In addition to adjuvants directly engaging cellular receptors, a plethora of evidence has emerged to suggest that many adjuvants, including alum and oil-in-water emulsions, elicit their immunogenicity in an indirect manner by inducing the extracellular release of DAMPs. For example, injection of alum- or MF59-adjuvanted formulations induces local cell death and the release of uric acid and ATP, respectively, with enzymatic depletion of either factor attenuating the potency of each respective adjuvant. Moreover, the release of uric acid, which can form monosodium urate (MSU) crystals and K+efflux, is now thought to be the factor driving activation of the NLRP3 inflammasome upon injection of alum rather than inflammasome activations being a direct property of alum itself. This may also propagate the response by inducing pyroptotic cell death via the cleavage and activation of Gasdermin D to form pores in cellular membranes. The release of endogenous mitochondrial and genomic DNA from dying cells and damaged tissues can also stimulate immune responses following adjuvant injection upon being sensed through cytosolic DNA sensors, including cGAS, IFI16, AIM2, and ZBP1, or via endosomal TLR9.
