Abstract Tick-associated diseases present challenges due to tridirectional interactions among host-specific responses, tick toxins and salivary proteins as well as microbes. We aimed to uncover molecular mechanisms in tick-bitten skin samples (cases) and contralateral skin samples (controls) collected simultaneously from the same participants, using spatial transcriptomics. Cases and controls analysed using NanoString GeoMx Digital Spatial Profiler identified 274 upregulated and 840 downregulated differentially expressed genes (DEGs), revealing perturbations in keratinization and immune system regulation. Samples of skin biopsies taken within 72 h post tick-bite DEGs had changes in protein metabolism and viral infection pathways as compared to samples taken 3 months post tick-bite, which instead displayed significant perturbations in several epigenetic regulatory pathways, highlighting the temporal nature of the host response following tick bites. Within-individual signatures distinguished tick-bitten samples from controls and identified between-individual signatures, offering promise for future biomarker discovery to guide prognosis and therapy. Keywords: Emerging diseases, Tick-borne diseases, Tick-host-pathogen, Spatial profiling, Spatial transcriptomics 1. Introduction Climate change and human usage of natural habitats are expected to increase tick-borne diseases (TBDs), which the Centers for Disease Control and Prevention of the United States of America have already described a two-fold spike within a span of 13 years, representing 77 % of all reported vector-borne diseases (VBDs) [[55]1,[56]2]. Throughout the world, tick-borne microbial pathogens (TBPs) such as bacteria, viruses and protozoa, have been rigorously investigated using the guidelines of Koch's postulates and have confirmed the causative agents of many TBDs [[57][3], [58][4], [59][5]]. In the last 50 years, considerable success has been achieved in identifying TBPs [[60]6], understanding tick envenomation [[61]7], and in the understanding of specific hypersensitivity reactions [[62]8]. Significant gaps in knowledge remain, however, and there are still tick-associated pathologies that have yet to be determined because current methodologies rely predominantly on the detection of TBPs in patients presenting tick-associated symptoms [[63]9,[64]10]. Ill-defined tick-associated illness, such as post-treatment Lyme disease syndrome (PTLDs) [[65]11] and Debilitating Symptom Complexes Attributed to Ticks (DSCATT) [[66]12], comprise heterogenous symptoms that can persist long after tick bite despite no detection of a pathogen. In Australia, there is an increasing urgency to bridge the knowledge gaps because there are many individuals suffering from tick-associated illnesses resembling Lyme disease symptomology but no evidence of Borrelia burgdorferi, the causative pathogen) [[67]12]. Host-tick and/or host-microbe interactions in the tick-bitten skin represent the initiating epicentre of the bite and can affect the trajectory of pathogenesis [[68][13], [69][14], [70][15], [71][16], [72][17], [73][18]]. Specifically, the skin is an important site of the complex and dynamic interactions between host-specific defences and territorial invasion by the biting arthropod and introduced pathogens [[74]13,[75]14,[76]17]. For example, the host adaptive immune response can be activated to elicit tick immunity, causing the arthropod to disengage prematurely (before feeding to repletion) and by mobilising immune cells into the bite site to phagocytose inoculated pathogens [[77]16,[78]19,[79]20]. However, the host immune response has also been shown to be modulated by tick saliva as part of an “arms race” to prevent disruption to the tick's blood meal, consequently enabling inoculated TBPs to replicate in resident skin cells, such as keratinocytes and epidermal dendritic cells [[80]13,[81]14,[82]17]. Indeed, the ability of ticks to dampen host immune responses and to evade immune detection, allowing the host to remain asymptomatic, has rendered the tick-bitten skin a biological reservoir for TBPs such as B. burgdorferi and tick-borne encephalitis virus [[83]18,[84]21]. The species-specific and host-specific nature of tick-mediated immune modulation and immune responses, respectively, further complicate the nature of the interaction at the tick bite site, resulting in heterogeneity in pathology and symptomology [[85]13]. Investigating host perturbations at the tick-bitten skin, which has thus far been inadequately studied, may add fresh knowledge of these processes and likely uncover molecular targets that have the potential to be exploited to benefit the human. For this technical pilot study, tick bitten participants were enrolled in Western Australia during the 2021/2022 tick season (spring and summer) and consented to provide skin samples for spatially resolved, untargeted transcriptomic analysis. Paired biopsies (at tick bite site and the unaffected corresponding contralateral site) presented a unique opportunity to investigate the pathophysiology of tick bites in the skin in comparison to healthy skin obtained from the same individual. Apart from elucidating between-individual heterogeneity in immune responses, tick bite differentially expressed genes (DEGs) and enriched pathways, our approach also allowed detection of spatially and temporally resolving host specific disturbances. The findings of this technical pilot study confirmed that the methodology could be applied in future studies with larger sample size to pinpoint pathogenic mechanisms as well as identify possible targets for diagnosis, prognosis, or therapeutic interventions. 2. Results 2.1. Participant cohort and power considerations In total, seven participants were enrolled. Four males and one female bitten by a tick in the preceding 72 h (‘acute’ bite) were enrolled for the technological pilot cohort and volunteered paired biopsies and blood samples. An additional female participant (AHP), enrolled under the same conditions as the main cohort, declined a control biopsy after the biopsy at the tick bite site was collected and was enrolled into sub-cohort 1. Furthermore, participant (AJK) who was bitten three months prior (long-term response) was enrolled into sub-cohort 2. The meta-data of all seven participants are summarised in [86]Table 1. All biting ticks were identified as Amblyomma triguttatum. With respect to the taxa of interest (TOI), bacterial 16S rRNA sequencing of the ticks revealed DNA from the genus Rickettsia and Francisella, as expected, in addition to other genera comprising the tick's microbiome. Only three tick-bitten skin biopsies had detectable DNA from the family Rickettsiaceae, and among them, two could be taxonomically classified within the genus Rickettsia ([87]Table 1). No other TOI were detected in the skin biopsies. Table 1. Tick-bitten participants details. Patient ID Main Cohort __________________________________________________________________ Sub-cohort 1 No control biopsy __________________________________________________________________ Sub-cohort 2 long-term response __________________________________________________________________ AHE AHL AHN AHR AJS AHP AJK Sex M M F M M F M Age 22 58 56 67 65 53 68 Local symptoms No No 1. Redness around the bite 1. Redness around the bite 1. Redness around the bite 1. Redness around the bite 1. Redness around the bite 2. Itchiness 2. Itchiness 2. Itchiness 2. Itchiness 3. Occasional discharge at tick bite site (clear or cloudy fluid, slightly blood stained at times) Generalised symptoms No No No “I appear to develop gut discomfort following a tick bite.” No No No Past tick bites 5+ 4 5 4 5+ 5+ 5+ Tick attachment location Abdomen Back/shoulders Back/shoulders Arm/armpit Legs Head/face Arm/armpit Tick attachment time 6–24 h >24 h >24 h >24 h >24 h >24 h 6–24 h (3 months previously) Tick species Amblyomma triguttatum Amblyomma triguttatum Amblyomma triguttatum Amblyomma triguttatum Amblyomma triguttatum Amblyomma triguttatum Not available Tick instar Nymph Adult female Nymph Adult male Larvae Nymph Not available CRP (mg/L) 2 8 36 <1 4 5 1 Haematology & Blood Chemistries interpretation no significant pathology no significant pathology no significant pathology no significant pathology pathological liver changes associated with tick bite. no significant pathology no significant pathology Serology to bacteria: interpretation no significant serology pre-existing antibodies to Spotted Fever Group Rickettsia. Seroconversion to Coxiella burnetii at 3 months post tick-bite no significant serology no significant serology no significant serology no significant serology no significant serology Serology to known tick virus Negative Negative Negative Weak positive at 1-week post-tick bite Negative Negative Negative MAVRIC on tick patient culture Negative Weak positive Negative Negative Negative Negative Negative Bacterial TOI in tick-bite skin Nil Rickettsiaceae Rickettsia Nil Rickettsia N/A Nil Bacterial TOI in control skin Nil Nil Nil Nil Nil N/A Nil Bacterial TOI in biting tick Rickettsia Francisella Rickettsia Francisella Rickettsia Francisella Rickettsia Francisella Rickettsia Francisella Rickettsia Francisella N/A [88]Open in a new tab ^aTaxa of interest, TOI; ^bmonoclonal antibodies to viral RNA intermediates in cells, MAVRIC; ^cnot applicable, N/A; ^dC reactive protein, CRP; ^edouble-stranded, ds; antibodies not detected, Negative; no DNA of TOI detected, Nil. Furthermore, we note that with our approach, where each participant is contrasted to themselves, statistically significant changes should be detectable in fewer than five participants [[89]22,[90]23]. Thus, while we consider this pilot of spatial genomics applied to tick bites strictly exploratory, it is likely to provide sufficiently robust insight to assess the feasibility of this approach for future studies. 2.2. Cellular infiltrates in tick bitten skin Visual comparison of H&E stains between paired sections (tick-bitten cases and contralateral controls) showed that tick-bitten sections had significant cellular infiltrates and increased cellular density when compared to the healthy controls ([91]Fig. 2, [92]Fig. 3). In the haematoxylin-rich staining in the dermal area close to the epidermis, depicted in [93]Fig. 3d, the cellular infiltrate appeared to be dominated by CD45 leukocytes and CD3 marker which successfully identified T cells ([94]Fig. 3f). Fig. 2. [95]Fig. 2 [96]Open in a new tab Cellular infiltrates in tick bitten skin with representative sections of tick bitten participants, top down, left to right: AHL Tick bitten; AHL Control; AHE Tick bitten; AHE Control; AHN Tick bitten; AHN Control; AJS Tick bitten; AJS Control. a) Haematoxylin & eosin stain for histological annotation; b) Region of Interest (ROI) selection slide with morphology markers: SYTO13 DNA (Blue), PanCK (Green), CD3 (Red) and CD45 (Cyan). (For interpretation of the references to colour