Molecular Profiling of Phagocytic Immune Cells in Anopheles gambiae Reveals Integral Roles for Hemocytes in Mosquito Innate Immunity

Abstract

The innate immune response is highly conserved across all eukaryotes and has been studied in great detail in several model organisms. Hemocytes, the primary immune cell population in mosquitoes, are important components of the mosquito innate immune response, yet critical aspects of their biology have remained uncharacterized. Using a novel method of enrichment, we isolated phagocytic granulocytes and quantified their proteomes by mass spectrometry. The data demonstrate that phagocytosis, blood-feeding, and Plasmodium falciparum infection promote dramatic shifts in the proteomic profiles of An. gambiae granulocyte populations. Of interest, large numbers of immune proteins were induced in response to blood feeding alone, suggesting that granulocytes have an integral role in priming the mosquito immune system for pathogen challenge. In addition, we identify several granulocyte proteins with putative roles as membrane receptors, cell signaling, or immune components that when silenced, have either positive or negative effects on malaria parasite survival. Integrating existing hemocyte transcriptional profiles, we also compare differences in hemocyte transcript and protein expression to provide new insight into hemocyte gene regulation and discuss the potential that post-transcriptional regulation may be an important component of hemocyte gene expression. These data represent a significant advancement in mosquito hemocyte biology, providing the first comprehensive proteomic profiling of mosquito phagocytic granulocytes during homeostasis blood-feeding, and pathogen challenge. Together, these findings extend current knowledge to further illustrate the importance of hemocytes in shaping mosquito innate immunity and their principal role in defining malaria parasite survival in the mosquito host.

Unbiased classification of mosquito blood cells by single-cell genomics and high-content imaging

“…Smith et al using magnetic bead isolation at 28°C only (Smith et al, 2016). Our analyses revealed 340 that similarities were the strongest when profiles were compared across all samples -PPO6 high , 341 PPO6 low , phagocytes and all cells, i.e.…”

Hemocyte-targeted gene expression in the female malaria mosquito using thehemolectinpromoter fromDrosophila

“…Transcriptomic and proteomic studies comparing gene and protein expression between hemocytes and carcass in An. gambiae identified some genes enriched in immune cells (Baton, Robertson et al 2009, Lombardo, Ghani et al 2013, Smith, King et al 2016, Kwon and Smith 2019. Promoters of these genes could therefore constitute other good candidates to drive expression in mosquito hemocytes.…”

“…gambiae adult females, bacterial or parasite challenges also trigger hemocyte differentiation (King and Hillyer 2012, King and Hillyer 2013, Ramirez, Garver et al 2014 and this is associated with differences in their gene and protein expression (Baton, Robertson et al 2009, Pinto, Lombardo et al 2009, Smith, King et al 2016. Moreover, hemocytes are involved in phagocytosis (Moita, Wang-Sattler et al 2005, King and Hillyer 2012, King and Hillyer 2013, Lombardo, Ghani et al 2013) and in immune responses against malaria parasites (Pinto, Lombardo et al 2009, Ramirez, Garver et al 2014, Smith, Barillas-Mury et al 2015, Lombardo and Christophides 2016, Smith, King et al 2016. Immune cell differentiation has also been implicated in innate immune memory (Ramirez, Garver et al 2014, Ramirez, de Almeida Oliveira et al 2015, Barletta, Trisnadi et al 2019.…”

Chemical depletion of phagocytic immune cells reveals dual roles of mosquito hemocytes in Anopheles gambiae anti-Plasmodium immunity

“…Following liposome treatments, hemolymph was perfused from individual mosquitoes (n=15) at naïve or 24 h P. berghei -infected mosquitoes using incomplete buffer (anticoagulant solution without fetal bovine serum) containing a protease inhibitor cocktail (Sigma) (10). Hemolymph protein concentrations were measured using Quick Start ™ Bradford Dye reagent (Bio-Rad).…”

“…Previous studies have demonstrated that Anopheles gambiae hemocytes respond to blood-feeding (7–10), as well as bacterial and malaria parasite challenge (3, 11), eliciting changes in their cellular profiles at the transcriptional (12, 13) and proteome levels (10). This has led to the identification of several mosquito hemocyte components with integral roles in phagocytosis (14, 15) and anti- Plasmodium immunity (10, 12, 16). In addition, hemocyte differentiation in response to malaria parasite challenge has been described as an integral requirement of mosquito immune priming (17–19) and the “late-phase” immune responses targeting Plasmodium oocyst survival (20–22).…”