Tick extracellular vesicles alter keratinocyte function in the skin epidermis

Abstract

Wound healing has been extensively studied through the lens of inflammatory disorders and cancer, but limited attention has been given to hematophagy and arthropod-borne diseases. Hematophagous ectoparasites, including ticks, subvert the wound healing response to maintain prolonged attachment and facilitate blood-feeding. Here, we unveil a strategy by which extracellular vesicles (EVs) ensure blood-feeding and arthropod survival in three medically relevant tick species. Through single cell RNA sequencing and murine genetics, we demonstrate that wildtype animals infested with EV-deficient Ixodes scapularis display a unique epidermal sub-population with a mesenchymal-like transcriptional program and an overrepresentation of pathways connected to wound healing. Furthermore, tick EVs inhibit proliferation and diminish the capacity of wound closure in keratinocytes. This occurrence was linked to phosphoinositide 3-kinase activity, keratinocyte growth factor 1 (KGF-1) and transforming growth factor β (TGF-β) levels. Collectively, we uncovered a strategy employed by a blood-feeding arthropod that disrupts the circuitry in cutaneous wound healing, contributing to ectoparasite fitness.

Figure 1:. Tick EVs affect hematophagy and survival.

(a) Graphical illustration of experimental design. (b-m) Vamp33 siRNA (siV33) (red) or vamp33 scramble control (scV33) (blue) microinjected nymphs were placed on C57BL/6 mice and allowed to feed for 3 days. On day 3, ticks were harvested and assessed for fitness measurements. Efficiency of Vamp33 silencing, tick attachment, weight, and survival curves for (b-e) I. scapularis, (f-i) A. americanum and (j-m) D. variabilis. Graphs represent at least three independent experiments combined. Statistical significance shown as *p<0.05, **p<0.01, *** p<0.001 ****p<0.0001. (b, f, j, d, h, l) Mann Whitney test with results presented as the mean +/− SD; (c, g, k) Fisher’s exact test and (e, i, m) Log-rank (Mantel-Cox) test. ns = not significant.

Figure 2:. Tick EVs alter epidermal immune surveillance.

(a) Schematic representation of the DETC-keratinocyte crosstalk at the skin epidermis. (b-f, h) I. scapularis scV33 (blue) or siV33 (red) ticks were placed on C57BL/6 mice and allowed to feed for 3 days. On day 3, biopsies were taken from the skin at the bite site and compared to the naïve treatment (gray). (b) DETC (Vγ5), (c) JAML, (d) NKG2D, (e) CD69, (f) CD25, and (h) CD100 cells were assessed by flow cytometry. Graphs represent 1 of 3 independent experiments. (g) Epidermis containing Langerhans cells (red), DETCs (green), and keratinocytes (white) imaged on day 3 after injection with phosphate buffered saline (PBS - mock) or EV (4×10⁷ particles) into the mouse ear. Cytochalasin D (100 μg) was applied topically on the mouse ear every 24 hours for 2 days to induce DETC rounding as a positive control. Langerhans cells, DETCs and epithelial cells were simultaneously visualized in the huLangerin-CreER; Rosa-stop-tdTomato; CX3CR1-GFP^+/−; K14-H2B-Cerulean mouse strain. Cre expression was induced with an intraperitoneal injection of tamoxifen (2 mg). The size of the scale bar represents 50 μm. Images from one out of three independent experiments. Statistical significance shown as *p<0.05, ns = not significant. Data are presented as a mean +/− SD. Significance was measured by One-way ANOVA followed by Tukey’s post hoc test.

Figure 3:. Epidermally-enriched scRNA-seq of the tick bite site.

(a) Overview of the experimental design. ScV33 and siV33 I. scapularis nymphs were placed on FVB-Jackson (FVB-Jax) or FVB-Taconic (FVB-Tac) mice and fed for 3 days. Skin biopsies at the bite site were digested with dispase and collagenase for epidermal cell isolation. Cells were sorted and prepared for scRNA-seq. (b) Composite tSNE plot of keratinocyte, T cell and antigen presenting cells in FVB-Jax and FVB-Tac mice in the presence or absence of I. scapularis nymphs microinjected with scV33 or siV33. tSNE plot represents 5,172 total cells following filtration as described in the materials and methods. (c) Heatmap depicting expression of the top 5 marker genes present in clusters from the epidermally enriched tSNE plot clusters (as shown in b). (d-i) Individual tSNE plots separated by mouse strain (FVB-Jax or FVB-Tac) in the presence or absence of I. scapularis nymphs microinjected with scV33 or siV33.

Figure 4:. Tick EVs impacts the keratinocyte transcriptional program.

(a) Composite tSNE plot of keratinocytes in FVB-Jax and FVB-Tac mice in the presence or absence of I. scapularis nymphs microinjected with scV33 or siV33. (b) Dot plot of the top 5 marker genes present in the keratinocyte clusters (as shown in a). Average gene expression is demarked by the intensity of color. Percent of gene expression within individual clusters is represented by the dot diameter. (c) Cells colored by clusters originated from the keratinocyte tSNE plot (as shown in a) ordered across pseudotime (x-axis) for naïve, scV33-, and siV33-tick bites of FVB-Jax and FVB-Tac mice. (d) Expression of Col1a1 across treatments ordered across pseudotime (x-axis) for naïve, scV33-, and siV33-tick bites of FVB-Jax and FVB-Tac mice. (e) Enriched pathways in the unidentified cell cluster based on functional annotation in DAVID. Fold enrichment is indicated in a Log2 scale. *p value and false discovery rate (FDR)<0.05 were set as threshold. KEGG, GO and InterPro were used as reference annotation databases. (f) The “AddModuleScore” function was used to generate an epithelial-to-mesenchymal transition (EMT) score across all keratinocytes based on 102 epithelial-to-mesenchymal transition genes. Selected genes were reported as indicative of keratinocytes in intermediary or mesenchymal transitioning states. (g) Ingenuity pathway analysis comparing keratinocytes of skin biopsies from FVB-Jax siV33 to FVB-Jax scV33. Blue denotes pathways predicted to be inhibited (negative z-score) whereas orange indicates pathways predicted to be activated (positive z-score) based on default parameters. Differential expression datasets were assessed for canonical pathway analysis. Results are shown in a −log (p-value) scale. *p value and FDR< 0.05 were set as threshold. (h) Volcano plot of genes representing the wound healing signaling pathway in keratinocytes of FVB-Jax siV33 compared to FVB-Jax scV33 datasets (highlighted in yellow; g). Blue denotes decrease whereas red indicates increase in the coefficient (coef) of expression. (i) Ingenuity pathway analysis derived from siV33 compared to the bite of scV33 ticks on FVB-Jax or FVB-Tac mice. Canonical pathways predicted to be inhibited (blue, negative z-score) or activated (orange, positive z-score) based on differential expression profile. The solid line indicates the p value significance threshold of 0.05 (−log=1.3). (j) The signaling cascade of EIF2 (highlighted in yellow, i), yielding (→) or inhibitory (┤) arrows. Orange indicates activation whereas blue shows inhibition according to the IPA prediction. Gene expression based on the scRNA-seq experiment is indicated in red (increased) or green (decreased). Gray – denotes no expression or prediction.

Figure 5:. Tick EVs disrupt the cutaneous wound healing circuitry in vivo.

(a) ScV33 (circle) or siV33 (square) injected I. scapularis nymphs were fed on FVB-Jax (white) or FVB-Tac (gray) mice for 3 days. Biopsies were taken from the skin at the bite site and assessed for EpCAM⁺ Ki67⁺ keratinocytes by flow cytometry. (b-i) ScV33 or siV33 ticks fed on C57BL/6 mice for 3 days. Biopsies were taken from the skin at the bite site and processed for flow cytometry and ELISA analysis. (b) EpCAM⁺ Ki67⁺ keratinocytes assessed by flow cytometry. Graph displays proliferation changes within the scV33 or siV33 treatments compared to the naïve skin. (c-d) Flow cytometry histogram plots of EpCAM⁺ Ki67⁺ keratinocytes. (c) scV33 or (d) siV33 treatments displayed according to the number of ticks bitten per biopsy. X-axis shows fluorescence intensity, and the Y-axis indicates the count of events in the fluorescence channel. (e) PI3K p85⁺, and (f) phospho-PI3K p85/p55⁺ keratinocytes were assessed by flow cytometry within the scV33 or siV33 treatments compared to the naïve skin. (g-h) ELISA analysis of (g) KGF-1 and (h) TGF-β levels normalized to total protein per 5 mm skin punch biopsy. (i) EpCAM⁺ Smad7⁺ keratinocytes assessed by flow cytometry. (a-b, e-i) Significance was measured by One-way ANOVA followed by Tukey’s post hoc test. All experiments have statistical significance shown as ***p<0.001, **p<0.01, *p<0.05, ns = not significant. Data are presented as the mean +/− SD.

Figure 6:. Tick EVs inhibit keratinocyte gap closure and proliferation.

Human keratinocytes (HaCaT) monolayers were inflicted a scratch wound and stimulated with I. scapularis EVs. (a) Experimental design. (b) HaCaT monolayers were imaged at 0, 12 and 24 hours. Dashed line denotes scratch area, and images are representative of medium (−) or EV stimulated (10⁶/mL) cells. (c) Scratch area was quantified using ImageJ software and the percentage of gap closure at each time point was determined based on the original area of scratch inflicted at 0 hours for each treatment. The size of the scale bar represents 400 μm. Significance was measured by non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons test. (d) Cells were treated with (−) medium, (+) mitomycin C or EVs. Ki67⁺ keratinocytes were assessed by flow cytometry 24 hours post-scratch infliction. Significance was measured by One-way ANOVA followed by Tukey’s post hoc test. All experiments have statistical significance shown as ****p<0.0001, **p<0.01, *p<0.05. Data are presented as the mean +/− SD.