Chitin is an abundant polysaccharide found in fungal cell walls, crustacean shells, and insect exoskeletons. The immunological properties of both chitin and its deacetylated derivative chitosan are of relevance because of frequent natural exposure and their use in medical applications. Depending on the preparation studied and the end point measured, these compounds have been reported to induce allergic responses, inflammatory responses, or no response at all. We prepared highly purified chitosan and chitin and examined the capacity of these glycans to stimulate murine macrophages to release the inflammasome-associated cytokine IL-1β. We found that although chitosan was a potent NLRP3 inflammasome activator, acetylation of the chitosan to chitin resulted in a near total loss of activity. The size of the chitosan particles played an important role, with small particles eliciting the greatest activity. An inverse relationship between size and stimulatory activity was demonstrated using chitosan passed through size exclusion filters as well as with chitosan-coated beads of defined size. Partial digestion of chitosan with pepsin resulted in a larger fraction of small phagocytosable particles and more potent inflammasome activity. Inhibition of phagocytosis with cytochalasin D abolished the IL-1β stimulatory activity of chitosan, offering an explanation for why the largest particles were nearly devoid of activity. Thus, the deacetylated polysaccharide chitosan potently activates the NLRP3 inflammasome in a phagocytosis-dependent manner. In contrast, chitin is relatively inert.

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Chitosan, the deacetylated derivative of chitin, can be found in the cell wall of some fungi and is utilized in translational applications. We have shown that highly purified preparations of chitosan, but not chitin, activate the NLRP3 inflammasome in primed mouse bone marrow-derived macrophages (BMMΦ), inducing a robust IL-1β response. Here, we further define specific cell types that are activated and delineate mechanisms of activation. BMMΦ differentiated to promote a classically activated (M1) phenotype released more IL-1β in response to chitosan than intermediate or alternatively activated macrophages (M2). Chitosan but not chitin induced a robust IL-1β response in mouse DCs, peritoneal macrophages, and human PBMCs. Three mechanisms for NLRP3 inflammasome activation may contribute: K+ efflux, reactive oxygen species (ROS), and lysosomal destabilization. The contributions of these mechanisms were tested using a K+ efflux inhibitor, high extracellular potassium, a mitochondrial ROS inhibitor, lysosomal acidification inhibitors, and a cathepsin B inhibitor. These studies revealed that each of these pathways participated in optimal NLRP3 inflammasome activation by chitosan. Finally, neither chitosan nor chitin stimulated significant release from unprimed BMMΦ of any of 22 cytokines and chemokines assayed. In conclusion, 1) chitosan, but not chitin, stimulates IL-1β release from multiple murine and human cell types; 2) multiple non-redundant mechanisms appear to participate in inflammasome activation by chitosan; and 3) chitin and chitosan are relatively weak stimulators of inflammatory mediators from unprimed BMMΦ. These data have implications for understanding the nature of the immune response to microbes and biomaterials that contain chitin and chitosan.

Allergens such as house dust mites (HDM) and papain induce strong Th2 responses, including elevated IL-4, IL-5 and IL-13 and marked eosinophilia in the airways. Histoplasma capsulatum (H. capsulatum) is a dimorphic fungal pathogen that induces a strong Th1 response marked by IFNγ and TNFα production, leading to rapid clearance in non-immunocompromised hosts. Th1 responses are generally dominant and overwhelm the Th2 response when stimuli for both are present, though there are instances when Th2 stimuli downregulate a Th1 response. We determined if the Th2 response to allergens prevents the host from mounting a Th1 response to H. capsulatum in vivo. C57BL/6 mice exposed to HDM or papain and infected with H. capsulatum exhibited a dominant Th2 response early, characterized by enhanced eosinophilia and elevated Th2 cytokines in lungs. These mice manifested exacerbated fungal burdens, suggesting that animals skewed towards a Th2 response by an allergen are less able to clear the H. capsulatum infection despite an intact Th1 response. On the other hand, secondary infection is not exacerbated by allergen exposure, indicating the memory response may suppress the Th2 response to HDM and quickly clear the infection. In conclusion, an in vivo skewing towards Th2 by allergens exacerbates fungal infection, even though there is a concurrent and unimpaired Th1 response to H. capsulatum.

Eosinophils contribute to type II immune responses in helminth infections and allergic diseases, however, their influence on intracellular pathogens is less clear. We previously reported that CCR2−/− mice exposed to the intracellular fungal pathogen Histoplasma capsulatum exhibit dampened immunity caused by an early exaggerated IL-4 response. We sought to identify the cellular source promulgating interleukin (IL)-4 in infected mutant animals. Eosinophils were the principal instigators of non-protective IL-4 and depleting this granulocyte population improved fungal clearance in CCR2−/− animals. The deleterious impact of eosinophilia on mycosis was also recapitulated in transgenic animals overexpressing eosinophils. Mechanistic examination of IL-4 induction revealed that phagocytosis of H. capsulatum via the pattern recognition receptor complement receptor (CR) 3 triggered the heightened IL-4 response in murine eosinophils. This phenomenon was conserved in human eosinophils; exposure of cells to the fungal pathogen elicited a robust IL-4 response. Thus, our findings elucidate a detrimental attribute of eosinophil biology in fungal infections that could potentially trigger a collapse in host defenses by instigating type II immunity.

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