Antiviral innate immune response in non-myeloid cells is augmented by chloride ions via an increase in intracellular hypochlorous acid levels

Abstract

Phagocytes destroy ingested microbes by producing hypochlorous acid (HOCl) from chloride ions (Cl-) and hydrogen peroxide within phagolysosomes, using the enzyme myeloperoxidase. HOCl, the active ingredient in bleach, has antibacterial/antiviral properties. As myeloperoxidase is needed for HOCl production, non-myeloid cells are considered incapable of producing HOCl. Here, we show that epithelial, fibroblast and hepatic cells have enhanced antiviral activity in the presence of increasing concentrations of sodium chloride (NaCl). Replication of enveloped/non-enveloped, DNA (herpes simplex virus-1, murine gammaherpesvirus 68) and RNA (respiratory syncytial virus, influenza A virus, human coronavirus 229E, coxsackievirus B3) viruses are inhibited in a dose-dependent manner. Whilst treatment with sodium channel inhibitors did not prevent NaCl-mediated virus inhibition, a chloride channel inhibitor reversed inhibition by NaCl, suggesting intracellular chloride is required for antiviral activity. Inhibition is also reversed in the presence of 4-aminobenzoic hydrazide, a myeloperoxidase inhibitor, suggesting epithelial cells have a peroxidase to convert Cl- to HOCl. A significant increase in intracellular HOCl production is seen early in infection. These data suggest that non-myeloid cells possess an innate antiviral mechanism dependent on the availability of Cl- to produce HOCl. Antiviral activity against a broad range of viral infections can be augmented by increasing availability of NaCl.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Dose dependent inhibition of HSV-1 by sodium chloride: (a) Time course analysis of HSV-1 in the presence of NaCl: HeLa cells were infected with HSV-1-GFP (MOI 0.5) for 1 hour before the inoculum was removed and replaced with increasing concentrations of NaCl in medium (in triplicate). NaCl (mM) values are over and above that found in DMEM (110 mM). Virus replication was monitored as a function of GFP fluorescence over time. (b) Viability of HeLa cells is not significantly impaired in the presence of NaCl. HeLa cells were treated with increasing concentrations of NaCl (in triplicate). 24 and 48 hours post-treatment cellular viability was determined with CellTiter-Blue (Promega) by the ability of cells to metabolise the substrate to produce a fluorescent end-product. Cell viability was normalised to untreated cells (0 mM NaCl). Viability below 70% is evidence of cytotoxicity. Error bars indicate the standard error of the mean of 3 independent experiments carried out in triplicate.

Figure 2

Confirmation of inhibition of HSV-1 by plaque forming assay: (a) HSV-1 virion release is inhibited by NaCl: HeLa cells were infected with HSV-1 (MOI 0.5) for 1 hour before inoculum was removed and replaced with increasing concentrations of NaCl. Supernatant was harvested after 48 hours and viral titer quantified by plaque assay on Vero cell monolayers, in the absence of NaCl, as plaque forming units (PFU) per ml. Error bars represent the standard error of the mean of three experiments carried out in duplicates. *p < 0.05 when compared to 0 mM NaCl (b) A representative image of NaCl inhibition of HSV-1 by plaque forming assay. NaCl (mM) values are over and above that found in DMEM (110 mM).

Figure 3

DNA and RNA viruses are inhibited by sodium chloride: Dose-dependent inhibition of (a) HSV-1, (b) RSV, (c) MHV68, (d) HCoV-229E, (e) CV-B3 and (f) IAV by NaCl. Cells were infected with virus for 1 hour before virus was replaced with media containing increasing concentrations of NaCl. Viral replication was quantified by measuring eGFP fluorescence intensity over multiple rounds of replication and determining the slopes of the growth curves in time course analyses, with the exception of IAV, which was quantified by qRT-PCR for the viral nucleoprotein (NP). With the exception of IAV, which represents one experiment of duplicates, all error bars represent the standard error of the mean of at least two independent experiments carried out in triplicate. *p < 0.05; **p < 0.01 and ***p < 0.001 when compared to 0 mM NaCl. NaCl (mM) values are over and above that found in DMEM (110 mM).

Figure 4

Viability of different non-myeloid cells in the presence of sodium chloride: Viability of A549 (a), 3T3 (b), HuH-7.5 (c), and HuH-7 (d) cells were not significantly impaired in the presence of NaCl. Cells were treated with increasing concentrations of NaCl (in triplicate). 48 hours post-treatment cellular viability was determined with CellTiter-Blue (Promega) by the ability of cells to metabolise the substrate to produce a fluorescent end-product. Cell viability was normalised to untreated cells (0 mM NaCl). Viability below 70% is evidence of cytotoxicity. While A549 and 3T3 cells were viable up to 100 mM NaCl, HuH-7.5 and HuH-7 cells were viable only up to 50 mM NaCl. Error bars represent the standard error of the mean of 3 independent experiments carried out in triplicate. NaCl (mM) values are over and above that found in DMEM (110 mM).

Figure 5

Sodium chloride inhibits HSV-1 infection of HeLa cells at a stage post-entry: (a) eGFP HSV-1 was pre-incubated with increasing concentrations of NaCl for 0, 1 or 2 hours before adsorption to HeLa cells (MOI 0.5). After adsorption, inoculum was replaced with media. Virus replication was monitored as a function of fluorescence over time. Error bars represent the standard error of the mean of three biological replicates. *p < 0.05 compared to 0 hours at the corresponding concentration of NaCl (b) HeLa cells were infected with eGFP HSV-1 (MOI 0.5) and treated with increasing concentrations of NaCl at different stages of infection: Adsorption (present during the 1 hour of adsorption alone), Post-infection (NaCl added following removal of inoculum), or Adsorption/Post-Infection (NaCl present both during adsorption and following removal of inoculum). Error bars represent the standard error of the mean of three independent experiments carried out in triplicate. ***p < 0.001 when compared to 0 mM NaCl. NaCl (mM) values are over and above that found in DMEM (110 mM).

Figure 6

Inhibition of Cl− but not Na+ transport restores HSV-1 replication in the presence of NaCl: HeLa cells were treated for 24 hours with increasing concentrations of ralfinamide (a,b) a voltage gated sodium channel blocker; or benzyl amiloride (c,d) an epithelial sodium channel blocker or (e,f) 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB) a chloride channel blocker and infected with HSV-1-GFP at a MOI of 0.5. After 1 hour the inoculum was removed and replaced with increasing concentrations of inhibitor and either 0 mM (blue) or 50 mM NaCl (grey). Virus replication was monitored as a function of GFP fluorescence over time and normalised to the no salt control for each channel blocker concentration. Error bars represent the standard error of the mean of multiple replicates. (b,d,f) HeLa cells were treated with increasing concentrations of ralfinamide (b), benzyl amiloride (d) or NPPB (f) with 0 mM or 50 mM NaCl. After 24 and 48 hours, cell viability was determined and normalised to untreated cells (0 mM NaCl, 0 mM inhibitor). Error bars represent the standard error of the mean of three experiments carried out in triplicate. **p < 0.01 when compared to 0 μM NPPB. NaCl (mM) values are over and above that found in DMEM (110 mM).

Figure 7

HSV-1 is inhibited in the presence of NaCl by increased production of intracellular hypochlorous acid: HeLa cells were infected with non-GFP labelled HSV-1 at MOI 0.5 for 1 hour before treating with increasing concentrations of NaCl. After 6 hours, cells were washed and stained with 10 μM HCSe (a BODIPY-based green fluorescent probe which is rapidly and specifically oxidized by HOCl to emit a fluorescence signal) or R-19S (a rhodamine fluorophore) for 30 minutes. Fluorescence was measured after cells were washed twice with PBS. Fluorescence values were normalized to uninfected cells treated with the corresponding NaCl concentration. NaCl (mM) values are over and above that found in DMEM (110 mM). Error bars represent the standard error of the mean of three biological replicates. *p 

Figure 8

Inhibition of myeloperoxidase restores HSV-1 replication in the presence of NaCl: (a) Production of HOCl requires a functional peroxidase enzyme. HeLa cells were treated for 24 hours with increasing concentrations of the myeloperoxidase (MPO) inhibitor 4-Aminobenzoic hydrazide (4ABAH) before infecting with HSV-1-GFP at MOI 0.5. Following 1-hour incubation, inoculum was removed and replaced with increasing concentrations of inhibitor and either 0 or 50 mM NaCl. Error bars represent the standard error of the mean of three experiments carried out in triplicate. (b) Effect of MPO inhibitor on cell viability was determined by treating HeLa cells with increasing concentrations of 4-Aminobenzoic hydrazide with either 0 mM or 50 mM NaCl. After 24 and 48 hours viability was determined and normalized to untreated cells (0 mM NaCl). Error bars represent the standard error of the mean of three experiments carried out in triplicate. **p < 0.01 when compared to 0 μM 4ABAH. NaCl (mM) values are over and above that found in DMEM (110 mM).