Defining the Role of Alpha-Macroglobulins in the Pathogenesis of Flavivirus Encephalitis

Abstract

West Nile Virus (WNV) and Japanese Encephalitis Virus (JEV) are positive sense, singlestranded RNA viruses belonging to the family Flaviviridae, where the primary mode of transmission occurs through arthropod vectors such as mosquitos. WNV and JEV are the leading etiological agents for arboviral encephalitis in humans, and sporadic outbreaks continue to occur over time resulting in symptomatic infections that account for thousands of deaths each year. Although a number of vaccines have been developed to prevent JEV infections in highly endemic regions, issues with efficacy exist and the vaccine often fails to protect at risk populations from the development of encephalitis, which can be fatal. For both WNV and JEV, no clinically approved therapies currently exist for treatment of the central nervous system (CNS) involvement, which can lead to encephalitis, the most severe, and highly fatal form of the disease. Alpha-macroglobulins, which are physiological proteinase inhibitors have been shown to bind to viral proteins and enhance viral infections in vitro. Moreover, in humans alphamacroglobulins such as pregnancy zone protein (PZP) and alpha-2-macroglobulin (A2M) also serve as immune-modulatory proteins where their normal functions within the body include binding and shuttling of protease inhibitors, growth factors, cytokines, hormones, disease factors, and various small molecule nucleophilic ligands. Previous reports from our laboratory have shown the up-regulation of alpha-macroglobulins in wild type (WT) mice after a lethal subcutaneous WNV infection. Therefore, to define the role of a-macroglobulins during flavivirus infection in vivo, we investigated the susceptibility of mice deficient in amacroglobulins (PZP and MUG-1 double knockout; DKO) against lethal subcutaneous infection with either WNV or JEV. Results of our study show that DKO mice are completely resilient to lethal flaviviral infections of WNV and JEV. Likewise, DKO mice had a significantly milder clinical disease through the course of the study, and this outcome can be coupled with the finding of significantly reduced viral burden in the blood, peripheral organs (kidney and spleen), and brains of the DKO mice when compared to WT mice. The DKO mice had a significantly reduced inflammatory response which was characterized by lower concentrations of pro-inflammatory cytokines and chemokines in the blood, spleen, and brain of DKO mice when compared to WT. Consistent with the multiplex immunoassay data, DKO mice also displayed a significantly decreased level of mRNA corresponding to immune genes in response to WNV infection when compared to WT. Overall, the data from this study demonstrates the significant impact that alpha-macroglobulins have in the pathogenesis of flavivirus-associated encephalitis in mice.