Cetylpyridinium chloride (CPC) reduces zebrafish mortality from influenza infection: Super-resolution microscopy reveals CPC interference with multiple protein interactions with phosphatidylinositol 4,5-bisphosphate in immune function

Abstract

The COVID-19 pandemic raises significance for a potential influenza therapeutic compound, cetylpyridinium chloride (CPC), which has been extensively used in personal care products as a positively-charged quaternary ammonium antibacterial agent. CPC is currently in clinical trials to assess its effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity. Two published studies have provided mouse and human data indicating that CPC may alleviate influenza infection, and here we show that CPC (0.1 μM, 1 h) reduces zebrafish mortality and viral load following influenza infection. However, CPC mechanisms of action upon viral-host cell interaction are currently unknown. We have utilized super-resolution fluorescence photoactivation localization microscopy to probe the mode of CPC action. Reduction in density of influenza viral protein hemagglutinin (HA) clusters is known to reduce influenza infectivity: here, we show that CPC (at non-cytotoxic doses, 5-10 μM) reduces HA density and number of HA molecules per cluster within the plasma membrane of NIH-3T3 mouse fibroblasts. HA is known to colocalize with the negatively-charged mammalian lipid phosphatidylinositol 4,5-bisphosphate (PIP₂); here, we show that nanoscale co-localization of HA with the PIP₂-binding Pleckstrin homology (PH) reporter in the plasma membrane is diminished by CPC. CPC also dramatically displaces the PIP₂-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) from the plasma membrane of rat RBL-2H3 mast cells; this disruption of PIP₂ is correlated with inhibition of mast cell degranulation. Together, these findings offer a PIP₂-focused mechanism underlying CPC disruption of influenza and suggest potential pharmacological use of this drug as an influenza therapeutic to reduce global deaths from viral disease.

Keywords: Cetylpyridinium chloride; Influenza; Phosphatidylinositol 4,5-bisphosphate; Quaternary ammonium compound; Super-resolution microscopy; Zebrafish.

Fig. 1

Zebrafish influenza infection and CPC exposure workflow. Zebrafish embryos were collected 15 min post-fertilization and grown at 33 °C. At 48 h post fertilization (hpf), embryos were dechorionated (spontaneously or manually) and injected with IAV. CPC treatment of 0.1 μM in egg water was administered to the embryos at 6, 24, or 48 h post infection (hpi) for a duration of ~60 min before being washed two times to remove remaining traces of CPC from the egg water. Mortality and viral load were assessed every 24 h for five days post-infection (dpi).

Fig. 2

CPC reduces the PM/Cytosolic ratio of PIP₂-binding protein MARCKS in RBL-2H3 cells and reduces the mean density of the PIP₂-binding protein PAmKate-PH in PM of NIH-3T3 cells. (A) Representative live-cell confocal microscopy images of RBL-2H3 cells expressing mRFP-MARCKS-ED, ± CPC exposure: Control (0 μM CPC), 5 μM CPC, or 10 μM CPC for 30 min. After washing off the CPC with BT, confocal images were taken. (B) The ratio of the mean fluorescence per pixel of MARCKS at the PM and at the cytosol. Values represent mean ± SEM of three independent experiments that were derived from analysis of n = 143 cells for Control (0 μM CPC), n = 90 cells for 5 μM CPC, and n = 65 cells for 10 μM CPC. (C) Quantification of PH levels at the PM using super-resolution microscopy. NIH-3T3 cells expressing PAmKate-PH, ± CPC exposure for one hour, then fixed by 4% PFA. FPALM imaging of the PM of the transfected cells was carried out with TIRF excitation. The mean density of PH molecules was plotted as a function of CPC treatment. Values represent mean ± SEM of three independent experiments from analysis of n = 32 cells for BT Control (0 μM CPC), n = 30 cells for 5 μM CPC, and n = 31 cells for 10 μM CPC. Statistically significant results, as compared to Control (0 μM CPC), are represented by *p < 0.05 and **p < 0.01, as determined by one-way ANOVA followed by Dunnett's post-test.

Fig. 3

Non-cytotoxic doses of CPC inhibit RBL-2H3 mast cell degranulation. (A) RBL-2H3 mast cells were sensitized with IgE and stimulated to degranulate with 0.0004 μg/mL Ag for 1 h, ± CPC in BT; the spontaneous (“spont.”) response was determined by cells which were not exposed to Ag or CPC. Normalized degranulation response is plotted against CPC exposure concentrations. Effects of CPC on RBL-2H3 cell survivability were assessed by LDH assay (B) and by trypan blue-exclusion (C). Values presented are means ± SEM of three independent experiments; three replicates were used in each experiment. Statistical significance, compared to the Control (0 μM CPC), is represented by **p < 0.01, ***p < 0.001 and determined by one-way ANOVA followed by Tukey's post-test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

CPC alters HA cluster properties in NIH-3T3 cells. Two-color TIRF FPALM was used to obtain images from fixed NIH-3T3 cells co-expressing HA-Dendra2 and PAmKate-PH constructs. NIH-3T3 cells were exposed to Control (0 μM CPC), 5 μM, and 10 μM CPC for 1 h at 37 °C, then were fixed with 4% PFA. HA cluster properties (A) Mean density of an HA cluster (B) Mean number of HA molecules forming an HA cluster (C) Mean Area of an HA cluster (D) and Mean Perimeter of an HA cluster were quantified using SLCA. (E) The mean density of HA molecules in each cell was quantified by taking the ratio of the total number of HA molecules (HA localizations) over the cell area. Values represent mean ± SEM of three independent experiments from analysis of n = 24 cells for BT Control (0 μM CPC), n = 20 cells for 5 μM CPC, and n = 22 cells for 10 μM CPC. Statistically significant results are represented by *p < 0.05, as compared to Control by one-way ANOVA followed by Dunnett's multiple comparison test against the Control.

Fig. 5

CPC affects PIP₂-binding protein cluster properties in NIH-3T3 cells. Two-color TIRF FPALM was used to obtain images from fixed NIH-3T3 cells co-expressing HA-Dendra2 and PAmKate-PH. NIH-3T3 cells were exposed to Control (0 μM CPC), 5 μM, and 10 μM CPC for 1 h at 37 °C, then were fixed with 4% PFA. PH domain cluster properties (A) Mean density of a PH domain cluster, (B) Mean number of PH domain molecules forming a cluster, (C) Mean Area of a PH domain cluster, (D) Mean perimeter of a PH domain cluster for Control, 5 μM, and 10 μM CPC-treated cells were quantified using SLCA. Values represent mean ± SEM of three independent experiments from analysis of n = 25 cells for BT Control (0 μM CPC), n = 26 cells for 5 μM CPC, and n = 29 cells for 10 μM CPC. Statistically significant results are represented by **p < 0.01, as compared to the Control by one-way ANOVA followed by Dunnett's multiple comparison test against the Control.

Fig. 6

CPC disrupts nanoscale colocalization of HA-Dendra2 and PAmKate-PH imaged in fixed NIH-3T3 cells. Two-color FPALM images of fixed NIH-3T3 cells expressing HA-Dendra2 (green) and PAmKate-PH (magenta) of (A) Control and (B) 10 μM CPC-treated cells. Arrows (red with yellow outline) point to areas of colocalization (white). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

CPC alters co-clustering of HA-Dendra2 and PAmKate-PH in NIH-3T3 cells. Co-clustering was quantified from the distribution of Dendra2-HA and PAmKate-PH molecules localized within a grid of 35 nm × 35 nm pixels, after bleed-through correction, in fixed NIH-3T3 cells. (A) Pixels identified as HA-Dendra2 clusters (containing at least 5 HA localizations) were selected. The number of PAmKate-PH localizations was then summed over all pixels within the selection, and then averaged over all cells, and is plotted as “mean pixel sum” (see Methods) as a function of CPC treatment (10 μM). (B) Pixels identified as PAmKate-PH clusters (at least 5 PH domain localizations) were selected. The number of HA-Dendra2 localizations was then summed over all pixels within that selection and then averaged over all cells and is plotted as “mean pixel sum” as a function of CPC treatment (10 μM). Values represent mean ± SEM of three independent experiments from analysis of n = 31 cells for BT Control (0 μM CPC) and n = 30 cells for 10 μM CPC. Statistically significant differences are represented by *p < 0.05 as compared to Control, determined by unpaired t-test.

Fig. 8

Cytotoxic doses of CPC for the extensive evaluation of zebrafish embryo survival. Zebrafish embryos injected with HBSS and exposed to various doses of CPC were monitored for cumulative survival over the course of 5 days (no observable death occurred after 3 days). Survival curve values presented are for a total of 204 zebrafish, assayed via four independent experiments. Statistically significant differences are represented by ****p < 0.0001, determined by the Mantel-Cox test, the Logrank test, and the Gehan-Breslow-Wilcoxon test of survival curves in GraphPad Prism.

Fig. 9

CPC increases zebrafish embryo survival rate following influenza infection. Zebrafish embryos (28 hpf) were injected with 1.0 nL (~ 8.7 × 10² EID50) of APR8 IAV into the duct of Cuvier, for all groups in this figure. Survival curves of IAV-infected zebrafish, ± 0.1 μM CPC treatment for one hour at 6 hpi (A), 24 hpi (B), or 48 hpi (C). Values presented are for a total of ~150 zebrafish, assayed in three independent experiments. Statistically significant differences are represented by ***p < 0.001, ****p < 0.0001, determined by the Mantel-Cox test, the Logrank test, and the Gehan-Breslow-Wilcoxon test of survival curves in GraphPad Prism.

Fig. 10

CPC treatment reduces viral load in AB zebrafish embryos. Zebrafish embryos (2 dpf) were injected with IAV as in Fig. 9 (duct of Cuvier, 1.0 nL, ~ 8.7 × 10² EID50 of APR8 IAV). TCID₅₀ comparison between groups with (green) and without (blue) 0.1 μM CPC administered for one hour at 6 hpi, in three replicate experiments. Statistical analysis of the difference between CPC-treated and untreated yielded p < 0.0005 using a two-way ANOVA. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)