As expected, no significant reduction of I/R injury was observed in BMTWT to ASC2/2 and BMTASC2/2 to WT mice. Neutrophil migration activity Because neutrophil infiltration was dramatically reduced in I/R liver of NLRP32/2 mice, we hypothesized that neutrophil functions differ between WT and NLRP32/2 mice. To test this hypothesis, we i.p. injected casein sodium, which is frequently used

as a chemoattractant for neutrophils, into the mice and evaluated the i.p. accumulation of neutrophils at 15 h after injection. In WT and NLRP32/2 mice, a similar number of neutrophils was recruited to the peritoneum, and the purity of neutrophils was ∼90% in both mice (Fig. 6A). Because it is known that neutrophils can induce hepatocyte injury during hepatic I/R injury (1), we next tested the effect of neutrophils on hepatocyte death using the coculture system. To mimic the condition during I/R in vivo, we cultured hepatocytes for 3 h under hypoxic conditions and then cocultured them with various numbers of neutrophils. Although cell death activity, as determined by lactate dehydrogenase release, tended to increase according to the number of neutrophils, no significant difference of its activity was observed between WT and NLRP32/2 neutrophils (Fig. 6B). I/R stimuli stimulate the induction of chemokines, such as CXCL1 (KC) and CXCL2 (MIP-2), for neutrophils and led to tissue injury in the liver (1). Therefore, we assessed neutrophil migration in response to KC using a transwell migration assay, and found that migration activity of NLRP32/2 neutrophils was significantly lower than that of WT neutrophils under baseline and KC stimulation conditions (Fig. 6C). We also confirmed that migrated cells in the lower wells were neutrophils using flow cytometry and morphological examination (Fig. 6D, 6E). Mechanism of neutrophil migration Because it has been well established that KC functions as a specific ligand for G protein–coupled receptors, CXCR1 and CXCR2, we tested the expression of CXCR1 and CXCR2 in WT and NLRP32/2 neutrophils. Flow cytometry analysis showed that there was no difference in the expression levels of CXCR1 and CXCR2 between WT and NLRP32/2 neutrophils (Fig. 7A). Therefore, we prepared cell membrane fractions and assessed their basal and KC-stimulated [35S]GTPgS binding activities. In WT neutrophil membrane, stimulation with KC significantly enhanced the GTPgS binding activity (Fig. 7B). However, KC stimulation failed to enhance the GTPgS binding activity of NLRP32/2 neutrophil membrane. These data indicate that chemokine-mediated signaling pathway is impaired in NLRP32/2 neutrophils. Because KC regulates neutrophil migration through the mobilization of [Ca2+]i, activation of the small G protein Rac, and subsequent F-actin assembly, we examined these molecular events that participate in the process of neutrophil migration. KC stimulation induced elevation of [Ca2+]i in WT neutrophils (Fig. 7C). However, in NLRP32/2 neutrophils, the elevation of [Ca2+]i in response to KC stimulation was significantly suppressed and delayed (Fig. 7D). To assess Rac1 activity, we performed an affinity pull-down assay for GTP-bound Rac1 in WT and NLRP32/2 neutrophils, and found that KC clearly stimulated Rac1 activation in WT neutrophils in a time-dependent manner, and this activation was markedly diminished in NLRP32/2 neutrophils (Fig. 7E). Cell lysates from the HEK293 cells transfected with a constitutive activated mutant of Rac1 (Rac1 G12V) and a negative mutant of Rac1 (Rac1 S17N) were used as positive and negative controls, respectively. The assembly of F-actin was assessed by staining with an F-actin-specific dye (Acti-stain 488 phalloidin) and quantified by flow cytometry. The formation of the F-actin assembly was observed 10–30 s after KC stimulation in WT neutrophils, and it was significantly inhibited in NLRP32/2 neutrophils (Fig. 7F).

Discussion The major findings of this study are as follows: 1) I/R stimuli upregulated NLRP3, but not ASC, in the liver; 2) NLRP32/2 mice, but

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FIGURE 2. Expression of inflammatory cytokines. Liver samples were obtained from sham-operated mice (Sham), or WT and NLRP32/2 mice at 3 h after hepatic I/R injury. Hepatic mRNA levels of Il1b (A), Il6 (B), Tnfa (C), Ifng (D), and Ccl2 (E) were assessed using real-time RT-PCR analysis. Data are expressed as means 6 SEM (n = 4). *p , 0.05.

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not ASC2/2 and caspase-12/2 mice, had markedly less liver injury after hepatic I/R; 3) NLRP32/2 mice showed reduced neutrophil infiltration, inflammatory cytokine expression, ROS production, and number of TUNEL+ apoptotic cells in I/R liver; 4) BMT experiments showed the role of NLRP3 not only in bone marrow–derived cells but also in non–bone marrow–derived cells in hepatic I/R injury; 5) in vitro experiments revealed that KCenhanced GTP binding was diminished in neutrophils from NLRP32/2 mice; and 6) NLRP32/2 neutrophils decreased KCinduced [Ca2+]i elevation, Rac activation, and actin assembly

formation, thereby resulting in impaired migration activity. The results of this study clearly indicate that NLRP3 regulates neutrophil functions through affecting a chemokine-mediated signaling and contribute to the pathophysiology of hepatic I/R injury. Importantly, this role of NLRP3 is novel and independent of inflammasomes. Increasing evidence indicates that sterile inflammation contributes to I/R injury (3, 20); however, it remains unclear as to how sterile inflammation is triggered after I/R in the liver. In this study, we hypothesized that sterile inflammation and subsequent injury

FIGURE 4. Infiltration of inflammatory cells. Cells were isolated from the liver of sham-operated mice or WT and NLRP32/2 mice at 3 h after hepatic I/R injury. Infiltration of CD45+/Gr-1+/CD45R2 cells (neutrophils) (A) and CD45+/F4/80+/CD11b+ cells (macrophages) (C) was analyzed using flow cytometry. Quantitative analysis of CD45+/Gr-1+/CD45R2 cells (neutrophils) (B) and CD45+/F4/80+/CD11b+ cells (macrophages) (D) was performed. Data are expressed as means 6 SEM (n = 3 for each). *p , 0.05.

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FIGURE 3. ROS and apoptosis. Liver sections were obtained from sham-operated mice or WT and NLRP32/2 mice at 6 and 24 h after hepatic I/R injury. (A) Sections collected at 6 h after I/R were immunohistochemically analyzed by staining with Abs against the ROS markers 4-HNE and 8-OHdG. (B) Sections collected at 24 h after I/R were analyzed by TUNEL staining. (C) Quantitative analysis of TUNEL+ cells was performed. Data are expressed as means 6 SEM (n = 4). *p , 0.05.

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are mediated by the inflammasome. Surprisingly, however, prevention of hepatic I/R injury was observed in mice deficient in NLRP3, but not ASC and caspase-12/2 mice. Because the inflammasome is defined as a molecular platform that activates caspase-1, these findings suggest that NLRP3 contributes to hepatic I/R injury independently of the inflammasome. Recent investigations suggest that ASC or NLRP3 exhibits an inflammasome-independent function in certain conditions. For instance, Ippagunta et al. (21) reported that ASC regulates the migration of lymphocytes, as well as Ag uptake by dendric cells via Dock2 expression, independently of inflammasomes, and that ASC also contributes to adaptive immune responses. Furthermore, the authors described that loss of Dock2 expression in T cells was observed in certain strains of ASC2/2 mice (22). We examined Dock2 mRNA expression in neutrophils and found that ASC2/2 mice in our colony express Dock2 normally (data not shown). Taxman et al. (23) also demonstrated the inflammasome-independent role of ASC in MAPK activation and chemokine induction. Furthermore, supporting our findings, Shigeoka et al. (24) recently reported that reduction in renal I/R injury was observed in mice deficient in NLRP3, but not ASC or caspase-1, and they conclude that an NLRP3-dependent, inflammasome-independent pathway may contribute to the development of I/R injury in the kidney. Our data show a novel and inflammasome-independent role of NLRP3 and provide evidence for the neutrophil-intrinsic role of NLRP3 that contributes to I/R injury. Neutrophils play a pivotal role in excessive inflammatory responses and resultant tissue injury during I/R in the liver (1, 25). I/R stimuli trigger chemotactic signals (e.g., KC) from the parenchyma (e.g., hepatocytes), leading to the recruitment of neutrophils into the ischemic tissues. The recruited neutrophils are then activated, and they produce a large amount of ROS and inflammatory cytokines, which promote tissue injury. Because IL1b and IL-1R signaling is critical for the induction of adhesion molecules required for the attachment of neutrophils to vascular endothelial cells (26), the inflammasome activation may influence the recruitment of neutrophils. In this study, however, we demonstrated that NLRP32/2 mice exhibited dramatically less neutrophil migration and tissue injury after hepatic I/R independently of the inflammasomes, and verified that NLRP32/2 neutrophils exhibited an impairment of chemokine-mediated signaling and functions, including activation of heterotrimeric G proteins, [Ca2+]i elevation, Rac activation, and actin assembly formation, and migration. These findings provide a molecular basis for the prevention of hepatic I/R injury observed in NLRP32/2 mice. Recently, Kamo et al. (27) reported that after hepatic I/R injury, ASC-mediated inflammasome activation leads to IL-1b production and subsequently promotes high mobility group box one induction, which triggers a TLR4-driven inflammatory response. In their study, ASC2/2 mice showed less inflammation and injury after hepatic I/R. More recently, Huang et al. (28) reported that NLRP32/2 and caspase-12/2 mice are protected from hepatic I/R injury and extracellular histones activate the NLRP3 inflammasome during hepatic I/R through TLR9. These are somewhat inconsistent with our findings. Although the reason for this discrepancy is unclear, the differences between our study and the study by Kamo et al. are the hepatic I/R protocol used and the extent of injury. Compared with our study and prior studies on hepatic I/R injury (13, 29), it is likely that the I/R protocol by Kamo et al. induced excessive inflammation and injury in the liver. These data suggest that the contribution of the inflammasome might depend on the extent of liver injury and the status of inflammatory responses after I/R. Indeed, the role of IL-1b in

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FIGURE 5. Contribution of bone marrow–derived cells. (A) NPCs and hepatocytes were isolated from WT mice. Cell lysates were prepared and analyzed by Western blotting with Abs against NLRP3, ASC, or b-actin. J774 macrophages were used as a positive control. (B and C) BMT WT to WT, BMT WT to NLRP32/2 , BMTNLRP32/2 to WT, BMT NLRP32/2 to NLRP32/2 , BMT WT to ASC2/2 , and BMTASC2/2 to WT mice were developed, and hepatic I/R injury was produced in these mice 8 wk after BMT. Liver sections were obtained from these BMT mice at 3 h after hepatic I/R injury. (B) Representative photographs of H&E staining are shown. (C) Serum ALT levels were assessed. Data are expressed as means 6 SEM (n = 7–8). *p , 0.05.

hepatic I/R injury is controversial. Kato et al. (30) reported that there was no difference in serum ALT levels between WT and IL-

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NLRP3 IN HEPATIC I/R INJURY

1R–deficient mice after hepatic I/R injury and suggested a limited role of IL-1b in causing hepatic I/R injury. Conversely, Tan et al. (25) showed that I/R upregulates hepatic IL-1b, and that hepatic I/R injury, liver inflammation, and neutrophil infiltration were attenuated in mice deficient in IL-1R1 or treated with the IL-1R antagonist Anakinra. We also observed that hepatic I/R injury was attenuated in IL-1b2/2 mice, suggesting that the inflammasomeindependent, IL-1b–driven inflammatory responses appear to be important in hepatic I/R injury. In this regard, previous studies reported that neutrophil-derived serine proteases, such as neutrophil elastase and proteinase 3, can mainly induce IL-1b processing and suggest a redundant or minor role of caspase-1 in neutrophil IL-1b processing (31–34). Guma et al. (32) reported that IL-1b processing is observed in neutrophils isolated from caspase-12/2 mice. Menzel et al. (35) also observed no protective effects against liver injury and inflammatory responses during trauma and hemorrhagic shock in caspase-12/2 mice.

Thus, further investigation is necessary to elucidate the exact role of the inflammasomes and IL-1b in inflammatory responses and injury after I/R in the liver. Although previous studies reported that some hematopoietic cells such as mast cells and Kupffer cells are relatively irradiation resistant (18, 19), our data on BMT mice suggest that NLRP3 not only in bone marrow–derived cells (i.e., neutrophils), but also in non–bone marrow–derived cells, plays a role in hepatic I/R injury. Watanabe et al. (36) recently reported that the inflammasome components including NLRP3 and ASC are present in hepatic stellate cells and regulate the development of liver fibrosis. Consistent with this, we observed that inflammasome activation of cardiac fibroblasts plays an essential role in myocardial I/R injury and subsequent remodeling (5). Therefore, we assume that hepatic stellate cells may play a role in hepatic I/R injury. In conclusion, we demonstrate that NLRP3 regulates chemokine-mediated neutrophil signaling and functions, which can

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FIGURE 6. Neutrophil migration activity. (A) Casein-induced peritoneal exudates were prepared from WT and NLRP32/2 mice. The content of neutrophils (Gr-1+/CD45R2 cells) was analyzed using flow cytometry. Representative histograms are shown. Data are expressed as means 6 SEM (n = 3). (B) Hepatocytes were cocultured with indicated numbers of WT and NLRP32/2 neutrophils for 22 h. Released lactate dehydrogenase levels in culture supernatants were assessed. Data are expressed as means 6 SEM (n = 3). (C) Neutrophil migration in response to KC (10 and 100 ng/ml) was measured using a modified Boyden chamber transwell migration assay. (D) The purity of neutrophils (Gr-1+/CD45R2 cells) that responded to KC in the lower chamber was assessed using flow cytometry and Giemsa staining. Data are expressed as means 6 SEM (n = 4). (E) Representative photographs of Giemsa staining are shown. *p , 0.05.

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FIGURE 7. Neutrophil functions in NLRP32/2 mice. (A) Expression of CXCR1 and CXCR2 was assessed in WT and NLRP32/2 neutrophils using flow cytometry analysis. Data are expressed as means 6 SEM (n = 3). (B) [35S]GTPgS binding to the membrane fractions after treatment with KC (10 ng/ml) for 5 min in WT and NLRP32/2 neutrophils was assessed (n = 3). (C) Change in [Ca2+]i in response to KC (10 ng/ml) in WT and NLRP32/2 neutrophils was measured using the Ca2+-sensitive fluorescent dye Fura-2. (D) Maximum value and peak time of [Ca2+]i after KC stimulation were analyzed (n = 5). (E) WT and NLRP32/2 neutrophils were treated with KC (10 ng/ml) for the indicated periods. Cell lysates were prepared and a Rac1 pull-down assay was performed. HEK293 cells transfected with constitutive active (G12V) and negative (S17N) mutants of Rac1 were used as positive and negative controls. (F) WT and NLRP32/2 neutrophils were treated with KC (10 ng/ml) for 10–30 s. F-actin formation was visualized using Acti-stain 488 phalloidin. Data are expressed as means 6 SEM (n = 3). *p , 0.05.

contribute to neutrophil recruitment in the I/R liver and subsequent tissue injury. Our data identify a previously unknown role of NLRP3, which is independent of ASC-mediated inflammasome activation. These findings revealed a novel mechanism by which I/R triggers sterile inflammation after I/R in the liver.

Acknowledgments We thank Masako Sakurai, Sumiyo Watanabe, and Yumi Ohde for technical assistance; Dr. Vishva M. Dixit (Genentech) for providing Nlrp32/2 mice; and Fumiko Chikugo (Jichi Medical University) and Dr. Shinsuke Taki (Shinshu University) for invaluable suggestions.

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Disclosures The authors have no financial conflicts of interest.

References

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