Tick hemocytes have a pleiotropic role in microbial infection and arthropod fitness

Abstract

Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here we use and develop advanced techniques to describe immune cells (hemocytes) from the clinically relevant tick Ixodes scapularis at a single-cell resolution. We observe molecular alterations in hemocytes upon feeding and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We reveal hemocyte clusters exhibiting defined signatures related to immunity, metabolism, and proliferation. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, two I. scapularis hemocyte markers, impacting blood-feeding, molting behavior, and bacterial acquisition. Mechanistically, astakine alters hemocyte proliferation, whereas hemocytin affects the c-Jun N-terminal kinase (JNK) signaling pathway in I. scapularis. Altogether, we discover a role for tick hemocytes in immunophysiology and provide a valuable resource for comparative biology in arthropods.

Fig. 1. Blood-feeding induces alterations in I. scapularis hemocytes.

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 40–80 pooled ticks per sample). b, d Results are represented as mean ± SD. 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.

Fig. 2. scRNA-seq uncovers hemocytes with immune, proliferative and metabolic signatures in I. scapularis.

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.

Fig. 3. Bacterial infection alters hemocyte subtypes and their gene expression.

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 mean ± SD. A minimum of two independent experiments were conducted. Statistical significance was determined using an unpaired two-tailed t test with Welch’s 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.

Fig. 4. Phagocytic hemocytes contribute to A. phagocytophilum infection and fitness in 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 50 μm. 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 40–80 pooled ticks per sample). d–i Ticks were subjected to microinjection with clodronate (CLD) or empty liposomes (Control) and subsequently fed on either (d-f, h–i) 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 16 s 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 (c–h) mean ± SD or as (i) a percentage from the total. A minimum of two independent experiments were conducted. Statistical significance was evaluated by (c–f) an unpaired two-tailed t test with Welch’s correction, (g–h) two-tailed Mann–Whitney 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.

Fig. 5. Astakine (astk) promotes hemocyte proliferation and differentiation in 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 5 ng rAstk (orange) compared to BSA controls (grey). c–h Ticks were subjected to microinjection with astk siRNA (si-astk; blue) or scrambled RNA (sc-astk; grey) and subsequently fed on either (c–g) 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 16 s rRNA (Ap16S) gene. Results are represented as (a–f, h) mean ± SD 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 Dunnett’s multiple comparisons test; (b–f, h) an unpaired two-tailed t test with Welch’s 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.

Fig. 6. Hemocytin (hmc) positively impacts the JNK pathway in I. scapularis.

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). e–h Ticks were subjected to microinjection with hmc siRNA (si-hmc; blue) or scrambled siRNA (sc-hmc; grey) and subsequently fed on either (e–g) 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 16 s rRNA (Ap16S) gene. Results are represented as mean ± SD. A minimum of two independent experiments were performed. Statistical significance was evaluated by (a, b, d, e) an unpaired two-tailed t-test with Welch’s correction; (f, h) a two-tailed Mann–Whitney 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.