| In mosquitoes, the immune responses are divided into two main components: the humoral component that consists mainly of pattern recognition receptors and anti-microbial peptide production, and the cellular component, that consists of hemocytes or blood cells that circulate in the open body cavity or hemocoel. Hemocytes are involved in a variety of immune effector responses, including phagocytosis and melanization in mosquitoes. Although humoral responses in mosquitoes are well studied, the cellular component of the innate immunity still remains under-explored. This is particularly evident in the case of mosquito-arbovirus interactions, where the role of hemocytes, the primary immune cells, in establishing vector competence is unclear. In the case of phagocytosis of microbes, while most of the research efforts concentrate on identifying pathogen-specific receptors, there is a paucity of information on the use of the endocytic machinery for pathogen uptake or the events that leads up to the degradation of the phagocytosed microbe.;In the current study, the role hemocytes might play in mosquito-arbovirus interactions was explored. Adult female mosquitoes from four different species were injected with the prototype alphavirus - Sindbis virus (SINV), and infection status of hemocytes was examined. Hemocytes, particularly granulocytes, which are the phagocytic cells, exhibited virus infection from 6 hours post-infection (hpi) for up to 96 hpi, with two strains of SINV, the transducing TE/5'2J/GFP and the orally infectious MRE16-eGFP, in four different species of mosquito. Similar results were obtained when the mosquitoes were exposed to the virus via per os infection, i.e. the natural route using SINV MRE16-eGFP. SINV was shown to use hemocytes as a site for amplification. Additionally, upon examining the virus entry mechanisms into mosquito hemocyte-like cells, SINV was shown to use the clathrin-mediated endocytosis machinery for entry, aided by the use of acidic compartments, cytoskeletal network and signaling cascades. In vivo, fewer hemocytes are infected with SINV when clathrin-mediated endocytosis is pharmacologically suppressed; these studies also showed that virus replication in hemocytes is important for dissemination into other tissues. Additionally, pathogen uptake by hemocytes was assessed using latex bead exposure to the same hemocyte-like cell line. Signaling cascades that govern the vesicular trafficking via cytoskeletal filament-polymerization and acidification of endosomes were found to be important for particle uptake. These assays showed that significant differences exist between virus entry and particle uptake in hemocyte-like cells. Moreover, a novel protein, Kill me now (KMNP), was characterized and its role in phagocytosis of bacteria analyzed. This protein was found to be specifically involved in the degradation of Gram-negative bacteria post phagocytosis. KMNP suppression had no effect on phagocytosis and degradation of Gram-positive bacteria or on amplification of SINV in hemocytes.;Overall, these studies further highlight the importance of hemocytes and cellular immunity in vector-pathogen interaction in the processes of phagocytosis and arbovirus amplification and dissemination; moreover, this work adds a new dimension in the form of mechanistic understanding of the cellular machinery involved in these mosquito-pathogen interactions. |
