Transcriptional profiling of the murine cutaneous response during initial and subsequent infestations with Ixodes scapularis nymphs

Abstract

Background: Ixodes scapularis ticks are hematophagous arthropods capable of transmitting many infectious agents to humans. The process of blood feeding is an extended and continuous interplay between tick and host responses. While this process has been studied extensively in vitro, no global understanding of the host response to ticks has emerged.

Methods: To address this issue, we used PCR-arrays to measure skin-specific expression of 233 discrete genes at 8 time points during primary and secondary infestations of mice with pathogen-free I. scapularis nymphs. Selected results were then validated at the mRNA and protein levels by additional real-time PCR and bioplex assay.

Results: Primary infestation was characterized by the late induction of an innate immune response. Lectin pattern recognition receptors, cytokines, and chemokines were upregulated consistent with increased neutrophil and macrophage migration. Gene ontology and pathway analyses of downregulated genes suggested inhibition of gene transcription and Th17 immunity. During the secondary infestation, additional genes were modulated suggesting a broader involvement of immune cells including CD8 and CD4 positive T lymphocytes. The cytokine response showed a mixed Th1/Th2 profile with a potential for T regulatory cell activity. Key gene ontology clusters observed during the secondary infestation were cell migration and activation. Matrix metalloproteinases were upregulated, apoptosis-related genes were differentially modulated, and immunoreceptor signaling molecules were upregulated. In contrast, transcripts related to mitogenic, WNT, Hedgehog, and stress pathways were downregulated.

Conclusions: Our results support a model of tick feeding where lectin pattern recognition receptors orchestrate an innate inflammatory response during primary infestation that primes a mixed Th1/Th2 response upon secondary exposure. Tick feeding inhibits gene transcription and Th17 immunity. Salivary molecules may also inhibit upregulation of mitogenic, WNT, Hedgehog, and stress pathways and enhance the activity of T regulatory cells, production of IL-10, and suppressors of cytokine signaling molecules (SOCS). This study provides the first comprehensive transcriptional analysis of the murine host response at the I. scapularis bite site and suggests both a potential model of the host cutaneous response and candidate genes for further description and investigation.

Figure 1

Number of modulated genes at each time point compared to tick-free mice. Number of significantly up and downregulated genes measured at each time point during primary and secondary infestations of mice with I. scapularis nymphs compared to tick-free mice.

Figure 2

Genes modulated during primary infestations of mice compared to tick-free mice. Gene expression was measured using PCR arrays at 12, 48, 72, and 96 hr p.i. during a primary exposure of BALB/cJ mice to I. scapularis nymphs. Significantly modulated genes were divided into biologically meaningful groups (Methods) to allow direct comparison between time points. Official gene symbols and fold changes for all results at 48, 72, and 96 hr p.i. are shown. No significant gene modulation was apparent at 12 hr p.i.

Figure 3

Genes modulated during secondary infestations of mice compared to tick-free mice. Gene expression during a secondary exposure of BALB/cJ mice to I. scapularis nymphs was measured using PCR arrays. Significant results were divided into groups as before. Official gene symbols and fold changes are shown at 12, 48, 72, and 96 hr post secondary infestation. The figure is split into sections A and B to aid viewing.

Figure 4

Quantitative real-time PCR validation of PCR array data. Based on the PCR array results, 25 genes were chosen for validation in a separate infestation experiment. Gene expression was measured at 48 and 96 hr p.i. during primary infestations and 48 and 72 hr p.i. during secondary exposures. All significant results are shown. In general, gene modulation supported the array study.

Figure 5

Cytokine analysis of tick bite sites during primary and secondary infestations. Concentrations of IL-1β, IL-3, IL-4, IL-6, IL-10, IL-17a, IFN-γ, and CCL2 were measured in skin biopsies from tick feeding sites at 48 and 96 hr p.i. primary infestation (48p and 96p) and 48 and 72 hr p.i. secondary infestation (48s and 72s) and compared to normal mouse skin using a two-tailed T-test; * indicates a p-value ≤ 0.05 compared to tick-free mice.