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Obesity Exacerbates Coxsackievirus Infection via Lipid-Induced Mitochondrial Reactive Oxygen Species Generation

Coxsackievirus B3 (CVB3) infection causes acute pancreatitis and myocarditis. However, its pathophysiological mechanism is unclear. Here, we investigated how lipid metabolism is associated with exacerbation of CVB3 pathology using high-fat diet (HFD)-induced obese mice. Mice were intraperitoneally inoculated with 1×106 pfu/mouse of CVB3 after being fed a control or HFD to induce obesity. Mice were treated with mitoquinone (MitoQ) to reduce the level of mitochondrial ROS (mtROS). In obese mice, lipotoxicity of white adipose tissue-induced inflammation caused increased replication of CVB3 and mortality. The coxsackievirus adenovirus receptor increased under obese conditions, facilitating CVB3 replication in vitro. However, lipid-treated cells with receptor-specific inhibitors did not reduce CVB3 replication. In addition, lipid treatment increased mitochondria-derived vesicle formation and the number of multivesicular bodies. Alternatively, we found that inhibition of lipid-induced mtROS decreased viral replication. Notably, HFD-fed mice were more susceptible to CVB3-induced mortality in association with increased levels of CVB3 replication in adipose tissue, which was ameliorated by administration of the mtROS inhibitor, MitoQ. These results suggest that mtROS inhibitors can be used as potential treatments for CVB3 infection.


INTRODUCTION
With the recent surge in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections worldwide, there is an increasing interest in differences in individual responses to viral infections in people with underlying health conditions. In particular, obesity is an important factor causing diabetes and high blood pressure, and viral infection in obese patients dehydrated in ethanol and xylene, then embedded in paraffin. Tissue section were sliced into 5 μm, and stained with H&E. The stained tissues were evaluated to determine the degree of inflammation under light microscope (100×) (Olympus CX41; Olympus, Tokyo, Japan).

Western blot analysis
Total protein lysates from cells or tissues were prepared by sonication with PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology, Seongnam, Korea). Protein levels were determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA). Equivalent amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to membranes. The membranes were subsequently incubated with primary Abs, including anti-coxsackievirus and adenovirus receptor (CAR) Abs (Santa Cruz Biotechnology, Dallas, TX, USA) and anti-β-actin Abs (cat. No. SC-47778; Santa Cruz Biotechnology) for 16 h at 4°C. Thereafter, secondary Abs, including goat anti-rabbit IgG (H+L)-HRP conjugate (Bio-Rad Laboratories, Hercules, CA, USA) and goat anti-mouse IgG F(ab′)2, polyclonal Abs (HRP conjugate) (Enzo Life Sciences, Farmingdale, NY, USA), were added for 1 h at 20°C. Proteins were detected using a femtoLUCENT PLUS-HRP Kit (G-Biosciences, Maryland Heights, MO, USA) and visualized with an ImageQuant LAS 4000 mini system (Cytiva, Marlborough, MA, USA). Chemiluminescence intensity was analyzed using Image J software (NIH, Bethesda, MD, USA).

Surgical lipectomy
Surgical lipectomy was performed as previously reported (19). In surgical removal of epididymal white adipose tissue (eWATx) mice, the test was carefully performed to avoid damage to the testicular blood supply. Both left and right epididymal white adipose tissue (eWAT) fat pads were surgically removed via a mid-abdominal incision. For the sham eWATx group, both left and right eWAT fat pads were pulled; however, they remained intact and were placed back into the abdominal cavity.

Measurement of intracellular ROS
Vero cells were seeded in 24-well cell culture plates at a density of 1.5×10 5 cells/well 1 day prior to the experiment. After 24 h, the remaining medium was removed, and 0.1% BSA only or 50 μM oleic acid (OA) in 0.1% BSA was added. Cells were further incubated for 24 h, washed twice with 1× PBS, and infected with CVB3 for 1 h in DMEM containing 1% heat-inactivated FBS. Cells were washed twice with 1× PBS and stained with 5 μM MitoSOX (Invitrogen) for 30 min at 37°C and 5% CO 2 . Thereafter, cells were washed twice with 1× PBS and fixed with 4% paraformaldehyde. Cells were assessed using FACSVerse instruments (BD Bioscience, San Diego, CA, USA), and the data were analyzed using FlowJo (FlowJo, Ashland, OR, USA).

Transmission electron microscopy (TEM)
Vero cells (5×10 6 cells/well) were seeded in Falcon 100 mm cell culture dishes (Corning). After 24 h, cells were treated with or without 100 μM OA (0.1% BSA) and incubated for 24 h at 37°C in a 5% CO 2 incubator. Cells were infected with CVB3 for 12 h, fixed with 0.1% glutaraldehyde and 2% paraformaldehyde in phosphate buffer (pH 7.4) for 1 h at 4°C, and subsequently post-fixed with osmium tetroxide for 40 min at 4°C. Samples were dehydrated in a graded ethanol series, treated with a graded propylene oxide series, and embedded into Epon (TED Pella Inc., CA, USA). Thereafter, cells were sliced into ultra-thin sections (80 nm) and placed on copper grids. Samples were stained with uranyl acetate and lead citrate and observed using a transmission electron microscope (JEM-2100F; Jeol, Tokyo Japan) at 200 kV (KBSI Chuncheon Center).

Statistical analysis
All results are expressed as means±SEMs and were analyzed using one-way analysis of variance followed by Newman-Keuls multiple comparison test. Differences between groups were assessed using unpaired 2-tailed Student's t-tests. Statistical analyses were performed using GraphPad Prism software version 5 (GraphPad Software, La Jolla, CA, USA). Results with p-values of less than 0.05 were considered statistically significant.

Mice fed a HFD showed increased severity of CVB3 infection
To assess the effects of HFD-induced obesity on CVB3 infection, mice fed a regular diet (RD) or HFD were infected with CVB3. Mice fed the HFD showed a significant reduction in survival after CVB3 infection compared with RD-fed mice, and mice fed an HFD for 4 wk had a lower survival rate than mice fed an HFD for 2 wk following CVB3 infection (Fig. 1A). In addition, the body temperature of HFD-fed mice decreased significantly compared with that of RD-fed mice at 3 days after CVB3 infection (Fig. 1B).
Because CVB3 initially replicates in the pancreas following intraperitoneal infection (20), we further screened several other tissues to assess tissue-specific replication (as determined by negative-strand viral RNA) and viral burden (as determined by positive-strand viral RNA) of CVB3 in mice. The replication of CVB3 facilitated in the pancreas of HFD-fed mice was higher on day 1 following CVB3 infection as compared to that of RD-fed mice. Notably, CVB3 replication was significantly increased in the WAT of HFD-fed mice on day 2 following viral Obesity Aggravates CVB3 Infection via mtROS Production infection. This data suggests that WAT in HFD-fed mice may be important place for CVB3 replication (Fig. 1C). In addition, we confirmed that the CVB3 burden in the pancreas, WAT, liver, and heart was profoundly increased in HFD-fed mice compared with RD-fed mice on day 3 after infection. Collectively, these results suggested that CVB3 replicated in several tissues, including the WAT, under obese conditions.

Obesity increased lipotoxicity and inflammation in the WAT of CVB3-infected mice
In order to further understand the enhanced severity of CVB in HFD-fed mice, we examined CVB3-infected obese mice for diagnostic necropsy. Gross anatomical analysis of abdominal organs in mice suggested the presence of lipotoxicity in the WAT of HFD-fed mice following CVB3 infection ( Fig. 2A). H&E staining of WAT revealed that cell infiltration involved adipose tissue macrophage (ATM)-like cells expressing F4/80 ( Fig. 2B and C). More ATM-like cells infiltrated into eWAT of HFD-fed mice at 3 days after CVB3 infection compared to RD-fed mice. In contrast, we did not observe any marked difference in the pancreas of RD-and HFD-fed mice on day 3 post-infection as both groups showed similar levels of acinar cell  hypochromicity and inflammatory cell infiltration (Supplementary Fig. 1A). Similarly, there were no pathological differences in the livers and hearts of both groups (Supplementary Fig.  1B and C). To assess whether the WAT was critical for CVB3-induced mortality in HFD-fed mice, we surgically removed the eWAT of mice (eWATx mice). The survival of eWATx mice was significantly higher than that of non-eWATx mice following CVB3 infection (Fig. 2D). These data indicated that the increased mortality of CVB3-infected obese mice was associated with severe inflammation in WAT.

OA treatment increased CVB3 replication in vitro
Fatty acids are known to enhance replication of several viruses, such as influenza virus (21) and Singapore grouper iridovirus (22). However, the relationship between fatty acids and CVB3 replication has not yet been reported. To investigate the role of fatty acids in CVB3 replication, we treated Vero cells with OA, the main free fatty acid of the HFD, to induce lipid accumulation in cells prior to CVB3 infection. We measured CVB3 gene expression levels using RT-qPCR to assess the effects of OA accumulation on host cells and CVB3 infection replication. The levels  of CVB3 positive-sense, single-stranded RNA in Vero cells were significantly increased with 100 μM OA (0.1% BSA) treatment for 24 h (Fig. 3A and Supplementary Fig. 2). In addition, OA treatment increased CVB3-mediated cytotoxicity (Fig. 3B), whereas 100 μM OA (0.1% BSA) treatment alone did not induce cytotoxicity in Vero cells at 48 h (Fig. 3C). We also confirmed that OA accumulation in HeLa cells increased CVB3 replication (Fig. 3D). The number of CVB3-GFP puncta representing viral gene expression in Vero cells increased significantly after OA treatment (Fig. 3E). Collectively, these results demonstrated that increased replication of CVB3 was associated with accumulated OA in host cells.

Valsartan treatment did not inhibit CVB3 infection upon OA treatment
The CAR plays important roles in the entry of both coxsackie B viruses and adenoviruses into host cells (23). Because CAR expression is increased in obese adipose tissue (24), we assessed   changes in CAR expression in Hela cells following OA treatment. CAR expression in HeLa cells increased following 100 μM OA (0.1% BSA) treatment (Fig. 4A). Notably, the expression levels of CAR in the heart, pancreas, liver, and WAT increased significantly in HFD-fed mice compared with those in RD-fed mice (Fig. 4B).  Valsartan is a well-known antihypertensive drug that exhibits antiviral activity against CVB3 in association with its effect on downregulating CAR in human endothelial cells (25). Similarly, Vero cells pretreated with 250 or 500 μM of valsartan for 48 h showed significant antiviral activity against CVB3, as demonstrated by reduced CVB3-mediated cytotoxicity (Fig. 4C). However, valsartan did not significantly reduce the enhanced cytotoxicity of CVB3 infected cells with OA accumulation (Fig. 4D). These results suggested that the upregulation of CAR by OA treatment was independent from the increased CVB3 replication in lipid-accumulated host cells.

OA treatment was correlated with RO formation upon CVB3 infection
MDVs are cargo systems that deliver mitochondrial contents, such as membrane components and mtDNA, to other intracellular organelles, including multivesicular bodies (MVBs) or late endosomes (26). We hypothesized that the RO of CVB3 could be derived from MDVs because MDVs can be transferred to MVBs (27,28) and MVB-like structures found during viral infection was similar to ROs (29). Assessment of TEM images of thin sections of OA-treated and untreated Vero cells showed that the number of MVBs was higher in Vero cells treated with OA than in untreated cells (Fig. 5A). Previous reports showed that OA treatment induced mitochondrial dysfunction in human umbilical vein endothelial cells (30). Similarly, OA treatment induced sequential mitochondrial modifications (Fig. 5B) including a decrease in average length (Fig. 5C). However, OA did not alter the number of mitochondria compared with that in untreated cells (Fig. 5D). MDVs can be identified by analysis of the mitochondrial marker translocase of the outer mitochondrial membrane (TOM20) and mitochondrial matrix protein (PDH) (31) and can be discriminated for the mitochondrial markers of PDH + / TOM20 − (matrix) or TOM20 + /PDH − (outer mitochondrial membrane) by selective and high enrichment (32,33). We also confirmed that OA treatment of Vero cells increased MDV formation, as determined by analysis of TOM20 + or PDH + enriched vesicles (Fig. 5E) (34). This finding suggested that OA treatment triggered mitochondrial dysfunction with enhanced MDV formation, which in turn provided a nest for CVB3 replication.

mtROS induced by OA treatment increased CVB3 replication
Previous reports have suggested that free fatty acids, such as palmitic acid and OA, can increase the generation of mtROS in various cells, including endothelial and epithelial cells (35). Thus, we assessed the level of mtROS using MitoSOX Red staining followed by flow cytometry. The level of mtROS increased in Vero cells treated with OA (0.1% BSA) compared with that in untreated cells. This increase was blocked by treatment with 50 μM MitoTEMPO, which inhibits mtROS (Fig. 6A). A previous study suggested that the replication of several viruses, including influenza (13), enterovirus 71 (36), and respiratory syncytial virus (16), is influenced by increased mtROS generation. Thus, we hypothesized that increased CVB3 replication upon OA treatment may be related to increased mtROS production. Notably, the reduced survival of OA-pretreated Vero cells following CVB3 infection was significantly increased by MitoTEMPO treatment (Fig. 6B). Similarly, CVB3 gene expression increased upon OA treatment and decreased significantly with MitoTEMPO treatment (Fig. 6C).
In contrast, CVB3-induced cytotoxicity in OA-pretreated cells did not improve following treatment with NAC, which inhibits cytosolic ROS (Supplementary Fig. 3), suggesting that the increase in mtROS upon OA treatment was crucial for enhanced CVB3 replication in vitro.

MitoQ treatment protected mice from CVB3 infection
MitoQ specifically inhibits mtROS accumulation in vivo (37) and exhibits antiviral activity against respiratory syncytial virus in human alveolar epithelial cells (16). Therefore, we next evaluated the antiviral activity of MitoQ in vivo. Mice were intraperitoneally infected  with CVB3, and MitoQ was administered once every day for 14 days starting on the day of infection. HFD-fed mice in the CVB3 infection group treated with vehicle died within 5 days after infection, whereas more than 50% of mice treated with MitoQ survived until 14 days post-infection (Fig. 7A). In addition, MitoQ treatment ameliorated the infiltration of ATMlike cells in the WAT of HFD-fed mice at 3 days after CVB3 infection ( Fig. 7B and C) and inhibited CVB3 gene expression in WAT. Collectively, these results suggested that increased mtROS induced by HFD feeding may aggravate CVB3 infection in mice and that inhibition of mtROS using MitoQ was attributed to its antiviral effects against CVB3 in obesity.

DISCUSSION
Obesity is a major risk factor for infections, and the incidence rates of several infections have been shown to be increased in obese individuals (38,39). In particular, obese individuals are more susceptible to various viral infections, including influenza A virus, rhinovirus, and severe acute respiratory syndrome coronavirus, compared with nonobese individuals (40). Several studies have shown that obesity impairs immune responses to viral infections (41). For example, in obese mice, influenza virus reduces the levels of antiviral cytokines, such as IFN-α and IFN-β, which are essential for infection control (42). Few studies have addressed cell-intrinsic factors regulating viral infection in association with obesity.  In the current study, we found that obese mice fed an HFD were more susceptible to CVB3 infection and showed increased mortality. Notably, increased mtROS was detected in MDVs of obese-conditioned host cells, and inhibition of mtROS using MitoQ successfully ameliorated HFD-induced CVB3 infection in the WAT and enhanced the survival rates of CVB3-infected obese mice. Collectively, our findings demonstrated that obesity increased the severity of CVB3 infection, which induced lethality in mice. Thus, we proposed that mtROS inhibitors could be adapted to ameliorate CVB3 infection, particularly in the context of obesity.
CVBs can cause both acute and chronic infectious diseases in humans, are the most common causes of infectious myocarditis (43), and are known to cause pancreatitis and aseptic meningitis (5,44). However, CVB3 replication in the WAT of mice has not been reported. In the current study, we found that the WAT was another important replication site for CVB3 and may be associated with increased disease severity in obese mice following CVB3 infection.
For CVB3 replication, the generation of the RO, a specialized membranous complex inside eukaryotic cells, is required for CVB3 replication (45). Enteroviruses trigger the recruitment of lipid droplets to provide lipids required for the development of ROs (46). Viral protein 3A of enteroviruses is a critical viral factor triggering the formation ROs via the interaction of the phosphatidylinositol kinase PI4KB and the sterol transporter OSBP to elicit the shuttling of lipids between lipid droplets and ROs (47). Free fatty acids in cells are stored as lipid droplets and are associated with metabolic diseases, such as obesity and diabetes (48). Obesity can  induce lipid-mediated metabolic or cell signaling malfunction, resulting in increased RO formation, which has advantages for CVB3 replication.
Lipids are important constituents of the plasma membrane and other cellular compartments (49). OA and palmitic acid increase mtROS generation in endothelial cells (35); thus, we confirmed that OA treatment in Vero cells significantly increased mtROS generation. In several studies, infection with many viruses, including hepatitis B virus, hepatitis C virus (HCV), HIV, respiratory syncytial virus, and influenza virus, was found to trigger mtROS production (50). Additionally, ROS produced after viral infection can help fight against viral infections; however, antioxidants exhibit antiviral effects against several viruses by reducing oxidative stress (51). In this study, MitoTEMPO inhibited CVB3 replication in obeseconditioned Vero cells by reducing the level of mtROS. This result implied that lipid-treated conditions exacerbated mtROS production following viral infection and further increased the formation of MDVs, which in turn enhanced viral replication.
MDVs resembles DMVs, which are sites of viral RNA replication for HCV (52) and provide substantial support for viral RNA replication in enteroviruses (53). MDVs are generated during oxidative stress (54) MitoQ and MitoTEMPO were originally designed as mitochondria-targeted antioxidants that block mtROS production (58,59); these reagents have been used in the context of several human diseases and do not induce toxicity (60). Mitochondrial superoxide and ROS production by mitochondrial dysfunction in WAT are associated with obesity, hepatosteatosis, and subsequent complications; accordingly, inhibition of mtROS may be a novel treatment strategy for obesity-associated disorders (37). However, antioxidant drugs differ in the ability to reduce virus replication. NAC inhibits hepatitis B virus replication (61), but does not prevent apoptosis in cytopathic human rhinovirus and bovine viral diarrhea (62,63). Although a previous report showed that CVB3 infection caused the generation of mtROS, NAC treatment, which blocks the generation of cytosolic ROS, did not inhibit CVB3 replication (64). Similarly, we confirmed that NAC did not inhibit CVB3 replication in vitro. NAC may only partially decrease mtROS generation and was not sufficient to diminish the effects of mtROS (65).
CAR is a transmembrane protein of the immunoglobulin superfamily and is involved in cell adhesion, thereby mediating the entry of CVB3 into host cells (66). In the current study, we found that CAR expression increased in cells upon OA treatment and that treatment of cells with valsartan reduced CAR expression. A previous report showed that valsartan-induced CAR downregulation decreased coxsackievirus infection in human endothelial cells (25). However, our data showed that valsartan did not significantly inhibit CVB3 replication in OA-

Obesity Aggravates CVB3 Infection via mtROS Production
treated Vero cells, suggesting that the enhanced susceptibility of CVB3 in OA treatment may not be related to increased CAR expression upon OA treatment.
Although we sought to identify the mechanisms through which lipid accumulation increases viral replication in CVB3 infection, we were unable to determine the direct effects of mtROS through RO formation. Therefore, further studies are needed to establish the specific mechanisms through which accumulated lipids and increased mtROS directly affect viral replication in cells.
OA treatment increases lipid droplet formation in cells (48), and which induces mtROS generation. The formation of MDVs is increased by oxidative stress (67), and it transfers membrane components and mitochondrial contents including mtDNA to MVB (26). Increase of CVB3 replication in ROs was observed in cells with elevated mtROS levels, and inhibition of mtROS suppressed CVB3 replication. These results suggest that lipid-induced mtROS increase viral replication, presumably by increasing MDV formation (Supplementary Fig. 4).
In summary, we found that CVB3 infection increased with obesity. OA treatment aggravated CVB3 infection in vitro because of increased mtROS production. We showed that the treatment of MitoQ inhibited mortality induced by CVB3 infection in HFD-fed mice and suggest that mtROS inhibitors act as potential therapeutic agents against severely increased CVB3 infection caused by obesity. In addition, this study suggests that mitochondrial changes related to fat metabolism are associated with enhanced viral infection and are expected to be applied in the development of new strategies to fight viral infection in the future.
the quantification. RT-PCR amplification were run in a Quantstudio 5 system (Thermo Fisher Scientific, Inc., Waltham, MA, USA) under the following the conditions: 50°C for 30 min at reverse transcription, 95°C at 10 min for initial denaturation then followed by 45 cycles of amplification with denaturation at 95°C for 15 s and annealing and extension at 55°C for 30 s. A standard curve was generated using 10-fold serial dilution (10 7 to 10 3 copy/test of in vitro transcript RNA).