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Dynamin-related protein 1 deciency impairs mitophagy and accelerates lipopolysaccharide-induced inammation in mice

Mitochondrial fusion and ssion, which are strongly related to normal mitochondrial function, are referred to as mitochondrial dynamics. Mitochondrial fusion defects in the liver cause a non-alcoholic steatohepatitis-like phenotype and liver cancer. However, whether mitochondrial ssion defect directly impair liver function and stimulate liver disease progression, too, is unclear. Dynamin-related protein 1 (DRP1) is a key factor controlling mitochondrial ssion. We hypothesized that DRP1 defects are a causal factor directly involved in liver disease development and stimulate liver disease progression. We administered lipopolysaccharide (LPS) to liver-specic Drp1-knockout (Drp1LiKO) mice. We observed an enhanced inammatory response accompanied by mitophagy impairment. Drp1 defects directly promoted hepatocyte apoptosis and subsequently induced inltration of inammatory macrophages enhanced inammasome activation in the liver and increased pro-inammatory cytokine expression in the liver and serum. Drp1 deletion increased the expression of numerous genes involved in the immune response and DNA damage in Drp1LiKO mouse primary hepatocytes. This is a novel mechanism of liver disease development in which Drp1 defect-induced mitochondrial dynamics dysfunction directly regulates the fate and function of hepatocytes and enhances LPS-induced acute liver injure in vivo.


Introduction
Mitochondria play a critical role in maintaining hepatocyte integrity and function, and mitochondrial dysfunction leads to liver diseases 1 2 3 4 . Mitochondrial function and morphology are interdependent, and the latter is shaped by ongoing mitochondrial fusion and ssion (mitochondrial dynamics) 5 6 7 . In vertebrates, mitofusion-1/2 (MFN1/2) and optic atrophy 1 (OPA1) control mitochondrial fusion, while dynamin-related protein 1 (DRP1) and its receptors control mitochondrial ssion. Mitochondrial dynamics research in recent years has shown the functional importance of mitochondrial dynamics in liver diseases. Kim et al. (2013Kim et al. ( , 2014 reported the impact of viral infection on mitochondrial dynamics, and mitochondrial dynamics alterations are used by hepatitis B virus 8 and hepatitis C virus 9 for maintenance of persistent infection. Overall, mitochondrial dynamics disruption promotes viral pathogenesis. Mitochondrial dynamics and liver diseases are at a crossroads. Studies have reported abnormal mitochondrial dynamics in other liver pathophysiological conditions, too. Cadmium is a long-lived environmental and occupational pollutant, and cadmium hepatotoxicity induces DRP1-dependent mitochondrial fragmentation by disturbing calcium homeostasis 10 . Mitochondrial dynamics are also related to the mechanism underlying acetaminophen-induced acute liver damage. Acetaminophen changes mitochondrial DRP1 levels, and when the DRP1 inhibitor Mdivi-1 inhibits mitochondria ssion, acetaminophen induces greater hepatic impairment 11 . Alcoholic animal models also show mitochondrial morphology alteration. Mitochondria in normal hepatocytes usually show relatively slow dynamics, which are sensitive to inhibition by ethanol exposure 12 . MFN2 ablation in the liver causes endoplasmic reticulum (ER)-mitochondrial phosphatidylserine transfer defects, leading to a non-alcoholic steatohepatitis (NASH)-like phenotype and liver cancer 13 .

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We have reported that a mitochondrial ssion defect in liver-speci c Drp1-knockout (Drp1LiKO) mice, which demonstrate ER stress-promoted broblast growth factor 21 expression, subsequently functions as a metabolic regulator with anti-obesity and anti-diabetes effects. In addition, hematoxylin and eosin (H&E) staining of Drp1LiKO mouse liver sections shows a disorganized lobular parenchyma with in ammatory cell in ltration 14 . Our previous studies have also reported that Drp1 disruption in the liver changes the expression of genes involved in the immune system in the liver. Gene Ontology (GO) biological studies have reported that 7 of the top 10 clusters are related to the immune system. These clusters include terms such as "immune response," "phagocytosis," "antigen processing," and "presentation" 15 .
In this study, we hypothesized that DRP1 de ciency-induced mitochondrial ssion defects directly lead to liver disease development. In addition, we hypothesized that Drp1 defects stimulate liver disease progression. The aim was to determine the relationship between mitochondrial ssion defects and liver disease progression. We tested our hypotheses in an acute liver injury experimental mouse model via lipopolysaccharide (LPS)-induced endotoxin shock. This is a novel mechanism involved in DRP1 defectinduced liver in ammation progression, which will provide insight into the role of DRP1 in liver function and acute liver injury.
Next, we compared the responses to LPS in control and Drp1LiKO mice after LPS injection. Drp1LiKO mice developed more severe symptoms of endotoxin shock, such as lack of activity and hunched back posture. Consistent with this observation, we also observed a signi cant increase in serum TNF levels at 1 h and IL-6, IL-1β, MCP1, and IFN-γ levels at 8 h after LPS injection in Drp1LiKO mice compared to control mice (Fig. 1a). In addition, liver Tnfa, Il6, Il1b, Ifnb1, and Mcp1 mRNA expression levels signi cantly increased in LPS-treated Drp1LiKO mice compared to control mice. An interesting nding was a decrease in Drp1 mRNA levels in control mice, probably through transcriptional inhibition (Fig. 1b). We also detected a signi cant increase in liver TNF, IL-10, IL-1β, MCP1, and IFN-γ levels in LPS-treated Drp1LiKO mice ( Supplementary Fig. 2a).
To evaluate the degree of functional damage in the liver, we further evaluated the serum levels of alanine aminotransferase (ALT) and aspartate transaminase (AST), which are two markers of hepatocellular injure or necrosis. Both alanine aminotransferase (ALT) and aspartate transaminase (AST) signi cantly increased in LPS-treated Drp1LiKO mice (Fig. 1c), indicating that a high LPS concentration induces hepatocyte death in the absence of DRP1. Western blot analysis showed that activation of NLRP3 in ammasome pathways during the LPS-induced in ammatory response in the liver, and NLRP3 in ammasome marker, IL-1β, and NLRP3 levels signi cantly increased in LPS-treated Drp1LiKO mice compared to control mice (Figs. 1d and 1e). Western blot analysis of the liver protein samples showed that LPS induces an in ammatory response via nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, in addition to the NLRP3 in ammasome pathway, while there was no difference in NF-κB and MAPK pathways between control and Drp1LiKO mice (Supplementary Figs. 2b and 2c). In addition, phosphorylation of eukaryotic translation factor 2α (P-eIF2α) levels signi cantly increased in LPS-treated Drp1LiKO mice, indicating an increase in ER stress in Drp1LiKO mice (Supplementary Figs. 2b and 2c), which is consistent with our previous study which reported that DRP1 deletion in the liver causes ER stress via the eIF2α pathway 14 .
Increased apoptosis and in ammasome overactivation in LPS-treated Drp1LiKO mice Macrophages represent a key cellular component of the liver, and are essential for maintaining tissue homeostasis and ensuring rapid response to hepatic injure 16 17 . Considering the observation on enhanced in ammasome pathway in Drp1LiKO mice, we went on to investigate the in ammasome activation in macrophages. To induce a maximum release of IL-1β, these mice were intraperitoneal injected (IP) with 10 mg/kg body weight LPS for 4 hours and received an additional 50 µl of 100 mM ATP 30 minutes before sacri ce. H&E staining con rmed in ammatory cell in ltration in both saline-and LPS/adenosine triphosphate (ATP)-treated livers of Drp1LiKO mice (Fig. 2a). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining showed that apoptotic hepatocytes signi cantly increased in saline-treated (Ctrl 8.67 ± 1.86 vs. KO 47.33 ± 6.17) and LPS/ATP-treated (Ctrl 42.00 ± 6.08 vs. KO 113.33 ± 10.87) livers of Drp1LiKO mice (Figs. 2a and 2b). Consistent with this observation, serum ALT and AST levels also signi cantly increased in LPS/ATP-treated Drp1LiKO mice ( Supplementary  Fig. 3a). Consistent with our previous observations (Fig. 1b), liver Tnfa, Il6, Il1b, F4/80, Mcp1, and Nlrp3 mRNA expression signi cantly increased in Drp1LiKO mice compared to control mice after LPS/ATP treatment ( Supplementary Fig. 3b).
Macrophages, neutrophils, T cells and B cells are cell types known for their role in the process of liver in ammation, to determine the composition of in ammatory cell clusters in Drp1LiKO mice especially which cell types are predominantly expressed in these cell clusters, we performed immunostaining in liver tissues with various immune markers including adhesion G protein-coupled receptor E1 (F4/80) for macrophages, lymphocyte antigen 6 complex, locus G (Ly6G) for neutrophils, CD3 antigen, epsilon polypeptide (CD3) for T cells and protein tyrosine phosphatase, receptor type, C (B220) for B cells ( Fig. 2a and Supplementary Fig. 4a). Cell-counting results are summarized in Fig. 2c and Supplementary   Figs. 4b-d. We found no differences in the number of T-cells ( Supplementary Fig. 4b) and neutrophils ( Supplementary Fig. 4d), but the number of B-cells increased (saline Ctrl 183. 33 (Fig. 2c). Interestingly, immunostaining analysis also revealed co-immunostaining of IL-1β with F4/80-positive macrophages, indicating speci c co-localization of IL-1β-positive cells with macrophages, (Fig. 2d and Supplementary Fig. 4e), that is, high IL-1β expression in macrophages. We also observed a clear line of liver-resident Kupffer cells along the sinusoid wall, which constituted the main macrophage population in the livers of control mice (Fig. 2d, Zoom1). In contrast, macrophages in Drp1LiKO mice showed an abnormal morphology with irregular location and distribution patterns. In addition to main macrophages, we found two other distinct macrophage populations, either derived from in ltrating monocytes or derived from activated Kupffer cells. The majority of these macrophages showed low F4/80 expression but signi cantly high IL-1β expression after LPS/ATP treatment (Fig. 2d, Zoom2 and Zoom3), and only a few macrophages showed high F4/80 expression but no IL-1β expression after LPS/ATP treatment (Fig. 2d, Zoom4).

In ammatory M1 macrophage in ltration in Drp1LiKO mice
It is possible that different populations of hepatic macrophages in control and Drp1LiKO mice exert distinct functions and contribute to differences in the response to an acute LPS challenge. During acute in ammation, M1-polarized macrophages express high IL-1β levels, while IL-1β levels are undetectable in M2-polarized macrophages 18 . To further explore the polarization signature of these macrophages in the liver of control and Drp1LiKO mice, we performed ow cytometry assay and sequential gating analysis using isolated non-parenchymal liver cells without LPS injection. M1 and M2 macrophages were identi ed as CD45 + CD11b + F4/80 + Ly6C High and 7AAD − CD45 + CD11b + F4/80 + CD206 High macrophages, respectively (Figs. 3a and 3b). Sequential gating analysis showed a signi cant increase in Ly6C High M1 macrophages (Ctrl 24.96% ± 1.16% vs. KO 44.10% ± 3.77%) and a signi cant decrease in the CD206 High M2 macrophages in Drp1LiKO mice (Ctrl 49.16% ± 1.07% vs. KO 37.42% ± 2.53%) (Fig. 3c). IL-1β with F4/80 co-immunostaining con rmed that liver-resident Kupffer cells, which lined the sinusoid wall in control mice and macrophages in Drp1LiKO mice, have different location and distribution patterns. Some macrophages with low F4/80 expression showed IL-1β production even without any stimulation in Drp1LiKO mice but not in control mice (Fig. 3d). Therefore, Drp1 loss in hepatocytes induces macrophage activation, and most of these activated macrophages are prone to differentiating into class M1 macrophages.

Decreased autophagy formation in LPS-treated Drp1LiKO mice
Autophagy is reported to be involved in the mitochondrial dynamics and mitochondrial quality control, furthermore, it is reported that autophagosomes form at the mitochondria-ER contact site (MAM) in mammalian cells 19 . In our previous study, we have demonstrated that loss of DRP1 leads to MAM defect, therefore, we investigated whether DRP1 defect alters autophagy formation in LPS treated hepatocytes.

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The autophagy pathways in the LPS-induced in ammatory response were activated, and the autophagy activation marker light chain 3-phospholipid-conjugated (LC3-II) expression signi cantly increased and peaked at 8 h after LPS treatment in control mice, while LC3-II expression signi cantly decreased in Drp1LiKO mice compared to control mice (Figs. 4a and 4b). It is known that the degradation of sequestosome1 (SQSTM1/P62) by the autophagic-lysosome pathway and the de cient autophagy could lead to P62 accumulation at protein levels 20 21 . In addition, western blot analysis showed a signi cant increase in P62 accumulation in Drp1LiKO mice (Figs. 4a and 4c). Mitophagy is the selective pathway degradation of mitochondria by autophagy. phosphatase and tensin homolog-induced putative kinase 1 (PINK1) leads to mitophagy of damaged mitochondria 22 23 24 . In healthy mitochondria, which have a well-maintained mitochondrial membrane potential (ΔΨ m ), PINK1 levels remain low or undetectable. An acute LPS challenge led to PINK1 accumulation in both control and Drp1LiKO mice. There was a greater increase in PINK1 levels in Drp1LiKO mice at 1 and 8 h, which was sustained to 24 h after LPS treatment (Figs. 4a and 4d), indicating that LPS treatment induces mitophagy impairment and that mitophagy impairment is further disturbed by Drp1 defects in Drp1LiKO mice.
Immuno uorescence microscopy showed a signi cant decrease in LC3-positive dots in Drp1LiKO mice compared to control mice after LPS/ATP treatment (Fig. 4e). Of note, localization of LC3-positive cells was different from that of IL-1β-positive macrophages, indicating that autophagy occurs in hepatocytes but not in macrophages. Western blot analysis also con rmed that the LC3-II in hepatocytes signi cantly decreased in Drp1LiKO mice compared to control mice after LPS/ATP treatment (Fig. 4f).
Increased cell death, ER stress, in ammatory response and decreased ΔΨ m in Drp1LiKO mouse primary hepatocytes So far, our results showed that DRP1 disruption in the liver can lead to mitophagy defects and hepatocyte apoptosis, which might trigger enhanced in ltration and polarization of macrophages. To gain further insight into this mechanism, we carried out further experiments with primary hepatocytes from control and Drp1 knockout mice. Isolation and culture of the hepatocytes were performed using a two-step collagenase perfusion method as described previously 25 . When examined and counted under a light microscope using a hemocytometer chamber, the cell yield and viability were less in Drp1LiKO mice (11.43% ± 1.38%) compared to control mice (100.00% ± 12.20%), indicating that DRP1 disruption in the liver impairs hepatocyte survival (Fig. 5a).
ER stress evokes upregulation of the nuclear protein 1 (Nupr1; p8), Tribbles homolog 3 (Trib3), and DNA damage-inducible transcript 3 (Ddit3; Chop) in Drp1LiKO mice 14 , and these genes are related to ER stress and ER stress-mediated cell apoptosis 26 27 28 . Real-time polymerase chain reaction (PCR) analysis con rmed the mRNA expression of ER stress genes signi cantly increased in Drp1LiKO mouse primary hepatocytes (Fig. 5b). Next, to fully reveal the gene expression altered by Drp1 deletion, we conducted an independent microarray analysis with primary hepatocyte harvested 24 hours after seeding. Genes were selected using the criterion of a Z score of ≥ 2, which identi ed 680 up-regulated and 559 down-regulated genes in Drp1LiKO cells. We use the DAVID tool to functionally cluster up-regulated and down-regulated genes by similarly annotated gene ontology (GO) biological process terms, respectively. The top 5 signi cantly enriched annotation clusters of upregulated genes are shown in Fig. 5c. All 5 clusters were related to the immune system: the immune system process, defense response to virus, innate immune response, response to virus, and immune response. Genes involved in the immune system process are shown in Supplementary Table 4. The most signi cant 5 clusters for downregulated genes were related to mitotic nuclear division, cell cycle, cell division, chromosome segregation, and mitotic chromosome condensation. Genes involved in mitotic nuclear division are shown in Supplementary Table 5. These data are consistent with previous studies that reported that DRP1 loss leads to cell arrest, replication stress, centrosome overduplication, and chromosomal instability, subsequently increasing DNA damage and cell apoptosis 29 . Overall, these ndings showed that Drp1 deletion increases the expression of numerous genes involved in the immune response and induces DNA damage in primary hepatocytes.
We further compared the mitochondrial morphology using Mito Tracker Red, a speci c mitochondria uorescence probe. In control mouse primary hepatocytes (Fig. 5d), 57.17% ± 4.00% of the mitochondria were fragmented, with only 14.80% ± 2.35% hepatocytes showing tubular morphology. Drp1LiKO mouse primary hepatocytes containing fragmented mitochondria decreased to 10.50% ± 2.66%, while those containing tubular mitochondria increased to 65.70% ± 3.77%. These results indicated less mitochondrial ssion and more mitochondrial fusion in Drp1LiKO mouse primary hepatocytes compared to control mouse primary hepatocytes.
The mitochondrial respiratory chain generates a membrane potential across the mitochondrial inner membrane as protons are pumped across the inner membrane. ΔΨ m regulates matrix con guration and cytochrome c release during apoptosis 30 . As shown in Fig. 5e, a greater percentage of hepatocytes with low ΔΨ m were found in the Drp1LiKO hepatocyte culture compared to the control mouse primary hepatocyte culture (Ctrl 17.68% ± 1.45% vs. KO 42.46% ± 3.88%).
Decreased mitophagy formation and increased in ammatory response in LPS-treated Drp1LiKO mouse primary hepatocytes OPA1 is a mitochondrial fusion protein that resides in the inner mitochondrial membrane, and OPA1 and LC3 co-localization indicates the sequestration of mitochondria inside autophagosomes (Fig. 6a). We observed that 5.07% ± 0.50% of mitochondria labeled with optic atrophy 1 (OPA1) co-localized with LC3 punctate foci in control mouse primary hepatocytes, indicating that mitophagy occurs in 5.07% ± 0.50% of the total mitochondria in control mouse primary hepatocytes after LPS treatment. In contrast, mitophagy decreased to 0.22% ± 0.10% of the total mitochondria in Drp1LiKO mouse primary hepatocytes (Fig. 6b). In addition, 49.43% ± 1.96% of positive LC3 signals were also positive for OPA1 in control mouse primary hepatocytes, indicating that around half of the total autophagy is mitophagy. The percentage of mitophagy in total autophagy signi cantly decreased to 6.92% ± 2.23% in Drp1LiKO mouse primary hepatocytes (Fig. 6c).
We also detected signi cantly increased TNF, IL-6, and IL-10 levels in Drp1LiKO hepatocyte culture supernatants (Fig. 6d). Consistent with these observations, we found a signi cant increase in Tnfa, Il6, Il1b, and Ifnb1 mRNA expression in Drp1LiKO mouse primary hepatocytes compared to control mouse primary hepatocytes (Fig. 6e).

Discussion
Many studies have reported the abnormal function of mitochondrial dynamics in liver disease. Although mitochondrial fusion defects in the liver cause a NASH-like phenotype and liver cancer, whether mitochondrial ssion defects directly impair liver function and stimulate liver disease progression is unclear. In the present study, DRP1 defects lead to elongated mitochondria with low ΔΨ m , DNA damage, and high expression of ER stress genes P8, Chop, and Trib3, which, in turn, increase hepatocyte apoptosis in Drp1LiKO mice; the dead hepatocytes then release danger signals that lead to M1 in ammatory macrophage in ltration (Fig. 7). ER stress pathway protein-folding and quality control functions maintain cell homeostasis and are closely linked to mechanisms underlying immunity and in ammation 26 31 .
Potent and prolonged ER stress produces excess Trib3 and leads to apoptosis 28 . ER stress-inducible P8 is involved in several physiological and pathological processes 32 . CHOP promotes liver damage during acute liver failure by activating a key mediator to link ER stress and reactive oxygen species (ROS) 33 . Drp1LiKO mice evoke the ER stress pathway, in addition to DNA damage, to promote hepatocyte apoptosis and hepatitis. DRP1 deletion increases the expression of numerous genes involved in the immune response and DNA damage in Drp1LiKO mouse primary hepatocytes. A lack of ssion by DRP1 downregulation leads to loss of mitochondrial DNA and a decrease in ΔΨ m in Hela cells 29 , since loss of ΔΨ m is closely linked to cell apoptosis by various insults, we believe the increased population with low ΔΨ m in Drp1LiKO mouse primary hepatocytes might be an insult and/or a consequence induced by increased cell apoptosis. Furthermore, DRP1 loss in the liver leads to an accelerated in ammatory response induced by LPS. Compared to control mice, Drp1LiKO mice show increased expression of proin ammatory cytokines in the liver and serum, increased in ltration of M1 in ammatory macrophages, enhanced in ammasome activation in liver, increased hepatocyte apoptosis, and decreased mitophagy formation in hepatocytes. Overall, these ndings showed that upon an LPS challenge, mitophagy defects in hepatocytes and NLRP3 in ammasome overactivation in M1 in ammatory macrophages jointly contribute to the elevated in ammatory response in Drp1LiKO mice (Fig. 7). In conclusion, using acute liver injury experimental mouse model via LPS-induced endotoxin shock, our work provides a novel approach to study DRP1 defect-induced liver in ammation progression, which will provide new insight into the role of DRP1 in liver function and acute liver injure.
Mitochondrial dynamics and mitophagy have gained signi cant interest because these events modulate mitochondrial function during many physiological and pathological conditions. In a mouse embryonic broblast model, Drp1 mutant cells showed abnormal mitochondrial morphology and defective mitophagy, leading to the activation of sterile myocardial in ammation, resulting in heart failure 34 . In decreases mitochondrial autophagy and results in the accumulation of damaged mitochondrial material 35 . In a transverse aortic constriction mouse model, DRP1-dependent mitochondrial autophagy plays a protective role against pressure overload-induced mitochondrial dysfunction and heart failure 36 . In addition, Parkin-independent mitophagy requires DRP1 and maintains the integrity of the mammalian heart and brain 37 . Insu cient mitophagy formation induced by DRP1 defects might increase the in ammatory response and enhance the severity of liver injury in response to multiple pathogens.
Hepatitis, refers to an in ammatory condition of the liver, is a serious global public health problem.
Although it is usually caused by a viral infection, there are several other possible risk factors, including: infections, alcohol, toxins, drugs and autoimmune diseases. In this article, we demonstrated that mitochondrial ssion defect caused by the lack of DRP1 lead to the tissue damage associated with hepatitis, suggesting that DRP1 could be a new therapeutic target for in ammatory liver diseases. Taken together, our study identi es mitochondrial ssion defect directly impairs liver function and stimulates liver disease progression. Our study with Drp1LiKO mice disclosed an essential role of mitochondrial ssion in mitochondrial quality control for prevention of hepatic in ammation. This is an exciting discovery which offers a promising alternative approach to developing new therapeutic strategies for in ammatory liver disease.
All mouse procedures and protocols were approved by the Ethics Committees on Animal Experimentation (Kyushu University, Graduate School of Medicine, Japan) and performed in accordance with the Guide for the Care and Use of Laboratory Animals.

Mouse Primary Hepatocyte Isolation And Culture
We performed a rapid two-step method of isolating mouse primary hepatocytes, as previously described 25 . Brie y, we anesthetized the mice with pentobarbital sodium (50 mg/kg body weight) and perfused their liver tissue with prewarmed Hank's balanced salt solution supplemented with 0.5 mM ethylene glycol tetraaceticacid (EGTA) and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) for 5 min, followed by digestion buffer (1000 mg/L of low-glucose Dulbecco's modi ed Eagle's medium [DMEM] supplemented with 0.8 mg/mL of collagenase type 1; Worthington Biochemical Corporation, Lakewood, NJ, USA). Next, we ltered single-cell suspensions through a 70 µm cell strainer (BD Falcon, Bedford, MA, USA) and centrifuged them at 50 × g for 1 min and collected the cell supernatants separately for the preparation of nonparenchymal cells, as described later. We washed the hepatocytes in the pellet twice, suspended them in 4500 mg/L of high-glucose DMEM supplemented with 1 µM insulin, 2 mM L-glutamine, 10 IU/mL of penicillin, 10 IU/mL of streptomycin, and 10% fetal bovine serum, and seeded them on 6-well plastic plates coated with collagen type I (Cellmatrix type I-P, Nitta Gelatin, Osaka, Japan) at a density of 300,000 cells/well. After 4 h incubation in a 5% CO 2 incubator at 37 °C, we changed the culture medium to discard oating unattached cells, and the growth medium was changed daily.

Mouse Hepatic Nonparenchymal Cell Isolation And Flow Cytometry
To further explore the polarization signature of macrophages in the liver of control and Drp1LiKO mice, we performed ow cytometry assay and sequential gating analysis using isolated nonparenchymal hepatocytes without LPS injection. We prepared hepatic nonparenchymal cell containing suspensions from the liver, as described before. The suspensions were centrifuged at 50 × g for 5 min to remove the remaining hepatocytes, and the resulting cell suspensions were pelleted by centrifugation at 800 × g for 5 min and resuspended in phosphate-buffered saline (PBS) supplemented with 5% bovine serum albumin. Next, we stained cells with appropriate antibodies (Supporting Table 1 For real-time-PCR assays, we converted 500 ng of total RNA into rst-strand complementary DNA (cDNA) using the QuantiTect Reverse Transcription Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Next, we used the cDNA for quantitative real-time PCR using the power 2 × SYBR Green PCR Master Mix and monitored the process using an ABI Prism 7500 sequence detection system (Thermo Fisher Scienti c, Rockford, IL, USA). Supporting Table 2 lists the primer sequences of the selected genes. We normalized relative gene expression versus control to Gapdh expression.

Western blot analysis
We homogenized fresh mouse liver tissue in western lysis buffer (20 mM Tris-HCl pH 7.6, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid [EDTA], 0.5% NP40) containing protease and phosphatase inhibitor tablet (Roche Applied Science, Mannheim, Germany) and centrifuged it at 10,000 × g for 5 min at 4 °C. We discarded the pellet and measured the protein content using a bicinchoninic acid (BCA) Protein Assay kit (Thermo Fisher Scienti c). Next, the samples were mixed with Laemmli sample buffer (1:1; Bio-Rad, Hercules, CA, USA) and heated for 5 min at 95 °C. We performed sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on an equal amount of protein from each sample, transblotted it on a polyvinylidene di uoride (PVDF) membrane, and subjected it to immunoblot assay with primary antibodies, followed by horseradish peroxidase (HRP)-linked secondary antibodies. Supporting Table 1 lists the primary and secondary antibodies used. Finally, we visualized protein bands using the electrochemiluminescence (ECL) Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK).
Histology, Immunohistochemistry, And Live-cell Imaging We xed liver tissue and primary hepatocytes by immersion in 4% (w/v) paraformaldehyde (PFA) overnight at 4 °C, embedded the liver tissue in para n according to standard methods, and cut it into 5µm-thick sections. For H&E staining, we stained the para n sections with Mayer's hematoxylin solution for 5 min, followed by counterstaining with 0.5% (v/v) eosin alcohol solution for 2 min. For immunostaining, we diluted primary and secondary antibodies in Can Get Signal™ Immunoreaction Enhancer Solutions A and B, respectively (Toyobo, Osaka, Japan). Supporting Table 1 lists the primary and secondary antibodies used. Next, we analyzed hepatocyte apoptosis using the TUNEL assay kit (Burlington, Boston, MA, USA) according to the standard protocol. The tissue sections were analyzed under a BZ-8000 microscope (Keyence, Osaka, Japan) or a confocal microscope LSM700 (Zeiss, Oberkochen, Germany).
For live-cell imaging, we seeded hepatocytes on a type I-coated 35 mm glass-based dish (Iwaki, Tokyo, Japan) preloaded with MitoTracker® Red, a speci c mitochondria uorescence probe. We further measured ΔΨ m using a uorescence probe tetramethylrhodamine ethyl ester perchlorate (TMRE). Next, we examined the hepatocytes under the confocal microscope LSM700 and analyzed image data using ZEN software (Zeiss, Oberkochen, Germany). A hepatocyte was judged to have fragmented mitochondria if < 25% of the mitochondria visible had a length ve times their width and tubular if > 75% of the mitochondria had a length ve times their width.
Measurement of cytokine levels in the liver, serum, and cell culture supernatants LPS was injected intraperitoneally into mice at 5 mg/kg body weight, and liver and serum samples were collected from the inferior vena cava at different time points (0, 1, 4, 8, 24, and 48 h). The liver tissue was perfused with cold PBS through the portal vein and then chopped into 1-2 mm pieces. Next, we added 100 mg of liver tissue to 1 mL of cell lysis buffer (R&D System, Minneapolis, MN, USA) and homogenized it using a tissue homogenizer. Finally, we diluted liver, serum, and cell culture supernatant samples with known high protein concentrations at 1:50 using an assay diluent and quanti ed the levels of various types of cytokines using the BD Cytometric Bead Array (CBA) Cytokine kit (Becton-Dickinson, Franklin Lakes, NJ, USA) and a NovoCyte ow cytometer (ACEA Biosciences, San Diego, CA, USA) and NovoExpress software.

Biochemical assays
To evaluate the degree of functional damage in the liver, we measured serum ALT and AST levels (two markers of hepatocellular injure or necrosis) using the DRI-CHEM3500 Chemistry Analyzer (Fuji lm, Tokyo, Japan).

Statistical analysis
All data were expressed as the mean ± standard error of the mean (SEM). Two-tailed Student's t-test was performed to compare two groups using Microsoft Excel (Mac 2011 version 14.3.5; Microsoft Japan, Tokyo, Japan). Two-way analysis of variance (ANOVA) with Tukey's post hoc test or ordinary one-way ANOVA was performed to compare multiple groups using GraphPad Prism 6.0 software (GraphPad, San Diego, CA, USA). To detect an outlier, we performed Grubbs' test using a free statistical calculator (QuickCals, http://www.graphpad.com/quickcalcs/). Signi cance levels were set at p < 0.05, p < 0.01, and p < 0.001.

Data Availability
The data that support the ndings of this study are available from the corresponding authors upon reasonable request.