Attenuation of neutrophil driven inflammation and fungal growth by pilocarpine hydrochloride

Abstract Background An increasing incidence of fungal infections and antifungal resistance highlight the urgent need for novel treatment approaches. Current antifungal treatment primarily targets the pathogen and does not address modulation of the host immune response. However, infections trigger a rapid and intense recruitment of immune cells, particularly neutrophil granulocytes. Although these first responders play a critical role in pathogen clearance, their activation can concurrently cause damage to surrounding tissues. This is especially detrimental in tissues like the cornea, where scarring and opacification induced by inflammation can lead to permanent visual impairment. A drug with simultaneous effects on fungal growth and immune reaction could reduce immune-mediated tissue damage while improving antifungal efficacy. Pilocarpine hydrochloride is a muscarinic receptor agonist previously shown to inhibit C. albicans hyphae formation and reduce granulocyte-mediated inflammation in Galleria mellonella larvae, which could be repurposed for the localised treatment of fungal infection. Results We investigated the effect of pilocarpine hydrochloride on fungal growth and on neutrophil responses in vitro . Growth of not only C. albicans , but also filamentous fungi A. flavus and F. keratoplasticum was reduced upon treatment. In neutrophils, pilocarpine induced apoptosis in a concentration-dependent manner. It further downregulated neutrophil activation as measured by CD11b and CD66b receptor expression, independent of calcium flux. Shape changes and chemotaxis in response to the chemoattractant N-formylmethionine-leucyl-phenylalanine (fMLF) were reduced by pilocarpine treatment. The drug further influenced neutrophil-mediated antimicrobial activities against C. albicans , including killing, reactive oxygen species (ROS) production, degranulation and phagocytosis. While pilocarpine itself did not induce NETs, it was able to downregulate ROS-dependent NETosis. We further investigated NET-release in response to heat-inactivated yeast, dead hyphae, and live C. albicans during its transition from yeast to hyphae under pilocarpine treatment. In a novel three-dimensional in vitro cornea infection model, neutrophil chemotaxis was reduced by pilocarpine, while fungal growth into the model was inhibited. Conclusions In summary, pilocarpine inhibits fungal metabolism while downregulating neutrophil activities and inducing apoptosis. Hence, pilocarpine could serve as a therapeutic option for local fungal infections, as it provides a dual benefit by targeting fungi and immune regulation.