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 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 neutrophils 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.

Hepatic ischemia-reperfusion (I/R) injury is a complication of trauma, shock, liver resection, and transplantation (1, 2). In the ischemic liver, an imbalance of the metabolic supply and demand results in severe tissue hypoxia, mitochondrial dysfunction, and the generation of reactive oxygen species (ROS). Subsequent reperfusion causes the activation of innate and adaptive immune responses and cell death programs, leading to liver dysfunction and failure.

Cellular injury can release endogenous damage-associated molecular patterns (DAMPs), which activate innate immunity (3, 4). Several DAMPs have been identified in which intranuclear high-mobility group box 1, DNA, and histones have been shown to play critical roles in hepatic I/R through a specific pattern recognition receptor (58). In addition, cellular injury also releases mitochondrial DAMPs, including formyl peptide and mitochondrial DNA, which are recognized by formyl-peptide receptor 1 (FPR1) and TLR9, respectively (3). Endogenous formyl peptides are released secondarily from the N terminus of mitochondrial NADH dehydrogenase and cytochrome c oxidase in dead cells, and mainly exert a chemotactic function by promoting neutrophil Ca2+ flux and MAPKs (9). Signals through FPR1 in neutrophils overcome signals hierarchically through CXCR2, which is known as a key chemokine receptor, allowing neutrophils to migrate toward end-target chemoattractants (10). We therefore hypothesized that hepatic I/R releases mitochondrial DAMPs, thereby inducing the accumulation of neutrophils in the ischemic liver in an FPR1-dependent manner. Recent studies have shown the therapeutic potential of FPR1 blockade in acetaminophen-induced acute liver failure and smoking-induced lung emphysema (11, 12); however, the impact of the formyl-peptide–FPR1 interaction on hepatic I/R injury is unknown.

Advances in intravital microscopy have enabled the visualization and quantification of real-time biological processes in situ (13, 14). Above all, the monitoring of immune cell recruitment has an especially broad range of applications. We recently showed a novel method for examining in vivo real-time neutrophil recruitment during hepatic I/R using two-photon laser-scanning microscopy (TPLSM) (15). TPLSM is one of the most progressive developments in imaging technology and has numerous advantages, including high-resolution deep-site imaging, less phototoxicity, and less photobleaching in comparison with conventional confocal laser-scanning microscopy. These major advantages are especially suited to imaging live tissues and organs in their native environment (16, 17). Using this method, we could investigate the structure of hepatic lobes, circulation of the sinusoids, cell death, and changes in the number, velocity, and morphology of recruited neutrophils. Because these findings provided valuable new insights into the functional-anatomical changes that occur during hepatic I/R, we applied the technique to test the dynamic effect of FPR1 blockade.

By combining the information of intravital TPLSM imaging and conventional biological parameters, we provided visual and quantitative evidence of the therapeutic effects of FPR1 blockade in hepatic I/R injury. FPR1 blockade inhibited the chemotaxis of neutrophils into the necrotic area and subsequent innate immune-mediated inflammation. FPR1 blockade also prevented the retention of neutrophils in spleen, which is the major supplier of neutrophils. Our results suggest that mitochondrial DAMPs and their specific receptors are potential targets for therapeutic intervention in hepatic I/R.

LysM-eGFP mice were a gift from Dr. T. Graf (Center for Genomic Regulation, Barcelona, Spain). These mice express eGFP under the lysozyme M promoter, and neutrophils are visualized by the expression of eGFPhi (18). C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan). All mice were maintained in a specific pathogen-free environment with ad libitum access to food and water at the Animal Resource Facility at Kumamoto University. A 12/12 h light/dark cycle was maintained with light from 07:00 am to 7:00 pm. Temperature was maintained at 22 ± 2°C. All experiments were performed according to guidelines of the Institutional Animal Committee of Kumamoto University.

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 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. After a midline laparotomy followed by an injection of heparin (100 U/kg), mice underwent a sham operation or the induction of I/R. In the I/R group, 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 Corporation, Tokyo, Japan). After 60 min of ischemia, the clip was removed and reperfusion was initiated. Sham operation was performed using the same protocol but without vascular occlusion. Mice received a selective FPR1 antagonist, cyclosporine H (CsH; 50 μg per mouse; LKT Laboratories), or PBS via an i.p. injection at 30 min before ischemia. In the other experiments, the synthetic N-formyl peptide, fMLF (3 μg per mouse; Sigma-Aldrich, Tokyo, Japan), was injected i.p. To visualize neutrophils, we injected i.v. FITC- or PE-labeled anti-mouse Ly6G (2.0 μg per mouse; BioLegend). To visualize monocyte/macrophage, C57BL/6 mice received the injection of FITC-labeled anti-mouse F4/80 (2.0 μg per mouse; eBioscience) i.v. Mice were set up for intravital imaging or sacrificed at indicated time points to collect blood and tissue samples.

Mice were prepared for intravital microscopy using a previously described protocol (15, 19) (Supplemental Fig. 1). In brief, after midline laparotomy, the left lateral lobe of the liver was exteriorized, and a cover ring was attached. The ring was fixed into a stereotaxic holder. General anesthesia was maintained with s.c. injections of xylazine and ketamine, and hydration was maintained by s.c. injections of warmed saline. They received an i.v. injection of tetramethylrhodamine isothiocyanate (TRITC)–labeled albumin (500 μg; Sigma-Aldrich) just before imaging to visualize the microvasculature. Experiments were performed using an Olympus BX61WI upright microscope and FV1000MPE (Olympus, Tokyo, Japan) laser-scanning microscope system. Mai Tai HP Deep See femtosecond-pulsed laser (Spectra Physics, Santa Clara, CA) was turned and mode locked at 840 nm. Liver was line scanned and fluorescence emission was captured by external non-descanned detectors. Time-lapse images were taken using FV10-ASW version 3.0 (Olympus). For low-magnification imaging, x-y planes spanning 1270 × 1270 μm were imaged using an Olympus UMPLFLN 10× W (numerical aperture 0.30) objective lens. For high-magnification imaging, images were recorded using an Olympus XLPLN 25× WMP (water-immersion, numerical aperture 1.05) objective lens with x-y planes spanning 508 × 508 μm. In some experiments, mice were placed in the right lateral recumbent position, and the spleen was exteriorized by a left flank incision. Spleen was fixed and imaged using TPLSM as described.

Selective hepatic necrosis was induced by high-power laser irradiation using TPLSM. Time-lapse imaging at low-magnification was zoomed up by six times, and the laser transmissivity was increased to 100% (it was ordinarily locked at 25% during low-magnification imaging). After 3 min of irradiation, the imaging conditions were returned to normal. Necrotic area was blacked out with x-y planes spanning 210 × 210 μm. Twenty-minute videos were recorded every 30 min for 3 h after laser irradiation. The 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 laser irradiation was evaluated.

Neutrophils were identified and distinguished from other myeloid subsets based on the characteristics of their LysM-eGFP fluorescence intensity (a threshold >40 in low-magnification imaging and >160 in high-magnification imaging, respectively) and cell size (dimensions of 5–15 μm). Monocytes/macrophages were identified based on the positivity of FITC-F4/80. The number of adherent cells was evaluated using the “analyze and measure” command in ImageJ software (National Institutes of Health, Bethesda, MD). Hue analysis was performed using the “Color Threshold Tool” in ImageJ software. Cell shape index of neutrophils was calculated as the ratio of the cell’s length to the cell’s width. To analyze the velocity of adherent neutrophils, we selected 30 neutrophils per low-magnification field for tracking and measured the crawling velocities at each time point using G-Track software (Olympus Medical Science). The velocity of adherent neutrophils within specific regions was measured separately using high-magnification imaging. Cell tracking was shown by the “Manual Tracking” command using ImageJ software. Meandering index was measured by Euclidian distance/accumulated distance.

Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, an indicator of hepatocellular injury, were measured using a Hitachi 7180 auto analyzer (Hitachi High-Technologies, Tokyo, Japan).

Liver samples were fixed in 10% buffered formalin, embedded in paraffin, and liver sections (4 μm) were stained with H&E. To calculate the percentage of the necrotic area, we evaluated 10 random sections per slide in a blind manner using ImageJ software.

The level of cytochrome c oxidase subunit III in serum samples was measured with ELISA according to the manufacturer’s instructions (Uscn Life Science, Houston, TX). Correlation analysis was performed by calculating the Pearson correlation coefficient between the serum cytochrome c oxidase and ALT levels at 24 h after I/R.

Apoptotic hepatocytes were stained using an ApopTag peroxidase in situ apoptosis detection kit (Chemicon International, Temecula, CA). The number of TUNEL-positive cells was counted in 10 random high-power fields per section.

Liver tissues were weighed and placed in 10 vol of a protease inhibitor mixture containing a tissue protein extraction reagent (Thermo Fisher Scientific, Kanagawa, Japan). The tissues were disrupted with a tissue homogenizer, and lysates were clarified by centrifugation at 10,000 × g for 5 min at 4°C. Serum levels of TNF-α, IL-1β, IL-6, and liver CXCL1/keratinocyte chemokine (KC) and CXCL2/MIP-2 were quantified by ELISA (TNF-α, IL-1β, IL-6 [eBioscience, San Diego, CA]; CXCL1/KC, CXCL2/MIP-2 [R&D Systems, Minneapolis, MN]).

To evaluate the oxidative stress induced by ROS, we measured 8-hydroxy-2′-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).

Data were expressed as mean ± SEM. Unpaired Student t test or Mann–Whitney U test was used to compare between two groups, as appropriate. ANOVA was 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).

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 upregulated within 2 h after reperfusion and remained elevated until 6 h after reperfusion (Fig. 1A). The serum level of cytochrome c oxidase was correlated with the serum level of ALT at 24 h after I/R (Fig. 1B).

FIGURE 1.

Expression of cytochrome c oxidase after hepatic I/R. (A) Serum concentration of cytochrome c oxidase after hepatic I/R. Serum samples were analyzed by ELISA. Data represent the mean ± SEM (n = 6 per group). *p < 0.05 in comparison with the sham group. (B) Serum level of cytochrome c oxidase was significantly correlated with ALT levels (R = 0.851, p < 0.05). Dotted lines indicate the 95% confidence interval.

FIGURE 1.

Expression of cytochrome c oxidase after hepatic I/R. (A) Serum concentration of cytochrome c oxidase after hepatic I/R. Serum samples were analyzed by ELISA. Data represent the mean ± SEM (n = 6 per group). *p < 0.05 in comparison with the sham group. (B) Serum level of cytochrome c oxidase was significantly correlated with ALT levels (R = 0.851, p < 0.05). Dotted lines indicate the 95% confidence interval.

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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, the CsH-treated group showed a significant decrease in serum transaminase levels at 6 h (AST: 4349 ± 1113 versus 7300 ± 1064 IU/l, p < 0.05; ALT: 9434 ± 2856 versus 16,902 ± 2595 IU/l, p < 0.05) and 24 h (AST: 669 ± 169 versus 1667 ± 457 IU/l, p < 0.05; ALT: 997 ± 298 versus 2094 ± 557 IU/l, p < 0.05) 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 ± 2.5 versus 33.5 ± 2.8%, p < 0.05) and 24 h (28.8 ± 2.9 versus 39.0 ± 2.7%, p < 0.05) (Fig. 2B, 2C). In addition, CsH treatment resulted in a decrease in the number of TUNEL-positive cells (11.0 ± 2.3 versus 33.1 ± 2.4, p < 0.001) at 6 h after I/R (Fig. 2D). We next examined the effects of fMLF in hepatic I/R; however, systemically administered fMLF showed no significant biochemical effects (Supplemental Fig. 2A).

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 significantly 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 μm. (C) Necrotic area was determined by a 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 μm. The numbers of TUNEL-positive 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.

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 significantly 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 μm. (C) Necrotic area was determined by a 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 μm. The numbers of TUNEL-positive 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.

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Six hours after I/R, the serum levels of TNF-α, IL-6, liver CXCL1/KC, and CXCL2/MIP2 were significantly lower in the CsH-treated group (Fig. 3A, 3B). Oxidative stress induced by hepatic I/R, as measured by the expression of 8-OHdG, was reduced in urine and liver DNA by CsH treatment (Fig. 3C). With the exception of liver CXCL1/KC levels, the systemic administration of fMLF had no significant effect on the levels of inflammatory cytokines or oxidative stress (Supplemental Fig. 2B–D).

FIGURE 3.

Blockade of FPR1 decreases the production of inflammatory cytokines and oxidative stress. (A) Serum levels of inflammatory cytokines (TNF-α, IL-1β, IL-6), (B) liver chemokine (CXCL1/KC, CXCL2/MIP-2), and (C) oxidative stress markers (8-OHdG in the serum, urine, and liver DNA) were analyzed by ELISA. Data represent the mean ± 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 control group.

FIGURE 3.

Blockade of FPR1 decreases the production of inflammatory cytokines and oxidative stress. (A) Serum levels of inflammatory cytokines (TNF-α, IL-1β, IL-6), (B) liver chemokine (CXCL1/KC, CXCL2/MIP-2), and (C) oxidative stress markers (8-OHdG in the serum, urine, and liver DNA) were analyzed by ELISA. Data represent the mean ± 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 control group.

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Selective liver necrosis was induced by laser irradiation in live LysM-eGFP mice, and time-lapse images were recorded using TPLSM (Fig. 4A, Supplemental Video 1), to confirm the role of FPR1 on the recruitment of neutrophils in sterile inflammation. Laser irradiation induced a square-shaped liver necrosis in the center of the low-magnification image. Neutrophils that were treated with CsH showed nondirectional random migration around the injury area at 2 h after laser irradiation (Fig. 4B). Meandering index of neutrophils treated with CsH was significantly lower compared with control (0.445 ± 0.054 versus 0.767 ± 0.030, p < 0.001) (Fig. 4C). In the control group, the number of adherent neutrophils around the necrosis gradually increased, whereas the number remained at the same level in the CsH-treated group (Fig. 4D). Their crawling velocity reached a maximum at 2 h after laser irradiation in both groups (5.21 ± 0.39 in the control group versus 4.67 ± 0.38 μm/m in the CsH-treated group) and gradually decreased (Fig. 4E). Their velocity was not significantly different at any of the time points. At 3 h after laser irradiation, a number of neutrophils accumulated in the necrotic area in the control group; in contrast, it was significantly inhibited in the CsH-treated group (Fig. 4F).

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 μm. (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.

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 μm. (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.

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We imaged the liver at low magnification using TPLSM at 6 and 24 h after I/R to examine the general view (Fig. 5A, Supplemental Video 2). The number of adherent neutrophils per field of view in the CsH-treated group was lower than that in the control group at 6 h (321.4 ± 40.9 versus 630.8 ± 106.3, p < 0.05) and 24 h (95.0 ± 17.1 versus 260.6 ± 45.3, p < 0.01) (Fig. 5B). Time-lapse imaging revealed the reduction of their crawling velocity in the CsH-treated group at 6 h (3.84 ± 0.17 versus 4.74 ± 0.20 μm/min, p < 0.001) and 24 h (2.70 ± 0.21 versus 3.08 ± 0.18 μm/min, p < 0.01) (Fig. 5C). At 24 h after I/R, the number and velocity of adherent neutrophils in the CsH-treated group were comparable with those in the sham group. Next, we confirmed that the number of neutrophils in the liver at 6 h after hepatic I/R did not differ significantly between wild type C57BL/6 mice and LysM-eGFP mice (Fig. 5D, 5E). In this experiments, we did not use TRITC-albumin to reveal that the TRITC-albumin as well as the genetic manipulation induce few changes to the chemotactic stimuli induced by hepatic I/R.

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 μm. (B) Quantification of 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) 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 μm. (E) Quantification 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. *p < 0.05, **p < 0.01 in comparison with the control group.

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 μm. (B) Quantification of 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) 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 μm. (E) Quantification 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. *p < 0.05, **p < 0.01 in comparison with the control group.

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To further explore the distinct migratory patterns of neutrophils, we imaged the liver at high magnification (Fig. 6A, Supplemental Video 3). According to the findings, in addition to being able to distinguish between the perfused and nonperfused (necrotic) areas, we could also analyze the detailed migratory pattern of neutrophils in each area. In the CsH-treated group, the number of neutrophils per 10,000 μm2 in the nonperfused area was significantly lower than that in the control group at 6 h (1.92 ± 0.28 versus 6.52 ± 0.68, p < 0.01) and 24 h (0.96 ± 0.23 versus 4.16 ± 0.39, p < 0.01) after I/R. In contrast, there were no significant differences in the perfused areas (Fig. 6B). CsH treatment reduced the crawling velocity in the nonperfused area at 6 h (1.76 ± 0.28 versus 2.70 ± 0.25 μm/min, p < 0.05) (Fig. 6C). Morphologically, neutrophils in the perfused areas had a more elongated shape than those in the nonperfused areas; however, the administration of CsH did not affect their cell shape index in any of the areas (Fig. 6D). To confirm that eGFP-labeled granule-containing cells are neutrophils, we injected PE-labeled Ly6G Ab to LysM-eGFP mice. In this model, LysM and Ly6G double-positive cells were visualized as yellow cells (Fig. 6E). Accordingly, 96.9% of eGFP+ cells in the sham group and 93.5% of eGFP+ cells in the I/R group (6 h) were Ly6G+.

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 μm. (B) Quantification of the number of adherent neutrophils/10,000 μm2 (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.

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 μm. (B) Quantification of the number of adherent neutrophils/10,000 μm2 (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.

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To examine whether FPR1 blockade affects monocyte/macrophage recruitment, we imaged the liver 6 h after hepatic I/R at high magnification (Supplemental Fig. 3A). Monocytes/macrophages were detected by FITC-F4/80 positivity. Hepatic I/R induced the increase of monocytes/macrophages in the liver after I/R, whereas FPR1 blockade did not affect monocyte/macrophage recruitment (Supplemental Fig. 3B, 3C). Most of the monocytes/macrophages were static, and a few of them infiltrated in the nonperfused area in each group.

Several reports have shown that immune cells recruited to ischemic tissue originate from a reservoir in the spleen (20, 21). We therefore studied the dynamics of neutrophils in the spleen during hepatic I/R using TPLSM. We first imaged the mouse native spleen to establish the baseline characteristics. Imaging of native spleen at low and high magnification (Fig. 7A, Supplemental Video 4) revealed greater numbers of neutrophils in the spleen; neutrophils also demonstrated lower motility and rounder shape in comparison with the native liver (Supplemental Fig. 4). We found that hepatic I/R promoted the retention of neutrophils and their active behavior including their higher velocity and a more elongated shape in the spleen at 6 h after I/R. In addition, some of the activated neutrophils transmigrated from the sinusoidal walls into circulation (Fig. 7B–F). Conversely, CsH treatment prevented the retention of neutrophils in spleen and restricted their active motility. Next, we injected PE-labeled Ly6G Ab to LysM-eGFP mice to confirm that eGFPhi cells in the spleen are neutrophils (Fig. 7G). Accordingly, 96.8% of eGFP+ cells in the sham group and 96.2% of eGFP+ cells in the I/R group (6 h) were Ly6G+.

FIGURE 7.

Hepatic I/R promotes the retention of neutrophils and their active behavior in the spleen. FPR1 blockade prevents these changes. Representative 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) at low (top; scale bar, 200 μm) and high magnification (bottom; scale bar, 100 μm). (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.

FIGURE 7.

Hepatic I/R promotes the retention of neutrophils and their active behavior in the spleen. FPR1 blockade prevents these changes. Representative 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) at low (top; scale bar, 200 μm) and high magnification (bottom; scale bar, 100 μm). (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.

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We demonstrated intravital imaging of neutrophil recruitment in response to FPR1 blockade during hepatic I/R using TPLSM. Our data show that the activation of neutrophil FPR1 via formyl peptides is a critical determinant of the chemotaxis in the necrotic area and subsequent inflammatory response after hepatic I/R. Consequently, FPR1 blockade prevented the accumulation of neutrophils in ischemic liver and attenuated hepatic I/R injury by inhibiting innate immune responses.

DAMPs are molecules that can initiate the immune response in sterile inflammation (22, 23). To date, several studies have reported that endogenous DAMPs are elevated during hepatic ischemia and that they contribute to liver damage (58). Formyl peptides are known as mitochondrial DAMPs; they are released in response to cellular injury (3). Formyl peptides are recognized by neutrophil FPR1 and mainly exert a chemotactic function. Recently, Hu et al. (24) demonstrated that hepatic I/R resulted in a significant increase of mitochondrial DAMPs both in vitro and in vivo. Our data also show that hepatic I/R results in hepatocyte death and the release of mitochondrial DAMPs into the circulation according to liver damage. We revealed that FPR1 blockade alleviated hepatic I/R injury, as evidenced by decreased serum levels of transaminase, reduced degree of hepatocyte necrosis/apoptosis, and diminished production of inflammatory cytokines, chemokines, and ROS. Using the TPLSM technique, we identified that these effects likely resulted from the inhibition of neutrophil chemotaxis to reach their final destination.

Accumulation of neutrophils at the site of inflammation is a multistep process (25). In this study, we first used the laser irradiation method to induce liver necrosis in live mice and imaged the process of neutrophil accumulation using TPLSM. This simple method allowed us to create a precise necrotic area and to perform subsequent noninvasive longitudinal imaging without refixation. Consistent with other studies, FPR1 blockade resulted in the significant inhibition of neutrophil migration into the liver necrotic area (10). Furthermore, CsH treatment did not affect the neutrophil crawling velocity in the perfused area. Next, we imaged the liver at 6 and 24 h after hepatic I/R. Combining low- and high-magnification imaging, we showed that the blockade of FPR1 signaling in hepatic I/R inhibited neutrophil recruitment, in particular, in the nonperfused area as laser irradiation model. Meanwhile CsH administration did not affect monocyte/macrophage recruitment in hepatic I/R. Thus, it was revealed that formyl-peptide–FPR1 signaling was responsible for the regulation of neutrophil chemotaxis, through which the neutrophils migrate to the necrotic area in hepatic I/R. Because of the arrest of neutrophils during their oxidative burst, it is possible that the nonmotile cells represent a population of activated neutrophils (26). Our data showed that neutrophils in the necrotic area moved more slowly, which suggested that neutrophils accumulating in the necrotic area made a significant contribution to the activation of inflammation. Recent studies showed that FPR1 signaling induces a rapid increase in the mitochondrial membrane potential within neutrophils, with an associated oxidative burst and extracellular release of ATP (27). In that sense, FPR1 blockade enables the effective alleviation of inflammatory activation derived from neutrophils in hepatic I/R.

Recent studies have revealed that FPR is also critical for the recruitment of neutrophils in bacterial infection (28, 29). Interstitial migration of neutrophils is mediated by PI3K and p38 MAPK signaling pathways. Under the condition of multiple signaling mediators, PI3K is inactivated by phosphatase and tensin homolog, allowing p38 and phospholipase A2 to activate neutrophil chemotaxis toward bacterial products (30). Increased neutrophils protect the organ against bacterial infection. Therefore, the blockade of FPR1 signaling could be a double-edged sword in the transplant setting. Further perspectives on the role of FPR1 in inflammation and infection would provide the exact treatment strategy in various settings.

Hypoxia-inducible factors, that is, composed of one of three hypoxia-regulated α subunits (-1α, -2α, and -3α) bound to an oxygen-independent β subunit, are oxygen-sensitive transcription factors that activate the hypoxia-responsive genes (31). Recent studies using pharmacological approaches to stabilize hypoxia-inducible factor have shown their protective effects in hepatic I/R injury (32). The α subunits are targeted for degradation by prolyl hydroxylase domain enzymes under normoxic conditions. Prolyl hydroxylase domain inhibitor has been shown to activate hypoxia-inducible factor-1α, thereby inhibiting mitochondrial permeability transition and mitochondrial polarization and attenuating hepatic I/R injury (33). These effects would reduce the release of mitochondrial DAMPs in response to cellular injury and contribute to inhibiting the interaction between formyl peptides and FPR1 indirectly.

Unexpectedly, systemic administration of fMLF showed a tendency to reduce hepatic I/R injury, although statistically significant difference was not shown. This phenomenon seems to be explained in connection with a preconditioning stimulus (2). Preconditioning can be induced by various stimuli and is applicable in different organ systems. Recent work has explored the potential use of endogenous danger molecules, such as heat shock protein and high-mobility group box 1, for preconditioning in hepatic I/R (34, 35). Because fMLF has been shown to function as a chemoattractant for neutrophils, the protective effect of fMLF might be through the competitive inhibition of the “find me” signal from the ischemic liver. The mechanism of preconditioning is complex and remains to be elucidated in complex interactions of molecules.

Spleen is one of the abdominal organs connected by the portal system. Hepatic ischemia decreases the blood flow in the spleen and increases vascular resistance; thus, congestion and ischemic injury might occur (21). A recent study using a massive partial hepatectomy model has shown the expression of numerous inflammatory-related genes occurred in the spleen (36). These expressions might increase the trafficking of the immune cells derived from bone marrow in the spleen. TPLSM imaging showed that the number and motility of splenic neutrophils was increased after hepatic I/R, which indicates that the spleen may play a harmful role and provide a negative impact during hepatic I/R as neutrophil supplier. Blockade of the FPR1 would prevent these chain reactions by inhibiting the primary immune response induced by neutrophil recruitment to the ischemic liver. Studies of immune cells in the spleen at the single-cell level may provide further molecular mechanisms and insights that broaden the understanding of hepatic I/R.

Development of fluorescent proteins and transgenic mice has enabled specific cells or molecules to be fluorescently labeled (37). Detailed monitoring of immune cell recruitment using TPLSM will open new doors for investigating the dynamic immune processes during hepatic I/R. It will also be important to analyze the cell–cell interaction in individual leukocyte subsets, platelets, and the endothelium, because these communications are indispensable in orchestration of the immune response in sterile inflammation (38). In addition, time-lapse TPLSM will be useful for identifying and validating new drug targets and evaluating how drugs work over time.

Our study has several limitations. First, motion artifact arising from breathing and the heartbeat could not be completely suppressed during the imaging. Modification of anesthesia management and the fixation procedure might work effectively to decrease the movements for prolonged periods. Second, surgical stress, exteriorization of the liver, and its fixation may cause hepatic microcirculatory disturbance. We made an attempt to minimize these effects and confirmed that the color of the liver had not changed because of compression; however, it is possible that the interventions needed for imaging influenced the behavior of immune cells. Development of novel methods that reduce physiological motion, deeper photon penetration, and easier access to the liver would be required.

In conclusion, this study revealed the essential role of formyl-peptide–FPR1 interaction on neutrophil recruitment to infiltrate into the necrotic area in hepatic I/R. FPR1 blockade using CsH alleviated hepatic I/R injury by inhibiting immune responses after the accumulation of neutrophils. TPLSM is a promising approach in in vivo study of cellular immune mechanisms during hepatic I/R injury.

We thank Dr. T. Graf (Center for Genomic Regulation, Barcelona, Spain) for providing LysM-eGFP mice. We also thank Dr. K. Tanaka and M. Kusunoki (Mie University, Mie, Japan) for the technical advice in microscopy.

This work was supported by the Ministry of Education, Culture, Sports, Sciences and Technology of Japan (Grants KAKENHI 22591410 and KAKENHI 25461954).

The online version of this article contains supplemental material.

Abbreviations used in this article:

ALT

alanine aminotransferase

AST

aspartate aminotransferase

CsH

cyclosporine H

DAMP

damage-associated molecular pattern

FPR

formyl-peptide receptor

I/R

ischemia-reperfusion

KC

keratinocyte chemokine

8-OHdG

8-hydroxy-2′-deoxyguanosine

ROS

reactive oxygen species

TPLSM

two-photon laser-scanning microscopy

TRITC

tetramethylrhodamine isothiocyanate.

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The authors have no financial conflicts of interest.