Acanthamoeba castellanii-Mediated Reduction of Interleukin-1β Secretion and Its Association With Macrophage Autophagy.

Abstract

Noncanonical autophagy including unconventional protein secretion has been extensively studied. Our work focused on a leaderless IL-1β protein secretion from human macrophage in response to Acanthamoeba castellanii components, Acanthamoeba culture supernatant (CS) and cell lysate (CL), as well as its association with macrophage autophagy. Phorbol 12-myristate 13-acetate (PMA)-induced THP-1 macrophages were treated with Acanthamoeba components of pathogenic (ATCC50739) and nonpathogenic (ATCC30010) strains in vitro. The data showed that Acanthamoeba treatment resulted in low IL-1β secretion from macrophages. In addition, Acanthamoeba CL of both strains was able to upregulate autophagy-related (Atg) protein 8, an autophagy marker, whereas Acanthamoeba CS downregulated Atg8 expression. We further manipulated autophagy and found that autophagy induction by starvation diminished IL-1β secretion while autophagy inhibition by 3-methyladenine (3MA) increased IL-1β secretion. Interestingly, in the presence of Acanthamoeba components either under starvation or 3MA treatment, IL-1β secretion was significantly reduced. Transcriptional expression of other ATG genes, i.e., ATG6, ATG7, and ATG5, were investigated and showed that their mRNA expression was maintained at the basal level under A. castellanii CS or CL treatment. Inflammasome-related genes, NLRP3 and CASPASE1, were upregulated following A. castellanii 50739 CS treatment but not in A. castellanii 50739 CL-treated condition. However, both conditions were able to increase IL-1β mRNA expression. TEM micrographs revealed that 3MA treatment induced the formation of large vacuoles and accumulation of autophagosome at the edge of THP-1 macrophages. However, the number and size of their structures were declined in the presence of A. castellanii 50739 CS with 3MA. Furthermore, immunofluorescence staining demonstrated the association between Atg8/LC3 and IL-1β expression, where downregulation of Atg8 by A. castellanii 50739 CS led to the upregulation of IL-1β. Altogether, the data indicate that Acanthamoeba can manipulate macrophage autophagy, thereby controlling low IL-1β secretion. The expression of autophagy- and inflammasome-related genes also indicates multiple mechanisms in IL-1β secretion in response to Acanthamoeba components. However, further characterization of Atg proteins and investigations into other intracellular pathways or defense mechanisms are needed to fully understand the unconventional secretion of IL-1β in macrophages. This knowledge could eventually lead to the development of innovative therapeutic strategies against Acanthamoeba infection by modulating autophagy or macrophage responses.