Coxsackievirus B (CVB) is a significant human pathogen that most commonly causes mild self-resolving symptoms such as fever, rash, and upper respiratory illness. In rare cases, the virus can cause a number of severe systemic inflammatory diseases such as meningo-encephalitis and myocarditis. CVB has particularly strong tropism to pancreatic acinar cells and can cause both mild and severe pancreatitis [1
]. Pancreatitis is marked by premature activation of digestive enzymes which causes destruction of the exocrine pancreas. This leads to acute inflammation, further damaging the tissue. Though acute pancreatitis is often mild, about 20% of acute pancreatitis cases manifest as severe pancreatitis which can lead to systemic inflammation and death in up to 30% of patients [3
]. Viral pancreatitis is more prevalent in children; however, adults are also susceptible to acute viral pancreatitis [5
]. CVB infections can result in chronic pancreatitis which is marked by long-term inflammation of the pancreas that progressively worsens over time. Chronic pancreatitis is a premalignant disease and a major risk factor for pancreatic cancer, which has a one-year survival rate of 20% [7
]. A five-year study showed that 34% of patients diagnosed with acute, recurrent, or chronic pancreatitis had elevated titers for coxsackievirus antibodies [8
]. This suggests that coxsackieviral infections may be a more prevalent cause of pancreatitis than previously thought. Treatment options for pancreatitis primarily focus on mitigating acute symptoms, thus effective interventions for potentially life-threatening viral pancreatitis are limited.
We and others have shown that enteroviruses including CVB induce autophagy during infection in order to support infection [9
]. Autophagy is a degradation pathway where cells package damaged or unneeded proteins into autophagosomes, the contents of which are degraded after fusing with acidic lysosomes [12
]. Autophagy is also a mechanism that can degrade faulty mitochondria, where it is referred to as “mitophagy” [13
]. This occurs when mitochondrial inner membrane potential is dissipated, and the organelle becomes depolarized. This is a trigger for selective autophagic degradation wherein mitochondrial fission proteins such as DRP1 and MFF mediate mitochondrial fragmentation [13
]. After faulty mitochondria are segregated from the mitochondrial network, membrane potential continues to decline which stabilizes PINK1 on the outer mitochondrial membrane. This allows for E3 ubiquitin ligases such as parkin to become recruited which ubiquitinate mitochondrial fragments to be trafficked to the autophagosome (herein called the mitophagosome), while healthy mitochondria are rejoined into the mitochondrial network [13
]. Several studies showed that impairing mitochondrial fission or promoting mitochondrial fusion suppresses selective mitophagy [13
]. It has also been shown that CVB and other picornaviruses activate autophagy and become engulfed in autophagosomes [9
]. While some intracellular pathogens are eliminated by autophagy, blocking autophagy suppresses CVB infection [18
]. We recently reported that CVB is engulfed by mitophagosomes which are expelled from cells as virus-laden extracellular vesicles (EVs) [19
]. Indeed, we saw that blocking mitochondrial fission protein DRP1 with either Mdivi-1 or siRNA or impairing mitophagy by silencing the mitophagy adaptor protein optineurin significantly reduced CVB infection. Thus, mitophagy serves as a non-lytic mode of viral dissemination that not only prolongs host cell survival, but also allows nonenveloped CVB to escape the cell in a hijacked membrane that may mask the virus from neutralizing antibodies.
Other viruses have also been shown to rely on mitochondrial fission and mitophagy to further infection. Hepatitis B virus induces fission and mitophagy as a method to impair antiviral apoptosis [20
]. Similar to CVB, the hepatitis C virus relies on fission and mitophagy to promote viral secretion [21
]. Though we have reported that CVB induces mitochondrial fission, which promotes the activation of proviral mitophagy, CVB-induced fission by itself may play a broader role in supporting infection. For example, cytomegalovirus has been shown to induce mitochondrial fission via the viral antiapoptotic protein vMIA [22
]. This induction of fission is thought to impair the association of mitochondrial antiviral signaling protein (MAVS) and the endoplasmic reticulum (ER)-associated antiviral protein stimulator of interferon genes (STING), which ultimately dampens the RIG-I-like receptor antiviral pathway. In all, these studies demonstrate the importance of considering mitochondrial dynamics when developing possible antiviral strategies.
It is unclear what mechanisms are responsible for CVB-mediated mitochondrial fission. We have found that members of the transient receptor potential (TRP) channels may be important for alterations in mitochondrial dynamics following CVB infection. TRPV1, commonly known as the heat and capsaicin-activated ion channel, has been shown to depolarize mitochondria and trigger mitochondrial fission following activation [23
]. Our preliminary findings suggest that CVB relies on TRPV1-mediated mitochondrial fragmentation, and treating cells with the specific TRPV1 inhibitor, SB-366791, potently reduces CVB infection. Inversely related to TRPV1 is TRPM8, which is known to be the primary TRP channel that is activated by hypothermia and the cooling compound menthol. It has been shown that TRPM8 is a TRPV1 antagonist [26
], and we see that subjecting cells to either hypothermia or menthol treatment results in significantly reduced CVB infection. We hypothesize that TRPM8 activation antagonizes TRPV1-mediated proviral mitochondrial fission via inhibition of mitochondrial fission proteins such as DRP1. When we deliver menthol orally to mice that have been infected with CVB, pancreatic viral titers are significantly reduced, and organ inflammation and tissue damage are also blunted. Because menthol is such a common and safe additive to both food and cough medicines, the notion that menthol could have a potent antiviral effect makes it an appealing potential therapeutic for viral diseases.
2. Material and Methods
2.1. Cell Culture and Treatments
HeLa RW human cervical cancer cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Waltham, MA, USA, 11996-073) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA, USA, 16010-159) and antibiotic/antimycotic cocktail (Life Technologies, 15240-062).
SB-366791 (Cayman Chemical, Ann Arbor, MI, USA, 11019) was dissolved in DMSO at a concentration of 10 mM. HeLa cells were treated with 10 µM SB-366791 or equivalent amount DMSO for 24 h prior to infection.
Menthol (Cayman Chemical Company, 25753) was dissolved in ethanol at a concentration of 1 M. HeLa cells were treated with 1 mM menthol or equivalent amount ethanol for one hour prior to infection.
TRPM8 siRNA (Santa Cruz Biotechnology, Dallas, USA, SC-95009) was reconstituted following manufacturer’s protocol. HeLa cells were transfected using Effectene Transfection Reagent (Qiagen, Hilden, Germany, B00118) according to manufacturer’s suggestions for reagent volumes. A total of 72 h following transfection, media was refreshed and cells were immediately infected.
2.2. Generation of Coxsackievirus B3 Expressing Enhanced Green Fluorescent Protein
Recombinant coxsackievirus B3 (pMKS1) expressing enhanced green fluorescent protein (EGFP-CVB) was generated as described previously. CVB clone (pH3) was engineered with SfiI restriction site allowing for DNA insertion. EGFP was amplified from expression plasmids with sequence-specific primers that add flanking SfiI sequences. Amplified products were cloned into linearized pMKS1 to create EGFP-CVB plasmid. HeLa RW cells were transfected with EGFP-CVB construct using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA, 12566014). When cells displayed ~50% cytopathic effect, cells were scraped and subjected to three rounds of freeze/thaw cycles. Freeze fractured cells were then centrifuged at 600 × g for 10 min. Clarified supernatant was collected and considered “passage 1” viral stock. “Passage 2” viral stocks were expanded by infecting new HeLa RW cells with “passage 1” and harvesting as described earlier. “Passage 2” viral stocks were used for all experiments in this study.
2.3. Western Blots
Cellular whole lysates were harvested by applying RIPA buffer containing protease inhibitor cocktail (Roche, Basel, Switzerland, 05056489001). Protein concentrations were measured using a bicinchoninic acid solution (Sigma-Aldrich, St. Louis, MO, USA, B9643). Equal amounts protein were loaded into 4–20% Tris-Glycine SDS PAGE gels (Life Technologies, EC6025) and transferred to nitrocellulose membranes (VWR, 27376-991). Prior to blocking, membranes were stained with Ponceau S (Sigma-Aldrich, P7170). Membranes were blocked in 5% nonfat dry milk dissolved in tris-buffered saline with 0.1% Tween-20 (TBS-T) for one hour at room temperature and then incubated in primary antibody diluted in 5% nonfat dry milk overnight at 4 °C. Primary antibodies used in this study were as follows: enterovirus VP1 (1:142, Dako, Santa Clara, CA, USA, M706401-1), TRPM8 (1:1000, Novus Biologicals, Littleton, CO, USA, NBP1-97311SS), phospho-DRP1Ser616 (1:1000, Cell Signaling Technology, Danvers, MA, USA, 3455), and MAVS (1:200, Santa Cruz Biotechnology, SC-166583). Membranes were then incubated in either anti-mouse secondary (1:3000, SeraCare, Milford, USA, CN, 5450-0011) or anti-rabbit secondary (1:3000, VWR, 95058-734). After applying enhanced chemiluminescence (ECL) (GE Healthcare, Chicago, IL, USA, RPN2235), membranes were imaged using Bio-Rad Chemidoc XRS+ System (Bio-Rad Laboratories, Hercules, CA, USA). Densitometry was performed using ImageJ software (National Institutes of Health).
2.4. Cell Immunostaining
HeLa cells grown on Permanox chamber slides (Thermo Scientific, 177437) were fixed in 4% formaldehyde (Ted Pella, Redding, CA, USA, 18505) diluted in phosphate buffered solution (PBS). Cells were then permeabilized in PBS with 0.25% Triton-X 100 for 10 min, washed in PBS and blocked for one hour in blocking solution (PBS + 0.1% Tween-20 + 1% bovine serum albumin). Cells were then incubated overnight at 4 °C in anti-TOM70 antibody (1:200, Proteintech, Rosemont, IL, USA, 14528-1-AP) diluted in blocking solution. Cells were then washed in PBS and incubated for one hour protected from light in Alexa Fluor 594-conjugated anti-rabbit secondary antibody (1:100, Invitrogen, Carlsbad, CA, USA, A11037) diluted in blocking solution. Cells were then washed, and coverslipped in Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA, H-1400).
2.5. RNA Isolation and Quantitative PCR
RNA isolation was carried out by using TRIsure (Bioline, London, England, BIO-38033) according to the manufacturer’s protocol, which was subsequently reverse transcribed using RevertAid First strand cDNA synthesis kit (ThermoScientific, K1691). qPCR was performed on CFX96 real time PCR system (Qiagen, Germany). Gene expression levels were quantified using ΔΔCt method with 18s rRNA used as normalization control. Following primer pairs were used for the qPCR analysis: IRF3-Forward 5`-AGAGGCTCGTGATGGTCAAG-3`, IRF3-Reverse 5`-AGGTCCACAGTATTCTCCAGG-3`; IRF7-Forward 5`-GCTGGACGTGACCATCATGTA-3`, IRF7-Reverse 5`-GGGCCGTATAGGAACGTGC-3`; 18s rRNA-Forward 5`-GTAACCCGTTGAACCCCATT-3`, 18s rRNA-Reverse 5`-CATCCAATCGGTAGTAGCGC-3`.
2.6. Mouse Treatments
Animal ethics: All mouse work adhered to the National Institutes of Health guidelines and was approved by Cedars-Sinai Medical Center’s Institutional Animal Care and Use Committee (IACUC008697, 11 October 2019–30 September 2022). Prior to sacrifice, animals were first anesthetized with isoflurane and underwent cervical dislocation.
Menthol treatments were prepared by dissolving menthol in water containing 0.2% Tween-80 at a concentration of 25 mg/mL. The 10-week-old male C57BL/6 mice were treated with 100 mg/kg menthol or equivalent volume vehicle via oral gavage. At the same time, mice were infected with 107 plaque forming units of EGFP-CVB with DMEM via intraperitoneal (IP) injection. The following day, mice were treated again with 100 mg/kg menthol or vehicle. Two days post-infection, mice were sacrificed and pancreata were harvested. Tissue was either flash frozen for plaque assay or fixed in 4% formaldehyde for histology.
Cerulein (Bachem, Bubendorf, Switzerland, H-3220) was dissolved in water containing 0.1% ammonium hydroxide at a concentration of 1 mg/mL. This was then diluted 1:100 in sterile saline to make working cerulein solution. The 10-week-old male C57BL/6 mice were first treated with menthol. The following day, mice were given additional dose of menthol and began receiving 50 µg/kg working cerulein or equivalent volume sterile saline via intraperitoneal (IP) injection hourly for 7 h (7 injections total). Animals were then sacrificed 1 h after the final injection. Pancreata were harvested and fixed in 4% formaldehyde for histology.
2.7. Plaque Assays
Frozen pancreatic tissue was weighed and then homogenized in DMEM using a TissueLyzer LT instrument (Qiagen, Hilden, Germany). Homogenates were then clarified by centrifuging at 1000 × g
for 10 min at 4 °C. Plaque assays on pancreatic homogenates were performed as previously described [27
]. Briefly, HeLa cells were grown to confluency in 6 well plates. Media were removed from cells, and 400 µL serially diluted pancreas sample was added on top of cells. After one hour of incubation with occasional rocking, infected cells were overlain with 4 mL 50:50 mixture of 1.2% molten agar combined with 2× DMEM. Plates were then incubated at 37 °C for 48 h and agar plugs were subsequently fixed for 20 min with 2 mL plaque fixative containing 25% acetic acid and 75% methanol. Plugs were removed and fixed cells were stained for one hour with 2.34% crystal violet solution. Cells were then washed and plaques were counted.
Fixed pancreatic tissue was embedded in paraffin and sectioned into 4 µm-thick sections. Tissue sections were then stained with hematoxylin and eosin. Sections were deparaffinized with xylene and rehydrated in decreasing concentrations of ethanol. Sections were stained with Gill 2 Hematoxylin (Richard-Allan Scientific, San Diego, CA, USA, 72504) and Eosin-Y (Richard-Allan Scientific, 71204) according to manufacturer’s protocols. Sections were dehydrated in increasing concentrations of ethanol, cleared in xylene, dried and coverslipped with Cytoseal Mounting Medium (Richard-Allan Scientific, 48212-187).
To date, few reports have established a connection between TRP channels and viral infections, and none have associated TRP channels with coxsackieviral infections. TRP channels have primarily been characterized in infections with respiratory viruses such as rhinovirus, measles, and respiratory syncytial virus which have been shown to upregulate TRPV1 causing hypersensitization to cough and promotion of airborne viral dissemination [36
]. A 2003 study demonstrated that menthol has direct virucidal effects on herpes simplex virus 1 and 2 [37
]. The authors attributed this to possible interference with the viral envelope or disruption of viral attachment to host cells. It is unclear if any of these aspects apply to non-enveloped viruses such as CVB. Because TRPV1 inhibition via SB-366791 significantly limits CVB infection, we hypothesize that TRPV1 stimulators such as heat or capsaicin amplify infection by inducing mitochondrial depolarization which leads to mitochondrial fission (Figure 8
). Not only could this support proviral mitophagy which we described recently, [19
] it could also potentially disrupt mitochondria-based antiviral machinery which was observed in the context of cytomegaloviral infection [22
]. Menthol and cold treatment indirectly inhibits TRPV1 activity by stimulating its antagonist, TRPM8. Indeed, we observed that treating HeLa cells with menthol reduced mitochondrial fission and led to formation of long filamentous mitochondrial networks. It is unclear if TRPM8 alters mitochondrial morphology directly or if it only does this by inhibiting TRPV1. Additionally, it is unknown if CVB infection itself upregulates TRPV1 activity, as is the case with a number of other respiratory viruses
We associate the antiviral effects of menthol with its ability to impair mitochondrial fission. In one of our earlier reports we showed that during CVB infection, the virus localizes to mitochondria, induces DRP1-mediated mitochondrial fission, and causes the autophagic engulfment of virus-laden mitochondria [19
]. These infectious mitophagosomes become released from the infected cell as a nonlytic mode of viral dissemination. We had also observed that these structures may contain proviral microRNA which amplifies downstream infection [38
]. Inhibiting mitochondrial fission by silencing DRP1 or treating with Mdivi-1 led to significant reductions in CVB infection. We hypothesize that a similar effect occurs with menthol treatment. This is evidenced by reduced phosphorylation of DRP1 and increased presence of long tubular mitochondria following menthol treatment. Additionally, CVB-induced fission by itself may play a broader role in supporting infection. As mentioned, cytomegalovirus has been shown to induce mitochondrial fission via the viral antiapoptotic protein vMIA which ultimately disrupts MAVS-based antiviral signaling [22
]. Because MAVS signaling also plays a central part in cellular defense against CVB infections, it is reasonable to believe that CVB-induced mitochondrial fission also impairs antiviral signaling.
Menthol could be an appealing therapeutic when treating CVB infections as it is relatively safe, cost-effective and commonly available. Our data also suggest that oral consumption of menthol could confer potent efficacy in reducing pancreatic infection. It is unclear as yet if oral menthol delivery could provide system-wide antiviral benefit such as in the case of meningitis and myocarditis; however, because the pancreas is a major initial sync for coxsackievirus prior to systemic infection, attenuating viral titers in the pancreas could potentially prevent further viral spread. It has been shown in mice that coxsackieviral myocarditis is preceded by pancreatitis, and if pancreatitis is absent, then infection generally does not progress to myocarditis [1
]. Further testing in other CVB disease models as well as with other viruses would greatly elucidate how treatment with menthol or other TRP channel effectors could be used to attenuate viral infections.