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Mar 13

Tick hemocytes have a pleiotropic role in microbial infection and arthropod fitness – Nature.com

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Blood-feeding induces molecular signatures in I. scapularis hemocytes related to immunity, metabolism, and proliferation

Ticks rely solely on blood as a source of essential metabolites, ingesting ~100 times their body mass per meal19. During feeding, extensive modifications and tissue rearrangements are necessary to accommodate and digest such large volumes of blood19. Considering that hemocytes are the circulating cells in the hemolymph, we hypothesized that these immune cells sense and respond to physiological and microbial exposure during tick infestation on vertebrate hosts. We optimized a protocol to collect hemocytes from I. scapularis nymphs, a clinically relevant stage in the blacklegged tick, and identified three common hemocyte morphotypes reported in the literature (Fig.1a, Supplementary Fig.1)16,17,18. Prohemocytes, considered the stem cell-like hemocyte population, were the smallest cells, with a round or oval shape between 5.7 and 12.5m (average diameter of 8.5m). The cytoplasm was minute (high nuclear/cytoplasmic ratio) and homogeneous, with no apparent protrusions or granules (Supplementary Fig.1a, d). Granulocytes were round or oval shaped cells with diameters ranging from 10.4 to 22.8m (average of 16.7m). The position of the nucleus varied, appearing most often near the periphery of the cell. Their cytoplasm was filled with dark-blue or violet stained granules or vacuoles (Supplementary Fig.1b, d). Plasmatocytes varied in size (ranged from 14.8 to 31.2m, with an average of 21.3m) and shape (oval, ameboid-like, pyriform), and had cytoplasmic protrusions or pseudopodia-like structures. The cytoplasm was clear and had few dark-blue or violet stained granules or vacuoles. The nucleus was located either near the center or the periphery of the cell (Supplementary Fig.1c, d). We then investigated the impact of blood-feeding on hemocytes originating from I. scapularis nymphs. We observed an increased quantity of total hemocytes upon mammalian feeding (Fig.1b). The percentage of plasmatocytes increased in engorged ticks, whereas we noticed a decline in the proportion of prohemocytes and granulocytes in repleted nymphs compared to unfed (Fig.1b). Altogether, we demonstrate the impact of hematophagy on the distribution of tick hemocyte morphotypes.

a Schematic representation of the hemocyte-enriched collection procedure. b Total number of hemocytes (n=40, 25 and 19) and percentages of different morphotypes (prohemocytes, plasmatocytes and granulocytes; n=20, 13 and 17 for all cases) from unfed (ivory), partially fed (light blue) or engorged (dark blue) nymphs. c Functional enrichment analysis of the differentially expressed genes (DEGs) in hemocyte-enriched samples from engorged ticks (blue; Up) compared to unfed ticks (red; Down). Fold enrichment of significant categories (FDR<0.05) is depicted. The number of DEGs per category is shown in parentheses. d The expression of key genes upregulated during feeding in hemocyte-enriched samples from unfed (ivory), partially fed (light blue) and engorged (dark blue) ticks was evaluated by RT-qPCR (n=11, 5 and 7 for all cases; with 4080 pooled ticks per sample). b, d Results are represented as meanSD. At least three independent experiments were performed. Statistical significance was evaluated by Brown-Forsythe ANOVA test, and significant p-values (<0.05) are displayed in the figure. Source data are provided as a Source Data file. 4cl1=4-coumarate-CoA ligase 1; ahcy = adenosylhomocysteinase B; g6pc = glucose-6-phosphatase 2; impdh = inosine-5-monophosphate dehydrogenase 1.

Next, we aimed to examine global transcriptional changes induced by hematophagy through bulk RNA-seq. We collected hemocyte-enriched samples from unfed and engorged nymphs and observed drastic changes in gene expression, with a total of 6134 differentially expressed genes (DEGs) (Fig.1c; Supplementary Data1). Samples from unfed I. scapularis nymphs were enriched for housekeeping genes, including those involved in mRNA transcription (e.g., RNA splicing, mRNA processing, histone modification), protein synthesis (e.g., peptide biosynthetic process, cellular protein modification process, ribosome biogenesis) and membrane receptor signaling pathways (e.g., transmembrane signaling receptor activity, G protein-coupled receptor signaling pathway, protein kinase activity) (Fig.1c; Supplementary Data2). In contrast, hemocyte-enriched samples from engorged ticks exhibited an overrepresentation of gene signatures related to immunity, metabolic pathways, cell proliferation/growth, and arthropod molting/development (Fig.1c; Supplementary Fig.2; Supplementary Data2). Key genes modulated during feeding, including 4-coumarate-CoA ligase 1 (4cl1), adenosylhomocysteinase B (ahcy), glucose-6-phosphatase 2 (g6pc), inosine-5-monophosphate dehydrogenase 1 (impdh), Brahma chromatin remodeling complex subunit osa (osa), runt-related transcription factor 1 (runx) and frizzled-5 (frizzled), were independently validated using RT-qPCR (Fig.1d; Supplementary Fig.3). Notably, the overall expression levels of these genes in hemocyte-enriched samples from partially fed ticks were intermediate compared to those in unfed and engorged ticks, suggesting a transitional phenotype. Collectively, this dataset indicated that I. scapularis hemocytes exhibit a dynamic genetic program during hematophagy.

To uncover whether the transcriptional changes observed through bulk RNA-seq are accompanied with heterogeneity among hemocytes, we performed scRNA-seq. We collected hemocyte-enriched samples from (1) unfed nymphs, (2) engorged nymphs fed on uninfected mice, and engorged nymphs fed on mice infected with either (3) the rickettsial pathogen A. phagocytophilum or (4) the Lyme disease spirochete B. burgdorferi. After stringent quality controls, we profiled a total of 20,432 cells (unfed = 4630; engorged uninfected = 6000; engorged A. phagocytophilum-infected = 6287; engorged B. burgdorferi-infected = 3515), with a median of 744 unique molecular identifiers (UMIs), 261 genes and 6.2% of mitochondrial transcripts per cell across conditions (Supplementary Fig.4). Consistent with the bulk RNA-seq results, the principal component analysis (PCA) identified distinct distributions between unfed and fed conditions, reinforcing the notion that significant cellular and/or transcriptional changes occur following a blood meal (Supplementary Fig.5).

Following unsupervised clustering, we identified seven clusters in unfed ticks and thirten clusters in engorged nymphs (Supplementary Fig.6). Based on similarities in marker gene profiles, two clusters in the unfed and two clusters in the engorged datasets were merged (Supplementary Data3, 4). Thus, six and twelve clusters remained, respectively, with each cluster expressing a unique set of cell type-defining genes (Fig.2ac, Supplementary Data57). One cluster in each dataset showed high expression of gut-associated genes cathepsinB, cathepsinD and boophilinH2 (Fig.2ac, Supplementary Data57) and two additional clusters in the engorged dataset had gene expression profiles indicative of cuticle and salivary glands tissues (Fig.2ac, Supplementary Data5 and 7). Therefore, these clusters were excluded from subsequent analysis. We assigned putative functions to the top 50 DEGs per cluster using information for tick genes in VectorBase, the sequence homology to D. melanogaster in FlyBase and the presence of functionally annotated protein domains in InterPro (Fig.2ce, Supplementary Data6, 7). We also performed functional enrichment analysis based on gene ontology (GO) using the entire list of DEGs for each cluster (Supplementary Data8, 9). Altogether, we defined five hemocyte clusters from unfed and nine clusters in engorged I. scapularis nymphs.

Hemocyte-enriched samples pooled from individual unfed (n=90) or engorged uninfected, A. phagocytophilum- or B. burgdorferi-infected I. scapularis ticks (n=50 for each) were collected immediately post-detachment from the host. t-Distributed Stochastic Neighbor Embedding (t-SNE) plot clustering of samples collected from (a) unfed (4,630 cells) and (b) engorged (15,802 cells) nymphs. The engorged t-SNE includes cells from uninfected (6000 cells), A. phagocytophilum-infected (6287 cells) and B. burgdorferi-infected (3515 cells) ticks. c Dot plot of the top 5 marker genes present in clusters from engorged ticks based on average gene expression. Color intensity demarks gene expression level, while the size of the dot indicates the percentage of cells within individual clusters expressing the corresponding gene. d Heatmap depicting the expression of marker genes for hemocyte subtypes from engorged ticks. The top 20 marker genes per cluster based on gene expression are included, with representative genes highlighted. e The top 50 marker genes from each hemocyte cluster were manually annotated using the VectorBase, FlyBase, and UniProt publicly available databases. The percentage of predicted functional categories, such as ncRNA/pseudogenes (yellow), protein synthesis (black), secreted/extracellular matrix (blue), unknown (orange), actin/cell rearrangement (brown), detoxification (white), cell proliferation/differentiation (grey), metabolism (green), hormone-related (purple), and immunity (red) are shown. f Pseudotime analysis using slingshot defined six hemocyte lineages (indicated by arrows) in engorged ticks. Tick images in (a, b) were created with BioRender.com.

The molecular features and differentiation process of I. scapularis hemocytes is currently unknown. Thus, based on GO enrichment and marker gene profiles, we were able to characterize clusters of hemocytes shared by both unfed and engorged ticks (Supplementary Figs.7, 8, Supplementary Data57). The Immune 1 cluster showed high expression of genes related to phagocytosis or cytoskeleton organization; coagulation and agglutination functions, such as lectins (hemocytin, techylectin-5A), chitin-binding and clotting proteins; and secreted proteins related to immunity, such as astakine, microplusin, mucins and cystatin domain peptides (Fig.2ce, Supplementary Data57). The Immune 2 cluster displayed genes encoding secreted proteins involved in immunity, such as antimicrobial peptides (AMPs) and clotting related peptides (Fig.2ce, Supplementary Data57). The Proliferative 1 cluster was enriched with mitochondrial genes, characteristic of stem cells in ancient arthropods, such as crayfish20. This cell cluster also had high expression of genes related to actin polymerization, cell proliferation and differentiation (Fig.2ce, Supplementary Data57). The Proliferative 2 cluster displayed a high percentage of transcription factors, RNA binding proteins and genes related to actin dynamics. Marker genes for this cluster included several genes involved in hormone-related responses, suggesting they may be responsive to ecdysteroids synthesized after a blood meal (Fig.2ce, Supplementary Data57). Lastly, both datasets had a Transitional cluster indicative of intermediate subtypes (Fig.2ce, Supplementary Data57).

Four hemocyte clusters were only observed in engorged I. scapularis ticks. The Immune 3 cluster displayed an enrichment in secreted proteins and genes related to immune functions. This cluster was enriched for chitinases, matrix and zinc metalloproteinases, peptidases, and actin binding proteins, which have roles related to wound healing or tissue rearrangement. Several glycine-rich proteins (GRPs), commonly associated with antimicrobial properties or structural proteins, were also present (Fig.2ce, Supplementary Data5, 7). The Immune 4 cluster showed an overrepresentation of genes related to protein degradation, immune function, and cell proliferation (Fig.2ce, Supplementary Data5, 7). Thus, we posit that the Immune 4 cluster represents an intermediate state between the Immune 2 and the Proliferative 2 clusters. Two clusters displayed an enrichment for genes related to metabolic functions. The Metabolism 1 cluster represented genes involved in sulfonation of proteins, lipids and glycosaminoglycans, transmembrane solute transporter, nucleotide, and protein metabolism (Fig.2ce, Supplementary Data5, 7). The Metabolism 2 cluster displayed genes related to detoxification, histamine binding, lipid metabolism, methionine and juvenile hormone metabolism (Fig.2ce, Supplementary Data5, 7).

Based on these findings, we predicted hemocyte ontogeny using pseudotime analysis, which orders hemocyte clusters based on their gene expression profiles, enabling us to infer developmental lineages (Fig.2f)21. We found six trajectories considering the cluster Proliferative 1 as a stem cell-like subpopulation. Lineage 1 and 2 ended with the Immune 2 and Proliferative 2 clusters, respectively, with the Immune 4 cluster serving as an intermediate state. Lineages 3 and 4 gave rise to the Immune 3 and Immune 1 clusters, respectively. Finally, two lineage trajectories ended with the metabolic clusters, Metabolism 1 and Metabolism 2. Overall, our findings suggest the presence of an oligopotent subpopulation that differentiates into more specialized subtypes involved in immune and metabolic functions, a process evoked by hematophagy.

The impact of bacterial infection on subtypes of tick hemocytes remains elusive. Thus, we collected hemocytes from I. scapularis nymphs fed on uninfected, A. phagocytophilum- or B. burgdorferi-infected mice and determined morphotype percentages. During infection with the rickettsial agent A. phagocytophilum, a relative decrease in prohemocytes and increase in plasmatocytes was noted (Fig.3a). Conversely, only a slight decrease in the proportion of prohemocytes was observed during infection with the Lyme disease spirochete B. burgdorferi (Fig.3a). No difference in total hemocyte numbers was observed across infection conditions (Supplementary Fig.9). However, partitioning the engorged scRNA-seq datasets by treatments revealed a reduction in the Transitional cluster with an expansion in the Metabolism 1 cluster during B. burgdorferi infection (Fig.3b, c). We next analyzed transcriptional changes at the cellular level in engorged uninfected nymphs compared to engorged infected ticks. Hemocyte clusters were grouped according to three molecular programs: Immune (Immune 1-4), Proliferative (Proliferative 1-2 and Transitional) and Metabolism (Metabolism 1-2). During A. phagocytophilum infection, we identified 177 DEGs within the Proliferative clusters, 53 DEGs in the Metabolism clusters, and 5 DEGs among the Immune clusters (Fig.3d, Supplementary Data10). Conversely, during B. burgdorferi infection, we detected 244 DEGs within the Proliferative clusters, 81 DEGs among the Metabolism clusters, and 12 DEGs associated with the Immune clusters (Fig.3d, Supplementary Data10). In total, we identified 188 and 257 unique DEGs across all hemocyte clusters during A. phagocytophilum and B. burgdorferi infection, respectively. Notably, 11 genes were differentially expressed during both bacterial infections and shared amongst all cluster groupings, in which 36.4% (4 out of 11) were marker genes of the Immune 1 cluster (Fig.3e). Collectively, our results defined specific hemocyte subpopulations and genes that are differentially expressed in I. scapularis in response to A. phagocytophilum or B. burgdorferi infection.

a Hemocyte morphotypes (prohemocytes, plasmatocytes and granulocytes) in I. scapularis nymphs fed on A. phagocytophilum- (Ap, pink) or B. burgdorferi- (Bb, green) infected mice compared to uninfected [(-), dark blue] (n=16 and 12; n=14 and 14, respectively). Results are presented as meanSD. A minimum of two independent experiments were conducted. Statistical significance was determined using an unpaired two-tailed t test with Welchs correction, and significant p-values (<0.05) are displayed in the figure. b t-Distributed Stochastic Neighbor Embedding (t-SNE) plot clustering of cells collected from the hemolymph of uninfected (6000 cells), A. phagocytophilum- (6287 cells) or B. burgdorferi-infected (3515 cells) I. scapularis nymphs. c Percentage of hemocyte clusters in uninfected, A. phagocytophilum- or B. burgdorferi-infected ticks. d Venn diagram illustrating the number of differentially expressed genes (DEGs) between groups of hemocyte clusters during A. phagocytophilum (top) or B. burgdorferi (bottom) infection compared to uninfected ticks. Hemocyte clusters were categorized into three molecular programs: Immune (Immune 1-4), Proliferative (Proliferative 1-2 and Transitional) and Metabolism (Metabolism 1-2). DEGs were determined using pairwise comparisons against uninfected. e Heatmap representing the average expression patterns of DEGs altered during infection and shared between all 3 cluster groups. For each DEG, the mean average across all experimental conditions was centered at zero for each hemocyte group. The # symbol indicates Immune 1 marker genes. Source data are provided as a Source Data file. Tick images in (b) were created with BioRender.com. (-)=Uninfected. Anaplasma=A. phagocytophilum. Borrelia=B. burgdorferi.

The preceding findings suggested that the Immune 1 hemocyte cluster represented a subpopulation of cells that responded to bacterial infections in ticks (Fig.3d). Therefore, our focus shifted to two marker genes from the Immune 1 hemocyte cluster: hemocytin and astakine (Fig.4a, Supplementary Data6, 7). Hemocytin is homologous to hemolectin in D. melanogaster and von Willebrand factors of mammals22,23. Hemocytin encodes for a large multidomain adhesive protein involved in clotting, microbial agglutination and hemocyte aggregation22,23,24,25. Conversely, astakine is a cytokine-like molecule present in chelicerates and crustaceans and is homologous to vertebrate prokineticins26,27,28,29. Astakine also induces hemocyte proliferation and differentiation of immune cells26,27,28,29. Hemocytin and astakine were broadly expressed in the Immune 1 cluster of I. scapularis hemocytes (Fig.4a). Validating our scRNA-seq results, we confirmed the expression of hemocytin and astakine in hemocyte-enriched samples obtained from unfed ticks using RNA-FISH, illustrating that these genes serve as markers for the Immune 1 cluster (Fig.4b, Supplementary Fig.10). Furthermore, we noted an upregulation of hemocytin and astakine in hemocyte-enriched samples collected from engorged ticks, a pattern also observed in other markers of the Immune 1 cluster (Fig.4c, Supplementary Data11). These findings suggested that blood-feeding expanded this hemocyte subtype and/or upregulated the expression of its marker genes in I. scapularis ticks.

a Expression patterns of hemocytin (left) and astakine (right) on t-Distributed Stochastic Neighbor Embedding (t-SNE) plots of hemocyte-enriched samples from engorged nymphs. Their highest expression is denoted in the Immune 1 cluster (outlined). b RNA FISH image of I. scapularis hemocytes labeled for hemocytin (hmc, green), astakine (astk, red), and nuclei (DAPI). White scale bars indicate a length of 50m. Refer to Supplementary Fig.10 for control images. c RT-qPCR evaluation of hemocytin (hmc; n=9 and 6) and astakine (astk; n=9 and 6) expression in hemocyte-enriched samples collected from unfed (ivory) or engorged (dark blue) ticks (with 4080 pooled ticks per sample). di Ticks were subjected to microinjection with clodronate (CLD) or empty liposomes (Control) and subsequently fed on either (d-f, hi) uninfected or (g) A. phagocytophilum-infected mice. d Total hemocyte counts (n=9 and 9) and (e) morphotype percentages (prohemocytes, plasmatocytes and granulocytes; n=8 and 9 in all cases) were assessed in the hemolymph of individual ticks. f RT-qPCR analysis of hemocytin (hmc; n=18 and 20) and astakine (astk; n=18 and 20) expression, and (g) A. phagocytophilum load in individual ticks (n=28 and 20). Bacterial quantification was based on the expression of A. phagocytophilum 16s rRNA (Ap16S) gene. h Weight measurements of engorged nymphs (n=34 and 25). i Percentage of nymphs that molted to adults. Results are represented as (ch) meanSD or as (i) a percentage from the total. A minimum of two independent experiments were conducted. Statistical significance was evaluated by (cf) an unpaired two-tailed t test with Welchs correction, (gh) two-tailed MannWhitney U test or (i) by a Fisher exact test, and significant p values (<0.05) are displayed in the figure. Source data are provided as a Source Data file.

Hemolectin is a known marker of phagocytic plasmatocytes in Drosophila30,31,32, and phagocytic hemocytes expressing hemocytin are present in crustaceans20,33,34. Therefore, we explored the phagocytic potential of the Immune 1 cluster by employing clodronate liposomes (CLD), which have been used to deplete phagocytic hemocytes in flies, mosquitoes, and, more recently, ticks35,36,37,38. We found that CLD treatment led to a 36% reduction in the total number of hemocytes in I. scapularis nymphs (Fig.4d). We observed an increase in the proportion of granulocytes along with a decrease in the percentages of prohemocytes and plasmatocytes compared to ticks treated with empty liposomes (Fig.4e). Interestingly, the expression of both Immune 1 marker genes, hemocytin and astakine, also decreased after CLD treatment, implying the Immune 1 cluster is phagocytic (Fig.4f). Taken together, CLD treatment effectively reduced the number of phagocytic hemocytes in I. scapularis, particularly impacting prohemocytes, plasmatocytes and cells associated with the Immune 1 cluster.

Phagocytosis serves as a pivotal immune mechanism against invading microbes. Previous studies have demonstrated that the depletion of phagocytic immune cells can influence the survival of arthropods following infection with either Gram-positive or Gram-negative bacteria35,36,37,38. However, the impact of depleting phagocytic hemocytes on the acquisition of tick-borne bacteria remains unclear. Thus, we further investigated the effects of CLD treatment during A. phagocytophilum infection, as this intracellular microbe is known to interact with I. scapularis hemocytes shortly after uptake39. Our findings revealed a reduction in A. phagocytophilum load in engorged ticks after injection with CLD compared to controls (Fig.4g), with no differences in tick attachment (Supplementary Fig.11), suggesting that phagocytic hemocytes promote either the acquisition or proliferation of A. phagocytophilum during blood-feeding.

Reports on Dipteran model organisms have demonstrated that hemocytes play roles beyond immunity, including tissue communication, clearing apoptotic cells during molting and development, and serving as vehicles for molecules40,41,42,43,44. However, whether hemocytes have non-immune roles in ticks remains unknown. Our sequencing results revealed an enrichment in functions related to metabolism, cell proliferation, and development after a blood meal, suggesting that hemocytes may participate in the feeding or molting processes in ticks (Figs.1, 2). To investigate this further, we recorded these physiological processes after injecting ticks with CLD. Although no differences in attachment were observed (Supplementary Fig.11), engorged ticks treated with CLD weighed significantly less compared to the control treatment, indicating that phagocytic hemocytes are required for proper hematophagy (Fig.4h). Furthermore, the number of nymphs that successfully molted to adults was significantly lower in CLD-treated ticks (Fig.4i). Collectively, these results indicated that phagocytic hemocytes pleiotropically impacted tick immunity, feeding, and molting in I. scapularis. Importantly, the observed correlation between changes in physiological parameters and the reduced expression of the Immune 1 markers, hemocytin and astakine, during CLD treatment suggested a potential regulatory role for these genes in various aspects of tick physiology, prompting us to explore this hypothesis further.

We found that hematophagy induces hemocyte proliferation and differentiation (Fig.1b) while upregulating astakine expression (Fig.4c). Thus, we investigated whether astakine was directly implicated in the proliferation or differentiation of I. scapularis hemocytes. We microinjected increasing amounts of recombinant astakine (rAstk) in unfed nymphs and observed a dose-dependent increase in the total number of hemocytes (Fig.5a). Specifically, we measured a decrease in the percentage of prohemocytes and an increase in plasmatocytes (Fig.5b). These findings matched alterations observed during normal blood-feeding (Fig.1b). Interestingly, we also detected an increase in tick IDE12 cell numbers in vitro following treatment with rAstk, supporting a role of astakine in inducing cell proliferation in hemocyte-like cells (Supplementary Fig.12). To corroborate these results, we then silenced astakine by microinjecting unfed nymphs with small-interfering RNA (siRNA) before feeding on uninfected mice. We utilized siRNA to knockdown gene expression, as genome editing through CRISPR has only recently been introduced for adult germline manipulation and possesses low efficiency in ticks45. We recovered 41% less hemocytes and observed an increase in the percentage of prohemocytes with a decrease in plasmatocytes from engorged ticks when astakine was silenced (Fig.5ce). Therefore, we determined that astakine acts on hematopoietic processes in the ectoparasite I. scapularis.

a Total hemocyte counts in the hemolymph of unfed I. scapularis nymphs subjected to microinjection with increasing amounts of rAstk (orange) or BSA (grey) as a control (n=21, 14, 14 and 24). b Percentage of hemocyte morphotypes (prohemocytes, plasmatocytes and granulocytes; n=10 and 10 in all cases) in the hemolymph of unfed nymphs following microinjection with 5ng rAstk (orange) compared to BSA controls (grey). ch Ticks were subjected to microinjection with astk siRNA (si-astk; blue) or scrambled RNA (sc-astk; grey) and subsequently fed on either (cg) uninfected or (h) A. phagocytophilum-infected mice. c Efficacy of astk silencing (n=20 and 16), (d) total number of hemocytes (n=9 and 8) and (e) percentage of hemocyte morphotypes in individual ticks (n=12 and 10 in all cases). f Weight measurements of engorged nymphs (n=29 and 19). g Percentage of nymphs that molted to adults. h RT-qPCR assessment of astk silencing efficiency (n=16 and 15) and A. phagocytophilum load (n=18 and 14) in individual infected ticks. Bacterial quantification was based on the expression of A. phagocytophilum 16s rRNA (Ap16S) gene. Results are represented as (af, h) meanSD or as (g) a percentage from the total. A minimum of two independent experiments were conducted. Statistical significance was evaluated by (a) one-way ANOVA with Dunnetts multiple comparisons test; (bf, h) an unpaired two-tailed t test with Welchs correction or (g) by a Fisher exact test, and significant p-values (<0.05) are displayed in the figure. Source data are provided as a Source Data file. rAstk = recombinant astakine; BSA = bovine serum albumin.

Our previous results showed alterations in the percentage of hemocytes by the CLD treatment, which influenced bacterial infection and tick physiology. Given that astakine also alters hemocyte composition in ticks, we then investigated whether decreasing the expression of astakine affects A. phagocytophilum infection, hematophagy or ecdysis in I. scapularis. Accordingly, we observed a significant reduction in weight, molting success, and A. phagocytophilum burden in ticks silenced for astakine compared to the control treatment, without differences in tick attachment (Fig.5fh; Supplementary Fig.13). Corroborating these findings, A. phagocytophilum load was also lower in astakine-silenced tick cells in vitro (Supplementary Fig.14). Overall, we demonstrated that astakine regulates hemocyte composition in I. scapularis, which affects not only bacterial infection but also feeding and ecdysis, supporting a pleiotropic role of hemocytes in ticks.

The broad expression of hemocytin detected in I. scapularis hemocytes (Fig.4a), and its homology to a Drosophila gene used as a marker of phagocytic immune cells, prompted us to explore whether hemocytin could be involved in hemocyte proliferation or differentiation, phagocytosis or immune signaling in ticks. Surprisingly, no differences were observed in hemocyte numbers or subtype proportions between ticks injected with siRNA targeting hemocytin, nor in the phagocytic capacity of hemocytin-silenced tick cells (Supplementary Figs.15, 16). We then asked whether hemocytin could act as an immune pathway regulator in I. scapularis. We focused on the IMD and the c-Jun N-terminal kinase (JNK) pathways, as previous reports indicated the importance of these molecular networks during bacterial infection of ticks5,6.

After transfection with siRNA targeting hemocytin, we found a decrease in JNK phosphorylation in hemocytin-silenced tick cells, without alteration in Relish cleavage (Fig.6a, b). To complement our findings, we overexpressed hemocytin in tick cells through CRISPR activation (CRISPRa). CRISPRa has been widely used to enhance the expression of an endogenous locus, employing a catalytically inactive Cas9 (dCas9) fused with transcription activators and single guide RNAs (sgRNAs) that direct the modified enzyme to the promoter region of a gene of interest46. However, so far none of the CRISPRa effectors have been tested in tick cell lines and no endogenous promoter has been identified for genetic expression of sgRNAs. We optimized reagents and developed a protocol to up-regulate hemocytin in ISE6 cells using two rounds of nucleofection with different expression plasmids. The first plasmid expressed dCas9-VPR paired with neomycin resistance under the control of the CMV promoter. The second plasmid expressed either a sgRNA specific to the promoter region of hemocytin (hmc-sgRNA) or a control sgRNA (ctrl-sgRNA) driven by an endogenous RNA polymerase III promoter (Fig.6c). Tick ISE6 cells were used as a platform for CRISPRa given the lower expression of hemocytin compared to IDE12 cells (Supplementary Fig.17). Strikingly, we detected a 277% increase in the expression of hemocytin in dCas9+ cells transfected with the hmc-sgRNA compared to dCas9+ cells transfected with the ctrl-sgRNA (Fig.6d). Importantly, we also noticed elevated levels of both jun (the transcription factor for the JNK pathway) and jnk expression (Fig.6d).

a, b Tick cells were transfected with either hmc siRNA (si-hmc) or scrambled RNA (sc-hmc). a Efficiency of hmc silencing in IDE12 cells (n=12 and 11). b Representative western blot (left) of N-Rel and p-JNK during treatment with sc-hmc (lane 1) or si-hmc (lane 2). N-Rel and p-JNK protein expression was quantified (right) in si-hmc (blue) or sc-hmc (grey) IDE12 cells (n=4). Data were normalized to the scrambled control, with N-Rel values relative to Actin and p-JNK values to JNK. A representative blot from four experiments is shown. Uncropped blots containing the molecular weight markers are supplied in the Source data. c Overview of CRISPRa-mediated hmc overexpression in ISE6 cells. d RT-qPCR analysis of hmc (left; n=9 and 10), jnk (middle; n=9 and 9) and jun (right; n=10 and 10) expression in dCas9+ ISE6 cells transfected with either a single guide RNA (sgRNA) specific to the promoter region of hemocytin (hmc-sgRNA, blue) or a random sgRNA (ctrl-sgRNA, grey). eh Ticks were subjected to microinjection with hmc siRNA (si-hmc; blue) or scrambled siRNA (sc-hmc; grey) and subsequently fed on either (eg) uninfected or (h) A. phagocytophilum-infected mice. e RT-qPCR analysis of hmc (left; n=19 and 18), relish (middle; n=17 and 18) and jun (right; n=17 and 18) expression in engorged ticks. f Weight measurements of engorged nymphs (n=20 and 23). g Percentage of nymphs that molted to adults. h RT-qPCR assessment of hmc silencing efficiency (n=11 and 10) and A. phagocytophilum load (n=11 and 10) in individual infected ticks. Bacterial quantification was based on the expression of A. phagocytophilum 16s rRNA (Ap16S) gene. Results are represented as meanSD. A minimum of two independent experiments were performed. Statistical significance was evaluated by (a, b, d, e) an unpaired two-tailed t-test with Welchs correction; (f, h) a two-tailed MannWhitney U test or (g) by a Fisher exact test, and significant p-values (<0.05) are displayed in the figure. Source data are provided as a Source Data file. N-Rel = cleaved Relish; p-JNK = phosphorylated JNK; JNK = c-Jun N-terminal kinase.

To corroborate our results in vivo, we microinjected unfed ticks with a scrambled control or siRNA targeting hemocytin and allowed them to feed on nave mice. Upon repletion, we measured the expression of jun and relish. Consistently, we found that reduction in hemocytin expression led to a decrease in jun levels in ticks, without affecting relish expression (Fig.6e), uncovering a role for hemocytin in the activation of the JNK pathway in I. scapularis.

Finally, building on our previous findings demonstrating that blood-feeding and infection alter the expression of hemocytin in ticks (Fig.4c, Supplementary Data9), coupled with the documented roles of hemocytin as an agglutinating factor and the JNK pathway in enhancing organismal growth and metabolism in insects47, we delved deeper into the relationship between hemocytin expression and physiological parameters in I. scapularis. We detected a decrease in weight and molting success to adulthood in hemocytin-silenced ticks that fed on uninfected mice compared to scrambled controls, with no differences in tick attachment (Fig.6f, g; Supplementary Fig.18). Additionally, we noted that silencing hemocytin increased A. phagocytophilum load both in vivo and in vitro (Fig.6h; Supplementary Fig.19), supporting an antimicrobial role for hemocytin. Collectively, our findings indicated that tick hemocyte subtypes and their associated marker genes play a critical role in I. scapularis immunophysiology.

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