Aedes aegypti Hemocytes Mediate Antiviral Immunity

Abstract

Hemocytes play several key roles in the mosquito's immune response. Despite most of our understanding regarding their immunological role concerns their responses against bacteria, fungi, and Plasmodium, our knowledge of hemocyte's role in antiviral defense is poorly understood. We performed a comprehensive comparative transcriptomic analysis between the dengue vector Aedes aegypti's two major immune cell types, hemocytes and fat body, revealing a plethora of differentially expressed immune genes that indicates a high level of functional specialization as well as complementation between the two immune cell types. Our transcriptomic approach yielded molecular insights into the antiviral immune response of Ae. aegypti hemocytes during systemic infection. In fact, hemocytes showed abundant expression of RNAi pathway genes under naive conditions and upregulated many of these upon dengue virus (DENV) infection. Furthermore, chemical depletion of phagocytic hemocytes resulted in a higher DENV systemic infection. Our results suggest that hemocytes possess mechanisms to control systemic viral infections.

Keywords: RNAi pathways; basal lamina; dengue; dissemination; hemocytes; mosquito; phagocytosis; systemic infection; virus.

Figure 1

Transcriptomic analysis of immunity-related genes in the fat body and hemocytes. (a) Experimental design for comparing fat body and hemocyte transcriptomic profiles. Fat body cells and hemolymph from 150 female mosquitoes (per replicate) were collected at 10 days post-blood feeding to analyze their transcriptomes. (b) Volcano plot of differentially expressed genes on hemocytes (p < 0.05). Dots in blue indicate downregulated genes in hemocytes (which are highly expressed in the fat body); dots in red show upregulated genes in hemocytes; black dots indicate non-differentially expressed genes. The X-axis indicates the Log2 of fold change, and the Y-axis indicates the -Log10 of p-value. (c) Scatter plot of KEEG pathway enrichment of highly expressed genes in hemocytes. The circle area indicates the number of upregulated genes in hemocytes; the color scale indicates the adjusted p-value. X-axis indicates gene ratio (count of core enrichment genes/count of pathway genes). (d) Scatter plot of immune genes classified by pathways and families. Blue dots indicate highly enriched gene mRNAs in the fat body (with lower expression in hemocytes); red dots denote highly expressed gene mRNAs in hemocytes (lower expression in the fat body). The X-axis indicates the Log2 fold change [Hemocytes/Fat body]. The number of genes is indicated on Y-axis as “n =”. (e) Heat map of Z-score values of hemocyte marker genes and antiviral immune genes. The red color indicates upregulated genes in hemocytes; the blue color shows downregulated genes. The heat map presents the values of each replicate of hemocytes (Hc1-3) and fat body (FB1-3) samples. (f) String protein–protein interaction analysis of antiviral genes highly expressed in hemocytes. Each circle represents a protein, and each line represents an interaction (physical or functional). All data were obtained from three biological replicates. Differentially expressed genes were determinate using DESeq2 package; the p-values were adjusted by Benjamini and Hochberg’s test, p ≤ 0.05 were assigned as differentially expressed. (a) was created by Victor Cardoso-Jaime in Microsoft PowerPoint Version 16.92.

Figure 2

Transcriptomic analysis of hemocytes from dengue virus-infected mosquitoes. (a) Experimental strategy for transcriptomic analysis of hemocytes. Comparison of transcriptomic profiles in hemocytes from DENV-infected mosquitoes was performed 10 days post-infection, using hemocytes from blood-fed mosquitoes as controls. (b) Volcano plot of differentially expressed genes in hemocytes from DENV-infected mosquitoes. The blue dots represent genes that are downregulated, and red dots denote genes that are upregulated in the hemocytes of DENV-infected mosquitoes. The X-axis indicates the Log2 of fold change, and the Y-axis indicates the -Log10 of p-value. (c) Scatter plot of KEEG pathway enrichment of upregulated genes in hemocytes of DENV-infected mosquitoes. The circle areas indicate the number of upregulated genes; the color scale indicates the adjusted p-value; X-axis indicates gene ratio (count of core enrichment genes/count of pathway genes). (d) Log2 of -fold change values, and z-score heat map of differentially expressed genes in the hemocytes of DENV-infected mosquitoes. The red color indicates upregulated genes in hemocytes, and the blue color represents downregulated genes. The heat map presents the values of each replicate of hemocytes from DENV-infected (HcDV1-3) and naive (Hc1-3) mosquitoes. All data were obtained from three biological replicates. Each experiment was performed using samples from 150 female mosquitoes. Differentially expressed genes were determined using DESeq2 package; the p-values were adjusted by Benjamini and Hochberg’s test, p ≤ 0.05 were assigned as differentially expressed. (a) was created by Victor Cardoso-Jaime in Microsoft PowerPoint Version 16.92.

Figure 3

Effect of phagocytic hemocyte depletion in dengue virus infection. (a,b) Non-treated [NT], control liposome-injected [LPSMs], and clodrosome-injected [CLDs] mosquitoes were fed on blood containing DENV, and then the viral load (a) and prevalence (b) were evaluated in the midgut at 7 days post-infection. (a,b) n= 57 (NT), n= 63 (LPSMs), n= 62 (CLDs). (c,d) NT, LPSMs, and CLDs mosquitoes were infected by directly injecting the same number of viral particles into the hemocoel. Viral titer (c) and prevalence (d) were then evaluated in the whole mosquitoes at 7 days post-infection. (c,d) n= 48 (NT), n = 48 (LPSMs), n = 48 (CLDs). Viral titers are expressed as a median with a min. to max. of plaque-forming units (PFUs), represented in a box and whiskers graph, where each dot represents an individual sample (midgut or whole mosquito). Statistical differences in viral titers between groups were analyzed using a Kruskal–Wallis test, followed by a Dunn’s multiple comparation test. Prevalence values are expressed as percentages in a bar graph. Statistical analysis of prevalence was performed using a two-sided Fisher’s exact test. *** p = 0.0001; **** p < 0.0001; ns, not significant. Experimental strategy diagrams were created by Victor Cardoso-Jaime in Microsoft PowerPoint Version 16.92.

Figure 4

Analysis of hemocyte capacity to take up nanobeads. (a) Experimental strategy: the basal lamina of tissues are permeable to particles smaller than 10 nm, while DENV and nanobeads are bigger than 10 nm, meaning that the tissues cannot take up nanobeads and viruses, except in the case of cells lacking a basal lamina, such as hemocytes. (b) Mosquitoes were injected with fluorescent nanobeads 30 nm sized, and then hemolymph was perfused to collect the hemocytes at 2, 4, and 24 h post-injection. (c) Images at 2, 4, and 24 h post-injection of hemocytes from mosquitoes injected with nanobeads; hemocytes from mosquitoes injected with the vehicle were used as controls. In blue, the nuclei of cells stained with DAPI; green, 30 nm fluorescent nanobeads; red, CM-Dil (a hemocyte-specific dye); scale bar = 20 μm. Images are representative of three biological replicates (original images (Raw Images) of replicates, Supplementary Materials). Experimental strategy diagrams were created by Victor Cardoso-Jaime in Microsoft PowerPoint Version 16.92.

Figure 5

Permeability of mosquito tissues to virus-sized nanobeads. (a) Experimental strategy for assessing tissue permeability to viruses. Epithelial tissues are surrounded by basal lamina permeable to particles smaller than 10 nm; the diameter of DENV is 50 nm, and the size of the similarly sized, virus-mimicking fluorescent nanobeads used is 30 nm. (b) Mosquitoes were injected with nanobeads, and next tissues were dissected at 2, 4, and 24 h post-injection (all time points are shown in Supplementary Figures S3–S5). (c–e) Representative images of the distribution of fluorescent nanobeads and hemocytes in the abdomen (c), ovaries (d), and midgut (e) at 4 h post-injection. Blue, nuclei stained with DAPI; red, hemocytes stained with CM-Dil; green, 30 nm fluorescent nanobeads; *, air sacs; #, trachea; arrowhead, tracheal end cells; arrows, tracheoles; bar = 25 μm. (f) Model depicting the permeability of epithelial tissues to virus-sized nanobeads. Nanobeads are absorbed by the tracheal end cells, which are located at the ends of the tracheal branches where the tracheoles converge. Circulating and sessile hemocytes also acquire nanobeads. Images are representative of three independent biological replicates (original Images (Raw Images) of replicates, Supplementary Materials). (a,b,f) were created by Victor Cardoso-Jaime in Microsoft PowerPoint Version 16.92.

Figure 6

Immune responses by Ae. aegypti hemocytes. (a) Hypothetical model of complementary functions of immunity-related genes expressed in the fat body and hemocytes. The fat body expresses the genes encoding CLIP-domain proteins (CLIPs) and serpins, which regulate the phenoloxidase (PO) cascade. However, hemocytes produce the precursors of POs, the prophenoloxidases (PPOs). The melanization of pathogens results from the combined function of these genes. The CTLs and LRR genes expressed in the fat body work with the TEP1 produced by hemocytes to activate melanization, the complement-like pathway, or phagocytosis. The fat body expresses the Vago1/2 cytokine-like proteins of the JAK/STAT pathway, and hemocytes highly express several other genes of the JAK/STAT pathway, including DOME, the putative receptor of Vago. Activation of the JAK/STAT pathway protects mosquitoes from viral infections. (b) Hypothetical model of hemocyte antiviral immune responses. Viruses circulating in the hemolymph infect only the hemocytes and tracheae. The tracheae serve as the route of infection for epithelial cells, since epithelial tissues are surrounded by a basal lamina that prevents virus entry by size exclusion. Hemocytes control virus dissemination through viral clearance. When hemocytes acquire the virus, they manage viral infection through the piRNA pathway. (a,b) were created by Victor Cardoso-Jaime in Microsoft PowerPoint Version 16.92.