NLRP3 Inflammasome Activation in Lung Vascular Endothelial Cells

NLRP3 Inflammasome Activation in Lung Vascular Endothelial Cells Contributes to Intestinal Ischemia/Reperfusion-Induced Acute Lung Injury This information is current as of September 28, 2021. Homare Ito, Hiroaki Kimura, Tadayoshi Karasawa, Shu Hisata, Ai Sadatomo, Yoshiyuki Inoue, Naoya Yamada, Emi Aizawa, Erika Hishida, Ryo Kamata, Takanori Komada, Sachiko Watanabe, Tadashi Kasahara, Takuji Suzuki, Hisanaga Horie, Joji Kitayama, Naohiro Sata, Kazuyo Yamaji-Kegan and Masafumi Takahashi Downloaded from J Immunol published online 29 July 2020 http://www.jimmunol.org/content/early/2020/07/28/jimmun ol.2000217 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 29, 2020, doi:10.4049/jimmunol.2000217 The Journal of Immunology

NLRP3 Inﬂammasome Activation in Lung Vascular Endothelial Cells Contributes to Intestinal Ischemia/ Reperfusion-Induced Acute Lung Injury

Homare Ito,*,† Hiroaki Kimura,* Tadayoshi Karasawa,* Shu Hisata,‡ Ai Sadatomo,*,† Yoshiyuki Inoue,*,† Naoya Yamada,* Emi Aizawa,* Erika Hishida,* Ryo Kamata,* Takanori Komada,* Sachiko Watanabe,* Tadashi Kasahara,* Takuji Suzuki,‡ Hisanaga Horie,† Joji Kitayama,† Naohiro Sata,† Kazuyo Yamaji-Kegan,x and Masafumi Takahashi*

Intestinal ischemia/reperfusion (I/R) injury is a life-threatening complication that leads to inflammation and remote organ damage. Downloaded from The NLRP3 inflammasome regulates the caspase-1–dependent release of IL-1b, an early mediator of inflammation after I/R injury. In this study, we investigated the role of the NLRP3 inflammasome in mice with intestinal I/R injury. Deficiency of NLRP3, ASC, caspase-1/11, or IL-1b prolonged survival after intestinal I/R injury, but neither NLRP3 nor caspase-1/11 deficiency affected intestinal inflammation. Intestinal I/R injury caused acute lung injury (ALI) characterized by inflammation, reactive oxygen species generation, and vascular permeability, which was markedly improved by NLRP3 deficiency. Bone marrow chimeric

experiments showed that NLRP3 in non–bone marrow–derived cells was the main contributor to development of intestinal http://www.jimmunol.org/ I/R-induced ALI. The NLRP3 inflammasome in lung vascular endothelial cells is thought to be important to lung vascular permeability. Using mass spectrometry, we identified intestinal I/R-derived lipid mediators that enhanced NLRP3 inflammasome activation in lung vascular endothelial cells. Finally, we confirmed that serum levels of these lipid mediators were elevated in patients with intestinal ischemia. To our knowledge, these findings provide new insights into the mechanism underlying intestinal I/R-induced ALI and suggest that endothelial NLRP3 inflammasome–driven IL-1b is a novel potential target for treating and preventing this disorder. The Journal of Immunology, 2020, 205: 000–000.

ntestinal ischemia/reperfusion (I/R) injury is a common life- endotoxins from ischemic intestine (4, 5). However, the mecha- by guest on September 28, 2021 threatening complication that can occur as a result of many nism by which intestinal I/R induces inflammation in the intestine I clinical conditions, such as mesenteric arterial thrombosis, and distant organs remains to be elucidated. strangulated ileus, trauma, abdominal aortic aneurysm surgery, Increasing evidence indicates that the nucleotide-binding olig- and intestinal transplantation (1). Intestinal I/R not only injures the omerization domain-like receptor (NLR) family pyrin domain intestine itself but also can severely damage distant organs. containing 3 inflammasome initiates an inflammatory response in In particular, the lung is susceptible to developing acute injury the development of various diseases (6–9). The NLR family pyrin after intestinal I/R (2, 3). One prominent feature of intestinal domain containing 3 (NLRP3) inflammasome is a large, intra- I/R-induced acute lung injury (ALI) is an excessive inflammatory cellular, multiprotein complex that consists of NLRP3, the adaptor response characterized by the release of proinflammatory cyto- protein apoptosis speck-like protein containing a caspase recruit- kines, the infiltration of inflammatory cells, and bacteria-derived ing domain (ASC), and cysteine protease caspase-1. In response to

*Division of Inflammation Research, Center for Molecular Medicine, Jichi Medical discussed the data and provided scientific advice. H.I. and M.T. wrote the manuscript. University, Tochigi 329-0498, Japan; †Department of Surgery, Jichi Medical Univer- All authors approved this study. sity, Tochigi 329-0498, Japan; ‡Division of Pulmonary Medicine, Department of x Address correspondence and reprint requests to Prof. Masafumi Takahashi, Division Medicine, Jichi Medical University, Tochigi 329-0498, Japan; and Department of of Inflammation Research, Center for Molecular Medicine, Jichi Medical University, 3311- Anesthesiology, University of Maryland School of Medicine, Baltimore, MD 21201 1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. E-mail address: [email protected] ORCIDs: 0000-0001-9523-1335 (H.I.); 0000-0001-5721-2619 (A.S.); 0000-0001- The online version of this article contains supplemental material. 6970-9657 (R.K.); 0000-0003-3360-3185 (T.Komada); 0000-0001-7687- 5477 (T.Kasahara); 0000-0002-5907-4050 (H.H.); 0000-0002-5708-7130 (J.K.); Abbreviations used in this article: AA, arachidonic acid; ALI, acute lung injury; ALT, 0000-0002-8500-972X (K.Y.-K.); 0000-0003-2716-7532 (M.T.). alanine aminotransferase; ASC, adaptor protein apoptosis speck-like protein containing a caspase recruiting domain; AST, aspartate aminotransferase; BALF, bronchoal- Received for publication February 27, 2020. Accepted for publication July 6, 2020. veolar lavage fluid; BMT, bone marrow transplantation; BUN, blood urea nitrogen; This work was supported by Japan Society for the Promotion of Science Grant-in-Aid EBD, Evans blue dye; Gr-1, granulocyte receptor 1; HETE, hydroxyeicosatetraenoic for Scientific Research 18K08112 (to M.T.), a Private University Research Branding acid; HMGB1, high-mobility group box 1; 4-HNE, 4-hydroxy-2-nonenal; HODE, Project (to M.T.), Japan Agency for Medical Research and Development Core Re- hydroxyoctadecadienoic acid; I/R, ischemia/reperfusion; KIM-1, kidney injury mol- search for Evolutional Science and Technology Grant 18gm0610012h0105 (to M.T.), ecule 1; LA, linoleic acid; LC/MS, liquid chromatography/mass spectrometry; LDH, the Takeda Science Foundation (to M.T.), a Jichi Medical University Graduate Stu- lactate dehydrogenase; LVEC, lung vascular endothelial cell; NLR, nucleotide- dent Start-up Award (to H.I.), and a Jichi Medical University Graduate Student binding oligomerization domain-like receptor; NLRP3, NLR family pyrin domain Research Award (to H.I.). containing 3; ROS, reactive oxygen species; SMA, superior mesenteric artery; SMV, superior mesenteric vein; VE, vascular endothelial; WT, wild-type. H.I. and M.T. designed the study concept and experiments. H.I., H.K., T. Karasawa, S.H., A.S., Y.I., N.Y., E.A., E.H., R.K., T. Komada, and S.W. performed the exper- Ó iments and analyzed the data. T. Kasahara, T.S., H.H., J.K., N.S., and K.Y.-K. Copyright 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50

www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000217 2 NLRP3 INFLAMMASOME IN INTESTINAL I/R-INDUCED ALI danger signals, NLRP3 inflammasome components are assembled the liver, heart, and kidney were assessed as described previously (29–31). to activate caspase-1 with subsequent maturation of IL-1b (pro- The severity of lung injury was graded according to the following pa- cessing to its active form), resulting in inflammation and tissue rameters (4): 1) periluminal infiltrates (around the airway/vessels), 2) pneumonitis (alveolar/interstitial), and 3) percentage of affected lung tis- damage. Our group has recently shown that the NLRP3 inflam- sue. Immunohistochemical analysis was performed as described previously masome is involved in the pathogenesis of several disease con- (32, 33) with Abs to the following proteins: Ly-6G (BioLegend, San ditions (10–15). In terms of I/R injury, we and other investigators Diego, CA), CD45 (BD Biosciences, Franklin Lakes, NJ), F4/80 (clone showed that the NLRP3 inflammasome mediates the initial in- A3-1; AbD Serotec), granulocyte receptor 1 (Gr-1; eBioscience, San Diego, CA), kidney injury molecule 1 (KIM-1; R&D Systems, Minneap- flammatory response after I/R in the heart, liver, kidney, and in- olis, MN), cleaved caspase-3 (Cell Signaling Technology, Boston, MA), testine (10, 16–18). Furthermore, several reports have shown that and 4-hydroxy-2-nonenal (4-HNE; clone HNEJ-2; Japan Institute for the NLRP3 deficiency enhanced survival and inhibited inflammatory Control of Aging, Nikken SEIL, Shizuoka, Japan). Isotype-matched IgG responses in a microbial sepsis model (19, 20). However, no in- (Vector Laboratories, Burlingame, CA) was used as a negative control. The formation is available on the role of NLRP3 inflammasome in stained sections were digitized and analyzed by microscopy (FSX-100; Olympus, Tokyo, Japan). We quantified the stained cells by counting the intestinal I/R injury. In the current study, we investigated the role number in five different fields of the lungs. We randomly selected 500 cells of NLRP3 inflammasome in the pathophysiology of intestinal I/R to calculate the percentage of positively stained cells. injury using mice deficient in NLRP3, ASC, caspase-1/11, and Measurement of serum parameters and cytokines IL-1b and found that deficiency in the inflammasome components and IL-1b prolonged survival after intestinal I/R. Contrary to our Serum levels of lactate dehydrogenase (LDH), alanine aminotransferase expectations, NLRP3 deficiency failed to inhibit inflammatory (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine were measured on a Fuji-DRYCHEM chemical analyzer (Fuji b Downloaded from responses and IL-1 production in the intestine, but it markedly Film, Tokyo, Japan). The levels of IL-1b, TNF-a, IL-6, and CCL2 were improved multiple features of ALI, including inflammation, reactive assessed with a mouse ELISA kit (R&D Systems). Serum levels of high- oxygen species (ROS) generation, and vascular permeability after mobility group box 1 (HMGB1) and citrullinated histone [3H] were measured with an HMGB1 ELISA Kit II (Shino-Test, Tokyo, Japan) and intestinal I/R. Furthermore, using mass spectrometry, we identified 3 intestine-derived lipid mediators 12–hydroxyeicosatetraenoic acid Circulating Histone [ H] Citrullination ELISA Kit (EpiGentek Group, Farmingdale, NY), respectively. (HETE), 11-HETE, and 13–hydroxyoctadecadienoic acid (HODE)

as potential modulators and showed that these lipid mediators Real-time RT-PCR http://www.jimmunol.org/ enhanced the activation of NLRP3 inflammasome in lung vas- We prepared total RNA using ISOGEN (Nippon Gene, Toyama, Japan) cular endothelial cells (LVECs). To our knowledge, these find- according to the manufacturer’s instructions. We carried out real-time RT- ings provide new insights into the mechanism underlying intestinal PCR analysis by using the Thermal Cycler Dice Real-Time System II I/R-induced ALI and suggest that NLRP3 inflammasome–driven (Takara Bio, Shiga, Japan) to detect mRNA expression. The primers b were as described previously (32). The expression levels of each target IL-1 is a novel potential target for treating this disorder. gene were normalized by subtracting the corresponding b-actin threshold cycle value; the DDthreshold cycle comparative method was used for normalization. Materials and Methods Neutrophil depletion Animals and intestinal I/R protocol by guest on September 28, 2021 All animal experiments were approved by the Use and Care of Experimental Mice were treated i.p. with anti–Gr-1 mAb (clone RB6-8C5; kindly pro- Animals Committee of the Jichi Medical University Guide for Laboratory vided by Dr. R. Coffman, DNAX Research Institute, Palo Alto, CA) or Animals (permit number 17172-01) and were carried out in accordance with isotype control IgG (Jackson ImmunoResearch Laboratories, West 2/2 2/2 Grove, PA) 24 h before intestinal I/R. The depletion of neutrophils the Jichi Medical University guidelines. NLRP3 , ASC , caspase-1/ + – 112/2, and IL-1b2/2 mice were kindly provided by Dr. V. M. Dixit (Ly-6G CD45R cells) was confirmed by flow cytometry (33). (Genentech, San Francisco, CA), Dr. S. Taniguchi (Shinshu University, Evans blue dye analysis Nagano, Japan), Dr. H. Tsutsui (Hyogo Medical College, Hyogo, Japan), and Dr. Y. Iwakura (Tokyo University of Science, Chiba, Japan), respec- To assess vascular leakage, we injected Evans blue dye (EBD) (30 mg/kg) tively (21–25). As control mice, we used either wild-type (WT) mice into the internal jugular vein 1 h prior to the harvest. The lungs were +/+ obtained from Japan SLC (Tokyo, Japan) or NLRP3 littermates. We perfused with PBS via the right ventricle and then harvested. Both lungs validated that I/R injury shortened survival time equally in WT mice and were homogenized and incubated with 500 ml of formamide (Wako +/+ NLRP3 littermates. For all experiments, we used 8–10-wk-old male Chemicals, Osaka, Japan) for 24 h at 55˚C and then centrifuged at 1000 mice on a C57BL/6J genetic background. Mice were housed four per cage rpm for 10 min. The OD of the supernatant was determined spectropho- (RAIR HD Ventilated Micro-Isolator Animal Housing Systems; Lab tometrically at 620 nm. The concentration of extravasated EBD in lung Products, Seaford, DE) in an environment maintained at 23 6 2˚C with ad homogenate was calculated against a standard curve. libitum access to food and water. They were exposed to a 12-h light/dark cycle with lights on from 8:00 to 20:00. Bronchoalveolar lavage fluid analysis The mice underwent intestinal I/R surgery according to a modified version of a previously described protocol (26, 27) or sham operations Bronchoalveolar lavage fluid (BALF) was obtained by cannulating the (Fig. 1A). We anesthetized the mice with isoflurane before performing a trachea with an 18-gauge catheter (34). After the whole lung was washed midline laparotomy. After folding the mesentery in the axial direction of four times with 0.8 ml of PBS, the lavage fluid was centrifuged at 1000 the superior mesenteric artery (SMA) and superior mesenteric vein (SMV), rpm for 10 min at 4˚C. The cell-free supernatants were processed for we clamped the SMA, SMV, and collateral vessels with an atraumatic clip measurement of protein concentration (Pierce BCA Protein Assay Kit; (Fine Science Tools, Foster City, CA) for 60 min to produce intestinal Thermo Fisher Scientific, Waltham, MA) and inflammatory cytokine ischemia; the clip was then released to allow reperfusion. The mice were levels. resuscitated with 1 ml of i.p. PBS before wound closure. Sham-operated Pulse oximetry mice underwent the same protocol without vascular occlusion. The mice were sacrificed at various periods of reperfusion, and samples were col- Real-time blood oxygen saturation (SpO2) in awake mice was measured lected from the intestine, lung, heart, liver, kidney, and blood. All efforts with a MouseOX Pulse Xximeter (Starr Life Sciences, Oakmont, PA). A were made to minimize animal discomfort and suffering. depilatory agent was used to remove the hair around the neck 1 d before the measurement. A sensory collar clip attached to the pulse oximeter was Histology and immunohistochemistry placed in a hairless area, and MouseOX software was used for analysis. The tissue samples were fixed overnight in 10% phosphate-buffered for- Bone marrow transplantation protocol malin and embedded in paraffin. Tissue sections (5-mm thick) were stained with H&E. To evaluate intestinal injury, we calculated the percentage of Whole bone marrow cells were harvested by flushing femurs and tibias with villi with severe damage as described previously (28). The injury scores of PBS. RBCs were lysed in hypotonic buffer. The cells were washed twice The Journal of Immunology 3 with PBS and resuspended. Recipient mice (6 wk old) were lethally irra- Results diated with a total dose of 9 Gy and were injected with bone marrow cells Effectiveness of the intestinal I/R model (2 3 106) via the cervical vein. Use of GFP mice as donors enabled us to verify the reconstitution of bone marrow after transplantation by this The most common model used to induce intestinal ischemia is protocol. We previously confirmed that peripheral blood cells consisted of occlusion of the SMA with an atraumatic clip; however, it has been .90% GFP+ cells in the chimeric mice 8 wk after bone marrow transplantation (BMT) (10). Using this protocol, we produced BMTWT→WT, suggested that this protocol is unreliable and difficult to reproduce BMTNLRP32/2→WT, BMTWT→NLRP32/2, and BMTNLRP32/2→NLRP32/2 (26, 27). To establish a highly reproducible protocol, we modified mice. the technique by occluding both superior mesenteric blood vessels Cell culture and in vitro experiments and collateral blood flow (Fig. 1A). This method created consistent and reproducible injury after intestinal I/R. The time- Primary mouse LVECs were isolated from the lungs of mice by using a dependent survival rate after intestinal I/R in WT mice showed previously described protocol with modifications (35). First, the lungs were $ perfused with PBS via the right ventricle to remove RBCs. Then, single- that 60 min of ischemia was fatal (Fig. 1B) and that serum LDH cell suspensions were prepared with the Lung Dissociation Kit (Miltenyi levels increased to a peak at 4 h after I/R and declined thereafter Biotec, Bergisch-Gladbach, Germany). LVECs (CD45–CD31+) were iso- (Fig. 1C). Based on these findings, we produced intestinal I/R by lated by negative selection with anti-CD45–conjugated magnetic beads 60 min of ischemia followed by reperfusion. and by positive selection with anti-CD31–conjugated magnetic beads. LVECs were cultured on a gelatin-coated dish in DMEM (Thermo Fisher Role of NLRP3 inflammasome in intestinal injury and survival m Scientific, Carlsbad, CA) supplemented with 20% FCS, 50 g/ml endo- after I/R thelial cell growth supplement (Sigma-Aldrich, St. Louis, MO), 50 mg/ml heparin (Sigma-Aldrich), 5 mM SB431542 (R&D Systems), and 1% an-

To investigate the role of the NLRP3 inflammasome, we examined Downloaded from 2 2 2 2 2 2 tibiotics at 37˚C in a 5% CO2 atmosphere for 72–96 h. Endothelial cells the survival time of WT, NLRP3 / , ASC / , caspase-1/11 / , were identified by a cobblestone-like morphology and specific expression b2/2 of vascular endothelial (VE)–cadherin. LVECs were used at passages 3–4. and IL-1 mice subjected to intestinal I/R. We found that the For inflammasome activation experiments, cells were primed with LPS survival of mice deficient in NLRP3 inflammasome components (1 mg/ml) for 24 h and then treated with ATP (3 or 5 mM) or nigericin and IL-1b was significantly prolonged compared with that of WT (5 mM) for 6 h. mice (Fig. 1D), indicating that the NLRP3 inflammasome greatly contributes to intestinal I/R-induced lethality. Because this le-

Western blotting http://www.jimmunol.org/ thality was almost completely absent in NLRP32/2 mice, we Western blotting was carried out as described previously (36) with pri- conducted experiments to assess the phenotypes of WT and mary Abs for NLRP3 (cryo-2; AdipoGen, San Diego, CA), ASC 2/2 (AL177; AdipoGen), caspase-1 (Casper-1; AdipoGen), IL-1b (no. 401- NLRP3 mice. Consistent with the survival data, histologic ML; R&D Systems), and VE-cadherin (no. 33168; Abcam, Cambridge, analysis showed that WT mice exhibited mucosal disruption in MA). Immunoreactive bands were visualized by Western BLoT Quant the jejunum and ileum that were moderately attenuated in the HRP Substrate and Ultrasensitive HRP Substrate (Takara Bio). The ex- intestine of NLRP32/2 mice (Fig. 1E). Indeed, the number of pression levels of b-actin served as an internal control for protein loading. severely damaged villi was partially, but significantly, decreased in NLRP32/2 mice (Fig. 1F). However, serum LDH levels did not Analysis of serum lipid mediators differ between these mice (Fig. 1G). The mRNA expression of by guest on September 28, 2021 Serum lipid mediators were analyzed by an LCMS-8060 (Shimadzu, NLRP3 inflammasome components (NLRP3 and ASC), inflam- Kyoto, Japan) as described previously (37). Briefly, the isolated serum matory cytokines (IL-1b, IL-6, IL-18, TNF-a, CCL2, CXCL1/2, samples were immediately frozen at –80˚C until use. Serum samples and CXCL5), and inflammatory cell markers (Gr-1 for neutrophils (30 ml) were mixed with internal standard and extracted by methanol containing 0.1% formic acid. The separated methanol phase was and EMR1 for macrophages) was markedly upregulated after in- extracted by a Strata X polymer reversed-phase column (33 mm, 10 mg; testinal I/R in WT mice compared with that in sham-operated Phenomenex, Torrance, CA). The cartridges were washed with 1 ml each mice (Fig. 1H, Supplemental Fig. 1A, 1B). However, Gr-1 was of 0.1% formic acid, 15% ethanol, and petroleum ether, and then the the only molecule to exhibit a difference in expression between lipid was eluted with 300 ml of methanol containing 0.1% formic acid. 2/2 The eluent was evaporated and reconstituted in 30 ml of methanol. WT and NLRP3 mice after intestinal I/R. Consistent with this The extracted lipids were analyzed by an LCMS-8060 using the lipid finding, immunohistochemical staining showed that the infiltration mediator version 2 software package (Shimadzu). Deuterium-labeled of macrophages (F4/80-positive cells) and neutrophils (Gr-1– 6-keto PG F1a-d4, PG F2a-d4, PG E2-d4, PG D2-d4, leukotri- positive cells) was increased in the intestine of WT mice with I/R ene B4-d4, 15(S)-HETE-d8, 12(S)-HETE-d8, 5(S)-HETE-d8, platelet- and that the infiltration of neutrophils, but not macrophages, was activating factor (PAF) C-16-d4, and oleoyl ethanolamide-d4 were 2/2 purchased from Cayman Chemical (Ann Arbor, MI) and used as internal reduced in the ileum of NLRP3 mice (Supplemental Fig. 1D, standards. 1E). Furthermore, the levels of IL-1b protein were elevated in the intestine of WT mice with I/R, but unexpectedly, they did not Patient sample collection differ between WT and NLRP32/2 mice (Fig. 1I). No significant Six patients (three male and three female) with intestinal ischemia who differences in intestinal IL-1b levels were observed between WT underwent an emergency operation at Jichi Medical University Hospital and caspase-1/112/2 mice after I/R (Fig. 1J). Changes in serum were enrolled. Preoperative blood samples were collected and the plasma b a was frozen at –80˚C for liquid chromatography/mass spectrometry (LC/ levels of IL-1 and TNF- were also similar (Fig. 1K). We also MS) analysis. Six healthy volunteers were enrolled as a control group. The assessed serum levels of HMGB1 and histone [3H], which were study was approved by the ethics committee of Jichi Medical University reported to be involved in NLRP3 inflammasome activation in (permit number A17-068), and written informed consent was obtained hepatic I/R injury (16, 38). However, levels in the I/R intes- from all subjects. tine of WT and NLRP32/2 mice did not differ significantly Statistical analysis (Supplemental Fig. 1C). In addition, intestinal injury, serum Data are expressed as the mean 6 SEM. The Mann–Whitney U test was LDH, and IL-1b levels at the later phase (12 h) after I/R were used to evaluate differences between two groups. For comparisons between assessed. Histologic analysis showed a significant reduction multiple groups, the significance of differences was determined by one- of intestinal injury in WT mice compared with those at the way ANOVA combined with the Kruskal–Wallis test. Survival curves were 2/2 analyzed by the log-rank test. All analyses were carried out in GraphPad early phase (4 h) after I/R, with no difference between NLRP3 Prism version 6 (San Diego, CA). A p value ,0.05 was considered to be mice (Supplemental Fig. 2A, 2B). Serum LDH and IL-1b levels statistically significant. also declined over time and did not differ between these mice 4 NLRP3 INFLAMMASOME IN INTESTINAL I/R-INDUCED ALI Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Role of NLRP3 inﬂammasome in intestinal injury and survival after I/R. (A) A murine model of intestinal I/R. After the mesentery was folded in the axial direction of the SMA and SMV (a and b), the SMA, SMV, and collateral vessels were clamped by an atraumatic clip to produce intestinal ischemia (c and d). (B) Survival of WT mice subjected to intestinal ischemia for the indicated periods followed by reperfusion were analyzed by the Kaplan–Meier method (n = 4–8 for each). (C) Time course of serum LDH levels in mice subjected to 1 h of intestinal ischemia followed by reperfusion for the indicated periods (n = 3 for each). (D) Survival rates of WT, NLRP32/2, ASC2/2, caspase-1/112/2, and IL-1b2/2 mice subjected to 1 h of intestinal ischemia followed by reperfusion were analyzed by the Kaplan–Meier method (n = 6–8 for each). Log-rank analysis showed the signiﬁcance of differences between WT and other groups (p , 0.01). (E–J) Intestine and serum samples were obtained from sham-operated, WT, and (Figure legend continues) The Journal of Immunology 5

(Supplemental Fig. 2C, 2D). These results suggested the possibility almost completely depleted neutrophils in the blood (data not that intestinal damage itself and serum inflammatory markers may shown), it had no effect on survival or lung damage after intestinal have not much effect on the difference of survival after I/R. I/R (Fig. 3G, 3H), indicating that neutrophils play little, if any, role in intestinal I/R-induced ALI. Intestinal I/R injury causes remote organ damage, particularly ALI NLRP3 deficiency reduces inflammatory cytokine expression Because intestinal I/R injury has been shown to induce remote and ROS generation organ damage (39), we hypothesized that remote organ(s) could Based on our finding that NLRP3 deficiency reduced the infiltration contribute to the cause of death after intestinal I/R. Considering of inflammatory cells into the lung after intestinal I/R, we next the time course of survival in WT mice subjected to intestinal I/R, assessed the expression of inflammatory cytokines. We found that we collected samples of remote organs after 12 h. Histologic the mRNA expression of IL-1b, IL-6, TNF-a, CCL2, CXCL1/2, analysis revealed significant ALI characterized by inflammatory and CXCL5 was considerably elevated in the lungs of WT mice cell infiltration, thickening of the alveolar septae, vascular con- subjected to intestinal I/R and that this increase in expression was gestion, and alveolar edema in WT mice. These changes were significantly inhibited in NLRP32/– mice (Fig. 4A, Supplemental 2/2 markedly reduced in NLRP3 mice (Fig. 2A). Slight tissue Fig. 3C). Similar expression patterns were observed for inducible damage was observed in the liver, heart, and kidney. The levels of NO synthase and ICAM-1. We also assessed apoptosis and ROS serum ALT, AST, BUN, and creatinine were elevated after intes- generation by immunostaining for cleaved caspase-3 and 4-HNE, tinal I/R, but these values and tissue injury did not differ between respectively. Only a few apoptotic cells were detected in the lungs 2/2 2 2 WT and NLRP3 mice (Fig. 2B, 2C, Supplemental Fig. 2E–G). of WT and NLRP3 / mice subjected to intestinal I/R, and the Downloaded from Furthermore, the expression of KIM-1 was clearly increased in the number did not differ between these genotypes (Fig. 4B, 4C). In kidneys of intestinal I/R, but there was no difference of the KIM-1 contrast, the number of 4-HNE–positive cells in the lungs was 2/2 expression between WT and NLRP3 mice (Supplemental Fig. increased to a much greater extent in WT mice than in NLRP32/2 2H). These findings suggest that intestinal I/R injury causes mice after intestinal I/R (Fig. 4D, 4E). damage in multiple organs and that intestinal I/R-induced ALI is NLRP3 deficiency improves lung vascular permeability

mediated through the NLRP3 inflammasome. http://www.jimmunol.org/ Increased vascular permeability in the lung is a hallmark of ALI NLRP3 deficiency reduces intestinal I/R-induced ALI and (40, 41). Extravasation of i.v. injected EBD showed that lung neutrophil infiltration permeability was increased in WT mice after intestinal I/R com- Consistent with the data described above, histologic analysis pared with that in sham-operated mice. This increase was almost revealed multiple features of ALI, including thickening of the completely eliminated in NLRP32/2 mice (Fig. 5A, 5B). We also alveolar septae, vascular congestion, alveolar edema, and infil- assessed hypoxemia, another cardinal feature of ALI, and found tration of inflammatory cells after intestinal I/R in WT mice, but all that arterial oxygen saturation fell below 95% at 4 h after intestinal 2/2 of these histologic manifestations were attenuated in NLRP3 I/R in WT mice, whereas it remained above 95% in NLRP32/2 mice (Fig. 3A). Accordingly, the lung injury score was signifi- mice (Fig. 5C). To assess alveolar–capillary barrier permeability, we by guest on September 28, 2021 2/2 cantly improved in NLRP3 mice (Fig. 3B). To investigate measured BALF protein and inflammatory cytokines. Total protein, whether the NLRP3 inflammasome is involved in intestinal I/R- BALF IL-1b, BALF IL-6, BALF TNF-a, and lung IL-1b increased induced ALI, we assessed the time-dependent mRNA expression after intestinal I/R in WT mice, but these increases were signifi- of NLRP3 inflammasome components and found that NLRP3 was cantly inhibited in NLRP32/2 mice (Fig. 5D, 5E). However, lung clearly upregulated in a time-dependent manner. It reached a peak IL-6 and TNF-a levels did not differ between WT and NLRP32/2 at 4 h and declined thereafter (Fig. 3C). This result was reasonable mice. Caspase-1/112/2 mice subjected to intestinal I/R also because NLRP3 upregulation is necessary to form the inflamma- exhibited reduced ALI and lung IL-1b levels when compared some assembly as a priming signal (7, 8). We further assessed with WT mice (Fig. 5F, 5G). These results indicate that the inflammatory cells that infiltrated the lung by immunostaining for alveolar–capillary barrier is disrupted after intestinal I/R and CD45 (pan-leukocyte marker), Ly-6G (neutrophil marker), and that NLRP3 inflammasome–driven IL-1b plays a pivotal role in F4/80 (macrophage marker). Compared with numbers in sham- this process. operated mice, the CD45+ and Ly-6G+ cells were more numerous in the lungs of mice with intestinal I/R (Fig. 3D, 3E). In LVECs contribute to the development of intestinal particular, the infiltration of neutrophils, but not macrophages, was I/R-induced ALI significantly inhibited in NLRP32/2 mice (Fig. 3F, Supplemental To determine the contributions of bone marrow– and non–bone Fig. 3A, 3B). Because we recently reported that NLRP3 protein marrow–derived cells in vivo, we generated four types of BMT regulates the migration of neutrophils during liver I/R injury and mice and evaluated survival after intestinal I/R injury. Consistent hyperoxia-induced ALI independent of the inflammasome and with the results in non-BMT mice, the survival after intestinal IL-1b (32, 34), our results prompted us to examine whether I/R injury was markedly improved in BMTNLRP32/2→NLRP32/2 neutrophil depletion could improve intestinal I/R-induced ALI. mice compared with that in BMTWT→WT mice (Fig. 6A). Inter- Although i.p. injection of anti–Gr-1 neutralizing Ab (RB6-8C5) estingly, BMTWT→NLRP32/2 mice showed improved survival after

NLRP32/2 mice 4 h after intestinal I/R. (E) Sections of the intestine (jejunum and ileum) were stained with H&E. The images are representative for three independent experiments. (F) The number of severely damaged villi was assessed (n = 7 for each). (G) Serum LDH levels were assessed (n = 3–6 for each). (H) Intestinal mRNA levels of IL-1b, IL-18, TNF-a, Gr-1, and EMR1 were assessed by real-time RT-PCR analysis (n = 4–6 for each). (I) Intestinal IL-1b protein levels were assessed (n = 4–7 for each). (J) Intestinal samples were obtained from sham-operated, WT, and caspase-1/112/2 mice 4 h after intestinal I/R for assessment of IL-1b protein levels (n = 4–7 for each). (K) Serum levels of IL-1b and TNF-a were assessed (n = 3–6 for each). (G–K) One representative experiment from three independent experiments is shown. Statistical signiﬁcance was calculated by the Kruskal–Wallis test (C) and Mann– Whitney U test (F–K). Data are expressed as mean 6 SEM. **p , 0.01, ****p , 0.0001. 6 NLRP3 INFLAMMASOME IN INTESTINAL I/R-INDUCED ALI

FIGURE 2. Intestinal I/R injury causes remote organ damage, particularly ALI. Lung, liver, heart, and kidney samples were obtained from WT and NLRP32/2 mice 12 h after intestinal I/R. (A) Sections of each tissue were stained with H&E. The images are representative for two independent experiments. (B and C) Serum levels of AST, ALT, BUN, and creatinine were obtained from sham-operated, WT, and NLRP32/2 mice 4 h after intestinal I/R (n = 3–6 for each). One representative experiment from two independent experiments is shown. Statistical signiﬁcance was calculated by Mann–Whitney U test (B and C).

Data are expressed as mean 6 SEM. Downloaded from http://www.jimmunol.org/ intestinal I/R, whereas BMTNLRP32/2→WT mice did not, sug- To address the clinical relevance of these findings, we measured gesting that non–bone marrow–derived cells rather than bone these metabolites in blood samples from patients with intestinal marrow–derived inflammatory cells play a role in intestinal ischemia by using LC/MS. The clinical diagnoses and extent of I/R-induced lethality. Based on our findings of lung vascular intestinal ischemia are provided in Fig. 7G. Consistent with the permeability, we speculated that LVECs would be important for results from mouse studies, AA and LA metabolites, including intestinal I/R-induced ALI. To test this possibility, we isolated 12-HETE, 11-HETE, and 13-HODE, were elevated in patients CD45+ cells (leukocytes), CD45–CD31– cells (resident cells ex- compared with those in healthy controls (Fig. 7F, 7G). Taken to- cept for endothelial cells), and CD45–CD31+ cells (LVECs) from gether, these results suggest that AA and LA metabolites derived the lungs of WT and NLRP32/2 mice by using immune-magnetic from I/R intestine contribute to NLRP3 inflammasome activation by guest on September 28, 2021 selection and confirmed VE-cadherin expression in LVECs in intestinal I/R-induced ALI (Fig. 8). (Fig. 6B). NLRP3 protein expression was greater in LVECs isolated from mice with intestinal I/R than in those isolated from Discussion sham-operated mice (Fig. 6C). To confirm that NLRP3 inflam- In this study, we found that intestinal I/R causes activation of the masome is activated in LVECs, we primed LVECs with low-dose NLRP3 inflammasome in LVECs and that subsequent IL-1b LPS to ensure the induction of pro–IL-1b, and treated them with production leads to ALI. Furthermore, our findings confirm that ATP and nigericin, well-known NLRP3 inflammasome activa- lipid mediators released by intestinal I/R amplify the activation of tors. ATP and nigericin induced the processing of IL-1b and IL-1b through these mechanisms. These results provide new in- produced its mature form in LVECs (Fig. 6D–F). These findings sights into the mechanism underlying intestinal I/R-induced ALI indicate that LVECs possess the machinery of NLRP3 inflam- and suggest that endothelial NLRP3 inflammasome–driven IL-1b masome and can produce IL-1b. is a potential target for treatment and prevention of this disorder. A growing body of evidence indicates that the NLRP3 b Intestinal I/R-derived lipid mediators enhance IL-1 inflammasome plays an important role in the pathophysiology processing in LVECs of early I/R injury in several organs, including the heart, liver, To identify potential contributors to NLRP3 inflammasome acti- kidney, and intestine (10, 16–18). Indeed, we have demonstrated vation during intestinal I/R-induced ALI, we focused on lipid previously that inflammasome activation in cardiac fibroblasts is mediators because they have been identified as key mediators of crucially involved in the initial inflammatory response after hepatic I/R injury (42). Blood samples collected from the portal myocardial I/R injury (10). Huang et al. (16) reported that vein immediately after intestinal I/R were analyzed by LC/MS NLRP3- or caspase-1–deficient mice are protected from hepatic (Fig. 7A). Several arachidonic acid (AA) and linoleic acid (LA) I/R injury and that inflammasome-mediated injury is dependent on metabolites were significantly elevated after intestinal I/R in both caspase-1 expression in liver nonparenchymal cells. Iyer et al. WT and NLRP32/2 mice compared with those in sham-operated (17) also showed that the NLRP3 inflammasome contributes to mice (Fig. 7B, 7C). Among them, we examined the effects of 12- kidney I/R-induced renal dysfunction and lethality. Jia et al. HETE, 11-HETE, and 13-HODE on the processing of IL-1b in (18) very recently reported that intestinal I/R induces NLRP3 LVECs. Although treatment with 12-HETE, 11-HETE, and 13- inflammasome-dependent pyroptosis at the early phase after I/R, HODE did not directly induce IL-1b maturation in LPS-primed which contributes to the development of intestinal injury. Because LVECs (Fig. 7D, Supplemental Fig. 4A), these metabolites en- we also observed that NLRP3 deficiency decreased intestinal hanced ATP-induced IL-1b maturation (Fig. 7E, Supplemental damage at 4 h after I/R, we assume that NLRP3 inflammasome Fig. 4B). As expected, IL-1b maturation was completely inhibi- contributes to some extent to the pathophysiology of intestinal ted in NLRP32/2 LVECs (Supplemental Fig. 4C). injury at the early phase of intestinal I/R. Interestingly, however, The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. NLRP3 deficiency reduces intestinal I/R-induced ALI and neutrophil infiltration. (A and B) Lung samples were obtained from sham-operated, WT, and NLRP32/2 mice 12 h after intestinal I/R. (A) Sections were stained with H&E. (B) Quantitative analysis of the lung injury score (n = 8 for each). (C) Time course of NLRP3 mRNA levels in the lungs of mice subjected to 1 h of intestinal ischemia followed by reperfusion for the indicated periods (n = 3 for each). One representative experiment from three independent experiments is shown (D and E) Lung samples were obtained from WT mice 12 h after intestinal I/R. Sections were analyzed by immunohistochemical staining for the pan-leukocyte marker CD45 and neutrophil marker Ly-6G. (F) Quantitative analysis of Ly-6G+ cells (n = 3–6 for each). (G and H) Mice were treated i.p. with anti–Gr-1 neutralizing or control Ab (50 mg/mouse) 24 h before intestinal I/R. (G) Survival of these mice subjected to intestinal I/R was analyzed by the Kaplan–Meier method (n = 6–8 for each). (H) Lung samples were obtained from these mice 12 h after intestinal I/R. Sections were stained with H&E. (A and E) The images are representative for three independent experiments. Statistical significance was calculated by Mann–Whitney U test (B and F) and Kruskal–Wallis test (C). Data are expressed as mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001. 8 NLRP3 INFLAMMASOME IN INTESTINAL I/R-INDUCED ALI Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. NLRP3 deficiency reduces inflammatory cytokine expression and ROS generation. (A) Lung samples were obtained from sham-operated, WT, and NLRP32/2 mice 4 h after intestinal I/R. Lung mRNA levels of IL-1b, IL-18, TNF-a, Gr-1, EMR1, and inducible NO synthase (iNOS) were assessed by real-time RT-PCR analysis (n = 3–6 for each). One representative experiment from three independent experiments is shown. (B–E) Lung samples were obtained from sham-operated, WT, and NLRP32/2 mice 12 h after intestinal I/R. Sections were analyzed by immunohistochemical staining for cleaved caspase-3 (B) and 4-HNE (D). Quantitative analysis of cleaved caspase-3+ cells (C) and 4-HNE+ cells (E)(n = 3–6 for each). (B and D) The images are representative for three independent experiments. Statistical significance was calculated by the Mann–Whitney U test (A and D). Data are expressed as mean 6 SEM. *p , 0.05, **p , 0.01.

NLRP3 inflammasome deficiency failed to inhibit intestinal and We showed that infiltration of macrophages and neutrophils into serum levels of IL-1b, suggesting that IL-1b is produced in the the intestine was increased in WT mice after intestinal I/R and that intestine after I/R, independent of the NLRP3 inflammasome. infiltration of neutrophils, but not macrophages, was reduced in Thus, the precise role of the NLRP3 inflammasome in I/R-induced the ileum of NLRP32/2 mice. These data suggest that neutrophils intestinal injury remains to be elucidated in future studies. may contribute to intestinal injury after I/R. Moreover, we found Indirect ALI, in which VE cells are initially injured, has a no difference in lung macrophage infiltration between WT and different pathogenic pathway from a direct lung injury, in which NLRP32/2 mice. Because neutrophil infiltration was significantly alveolar macrophages and alveolar epithelial cells are injured (43). inhibited in the lung of NLRP32/2 mice, we hypothesized that However, the NLRP3 inflammasome is expressed predominantly neutrophils contribute to the development of intestinal I/R- in innate immune cells, such as macrophages and neutrophils induced ALI. Unexpectedly, however, neutrophil depletion failed (6, 8), that are involved in the pathogenesis of ALI (44, 45). to improve lung damage and survival after intestinal I/R. BMT The Journal of Immunology 9

FIGURE 5. NLRP3 deﬁciency improves lung vascular permeability. WT and NLRP32/2 mice were subjected to 1 h of intestinal ischemia, followed by 12 h of reperfusion. (A) EBD was injected i.v. 1 h prior to organ collection. Representative images of whole lungs are shown. (B) Colorimetric quantitative analysis of EBD extravasated from the lungs (n = 3–6 for each). (C) Arterial oxygen saturation

(SpO2) was measured 4 h after intestinal I/R (n = 6 for each). (D and E) BALF and lung samples were obtained from sham- Downloaded from operated, WT, and NLRP32/2 mice 4 h after intestinal I/R. The levels of total protein, IL-1b, TNF-a, and IL-6 in were assessed in BALF (D)(n = 3–6 for each) and lung (E)(n = 3–7 for each). One representative experiment from three independent experiments is shown. (F) Lung http://www.jimmunol.org/ samples were obtained from sham-operated, WT, and caspase-1/112/2 mice 12 h after intestinal I/R. Sections were stained with H&E. The images are representative for three independent experiments. (G) IL- 1b protein levels were assessed 4 h after intestinal I/R (n = 3–7 for each). One representative experiment from three inde-

pendent experiments is shown. Statistical by guest on September 28, 2021 signiﬁcance was calculated by the Mann– Whitney U test (B–E and G). Data are expressed as mean 6 SEM. *p , 0.05, **p , 0.01.

experiments suggested that NLRP3 in resident cells, rather than as a primary target of NLRP3 inflammasome and IL-1b in in- bone marrow–derived cells (i.e., macrophages and neutrophils), testinal I/R. contribute to intestinal I/R-induced ALI. Interestingly, the sur- Lipid mediators are potent regulators of innate and adaptive vival rate of BMTNLRP32/2→NLRP32/2 mice was lower than that immune responses and have been implicated in numerous in- of nonirradiated WT mice, suggesting that irradiated mice are flammatory disorders, including I/R injury (42, 49, 50). Eicosa- more susceptible to damage by intestinal I/R. Additionally, noids, which are primarily derived from AA and include lipid NLPR3 deficiency considerably attenuated lung vascular per- mediators such as PGs, thromboxane, HETE, and leukotrienes, meability and BALF protein levels after intestinal I/R. These have a wide range of biological actions, including both pro- and observations suggest that LVECs are important for the activation anti-inflammatory effects (51). Lopategi et al. (52) reported that of NLRP3 inflammasome after intestinal I/R. In support of this anti-inflammatory lipid mediators inhibit IL-1b production in notion, Yang et al. (46) reported that cold-inducible RNA- LPS-stimulated bone marrow–derived macrophages, but no stud- binding protein activates the NLRP3 inflammasome and subse- ies have shown that these mediators themselves enhance the quently induces IL-1b production and pyroptosis in mouse production of IL-1b. We found that several AA and LA metabo- LVECs. Similarly, Yang et al. (47) reported caspase-1–mediated lites are produced from the intestine immediately after I/R. pyroptosis in LVECs during hemorrhagic shock–induced ALI. In Among them, 12-HETE, produced from AA by 12/15–lip- this study, we observed that LVECs possess NLRP3 inflamma- oxygenase (LOX), is dramatically elevated after intestinal I/R. It some machinery and can produce IL-1b. Because IL-1b in- enhances NLRP3 inflammasome activation and IL-1b maturation creased permeability in LVECs (48), our study highlights LVECs in both LVECs and macrophages. Furthermore, we showed that 10 NLRP3 INFLAMMASOME IN INTESTINAL I/R-INDUCED ALI Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. LVECs contribute to the development of intestinal I/R-induced ALI. (A) Survival of BMTWT→WT, BMTNLRP32/2→WT, BMTWT→NLRP32/2, and BMTNLRP32/2→NLRP32/2 mice subjected to intestinal I/R was analyzed by the Kaplan–Meier method (n = 6–8 for each). (B) CD45+ (leukocytes), CD45–CD31– (resident cells except for endothelial cells), and CD45–CD31+ (LVECs) cells from murine lungs were analyzed for VE-cadherin protein by Western blotting. b-Actin served as a loading control. (C) LVECs isolated from WT mice 4 h after intestinal I/R were analyzed for NLRP3 protein by Western blotting. Each lane indicates NLRP3 expression from an individual mouse. (D and E) LVECs from WT and NLRP32/2 mice were primed with LPS (1 mg/ml) for 24 h before treatment with ATP (5 mM) or nigericin (5 mM) for 6 h. Representative images of the LVECs are shown. Expression of NLRP3, ASC, caspase-1, and IL-1b was analyzed by Western blotting. Lys, lysate; Sup, supernatant. (F) IL-1b levels in the supernatants. Three independent experiments in triplicate were performed and representative data are shown. Data are shown as mean 6 SEM. (B, C, and E) The Western blot is representative for three independent experiments.

12-HETE is increased in the sera of patients with intestinal is- that arachidonate 12-LOX (ALOX12)–generated 12-HETE reg- chemia. Consistent with our results, Zarbock and coworkers (50, ulates several inflammatory pathways, including NLR signaling, 53) reported that inhibition of 12/15-LOX reduces neutrophil in- and greatly contributes to inflammation and cell death after he- filtration and improves vascular permeability and lethality in a patic I/R injury. Because12-HETE activates NF-kB and MAPK mouse model of LPS-induced ALI. Zhang et al. (42) also showed cascades to promote the production of inflammatory cytokines as The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. Intestinal I/R-derived lipid mediators enhance IL-1b maturation in LVECs. (A) Blood samples were collected from the portal vein immediately after intestinal I/R. (B) Blood samples were analyzed by LC/MS. A heatmap shows the levels of major AA and LA metabolites in WT and NLRP32/2 mice subjected to intestinal I/R. (C) Blood levels of 12-HETE, 11-HETE, and 13-HODE (n = 6 for each). Statistical signiﬁcance was calculated by the Kruskal–Wallis test. Data are expressed as mean 6 SEM. *p , 0.05, **p , 0.01. (D) LVECs were primed with LPS (1 mg/ml) for 24 h before treatment with 12-HETE (1, 100 nM), 11-HETE (1, 100 nM), or 13-HODE (1, 100 nM) for 6 h. Nigericin (Nig; 5 mM) was used as a positive control. Cell lysates (Lys) and supernatants (Sup) were analyzed for IL-1b by Western blotting. The Western blot is representative for three independent experiments. (E) LVECs were primed with LPS (1 mg/ml) for 24 h before treatment with ATP (3 mM). Cells were treated with or without 12-HETE (1 and 100 nM), 11-HETE (1 and 100 nM), or 13-HODE (1 and 100 nM) for 1 h before ATP treatment. Cell lysates and supernatants were analyzed for IL-1b by Western blotting. Expression of NLRP3, ASC, and caspase-1 are shown in Supplemental Fig. 4. The Western blot is representative for three independent experiments. (F) Blood samples from patients (PT) with intestinal ischemia and healthy controls (HC) were analyzed by LC/MS. A heatmap shows the levels of major AA and LA metabolites. (G) Levels of 12-HETE, 11-HETE, and 13-HODE in each of the subjects. 12 NLRP3 INFLAMMASOME IN INTESTINAL I/R-INDUCED ALI

FIGURE 8. Proposed model of intestinal I/R-induced ALI. I/R stimuli induce the release of lipid mediators (e.g., 12-HETE, 11-HETE, and 13-HODE) from the intestine. These mediators enhance NLRP3 inflammasome activation and subsequent IL-1b production in LVECs. NLRP3 inflammasome–driven IL-1b increases lung vascular permeability and the inflammatory response, resulting in ALI and lethality. Downloaded from well as leukocyte recruitment and activation (54), we speculate Disclosures that 12-HETE derived from I/R intestine promotes lung inflam- The authors have no financial conflicts of interest. mation at least in part by enhancing NLRP3 inflammasome activation. References

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Matsuzaka, et al. 2018. and Dr. Yoichiro Iwakura (Tokyo University of Science) for providing Saturated fatty acids undergo intracellular crystallization and activate the NLRP32/2, ASC2/2, caspase-1/112/2, and IL-1b2/2 mice, respectively, NLRP3 inflammasome in macrophages. Arterioscler. Thromb. Vasc. Biol. 38: 744–756. and we thank Drs. Masaki Yamada and Tsuyoshi Nakanishi (Shimadzu 16. Huang, H., H. W. Chen, J. Evankovich, W. Yan, B. R. Rosborough, G. W. Nace, Corporation) for providing technical assistance with LC/MS analysis. Q. Ding, P. Loughran, D. Beer-Stolz, T. R. Billiar, et al. 2013. Histones activate The Journal of Immunology 13

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