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.
Project description:The innate immune response to a broad class of pathogens is highly conserved across all eukaryotes and has been studied in great detail at the cellular and transcriptomic level in several insect species. However, the commensurate cellular proteomic response, especially of hemocytes, the primary immune cell population in insects, has remained poorly understood. We report on the comprehensive proteogenomic analysis of a phagocyte subpopulation from Anopheles gambiae, the primary malaria mosquito vector in Sub-Saharan Africa. We leveraged the innate phagocytic response of mosquito granulocytes to achieve targeted enrichment for these cells to facilitate the examination of their proteomic response profiles following sugar feeding, as well as non-infectious and infectious blood feeding with Plasmodium falciparum. A comparative integrative-OMICs analysis of existing transcriptomic profiles combined with these proteomic data permitted the delineation of the functional genome of anopheline granulocytes. We observed that phagocytosis, blood feeding, and P. falciparum infection induced dramatic shifts in granulocyte protein expression indicative of broad changes in cellular proliferation and innate immune response priming. Importantly, we identified a large number of hemocyte immune proteins that respond to blood feeding alone, suggesting that granulocytes may play an integral role in an anticipatory immune response prior to immune challenge.
Project description:Hemocytes are crucial players of the mosquito immune system and critically affect transmission of pathogens including malaria parasites. We and others discovered previously that a blood meal is a major immune stimulus for mosquito hemocytes. To determine whether these blood meal-induced hemocyte changes in Anopheles gambiae constitute steps in cell differentiation or demonstrate transient cell activation, we analyzed the temporal pattern of these changes over the first three days post blood meal (dpbm). Flow cytometry and immunofluorescence analyses revealed a global shift of the entire hemocyte population, peaking at 1 dpbm. All hemocyte activation markers returned to pre-blood meal baseline levels within the following 24-48 h. Our observations are consistant with An. gambiae hemocytes undergoing transient activation rather than terminal differentiation upon blood feeding. Interestingly, the temporal pattern followed the gonotrophic cycle of the mosquito, strongly suggesting hormonal control of mosquito hemocyte activation and deactivation.
Project description:Hemocytes, the immune cells in mosquitoes, participate in immune defenses against pathogens including malaria parasites. Mosquito hemocytes can also be infected by arthropod-borne viruses but the pro- or anti-viral nature of this interaction is unknown. Although there has been progress on hemocyte characterization during pathogen infection in mosquitoes, the specific contribution of hemocytes to immune responses and the hemocyte-specific functions of immune genes and pathways remain unresolved due to the lack of genetic tools to manipulate gene expression in these cells specifically. Here, we used the Gal4-UAS system to characterize the activity of the Drosophila hemocyte-specific hemolectin promoter in the adults of Anopheles gambiae, the malaria mosquito. We established an hml-Gal4 driver line that we further crossed to a fluorescent UAS responder line, and examined the expression pattern in the adult progeny driven by the hml promoter. We show that the hml regulatory region drives hemocyte-specific transgene expression in a subset of hemocytes, and that transgene expression is triggered after a blood meal. The hml promoter drives transgene expression in differentiating prohemocytes as well as in differentiated granulocytes. Analysis of different immune markers in hemocytes in which the hml promoter drives transgene expression revealed that this regulatory region could be used to study phagocytosis as well as melanization. Finally, the hml promoter drives transgene expression in hemocytes in which o'nyong-nyong virus replicates. Altogether, the Drosophila hml promoter constitutes a good tool to drive transgene expression in hemocyte only and to analyze the function of these cells and the genes they express during pathogen infection in Anopheles gambiae.
Project description:Malaria is a global public health problem, especially in sub-Saharan Africa, where the mosquito Anopheles gambiae Giles serves as the major vector for the protozoan Plasmodium falciparum Welch. One determinant of malaria vector competence is the mosquito's immune system. Hemocytes are a critical component as they produce soluble immune factors that either support or prevent malaria parasite development. However, despite their importance in vector competence, understanding of their basic biology is just developing. Applying novel technologies to the study of mosquito hemocytes, we investigated the effect of blood meal on hemocyte population dynamics, DNA replication and cell cycle progression. In contrast to prevailing published work, the data presented here demonstrate that hemocytes in adult mosquitoes continue to undergo low basal levels of replication. In addition, blood ingestion caused significant changes in hemocytes within 24 h. Hemocytes displayed an increase in cell number, size, granularity and Ras-MAPK signaling as well as altered cell surface moieties. As these changes are well-known markers of immune cell activation in mammals and Drosophila melanogaster Meigen, we further investigated whether a blood meal changes the expression of hemocyte-derived immune factors. Indeed, hemocytes 24 h post-blood meal displayed higher levels of critical components of the complement and melanization immune reactions in mosquitoes. Taken together, this study demonstrates that the normal physiological process of a blood meal activates the innate immune response in mosquitoes. This process is likely in part regulated by Ras-MAPK signaling, highlighting a novel mechanistic link between blood feeding and immunity.
Project description:All vector mosquito species must feed on the blood of a vertebrate host to produce eggs. Multiple cycles of blood feeding also promote frequent contacts with hosts, which enhance the risk of exposure to infectious agents and disease transmission. Blood feeding triggers the release of insulin-like peptides (ILPs) from the brain of the mosquito Aedes aegypti, which regulate blood meal digestion and egg formation. In turn, hemocytes serve as the most important constitutive defense in mosquitoes against pathogens that enter the hemocoel. Prior studies indicated that blood feeding stimulates hemocytes to increase in abundance, but how this increase in abundance is regulated is unknown. Here, we determined that phagocytic granulocytes and oenocytoids express the A. aegypti insulin receptor (AaMIR). We then showed that: 1) decapitation of mosquitoes after blood feeding inhibited hemocyte proliferation, 2) a single dose of insulin-like peptide 3 (ILP3) sufficient to stimulate egg production rescued proliferation, and 3) knockdown of the AaMIR inhibited ILP3 rescue activity. Infection studies indicated that increased hemocyte abundance enhanced clearance of the bacterium Escherichia coli at lower levels of infection. Surprisingly, however, non-blood fed females better survived intermediate and high levels of E. coli infection than blood fed females. Taken together, our results reveal a previously unrecognized role for the insulin signaling pathway in regulating hemocyte proliferation. Our results also indicate that blood feeding enhances resistance to E. coli at lower levels of infection but reduces tolerance at higher levels of infection.
Project description:Insects rely on the innate immune system for defense against pathogens, some aspects of which are under hormonal control. Here we provide direct experimental evidence showing that the juvenile hormone-binding protein (mJHBP) of Aedes aegypti is required for the regulation of innate immune responses and the development of mosquito blood cells (hemocytes). Using an mJHBP-deficient mosquito line generated by means of CRISPR-Cas9 gene editing technology we uncovered a mutant phenotype characterized by immunosuppression at the humoral and cellular levels, which profoundly affected susceptibility to bacterial infection. Bacteria-challenged mosquitoes exhibited significantly higher levels of septicemia and mortality relative to the wild type (WT) strain, delayed expression of antimicrobial peptides (AMPs), severe developmental dysregulation of embryonic and larval hemocytes (reduction in the total number of hemocytes) and increased differentiation of the granulocyte lineage. Interestingly, injection of recombinant wild type mJHBP protein into adult females three-days before infection was sufficient to restore normal immune function. Similarly, injection of mJHBP into fourth-instar larvae fully restored normal larval/pupal hemocyte populations in emerging adults. More importantly, the recovery of normal immuno-activation and hemocyte development requires the capability of mJHBP to bind JH III. These results strongly suggest that JH III functions in mosquito immunity and hemocyte development in a manner that is perhaps independent of canonical JH signaling, given the lack of developmental and reproductive abnormalities. Because of the prominent role of hemocytes as regulators of mosquito immunity, this novel discovery may have broader implications for the understanding of vector endocrinology, hemocyte development, vector competence and disease transmission.
Project description:Insect hemocytes mediate important cellular immune responses including phagocytosis and encapsulation and also secrete immune factors such as opsonins, melanization factors, and antimicrobial peptides. However, the molecular composition of these important immune cells has not been elucidated in depth, because of their scarcity in the circulating hemolymph, their adhesion to multiple tissues and the lack of primary culture methods to produce sufficient material for a genome-wide analysis. In this study, we report a genome-wide molecular characterization of circulating hemocytes collected from the hemolymph of adult female Anopheles gambiae mosquitoes--the major mosquito vector of human malaria in subSaharan Africa. Their molecular profile identified 1,485 transcripts with enriched expression in these cells, and many of these genes belong to innate immune gene families. This hemocyte-specific transcriptome is compared to those of Drosophila melanogaster and two other mosquitoes, Aedes aegypti and Armigeres subalbatus. We report the identification of two genes as ubiquitous hemocyte markers and several others as hemocyte subpopulation markers. We assess, via an RNAi screen, the roles in development of Plasmodium berghei of 63 genes expressed in hemocytes and provide a molecular comparison of the transcriptome of these cells during malaria infection.
Project description:Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy) and transmission electron microscopy (TEM). Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes) were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules) and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all) L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules). Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes. Extensive hemocyte aggregates ('islets') occupy most renal hemocoelic spaces and hyalinocyte-like cells are the most frequent component in them. Presumptive glycogen deposits were observed in most hyalinocytes in renal islets (they also occur in the circulation but less frequently) and may mean that hyalinocytes participate in the storage and circulation of this compound. Injection of microorganisms in the foot results in phagocytosis by hemocytes in the islets, and the different phagosomes formed are similar to those in circulating hyalinocytes. Dispersed hemocytes were obtained after kidney collagenase digestion and cell sorting, and they were able to phagocytize fluorescent beads. A role for the kidney as an immune barrier is proposed for this snail.
Project description:Honey bees as other insects rely on the innate immune system for protection against diseases. The innate immune system includes the circulating hemocytes (immune cells) that clear pathogens from hemolymph (blood) by phagocytosis, nodulation or encapsulation. Honey bee hemocyte numbers have been linked to hemolymph levels of vitellogenin. Vitellogenin is a multifunctional protein with immune-supportive functions identified in a range of species, including the honey bee. Hemocyte numbers can increase via mitosis, and this recruitment process can be important for immune system function and maintenance. Here, we tested if hemocyte mediated phagocytosis differs among the physiologically different honey bee worker castes (nurses, foragers and winter bees), and study possible interactions with vitellogenin and hemocyte recruitment. To this end, we adapted phagocytosis assays, which-together with confocal microscopy and flow cytometry-allow qualitative and quantitative assessment of hemocyte performance. We found that nurses are more efficient in phagocytic uptake than both foragers and winter bees. We detected vitellogenin within the hemocytes, and found that winter bees have the highest numbers of vitellogenin-positive hemocytes. Connections between phagocytosis, hemocyte-vitellogenin and mitosis were worker caste dependent. Our results demonstrate that the phagocytic performance of immune cells differs significantly between honey bee worker castes, and support increased immune competence in nurses as compared to forager bees. Our data, moreover, provides support for roles of vitellogenin in hemocyte activity.
Project description:Insects counter infection with innate immune responses that rely on cells called hemocytes. Hemocytes exist in association with the insect's open circulatory system and this mode of existence has likely influenced the organization and control of anti-pathogen immune responses. Previous studies reported that pathogens in the mosquito body cavity (hemocoel) accumulate on the surface of the heart. Using novel cell staining, microdissection and intravital imaging techniques, we investigated the mechanism of pathogen accumulation in the pericardium of the malaria mosquito, Anopheles gambiae, and discovered a novel insect immune tissue, herein named periostial hemocytes, that sequesters pathogens as they flow with the hemolymph. Specifically, we show that there are two types of endocytic cells that flank the heart: periostial hemocytes and pericardial cells. Resident periostial hemocytes engage in the rapid phagocytosis of pathogens, and during the course of a bacterial or Plasmodium infection, circulating hemocytes migrate to the periostial regions where they bind the cardiac musculature and each other, and continue the phagocytosis of invaders. Periostial hemocyte aggregation occurs in a time- and infection dose-dependent manner, and once this immune process is triggered, the number of periostial hemocytes remains elevated for the lifetime of the mosquito. Finally, the soluble immune elicitors peptidoglycan and ?-1,3-glucan also induce periostial hemocyte aggregation, indicating that this is a generalized and basal immune response that is induced by diverse immune stimuli. These data describe a novel insect cellular immune response that fundamentally relies on the physiological interaction between the insect circulatory and immune systems.