Murine cutaneous responses to the rocky mountain spotted fever vector, Dermacentor andersoni, feeding

Abstract

Tick salivary glands produce complex cocktails of bioactive molecules that facilitate blood feeding and pathogen transmission by modulating host hemostasis, pain/itch responses, wound healing, and both innate and adaptive immunity. In this study, cutaneous responses at Dermacentor andersoni bite-sites were analyzed using Affymetrix mouse genome arrays and histopathology at 12, 48, 96 and 120 h post- infestation (hpi) during primary infestations and 120 hpi during secondary infestations. The microarray data suggests: (1) chemotaxis of neutrophils, monocytes, and other cell types; (2) production and scavenging of reactive oxygen species; and, (3) keratin- based wound healing responses. Histological analysis supported the microarray findings. At 12 hpi, a mild inflammatory infiltrate was present in the dermis, especially concentrated at the junction between dermal connective tissue and underlying adipose tissue. A small lesion was located immediately under the hypostome and likely represents the feeding "pool." Surprisingly, at 48 hpi, the number of inflammatory cells had not increased from 12 hpi, perhaps mirroring the reduction in gene expression seen at this time point. The feeding lesion is very well defined, and extravasated erythrocytes are readily evident around the hypostome. By 96 hpi, the inflammatory infiltrate has increased dramatically and the feeding lesion appears to have moved deeper into the dermis. At 120 hpi, most of the changes at 96 hpi are intensified. The infiltrate is very dense, the epidermis is markedly thickened, the feeding lesion is poorly defined and the dermal tissue near the hypostome appears to be loosing its normal architecture. In conclusion, during D. andersoni feeding infiltration of inflammatory cells increases across time concurrent with significant changes in the epidermal and dermal compartments near the feeding tick. The importance of changes in the epidermal layer in the host response to ticks is not known, however, it is possible the host attempts to "slough off" the tick by greatly increasing epithelial cell replication.

Keywords: Dermacentor andersonii; Immunomodulation; tick; tick feeding; tick saliva.

Figure 1

Number of up and downregulated genes at each time point in the microarray data. Mice were infested with D. andersoni nymphs and 4 mm skin biopsies collected at 12, 48, 96, and 120 hpi during primary infestations and 120 hpi during secondary infestations. Gene expression profiling was measured using Affymetrix mouse genome 430A 2.0 arrays. Significance was assessed using iReports data analysis methods, and results were filtered for genes with fold changes greater than ±1.5 and p-values less than 0.05. DAP, D. andersoni primary; DAS, D. andersoni secondary; hpi, hours post infestation.

Figure 2

Heatmaps showing changes in gene expression across time. (A) Changes in genes significant at any time point in the microarray study. (B) Changes in genes significant during the primary infestation only.

Figure 3

Venn diagram showing the overlap of significantly modulated genes between time points in the microarray study. (A) Venn diagram for primary infestation time points. (B) Venn diagram for early time points (DAP12, 48, and 96 hpi), later time points (DAP120 hpi), and secondary infestation (DAS120 hpi). The Venn diagram in B was constructed based on variations in gene expression profiles suggested in the heatmaps. All Venn diagrams were created using (Oliveros, 2007).

Figure 4

Validation of microarray data by quantitative real-time PCR. A list of genes significantly modulated in the microarray study (Table 2) were validated using quantitative real-time PCR on bite site skin samples from a separate infestation experiment. Significance was assessed using LIMMA (linear models in microarray analysis) implemented in HTqPCR, and R based program for qrt-PCR analysis (see methods chapter). All the genes marked with an asterisk were significant (p < 0.05) as compared to tick-free mice.

Figure 5

Lymph node gene expression profile supports a systemic Th2 response. Gene expression changes between secondary infestation 120 hpi draining lymph nodes and lymph nodes from tick-free mice measured by quantitative real-time PCR. Significance was assessed using LIMMA (linear models in microarray analysis) implemented in HTqPCR, and R based program for qrt-PCR analysis (see methods chapter). All the genes shown were significant (p < 0.05).

Figure 6

Histological analysis of D. andersoni bites during primary infestations. Biopsies from tick feeding sites were fixed in zinc fixative and embedded in paraffin. Paraffin blocks were carefully sectioned and slides showing the entry of the hypostome into the skin were stained with H&E. In the top two rows, relevant structures such as the tick, tick cement, epidermis, palps, and feeding lesion have been labeled. The second row appears different that the others because the orientation of the tick in the block was different.