Intravital Imaging of Neutrophil Recruitment Reveals the Efficacy of FPR1 Blockade in Hepatic Ischemia-Reperfusion Injury

This information is current as Masaki Honda, Takayuki Takeichi, Shintaro Hashimoto, of September 29, 2021. Daiki Yoshii, Kaori Isono, Shintaro Hayashida, Yuki Ohya, Hidekazu Yamamoto, Yasuhiko Sugawara and Yukihiro Inomata J Immunol 2017; 198:1718-1728; Prepublished online 6

January 2017; Downloaded from doi: 10.4049/jimmunol.1601773 http://www.jimmunol.org/content/198/4/1718

Supplementary http://www.jimmunol.org/content/suppl/2017/01/06/jimmunol.160177 http://www.jimmunol.org/ Material 3.DCSupplemental References This article cites 37 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/198/4/1718.full#ref-list-1

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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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Intravital Imaging of Neutrophil Recruitment Reveals the Efﬁcacy of FPR1 Blockade in Hepatic Ischemia-Reperfusion Injury

Masaki Honda, Takayuki Takeichi, Shintaro Hashimoto, Daiki Yoshii, Kaori Isono, Shintaro Hayashida, Yuki Ohya, Hidekazu Yamamoto, Yasuhiko Sugawara, and Yukihiro Inomata

Neutrophils are considered responsible for the pathophysiological changes resulting from hepatic ischemia-reperfusion (I/R) injury, which is a complication of trauma, shock, liver resection, and transplantation. Recently, evidence is accumulating that formyl- peptide receptor (FPR) signaling constitutes an important danger signal that guides neutrophils to sites of inflammation. This study aimed to investigate dynamic neutrophil recruitment using two-photon laser-scanning microscopy (TPLSM) in response to FPR1 Downloaded from blockade during hepatic I/R. LysM-eGFP mice were subjected to partial warm hepatic I/R. They were pretreated with an FPR1 antagonist, cyclosporine H (CsH), or formyl peptide, fMLF. Liver was imaged after hepatic laser irradiation or I/R using the TPLSM technique. CsH treatment alleviated hepatic I/R injury, as evidenced by decreased serum transaminase levels, reduced hepatocyte necrosis/apoptosis, and diminished inflammatory cytokine, chemokine, and oxidative stress. In contrast, systemic administration of fMLF showed few effects. Time-lapse TPLSM showed that FPR1 blockade inhibited the accumulation of neutrophils in the necrotic area induced by laser irradiation in vivo. In the CsH-treated I/R group, the number and crawling velocity of neu- http://www.jimmunol.org/ trophils in the nonperfused area were lower than those in the control group. Meanwhile, FPR1 blockade did not affect monocyte/ macrophage recruitment. Hepatic I/R promoted the retention of neutrophils and their active behavior in the spleen, whereas CsH treatment prevented their changes. Intravital TPLSM revealed that formyl-peptide–FPR1 signaling is responsible for regulating neutrophil chemotaxis to allow migration into the necrotic area in hepatic I/R. Our findings suggest effective approaches for elucidating the mechanisms of immune cell responses in hepatic I/R. The Journal of Immunology, 2017, 198: 1718–1728.

epatic ischemia-reperfusion (I/R) injury is a complication to play critical roles in hepatic I/R through a speciﬁc pattern

of trauma, shock, liver resection, and transplantation (1, recognition receptor (5–8). In addition, cellular injury also re- by guest on September 29, 2021 H 2). In the ischemic liver, an imbalance of the metabolic leases mitochondrial DAMPs, including formyl peptide and mito- supply and demand results in severe tissue hypoxia, mitochondrial chondrial DNA, which are recognized by formyl-peptide receptor dysfunction, and the generation of reactive oxygen species (ROS). 1 (FPR1) and TLR9, respectively (3). Endogenous formyl peptides Subsequent reperfusion causes the activation of innate and adap- are released secondarily from the N terminus of mitochondrial tive immune responses and cell death programs, leading to liver NADH dehydrogenase and cytochrome c oxidase in dead cells, dysfunction and failure. and mainly exert a chemotactic function by promoting neutrophil Cellular injury can release endogenous damage-associated Ca2+ flux and MAPKs (9). Signals through FPR1 in neutrophils molecular patterns (DAMPs), which activate innate immunity (3, overcome signals hierarchically through CXCR2, which is known 4). Several DAMPs have been identified in which intranuclear as a key chemokine receptor, allowing neutrophils to migrate to- high-mobility group box 1, DNA, and histones have been shown ward end-target chemoattractants (10). We therefore hypothesized that hepatic I/R releases mitochondrial DAMPs, thereby inducing Department of Transplantation and Pediatric Surgery, Postgraduate School of Med- the accumulation of neutrophils in the ischemic liver in an FPR1- ical Sciences, Kumamoto University, Kumamoto 860-8556, Japan dependent manner. Recent studies have shown the therapeutic ORCIDs: 0000-0002-6102-3201 (T.T.); 0000-0002-7477-9614 (Y.I.). potential of FPR1 blockade in acetaminophen-induced acute liver Received for publication October 17, 2016. Accepted for publication December 5, failure and smoking-induced lung emphysema (11, 12); however, 2016. the impact of the formyl-peptide–FPR1 interaction on hepatic I/R This work was supported by the Ministry of Education, Culture, Sports, Sciences and injury is unknown. Technology of Japan (Grants KAKENHI 22591410 and KAKENHI 25461954). Advances in intravital microscopy have enabled the visualization Address correspondence and reprint requests to Dr. Yukihiro Inomata, Department of Transplantation and Pediatric Surgery, Postgraduate School of Medical Sciences, and quantification of real-time biological processes in situ (13, 14). Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan. E-mail Above all, the monitoring of immune cell recruitment has an es- address: [email protected] pecially broad range of applications. We recently showed a novel The online version of this article contains supplemental material. method for examining in vivo real-time neutrophil recruitment Abbreviations used in this article: ALT, alanine aminotransferase; AST, aspartate during hepatic I/R using two-photon laser-scanning microscopy aminotransferase; CsH, cyclosporine H; DAMP, damage-associated molecular pattern; FPR, formyl-peptide receptor; I/R, ischemia-reperfusion; KC, keratinocyte che- (TPLSM) (15). TPLSM is one of the most progressive develop- mokine; 8-OHdG, 8-hydroxy-29-deoxyguanosine; ROS, reactive oxygen species; ments in imaging technology and has numerous advantages, in- TPLSM, two-photon laser-scanning microscopy; TRITC, tetramethylrhodamine cluding high-resolution deep-site imaging, less phototoxicity, and isothiocyanate. less photobleaching in comparison with conventional confocal Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 laser-scanning microscopy. These major advantages are especially www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601773 The Journal of Immunology 1719 suited to imaging live tissues and organs in their native envi- Olympus UMPLFLN 103 W (numerical aperture 0.30) objective lens. For ronment (16, 17). Using this method, we could investigate the high-magnification imaging, images were recorded using an Olympus 3 structure of hepatic lobes, circulation of the sinusoids, cell death, XLPLN 25 WMP (water-immersion, numerical aperture 1.05) objective lens with x-y planes spanning 508 3 508 mm. In some experiments, mice and changes in the number, velocity, and morphology of recruited were placed in the right lateral recumbent position, and the spleen was neutrophils. Because these findings provided valuable new in- exteriorized by a left flank incision. Spleen was fixed and imaged using sights into the functional-anatomical changes that occur during TPLSM as described. hepatic I/R, we applied the technique to test the dynamic effect of Induction of selective hepatic necrosis FPR1 blockade. By combining the information of intravital TPLSM imaging Selective hepatic necrosis was induced by high-power laser irradiation using and conventional biological parameters, we provided visual and TPLSM. Time-lapse imaging at low-magnification was zoomed up by six quantitative evidence of the therapeutic effects of FPR1 blockade in times, and the laser transmissivity was increased to 100% (it was ordinarily locked at 25% during low-magnification imaging). After 3 min of irradi- hepatic I/R injury. FPR1 blockade inhibited the chemotaxis of ation, the imaging conditions were returned to normal. Necrotic area was neutrophils into the necrotic area and subsequent innate immune- blacked out with x-y planes spanning 210 3 210 mm. Twenty-minute mediated inflammation. FPR1 blockade also prevented the re- videos were recorded every 30 min for 3 h after laser irradiation. The tention of neutrophils in spleen, which is the major supplier of number and velocity of adherent neutrophils were assessed at each time point. The proportion of eGFP+ area within the necrotic area at 3 h after neutrophils. Our results suggest that mitochondrial DAMPs and laser irradiation was evaluated. their specific receptors are potential targets for therapeutic inter- vention in hepatic I/R. Analysis of TPLSM images Downloaded from Neutrophils were identified and distinguished from other myeloid subsets Materials and Methods based on the characteristics of their LysM-eGFP fluorescence intensity . . Mice (a threshold 40 in low-magnification imaging and 160 in high- magnification imaging, respectively) and cell size (dimensions of 5–15 LysM-eGFP mice were a gift from Dr. T. Graf (Center for Genomic mm). Monocytes/macrophages were identified based on the positivity of Regulation, Barcelona, Spain). These mice express eGFP under the lyso- FITC-F4/80. The number of adherent cells was evaluated using the “an- zyme M promoter, and neutrophils are visualized by the expression of alyze and measure” command in ImageJ software (National Institutes of eGFPhi (18). C57BL/6 mice were purchased from Japan SLC (Shizuoka, Health, Bethesda, MD). Hue analysis was performed using the “Color http://www.jimmunol.org/ Japan). All mice were maintained in a specific pathogen-free environment Threshold Tool” in ImageJ software. Cell shape index of neutrophils was with ad libitum access to food and water at the Animal Resource Facility at calculated as the ratio of the cell’s length to the cell’s width. To analyze the Kumamoto University. A 12/12 h light/dark cycle was maintained with velocity of adherent neutrophils, we selected 30 neutrophils per low- light from 07:00 AM to 7:00 PM. Temperature was maintained at 22 6 2˚C. magnification field for tracking and measured the crawling velocities at All experiments were performed according to guidelines of the Institu- each time point using G-Track software (Olympus Medical Science). The tional Animal Committee of Kumamoto University. velocity of adherent neutrophils within specific regions was measured separately using high-magnification imaging. Cell tracking was shown by Warm hepatic I/R injury model the “Manual Tracking” command using ImageJ software. Meandering index was measured by Euclidian distance/accumulated distance. Male mice 8–12 wk of age, weighing 22–26 g, were used. Mice were anesthetized with an i.p. injection of xylazine (10 mg/kg; Tokyo Kasei Assessment of liver damage by guest on September 29, 2021 Kogyo, Tokyo, Japan) and ketamine (100 mg/kg; Fujita, Tokyo, Japan). They were placed on a heating pad to maintain a body temperature of 37˚C. Serum aspartate aminotransferase (AST) and alanine aminotransferase After a midline laparotomy followed by an injection of heparin (100 U/kg), (ALT) levels, an indicator of hepatocellular injury, were measured using a mice underwent a sham operation or the induction of I/R. In the I/R group, Hitachi 7180 auto analyzer (Hitachi High-Technologies, Tokyo, Japan). blood supply to the left lateral and median liver lobes (comprising 70% of the liver) was interrupted using an atraumatic clip (0.29 N; BEAR Medic Liver histology Corporation, Tokyo, Japan). After 60 min of ischemia, the clip was re- moved and reperfusion was initiated. Sham operation was performed using Liver samples were fixed in 10% buffered formalin, embedded in paraffin, m the same protocol but without vascular occlusion. Mice received a selec- and liver sections (4 m) were stained with H&E. To calculate the per- tive FPR1 antagonist, cyclosporine H (CsH; 50 mg per mouse; LKT centage of the necrotic area, we evaluated 10 random sections per slide in a Laboratories), or PBS via an i.p. injection at 30 min before ischemia. In the blind manner using ImageJ software. other experiments, the synthetic N-formyl peptide, fMLF (3 mg per mouse; Sigma-Aldrich, Tokyo, Japan), was injected i.p. To visualize neutrophils, Measurement of cytochrome c oxidase subunit III we injected i.v. FITC- or PE-labeled anti-mouse Ly6G (2.0 mg per mouse; BioLegend). To visualize monocyte/macrophage, C57BL/6 mice received The level of cytochrome c oxidase subunit III in serum samples was the injection of FITC-labeled anti-mouse F4/80 (2.0 mg per mouse; measured with ELISA according to the manufacturer’s instructions (Uscn eBioscience) i.v. Mice were set up for intravital imaging or sacrificed at Life Science, Houston, TX). Correlation analysis was performed by cal- indicated time points to collect blood and tissue samples. culating the Pearson correlation coefficient between the serum cytochrome c oxidase and ALT levels at 24 h after I/R. TPLSM imaging TUNEL assay Mice were prepared for intravital microscopy using a previously described protocol (15, 19) (Supplemental Fig. 1). In brief, after midline laparotomy, Apoptotic hepatocytes were stained using an ApopTag peroxidase in situ the left lateral lobe of the liver was exteriorized, and a cover ring was apoptosis detection kit (Chemicon International, Temecula, CA). The attached. The ring was fixed into a stereotaxic holder. General anesthesia number of TUNEL-positive cells was counted in 10 random high-power was maintained with s.c. injections of xylazine and ketamine, and hydra- fields per section. tion was maintained by s.c. injections of warmed saline. They received an i.v. injection of tetramethylrhodamine isothiocyanate (TRITC)–labeled Analysis of cytokine/chemokine production albumin (500 mg; Sigma-Aldrich) just before imaging to visualize the microvasculature. Experiments were performed using an Olympus Liver tissues were weighed and placed in 10 vol of a protease inhibitor BX61WI upright microscope and FV1000MPE (Olympus, Tokyo, Japan) mixture containing a tissue protein extraction reagent (Thermo Fisher laser-scanning microscope system. Mai Tai HP Deep See femtosecond- Scientific, Kanagawa, Japan). The tissues were disrupted with a tissue pulsed laser (Spectra Physics, Santa Clara, CA) was turned and mode homogenizer, and lysates were clarified by centrifugation at 10,000 3 g for locked at 840 nm. Liver was line scanned and fluorescence emission was 5 min at 4˚C. Serum levels of TNF-a, IL-1b, IL-6, and liver CXCL1/ captured by external non-descanned detectors. Time-lapse images were keratinocyte chemokine (KC) and CXCL2/MIP-2 were quantified by taken using FV10-ASW version 3.0 (Olympus). For low-magnification ELISA (TNF-a, IL-1b, IL-6 [eBioscience, San Diego, CA]; CXCL1/KC, imaging, x-y planes spanning 1270 3 1270 mm were imaged using an CXCL2/MIP-2 [R&D Systems, Minneapolis, MN]). 1720 FPR1 IN HEPATIC I/R: INTRAVITAL IMAGING

Analysis of 8-hydroxy-29-deoxyguanosine To evaluate the oxidative stress induced by ROS, we measured 8-hydroxy- 29-deoxyguanosine (8-OHdG) of serum, urine, and ischemic liver DNA using ELISA kit (NIKKEN SEIL, Tokyo, Japan). Serum samples were ultraﬁltered using Vivaspin 500-10K (GE Healthcare UK) before analysis. Liver DNA was obtained using a DNA Extractor TIS kit, and prepared using an 8-OHdG Assay Preparation Reagent Set (Wako Chemicals, Osaka, Japan). Statistical analysis Data were expressed as mean 6 SEM. Unpaired Student t test or Mann– Whitney U test was used to compare between two groups, as appropriate. ANOVAwas used to compare more than two groups, followed by Tukey’s post hoc test. A p value ,0.05 was considered statistically signiﬁcant. All tests were two-tailed. All statistical analyses were performed using PASW Statistics 18 (IBM, Tokyo, Japan).

Results Mitochondrial DAMPs are released into the circulation after hepatic I/R Downloaded from To prove that hepatic I/R induces the release of mitochondrial DAMPs into the circulation, we measured the serum level of cytochrome c oxidase, which is specific for mitochondrial protein and also an indication of the release of mitochondrial formyl peptides (3). The serum level of cytochrome c oxidase was up- regulated within 2 h after reperfusion and remained elevated until 6 h after reperfusion (Fig. 1A). The serum level of cytochrome c http://www.jimmunol.org/ oxidase was correlated with the serum level of ALT at 24 h after I/R (Fig. 1B). Blockade of FPR1 alleviates hepatic I/R injury To examine whether the extracellular formyl-peptide–FPR1 signaling pathway contributes to hepatic I/R injury, we administered CsH to mice. In comparison with the PBS-treated control group, FIGURE 1. Expression of cytochrome c oxidase after hepatic I/R. (A) the CsH-treated group showed a significant decrease in serum Serum concentration of cytochrome c oxidase after hepatic I/R. Serum by guest on September 29, 2021 transaminase levels at 6 h (AST: 4349 6 1113 versus 7300 6 samples were analyzed by ELISA. Data represent the mean 6 SEM (n =6 1064 IU/l, p , 0.05; ALT: 9434 6 2856 versus 16,902 6 2595 IU/ per group). *p , 0.05 in comparison with the sham group. (B) Serum level l, p , 0.05) and 24 h (AST: 669 6 169 versus 1667 6 457 IU/l, of cytochrome c oxidase was significantly correlated with ALT levels (R = p , 0.05; ALT: 997 6 298 versus 2094 6 557 IU/l, p , 0.05) 0.851, p , 0.05). Dotted lines indicate the 95% confidence interval. after I/R (Fig. 2A). The proportion of necrotic area in ischemic liver was significantly reduced by treatment with CsH at 6 h (25.9 6 2.5 versus 33.5 6 2.8%, p , 0.05) and 24 h (28.8 6 2.9 versus Laser irradiation induced a square-shaped liver necrosis in the 39.0 6 2.7%, p , 0.05) (Fig. 2B, 2C). In addition, CsH treatment center of the low-magnification image. Neutrophils that were resulted in a decrease in the number of TUNEL-positive cells treated with CsH showed nondirectional random migration around (11.0 6 2.3 versus 33.1 6 2.4, p , 0.001) at 6 h after I/R the injury area at 2 h after laser irradiation (Fig. 4B). Meandering (Fig. 2D). We next examined the effects of fMLF in hepatic I/R; index of neutrophils treated with CsH was significantly lower however, systemically administered fMLF showed no significant compared with control (0.445 6 0.054 versus 0.767 6 0.030, p , biochemical effects (Supplemental Fig. 2A). 0.001) (Fig. 4C). In the control group, the number of adherent neutrophils around the necrosis gradually increased, whereas the Blockade of FPR1 decreases inflammatory cytokine/chemokine number remained at the same level in the CsH-treated group (Fig. production and oxidative stress after hepatic I/R 4D). Their crawling velocity reached a maximum at 2 h after laser Six hours after I/R, the serum levels of TNF-a, IL-6, liver CXCL1/KC, irradiation in both groups (5.21 6 0.39 in the control group versus and CXCL2/MIP2 were significantly lower in the CsH-treated 4.67 6 0.38 mm/m in the CsH-treated group) and gradually de- group (Fig. 3A, 3B). Oxidative stress induced by hepatic I/R, as creased (Fig. 4E). Their velocity was not significantly different at measured by the expression of 8-OHdG, was reduced in urine and any of the time points. At 3 h after laser irradiation, a number of liver DNA by CsH treatment (Fig. 3C). With the exception of liver neutrophils accumulated in the necrotic area in the control group; CXCL1/KC levels, the systemic administration of fMLF had no in contrast, it was significantly inhibited in the CsH-treated group significant effect on the levels of inflammatory cytokines or oxi- (Fig. 4F). dative stress (Supplemental Fig. 2B–D). FPR1 blockade decreases the accumulation of neutrophils in FPR1 signaling is indispensable for the recruitment of the liver after I/R neutrophils into liver necrosis induced by laser irradiation We imaged the liver at low magnification using TPLSM at 6 and Selective liver necrosis was induced by laser irradiation in live 24 h after I/R to examine the general view (Fig. 5A, Supplemental LysM-eGFP mice, and time-lapse images were recorded using Video 2). The number of adherent neutrophils per field of view in TPLSM (Fig. 4A, Supplemental Video 1), to confirm the role of the CsH-treated group was lower than that in the control group at FPR1 on the recruitment of neutrophils in sterile inflammation. 6 h (321.4 6 40.9 versus 630.8 6 106.3, p , 0.05) and 24 h (95. The Journal of Immunology 1721

FIGURE 2. Blockade of FPR1 alleviates liver injury after I/R. Mice were treated with PBS or CsH 30 min before ischemia. (A) Serum AST and ALT levels were measured at 6 and 24 h after I/R (n = 12 per group). Transaminase levels were signiﬁ- cantly decreased in the CsH-treated group. (B) Representative H&E staining of the control and CsH- treated livers at 6 and 24 h after I/R. Dotted lines indicate the necrotic areas. Scale bar, 200 mm. (C) Ne-

crotic area was determined by a Downloaded from quantitative analysis and was expressed as the percentage of total area examined (n = 5 per group). (D) Representative TUNEL staining of sham, control, and CsH-treated livers at 6 h after I/R. Scale bar, 50 mm.

The numbers of TUNEL-positive http://www.jimmunol.org/ cells were signiﬁcantly decreased in the CsH-treated liver in comparison with the control liver (n = 4 per group). *p , 0.05, **p , 0.01 in comparison with the control group. #p , 0.05, ##p , 0.01 in comparison with the sham group. by guest on September 29, 2021

0 6 17.1 versus 260.6 6 45.3, p , 0.01) (Fig. 5B). Time-lapse after I/R. In contrast, there were no significant differences in the imaging revealed the reduction of their crawling velocity in the perfused areas (Fig. 6B). CsH treatment reduced the crawling CsH-treatedgroupat6h(3.846 0.17 versus 4.74 6 0.20 mm/min, velocity in the nonperfused area at 6 h (1.76 6 0.28 versus 2.70 6 p , 0.001) and 24 h (2.70 6 0.21 versus 3.08 6 0.18 mm/min, 0.25 mm/min, p , 0.05) (Fig. 6C). Morphologically, neutrophils p , 0.01) (Fig. 5C). At 24 h after I/R, the number and velocity of in the perfused areas had a more elongated shape than those in the adherent neutrophils in the CsH-treated group were comparable nonperfused areas; however, the administration of CsH did not with those in the sham group. Next, we confirmed that the number affect their cell shape index in any of the areas (Fig. 6D). To of neutrophils in the liver at 6 h after hepatic I/R did not differ confirm that eGFP-labeled granule-containing cells are neutro- significantly between wild type C57BL/6 mice and LysM-eGFP phils, we injected PE-labeled Ly6G Ab to LysM-eGFP mice. In mice (Fig. 5D, 5E). In this experiments, we did not use TRITC- this model, LysM and Ly6G double-positive cells were visualized albumin to reveal that the TRITC-albumin as well as the genetic as yellow cells (Fig. 6E). Accordingly, 96.9% of eGFP+ cells in manipulation induce few changes to the chemotactic stimuli in- the sham group and 93.5% of eGFP+ cells in the I/R group (6 h) duced by hepatic I/R. were Ly6G+. Distinct migratory patterns of neutrophils within nonperfused FPR1 blockade does not affect monocyte/macrophage area of the liver after I/R recruitment in the liver after I/R To further explore the distinct migratory patterns of neutrophils, we To examine whether FPR1 blockade affects monocyte/macrophage imaged the liver at high magnification (Fig. 6A, Supplemental recruitment, we imaged the liver 6 h after hepatic I/R at high Video 3). According to the findings, in addition to being able to magnification (Supplemental Fig. 3A). Monocytes/macrophages distinguish between the perfused and nonperfused (necrotic) areas, were detected by FITC-F4/80 positivity. Hepatic I/R induced the we could also analyze the detailed migratory pattern of neutro- increase of monocytes/macrophages in the liver after I/R, whereas phils in each area. In the CsH-treated group, the number of neu- FPR1 blockade did not affect monocyte/macrophage recruitment trophils per 10,000 mm2 in the nonperfused area was significantly (Supplemental Fig. 3B, 3C). Most of the monocytes/macrophages lower than that in the control group at 6 h (1.92 6 0.28 versus 6.52 6 were static, and a few of them infiltrated in the nonperfused area in 0.68, p , 0.01)and24h(0.966 0.23 versus 4.16 6 0.39, p , 0.01) each group. 1722 FPR1 IN HEPATIC I/R: INTRAVITAL IMAGING

FIGURE 3. Blockade of FPR1 decreases the production of inﬂam- matory cytokines and oxidative stress. (A) Serum levels of inﬂam- matory cytokines (TNF-a, IL-1b, IL- 6), (B) liver chemokine (CXCL1/KC, CXCL2/MIP-2), and (C) oxidative stress markers (8-OHdG in the serum,urine,andliverDNA)were analyzed by ELISA. Data represent the mean 6 SEM (n = 6 per group). *p , 0.05, **p , 0.01 in comparison with the sham group. #p , 0.05, ## ,

p 0.01 in comparison with the Downloaded from control group. http://www.jimmunol.org/

FPR1 blockade prevents neutrophil retention in the spleen neutrophil FPR1 and mainly exert a chemotactic function. Re- during hepatic I/R cently, Hu et al. (24) demonstrated that hepatic I/R resulted in a Several reports have shown that immune cells recruited to ischemic significant increase of mitochondrial DAMPs both in vitro and tissue originate from a reservoir in the spleen (20, 21). We therefore in vivo. Our data also show that hepatic I/R results in hepatocyte studied the dynamics of neutrophils in the spleen during hepatic death and the release of mitochondrial DAMPs into the circulation I/R using TPLSM. We first imaged the mouse native spleen to according to liver damage. We revealed that FPR1 blockade al- establish the baseline characteristics. Imaging of native spleen leviated hepatic I/R injury, as evidenced by decreased serum levels by guest on September 29, 2021 at low and high magnification (Fig. 7A, Supplemental Video 4) of transaminase, reduced degree of hepatocyte necrosis/apoptosis, revealed greater numbers of neutrophils in the spleen; neutrophils and diminished production of inflammatory cytokines, chemo- also demonstrated lower motility and rounder shape in comparison kines, and ROS. Using the TPLSM technique, we identified that with the native liver (Supplemental Fig. 4). We found that hepatic these effects likely resulted from the inhibition of neutrophil I/R promoted the retention of neutrophils and their active behavior chemotaxis to reach their final destination. including their higher velocity and a more elongated shape in the Accumulation of neutrophils at the site of inflammation is a spleen at 6 h after I/R. In addition, some of the activated neu- multistep process (25). In this study, we first used the laser irra- trophils transmigrated from the sinusoidal walls into circulation diation method to induce liver necrosis in live mice and imaged (Fig. 7B–F). Conversely, CsH treatment prevented the retention of the process of neutrophil accumulation using TPLSM. This simple neutrophils in spleen and restricted their active motility. Next, we method allowed us to create a precise necrotic area and to perform injected PE-labeled Ly6G Ab to LysM-eGFP mice to confirm that subsequent noninvasive longitudinal imaging without refixation. eGFPhi cells in the spleen are neutrophils (Fig. 7G). Accordingly, Consistent with other studies, FPR1 blockade resulted in the 96.8% of eGFP+ cells in the sham group and 96.2% of eGFP+ cells significant inhibition of neutrophil migration into the liver necrotic in the I/R group (6 h) were Ly6G+. area (10). Furthermore, CsH treatment did not affect the neutrophil crawling velocity in the perfused area. Next, we imaged the Discussion liver at 6 and 24 h after hepatic I/R. Combining low- and high- We demonstrated intravital imaging of neutrophil recruitment in magnification imaging, we showed that the blockade of FPR1 response to FPR1 blockade during hepatic I/R using TPLSM. Our signaling in hepatic I/R inhibited neutrophil recruitment, in par- data show that the activation of neutrophil FPR1 via formyl ticular, in the nonperfused area as laser irradiation model. peptides is a critical determinant of the chemotaxis in the necrotic Meanwhile CsH administration did not affect monocyte/ area and subsequent inflammatory response after hepatic I/R. macrophage recruitment in hepatic I/R. Thus, it was revealed Consequently, FPR1 blockade prevented the accumulation of that formyl-peptide–FPR1 signaling was responsible for the reg- neutrophils in ischemic liver and attenuated hepatic I/R injury by ulation of neutrophil chemotaxis, through which the neutrophils inhibiting innate immune responses. migrate to the necrotic area in hepatic I/R. Because of the arrest of DAMPs are molecules that can initiate the immune response neutrophils during their oxidative burst, it is possible that the in sterile inflammation (22, 23). To date, several studies have nonmotile cells represent a population of activated neutrophils reported that endogenous DAMPs are elevated during hepatic is- (26). Our data showed that neutrophils in the necrotic area moved chemia and that they contribute to liver damage (5–8). Formyl more slowly, which suggested that neutrophils accumulating in the peptides are known as mitochondrial DAMPs; they are released in necrotic area made a significant contribution to the activation of response to cellular injury (3). Formyl peptides are recognized by inflammation. Recent studies showed that FPR1 signaling induces The Journal of Immunology 1723 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. FPR1 signaling is indispensable for the accumulation of neutrophils at sites of liver necrosis in sterile injury. (A) Representative imaging of the liver (neutrophil in green, LysM-eGFP, and microvasculature in red, TRITC) in control or CsH-treated mice at low magnification during 3 h after laser irradiation. Scale bars, 200 mm. (B) Migration paths. (C) Meandering index. (D) The number of adherent neutrophils and (E) velocity of neutrophils responding to sterile liver injury in control and CsH-treated mice. (F) At 3 h after laser irradiation, the accumulation of neutrophils in the necrotic area of the CsH-treated group was significantly inhibited. n = 3 per group; error bars indicate SEM. *p , 0.05, **p , 0.01 in comparison with the control group. a rapid increase in the mitochondrial membrane potential within protect the organ against bacterial infection. Therefore, the neutrophils, with an associated oxidative burst and extracellular blockade of FPR1 signaling could be a double-edged sword in the release of ATP (27). In that sense, FPR1 blockade enables the transplant setting. Further perspectives on the role of FPR1 in effective alleviation of inflammatory activation derived from neu- inflammation and infection would provide the exact treatment trophils in hepatic I/R. strategy in various settings. Recent studies have revealed that FPR is also critical for the Hypoxia-inducible factors, that is, composed of one of three recruitment of neutrophils in bacterial infection (28, 29). Interstitial hypoxia-regulated a subunits (-1a,-2a, and -3a) bound to an migration of neutrophils is mediated by PI3K and p38 MAPK oxygen-independent b subunit, are oxygen-sensitive transcription signaling pathways. Under the condition of multiple signaling factors that activate the hypoxia-responsive genes (31). Recent mediators, PI3K is inactivated by phosphatase and tensin homo- studies using pharmacological approaches to stabilize hypoxia- log, allowing p38 and phospholipase A2 to activate neutrophil inducible factor have shown their protective effects in hepatic chemotaxis toward bacterial products (30). Increased neutrophils I/R injury (32). The a subunits are targeted for degradation by 1724 FPR1 IN HEPATIC I/R: INTRAVITAL IMAGING

FIGURE 5. FPR1 signaling is indispensable for the accumulation of neutrophils into the liver in a mouse model of hepatic I/R injury. (A) Representative imaging of the liver (neutrophil in green, LysM-eGFP, and microvasculature in red, TRITC) in sham, control I/R, and CsH-treated I/R groups at low magnification. Scale bars, 200 mm. (B) Quantification of Downloaded from the number of adherent neutrophils per field of view and (C) velocity of neutrophils (at each time point, 30 neutrophils were selected per mouse) 6 and 24 h after hepatic I/R in sham, control, and CsH-treated mice. (D) http://www.jimmunol.org/ Representative low-magnification imaging of the liver (neutrophil in green) at 6 h after I/R in C57BL/6 and LysM- eGFP mice. Neutrophils were detected by FITC-Ly6G positivity in C57BL/6 mice. Scale bars, 200 mm. (E) Quan- tification of the number of adherent neutrophils per field of view in C57BL/6 and LysM-eGFP mice. n =5 per group; error bars indicate SEM. by guest on September 29, 2021 *p , 0.05, **p , 0.01 in comparison with the control group.

prolyl hydroxylase domain enzymes under normoxic conditions. attractant for neutrophils, the protective effect of fMLF might be Prolyl hydroxylase domain inhibitor has been shown to activate through the competitive inhibition of the “find me” signal from the hypoxia-inducible factor-1a, thereby inhibiting mitochondrial ischemic liver. The mechanism of preconditioning is complex and permeability transition and mitochondrial polarization and atten- remains to be elucidated in complex interactions of molecules. uating hepatic I/R injury (33). These effects would reduce the Spleen is one of the abdominal organs connected by the portal release of mitochondrial DAMPs in response to cellular injury and system. Hepatic ischemia decreases the blood flow in the spleen contribute to inhibiting the interaction between formyl peptides and increases vascular resistance; thus, congestion and ischemic and FPR1 indirectly. injury might occur (21). A recent study using a massive partial Unexpectedly, systemic administration of fMLF showed a ten- hepatectomy model has shown the expression of numerous dency to reduce hepatic I/R injury, although statistically significant inflammatory-related genes occurred in the spleen (36). These difference was not shown. This phenomenon seems to be explained expressions might increase the trafficking of the immune cells in connection with a preconditioning stimulus (2). Preconditioning derived from bone marrow in the spleen. TPLSM imaging showed can be induced by various stimuli and is applicable in different that the number and motility of splenic neutrophils was increased organ systems. Recent work has explored the potential use of after hepatic I/R, which indicates that the spleen may play a endogenous danger molecules, such as heat shock protein and harmful role and provide a negative impact during hepatic I/R as high-mobility group box 1, for preconditioning in hepatic I/R (34, neutrophil supplier. Blockade of the FPR1 would prevent these 35). Because fMLF has been shown to function as a chemo- chain reactions by inhibiting the primary immune response The Journal of Immunology 1725 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. Formyl-peptide–FPR1 signaling is responsible for the regulation of neutrophil chemotaxis to migrate into the necrotic area in hepatic I/R. (A) Representative imaging of the liver (neutrophil is green and microvasculature is red) in sham, control I/R, and CsH-treated I/R group at high magnification. Scale bars, 100 mm. (B) Quantification of the number of adherent neutrophils/10,000 mm2 (counted in five independent areas per mouse), (C) velocity of neutrophils (at each time point, 10 neutrophils were selected in each perfused and nonperfused area per mouse), and (D) cell shape index at 6 and 24 h after hepatic I/R in sham, control, and CsH-treated mice. (E) Representative high-magnification imaging of LysM and Ly6G double-positive cells at 6 h after I/R. Cells were distributed by their color (red, PE; yellow, double positive; green, LysM-eGFP). n = 5 per group; error bars indicate SEM. *p , 0.05, **p , 0.01 in comparison with the control group. 1726 FPR1 IN HEPATIC I/R: INTRAVITAL IMAGING Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 7. Hepatic I/R promotes the retention of neutrophils and their active behavior in the spleen. FPR1 blockade prevents these changes. Repre- sentative imaging of the spleen (neutrophil is green and microvasculature is red) at 6 h after hepatic I/R in sham, control, and CsH-treated mice (A)atlow (top; scale bar, 200 mm) and high magnification (bottom; scale bar, 100 mm). (B) Cellular tracking during 20 min of time-lapse imaging. (C) The white dashed arrow shows the direction of blood flow. Arrowheads indicate neutrophil migrating on the sinusoidal wall, followed by their release into the circulation (white and yellow arrows). Time scale is shown below the images. (D) Quantification of the number of adherent neutrophils per field of view in low-magnification imaging of spleen, (E) velocity of neutrophils (50 neutrophils per mouse) in low-magnification imaging of spleen, and (F) cell shape index (10 neutrophils per mouse) in high-magnification imaging of spleen 6 h after hepatic I/R in sham, control, or CsH-treated mice. (G) Representative high-magnification imaging of LysM and Ly6G double-positive cells at 6 h after I/R. Cells were distributed by their color (red, PE; yellow, double positive; green, LysM-eGFP). n = 5 per group; error bars indicate SEM; *p , 0.05, **p , 0.01 in comparison with the sham group. #p , 0.05, ##p , 0.01 in comparison with the control group. The Journal of Immunology 1727 induced by neutrophil recruitment to the ischemic liver. Studies of as alarmins in sterile inflammatory liver injury through Toll-like receptor 9 in mice. Hepatology 54: 999–1008. immune cells in the spleen at the single-cell level may provide 9. 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