The innate immune response is the first mechanism for host defense and is intended to prevent infection and remove the invading pathogens. Activation of the inflammasome is one of the inflammation-related pathways mediated by the innate immune system. Inflammasomes are responsible for producing inflammatory molecules and causing inflammatory cell death - pyroptosis [1]. There are a few inflammasome types, but they all have a common adapter protein – apoptosis-associated speck-like protein containing a CARD, ASC [2]. ASC is necessary for the inflammasome complex assembly and activation. During inflammasome activation, ASCs oligomerize to about 1 μM size particles called ASC specks, which are released to extracellular space and can further trigger inflammation [3]. Inflammasomes are linked to a variety of autoimmune and neurodegenerative diseases [4]. Thus, ASC proteins can be used as targets to inhibit the inflammatory response. This research aimed to investigate how ASC specks affect macrophage activation by viral antigens.
We prepared ASC speck fractions using the THP1-ASC-GFP cell line. We settled on an optimal technique to gather ASC specks using various cell lysis methods and centrifugation steps. THP-1 macrophage-like cells were treated with ASC specks and viral antigens – human KI polyomavirus virus-like particles (KIPyV VLPs). We assessed cell viability and inflammatory response via cytokine secretion to analyze the effect of ASC specks and KIPyV VLPs on macrophages. Cell viability was evaluated by lactate dehydrogenase (LDH) assay. Enzyme-linked immunosorbent assay (ELISA) was applied to measure the release of cytokines, including IL-8, IL-1β and CCL2.
Our results demonstrated that ASC specks and a mixture of ASC specks with KIPyV VLPs significantly increased LDH release in macrophages. We also observed the tendency - the higher the concentration of ASC specks used for macrophage activation, the higher the LDH concentration in the cell medium. Macrophages treated with KIPyV VLPs released a higher LDH concentration than the control group, but it was insignificant. We also found that KIPyV VLPs induced a higher IL-8 and CCL2 secretion in macrophages than ASC specks. However, macrophages treated with ASC specks released more IL-1β than viral antigen-activated cells. Interestingly, ASC specks significantly reduced the KIPyV VLPs-induced effect on macrophages regarding CCL2 secretion. A mixture of ASC specks and KIPyV VLPs induced different secretion of CCL2 and IL-1β than KIPyV VLPs alone. However, the opposite effect was observed in the case of IL-8 secretion - ASC specks alone had less impact on IL-8 secretion than the mixture of KIPyV VLPs and ASC specks.
Our results suggest that ASC specks induce inflammasome assembly and promote pyroptosis in macrophages. The specks also modulate KIPyV VLP-induced release of pro-inflammatory cytokines. In conclusion, ASC specks can affect viral antigen-mediated inflammatory response in macrophages.