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.

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.

Triclosan disrupts immune cell function by depressing Ca2+ influx following acidification of the cytoplasm.

Triclosan (TCS) is an antimicrobial agent that was effectively banned by the FDA from hand soaps in 2016, hospital soaps in 2017, and hand sanitizers in 2019; however, TCS can still be found in a few products. At consumer-relevant, non-cytotoxic doses, TCS inhibits the functions of both mitochondria and mast cells, a ubiquitous cell type. Via the store-operated Ca2+ entry mechanism utilized by many immune cells, mast cells undergo antigen-stimulated Ca2+ influx into the cytosol, for proper function. Previous work showed that TCS inhibits Ca2+ dynamics in mast cells, and here we show that TCS also inhibits Ca2+ mobilization in human Jurkat T cells. However, the biochemical mechanism behind the Ca2+ dampening has yet to be elucidated. Three-dimensional super-resolution microscopy reveals that TCS induces mitochondrial swelling, in line with and extending the previous finding of TCS inhibition of mitochondrial membrane potential via its proton ionophoric activity. Inhibition of plasma membrane potential (PMP) by the canonical depolarizer gramicidin can inhibit mast cell function. However, use of the genetically encoded voltage indicators (GEVIs) ArcLight (pH-sensitive) and ASAP2 (pH-insensitive), indicates that TCS does not disrupt PMP. In conjunction with data from a plasma membrane-localized, pH-sensitive reporter, these results indicate that TCS, instead, induces cytosolic acidification in mast cells and T cells. Acidification of the cytosol likely inhibits Ca2+ influx by uncoupling the STIM1/ORAI1 interaction that is required for opening of plasma membrane Ca2+ channels. These results provide a mechanistic explanation of TCS disruption of Ca2+ influx and, thus, of immune cell function.

Antimicrobial agent triclosan suppresses mast cell signaling via phospholipase D inhibition.

Humans are exposed to the antimicrobial agent triclosan (TCS) through use of TCS-containing products. Exposed tissues contain mast cells, which are involved in numerous biological functions and diseases by secreting various chemical mediators through a process termed degranulation. We previously demonstrated that TCS inhibits both Ca2+ influx into antigen-stimulated mast cells and subsequent degranulation. To determine the mechanism linking the TCS cytosolic Ca2+ depression to inhibited degranulation, we investigated the effects of TCS on crucial signaling enzymes activated downstream of the Ca2+ rise: protein kinase C (PKC; activated by Ca2+ and reactive oxygen species [ROS]) and phospholipase D (PLD). We found that TCS strongly inhibits PLD activity within 15 minutes post-antigen, a key mechanism of TCS mast cell inhibition. In addition, experiments using fluorescent constructs and confocal microscopy indicate that TCS delays antigen-induced translocations of PKCßII, PKCδ and PKC substrate myristoylated alanine-rich C-kinase. Surprisingly, TCS does not inhibit PKC activity or overall ability to translocate, and TCS actually increases PKC activity by 45 minutes post-antigen; these results are explained by the timing of both TCS inhibition of cytosolic Ca2+ (~15+ minutes post-antigen) and TCS stimulation of ROS (~45 minutes post-antigen). These findings demonstrate that it is incorrect to assume that all Ca2+ -dependent processes will be synchronously inhibited when cytosolic Ca2+ is inhibited by a toxicant or drug. The results offer molecular predictions of the effects of TCS on other mammalian cell types, which share these crucial signal transduction elements and provide biochemical information that may underlie recent epidemiological findings implicating TCS in human health problems.

Pharmaceutical agent cetylpyridinium chloride inhibits immune mast cell function by interfering with calcium mobilization.

Cetylpyridinium chloride (CPC) is an antimicrobial used in numerous personal care and janitorial products and food for human consumption at millimolar concentrations. Minimal information exists on the eukaryotic toxicology of CPC. We have investigated the effects of CPC on signal transduction of the immune cell type mast cells. Here, we show that CPC inhibits the mast cell function degranulation with antigen dose-dependence and at non-cytotoxic doses ∼1000-fold lower than concentrations in consumer products. Previously we showed that CPC disrupts phosphatidylinositol 4,5-bisphosphate, a signaling lipid critical for store-operated Ca2+ entry (SOCE), which mediates degranulation. Our results indicate that CPC inhibits antigen-stimulated SOCE: CPC restricts Ca2+ efflux from endoplasmic reticulum, reduces Ca2+ uptake into mitochondria, and dampens Ca2+ flow through plasma membrane channels. While inhibition of Ca2+ channel function can be caused by alteration of plasma membrane potential (PMP) and cytosolic pH, CPC does not affect PMP or pH. Inhibition of SOCE is known to depress microtubule polymerization, and here we show that CPC indeed dose-dependently shuts down formation of microtubule tracks. In vitro data reveal that CPC inhibition of microtubules is not due to direct CPC interference with tubulin. In summary, CPC is a signaling toxicant that targets Ca2+ mobilization.

Pharmaceutical agent cetylpyridinium chloride inhibits immune mast cell function by interfering with calcium mobilization.

Cetylpyridinium chloride (CPC) is an antimicrobial used in numerous personal care and janitorial products and food for human consumption at millimolar concentrations. Minimal information exists on the eukaryotic toxicology of CPC. We have investigated the effects of CPC on signal transduction of the immune cell type mast cells. Here, we show that CPC inhibits the mast cell function degranulation with antigen dose-dependence and at non-cytotoxic doses ∼1000-fold lower than concentrations in consumer products. Previously we showed that CPC disrupts phosphatidylinositol 4,5-bisphosphate, a signaling lipid critical for store-operated Ca 2+ entry (SOCE), which mediates degranulation. Our results indicate that CPC inhibits antigen-stimulated SOCE: CPC restricts Ca 2+ efflux from endoplasmic reticulum, reduces Ca 2+ uptake into mitochondria, and dampens Ca 2+ flow through plasma membrane channels. While inhibition of Ca 2+ channel function can be caused by alteration of plasma membrane potential (PMP) and cytosolic pH, CPC does not affect PMP or pH. Inhibition of SOCE is known to depress microtubule polymerization, and here we show that CPC indeed dose-dependently shuts down formation of microtubule tracks. In vitro data reveal that CPC inhibition of microtubules is not due to direct CPC interference with tubulin. In summary, CPC is a signaling toxicant that targets Ca 2+ mobilization.