Antiviral Effects of Menthol on Coxsackievirus B

Figures

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  Figure 1. Temperature significantly alters CVB infection- HeLa human cervical cancer cells were kept at either 37 °C, 39 °C or 25 °C for 24 h. Cells were then all incubated at 37 °C for 30 min and infected with enhanced green fluorescent protein coxsackievirus B (EGFP-CVB) at multiplicity of infection of 1 (MOI 1) for 6 h. (A) Fluorescence microscopy compares viral EGFP expression between cells kept at either 37 °C or 39 °C. Phase contrast images are shown to the right of respective fluorescence image fields. Scale bars represent 150 µm. (B) Western blot on cell lysates from A detecting VP1 viral capsid protein. Ponceau S is shown below. (C) Densitometric quantification of western blot in B. (** p < 0.01; Student’s t-test, n = 3). (D) Fluorescence microscopy comparing viral EGFP expression between cells kept at either 37°C or 25 °C. Phase contrast images of same field are shown to the right. Scale bars represent 150 µm. (E) Western blot on cell lysates from D detecting VP1 viral capsid protein. Ponceau S is shown below. (F) Densitometric quantification of western blot in E. (** p < 0.01; Student’s t-test, n = 3).

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  Figure 2. Treatment with TRPV1 inhibitor SB-366791 attenuates CVB infection- HeLa cells were treated with 10 µM of TRPV1 inhibitor SB-366791 or equivalent volume DMSO for 24 h and subsequently infected with EGFP-CVB at MOI 0.1 for 24 h. Fluorescence microscopy compares viral EGFP expression between cells treated with (A) DMSO or (B) SB-366791. Phase contrast images are shown below respective fluorescence image fields. Scale bars represent 150 µm. (C) Western blot on cell lysates from A and B detecting VP1 viral capsid protein. Ponceau S is shown below. (D) Densitometric quantification of western blot in C. (** p < 0.01; Student’s t-test, n = 3). (E) Plaque assay quantification of infectious virus in media from cells in A and B. (** p < 0.01; Student’s t-test, n = 3).

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  Figure 3. Treatment with TRPM8 agonist menthol impairs CVB infection- HeLa cells were treated with 1 mM menthol or equivalent volume ethanol for 1 h and subsequently infected with EGFP-CVB at MOI 0.1 for 24 h. Fluorescence microscopy compares viral EGFP expression between cells treated with (A) ethanol or (B) menthol. Phase contrast images are shown below respective fluorescence image fields. Scale bars represent 150 µm. (C) Western blot on cell lysates from A and B detecting VP1 viral capsid protein. Ponceau S is shown below. (D) Densitometric quantification of western blot in C. (** p < 0.01; Student’s t-test, n = 3). (E) Plaque assay quantification of infectious virus in media from cells in A and B. (* p < 0.05; Student’s t-test, n = 3).

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  Figure 4. Silencing TRPM8 bolsters CVB infection- HeLa cells were transfected with siRNA targeting TRPM8 (siTRPM8) or scrambled RNA (siSCRAMBLE) for 72 h and subsequently infected with EGFP-CVB at MOI 1 for 6 h. (A) Fluorescence microscopy comparing viral EGFP expression between cells transfected with either siSCRAMBLE or siTRPM8. Phase contrast images of same fluorescence image fields are shown to the right. Scale bars represent 150 µm. (B) Western blot on cell lysates from A detecting TRPM8 and VP1 viral capsid protein. Ponceau S is shown below. (C) Densitometric quantification of TRPM8 western blot in B. (* p < 0.05; Student’s t-test, n = 3). (D) Densitometric quantification of VP1 western blot in B. (** p < 0.01; Student’s t-test, n = 3).

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  Figure 5. Menthol treatment reduces mitochondrial fission basally and during CVB infection- HeLa cells were treated with 1 mM menthol and then examined after 6 h. (A) Western blot on cell lysates detecting mitochondrial fission marker phospho-DRP1Ser616 (pDRP1). Ponceau S is shown below. (B) Densitometric quantification of western blot in A. HeLa cells were treated with 1 mM menthol or equivalent dose of ethanol for 1 h and subsequently infected with EGFP-CVB at MOI 0.1 for 24 h. (C) Western blot on cell lysates detecting pDRP1. Ponceau S is shown below. (D) Densitometric quantification of western blot in C. (* p < 0.05, *** p < 0.001; Student’s t-test, n = 3–4).

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  Figure 6. Menthol enhances antiviral immunity during infection- HeLa cells were treated with 1 mM menthol or equivalent dose ethanol for 1 h and subsequently infected with EGFP-CVB at MOI 0.1 for 24 h. (A) Western blot on cell lysates detecting mitochondrial antiviral signaling (MAVS). Ponceau S is shown below. (B) Densitometric quantification of western blot in A. (** p < 0.01; Student’s t-test, n = 3). mRNA was extracted from these cells and quantitative PCR was performed to detect (C) IRF3 or (D) IRF7. (* p < 0.05, ** p < 0.01; Student’s t-test, n = 3).

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  Figure 7. Oral menthol treatment reduces pancreatic CVB titers and tissue destruction in CVB-infected mice- The 10-week-old C57BL/6 male mice were treated with 100 mg/kg menthol or equivalent volume vehicle each day for 2 days via oral gavage. At the same time as the first menthol dose, mice were infected IP with 107 plaque forming units (PFU) EGFP-CVB. A total of 2 days post-infection (PI), mice were sacrificed, and pancreata were assessed. (A) Pancreatic viral titers as measured by plaque assays on pancreatic homogenates. (** p < 0.01; Student’s t-test, n = 6–7). (B) Hematoxylin and eosin staining on representative pancreas sections. Scale bars represent 150 µm.

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  Figure 8. Hypothetical model of TRP channels and CVB infection- CVB has previously been shown to rely on mitochondrial fission and mitophagy to promote infection. Activity of the capsaicin and heat receptor TRPV1 appears to be important for CVB infection, and this may be due to its ability to cause mitochondrial depolarization and subsequent mitochondrial fission. It is unclear if CVB itself upregulates TRPV1 activity. The cold and menthol receptor TRPM8 antagonize TRPV1, thus menthol treatment indirectly suppresses TRPV1-mediated mitochondrial fission and blunts CVB infection.