Respiratory virus infection up-regulates TRPV1, TRPA1 and ASICS3 receptors on airway cells

Abstract

Receptors implicated in cough hypersensitivity are transient receptor potential vanilloid 1 (TRPV1), transient receptor potential cation channel, Subfamily A, Member 1 (TRPA1) and acid sensing ion channel receptor 3 (ASIC3). Respiratory viruses, such as respiratory syncytial virus (RSV) and measles virus (MV) may interact directly and/or indirectly with these receptors on sensory nerves and epithelial cells in the airways. We used in vitro models of sensory neurones (SHSY5Y or differentiated IMR-32 cells) and human bronchial epithelium (BEAS-2B cells) as well as primary human bronchial epithelial cells (PBEC) to study the effect of MV and RSV infection on receptor expression. Receptor mRNA and protein levels were examined by qPCR and flow cytometry, respectively, following infection or treatment with UV inactivated virus, virus-induced soluble factors or pelleted virus. Concentrations of a range of cytokines in resultant BEAS-2B and PBEC supernatants were determined by ELISA. Up-regulation of TRPV1, TRPA1 and ASICS3 expression occurred by 12 hours post-infection in each cell type. This was independent of replicating virus, within the same cell, as virus-induced soluble factors alone were sufficient to increase channel expression. IL-8 and IL-6 increased in infected cell supernatants. Antibodies against these factors inhibited TRP receptor up-regulation. Capsazepine treatment inhibited virus induced up-regulation of TRPV1 indicating that these receptors are targets for treating virus-induced cough.

Fig 1. BEAS-2B and SHY5Y5 cells express TRPV1 and ASIC3 and support virus replication.

(A) BEAS-2B and (B) SHSY5Y cells were stained with anti-TRPV1, anti-ASIC3 antibodies or non-immune rabbit serum and DAPI. HEK293T TRPV1 transfected cells were used as positive control for TRPV1 expression. Receptors (green), nuclei (blue). (C) BEAS-2B cells were mock infected or infected with RSV or MV at an MOI of 1 for 48 (D) SHSY5Y cells were mock infected or infected with RSV or MV at an MOI of 1 for 72 hours and stained with anti-viral antibodies and DAPI. RSV (green), MV (red), nuclei (blue). Magnification X 200.

Fig 2. RSV and MV induced TRPV1 and ASIC3 mRNA up-regulation is dependent on virus titre and time post infection.

BEAS-2B cells were infected at MOIs of 0.1, 0.5 and 1 (RSV) and 0.1, 1 and 5 (MV) for 12, 24 and 36 hours. Capsaicin (10μM) was used as positive control for stimulation of TRPV1 and acidified medium (pH 5.9) for stimulation of ASICS3. SHSY5Y were also infected with RSV (MOI 0.1) and MV (MOI 1) for 12 and 24 hours. TRPV1 and ASIC3 mRNA levels were determined by qRT-PCR. (A), (B) (C) and (D) BEAS-2 B cells; (E) and (F) SHSY5Y cells. (A) RSV TRPV1, (B) RSV ASCI3, (C) MV TRPV1, (D) MV ASICS3, (E) RSV and (F) MV. Data are presented as relative fold changes to TRPV1 and ASIC3 mRNA levels in uninfected/untreated control cultures.

Fig 3. TRPV1, TRPA1 and ASICS3 protein expression is up-regulated by virus infection.

(A to D) BEAS-2B cells were infected with RSV (MOI 0.1) and MV (MOI 1). (E and F) SHSY5Y cells were infected with MV (MOI 1). TRPV1, TRPA1 or ASIC3 expression was determined by flow cytometry at 12 and 24 hpi. The levels were quantified by geometric mean fluorescence intensity (GMFI). (A) RSV TRPV1, (B) RSV ASCI3, (C) MV TRPV1, (D) MV TRPA1. The results are representative of 3 independent experiments.

Fig 4. UV inactivated virus preparations up-regulate TRPV1 and ASICS3 mRNA levels.

BEAS-2B cells were infected with RSV (MOI 0.1) or MV (MOI 1), or were treated with equivalent virus preparations that had been UV inactivated. SHSY5Y were also infected with MV or treated with UV-inactivated virus. (A, B, C and D) BEAS-2B cells, (E) and (F) SHSY5Y cells. (A) RSV TRPV1, (B) RSV ASIC3, (C) and (E) MV TRPV1, (D) and (F) MV ASIC3. Data is presented as relative fold change to TRPV1 and ASIC3 mRNA levels relative to uninfected/untreated control cultures.

Fig 5. UV Inactivated virus preparations increase TRPV1 and ASICS3 protein expression in BEAS-2B cells.

BEAS-2B cells were infected with RSV (MOI 0.1) or MV (MOI 1), or were treated with equivalent virus preparations that had been UV inactivated. TRPV1 and ASIC3 expression was determined by flow cytometry at 12 and 24 hpi. Protein expression levels were quantified by geometric mean fluorescence intensity (GMFI) using Flow cytometry. The results are representative of 3 independent experiments.

Fig 6. TRPV1 and ASIC3 mRNA levels are up-regulated by virus-induced soluble factors and UV inactivated pelleted virus.

BEAS-2B cells were infected with RSV (MOI 0.1) and MV (MOI 1) or treated with virus free supernatant, supernatant from mock infected BEAS-2B cells or UV inactivated pelleted virus. TRPV1 and ASIC mRNA levels were quantified by qRT-PCR. (A) RSV; TRPV1, (B) RSV; ASICS3 (C), MV; TRPV1 (D) MV; ASIC3. Data is presented as relative fold change to TRPV1 and ASIC3 mRNA levels in uninfected/untreated control cultures.

Fig 7. Antibodies to TRPV1 and ASIC3 inhibit up-regulation of these receptors by virus infection.

BEAS-2B and SHY5Y5 cells were treated for 1 hour with either anti-TRPV1 or anti-ASIC3 antibodies or with non-immune rabbit serum before infection with MV (MOI 1) or mock infection. Antibodies were left in the culture throughout the experiment. TRPV1 and ASICS3 mRNA levels were determined by qRT-RT. (A) and (B) BEAS-2B cells; (C) and (D) SHSY5Y. (A) and (C) TRPV1, (B) and (D) ASIC3. Fold change was determined relative to uninfected, untreated cells.

Fig 8. RSV and MV infection induce cell surface TLRs in BEAS-2B and SHSY5Y cells.

BEAS-2B and SHSY5Y cells were infected with RSV (MOI 0.1) and MV (MOI 1) or treated with capsaicin (10 μM) for 12 and 24 hours. TLR2, TLR3 and TLR4 levels were determined by qRT-PCR. Data is presented as relative fold change to TLR mRNA levels in uninfected/untreated control cultures. (A) and (B) BEAS-2B cells, (C) and (D) SHSY5Y cells. (A) and (C) RSV, (B) and (D) MV.

Fig 9. RSV and MV infection induce TRPV1 and ASIC3 mRNA up-regulation in PBEC.

PBEC were infected with RSV (MOI 0.1) and MV (MOI 1). TRPV1 and ASIC3 mRNA levels were determined by qRT-PCR at 12 and 24 hpi. (A) RSV), (B) MV. Fold change was determined relative to uninfected/untreated cells.

Fig 10. Neutralisation of IL-6 and IL-8 in virus induced supernatant preparations inhibits increase in expression of TRPA1 in dIMR-32 cells.

(A) Virus free supernatants from BEAS-2B cells and PBECs infected with RSV (MOI 0.1) and MV (MOI 1) for 24 hours were examined using the Human Inflammatory Cytokines & Chemokines Multi-Analyte ELISArray Kit MEH-004A. Optical density measurements were made for triplicate samples. Significance levels in infected samples were determined in comparison to supernatants from mock infected controls. Virus free supernatants were treated with 0.1, 1 or 5μg /ml of (B) anti- IL-6 or (C) anti-IL-8 or with 5 μg/ml of isotype control antibody. The results are representative of 3 independent experiments.

Fig 11. Capazepine treatment of RSV infected BEAS-2B cells inhibits up-regulation of TRPV1.

BEAS-2B cells were infected with RSV (MOI 0.1) and treated with 10, 20 or 100 uM capazepine or buffer. TRPV1 RNA levels were determined by qRT-PCR at 12 and 24 hpi.