Disparate Activation of the Inflammasome by Chitin and Chitosan: A Dissertation

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 due to frequent natural exposure and their increasing use in translational applications. Depending on the preparation studied and the endpoint measured, these compounds have been reported to induce allergic responses, inflammatory responses, or no response at all. Highly purified chitosan and chitin were prepared and the capacity of these glycans to stimulate the release of the inflammasomeassociated cytokine IL-1β was examined. Chitosan was shown to be a potent inflammasome activator in mouse bone marrow macrophages, macrophages polarized towards a M1 or M2 phenotype, dendritic cells, peritoneal cells, and human PBMCs. Acetylation of the chitosan to chitin resulted in a near total loss of IL-1β activity in all cell types tested. 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. The reason for chitin’s inability to elicit IL-1β is unknown, but it does not appear to be due to active inhibition of the inflammasome and while chitin appears to be more readily digested by macrophage cell lysates, it does not occur at a rate which would likely impact inflammasome activation. There are three proposed mechanisms for NLRP3 inflammasome activation: K+ efflux, ROS, and lysosomal destabilization. The contributions of these mechanisms were tested and it was revealed that each of these pathways participated in optimal NLRP3 inflammasome activation by chitosan. Finally, the laminin receptor was evaluated as a potential chitin receptor. These studies provide insight into the activating properties of chitin and chitosan and highlight the importance of matching particle size and degree of acetylation to the level of activity desired for translational applications

Innate sensing of chitin and chitosan

Chitin is the second most common polysaccharide found in nature. It is present in crustacean shells, insect exoskeletons, parasitic nematode eggs and gut linings, and in the cell wall of fungi. The deacetylated derivative of chitin, chitosan, is less common but is particularly evident in certain species of fungi, such as Cryptococcus, and the cyst wall of Entamoeba. How mammals sense and respond to these polymers is not well understood, and conflicting reports on their immunological activity have led to some controversy. Despite this, promising translational applications that exploit the unique properties of chitin and chitosan are being developed

Chitosan but not chitin activates the inflammasome by a mechanism dependent upon phagocytosis

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-1beta. 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-1beta 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

Chitin and chitosan: from source to consequence.

<p>Chitin and chitosan are naturally found in fungal cell walls, crustacean shells, nematodes eggs and gut linings, and insect exoskeletons. These polymers consist of long chains of N-acetylglucosamine (chitin) or glucosamine (chitosan). Conversion between the two polysaccharides can be performed chemically or happen within the organisms via chitin deacetylases. Mammalian exposure to the polymers has been linked to both upregulation and downregulation of inflammatory responses, including those involved in asthma. Despite this, chitin and chitosan are being utilized in a variety of biomedical applications, including tissue engineering and drug delivery.</p

Spectrum and mechanisms of inflammasome activation by chitosan

Chitosan, the deacetylated derivative of chitin, can be found in the cell wall of some fungi and is used in translational applications. We have shown that highly purified preparations of chitosan, but not chitin, activate the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in primed mouse bone marrow-derived macrophages (BMMPhi), inducing a robust IL-1beta response. In this article, we further define specific cell types that are activated and delineate mechanisms of activation. BMMPhi differentiated to promote a classically activated (M1) phenotype released more IL-1beta in response to chitosan than intermediate or alternatively activated macrophages (M2). Chitosan, but not chitin, induced a robust IL-1beta response in mouse dendritic cells, peritoneal macrophages, and human PBMCs. Three mechanisms for NLRP3 inflammasome activation may contribute: K(+) efflux, reactive oxygen species, and lysosomal destabilization. The contributions of these mechanisms were tested using a K(+) efflux inhibitor, high extracellular potassium, a mitochondrial reactive oxygen species 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 BMMPhi of any of 22 cytokines and chemokines assayed. This study has the following conclusions: 1) chitosan, but not chitin, stimulates IL-1beta release from multiple murine and human cell types; 2) multiple nonredundant mechanisms appear to participate in inflammasome activation by chitosan; and 3) chitin and chitosan are relatively weak stimulators of inflammatory mediators from unprimed BMMPhi. These data have implications for understanding the nature of the immune response to microbes and biomaterials that contain chitin and chitosan