T the transcriptional level, the histopathological analysis clearly shows tissue damage from the insertion of the hypostome and degranulating mast cells (Figure S1) as early as 1 hr post attachment. Minor inflammatory changes consisting of a few inflammatory cells and a small amount of eosinophilic material near the tick hypostome were also observed. By 3 hrs post-infestation, inflammatory cells were readily evident, the eosinophilic material near the hypostome was much more pronounced, and the tissue architecture had the appearance of streaming toward the bite site, even in hypodermal muscle layers. This appearance suggests that ticks may initially insert the hypostome deeply and then retract it, pulling deeper tissues towards 
the epidermis. These changes intensify at 6 hrs post-infestation, leading to a very intense, neutrophil dominated inflammatory lesion by 12 hrs of tick feeding. Also visible at 12 hrs were potential areas of myositis, muscle necrosis, and increased congestion in blood vessels near the hypostome (Figure 5).Early Immunologic Response to Tick BitesThe early appearance of pro-inflammatory changes in transcription and histopathology was unexpected. Previous studies in our laboratory had suggested a minimal early host response [13], supporting many studies that have shown tick salivary components are capable of inhibiting nearly every aspect of cell recruitment. Ixodes ricinus saliva contains leukotriene B4 binding proteins that have been shown to reduce neutrophil migration [35], histamine binding proteins have been described from Rhipicephalus appendiculatus saliva [36], and numerous tick anti-complement molecules have been described [37,38,39]. The release of histamine, eicosanoids, and complement fragments are likely some of the earliest events in the chemotactic cascade. In addition, I. scapularis saliva has been shown to downregulate neutrophil beta-2 integrins, reduce phagocytic efficiency, and inhibit intracellular killing of Borrelia burgdorgeri [40]. The reduction in intracellular killing may be explained by salivary proteins that block super-oxide formation [41], and detoxify reactive oxygen species [42]. Tick salivary proteins have also been shown to bind human IL-8 [43] and chemokines such as Cxcl8 [44]. These studies show tick saliva can inhibit later events in the chemotactic cascade and also effector functions of neutrophils. Against this backdrop, the present study shows leukocytes such as neutrophils and pro-inflammatory geneTick-Host InterfaceFigure 5. Histopathology of Ixodes scapularis nymphal bite sites at 1, 3, 6, and 12 hrs PI. Skin biopsies were fixed in formaldehyde 117793 followed by decalcification prior to paraffin embedding. Five micron sections were stained with hematoxylin and eosin, as described in the methods section. The arrowhead marks a 
marginating neutrophil at 6 hrs PI 1000x, while the arrow marks 1326631 areas of putative myositis/muscle necrosis at 12 hrs PI 100x. doi:10.1371/journal.pone.58-49-1 0047301.gtranscription was initiated before 3 hours post-infestation. Thus despite the impressive arsenal of inhibitory tick salivary proteins, the host is able to mount a surprisingly timely immune response.Studies in mice with labeled neutrophils (enhanced GFP expression under the control of the lysozyme M promoter) have shown that neutrophils migrate into sites of sterile cutaneous injuryTick-Host Interfaceas soon as 20 minutes post-injury. Neutrophil numbers then increased rapidly for 2 hrs when a plateau.T the transcriptional level, the histopathological analysis clearly shows tissue damage from the insertion of the hypostome and degranulating mast cells (Figure S1) as early as 1 hr post attachment. Minor inflammatory changes consisting of a few inflammatory cells and a small amount of eosinophilic material near the tick hypostome were also observed. By 3 hrs post-infestation, inflammatory cells were readily evident, the eosinophilic material near the hypostome was much more pronounced, and the tissue architecture had the appearance of streaming toward the bite site, even in hypodermal muscle layers. This appearance suggests that ticks may initially insert the hypostome deeply and then retract it, pulling deeper tissues towards the epidermis. These changes intensify at 6 hrs post-infestation, leading to a very intense, neutrophil dominated inflammatory lesion by 12 hrs of tick feeding. Also visible at 12 hrs were potential areas of myositis, muscle necrosis, and increased congestion in blood vessels near the hypostome (Figure 5).Early Immunologic Response to Tick BitesThe early appearance of pro-inflammatory changes in transcription and histopathology was unexpected. Previous studies in our laboratory had suggested a minimal early host response [13], supporting many studies that have shown tick salivary components are capable of inhibiting nearly every aspect of cell recruitment. Ixodes ricinus saliva contains leukotriene B4 binding proteins that have been shown to reduce neutrophil migration [35], histamine binding proteins have been described from Rhipicephalus appendiculatus saliva [36], and numerous tick anti-complement molecules have been described [37,38,39]. The release of histamine, eicosanoids, and complement fragments are likely some of the earliest events in the chemotactic cascade. In addition, I. scapularis saliva has been shown to downregulate neutrophil beta-2 integrins, reduce phagocytic efficiency, and inhibit intracellular killing of Borrelia burgdorgeri [40]. The reduction in intracellular killing may be explained by salivary proteins that block super-oxide formation [41], and detoxify reactive oxygen species [42]. Tick salivary proteins have also been shown to bind human IL-8 [43] and chemokines such as Cxcl8 [44]. These studies show tick saliva can inhibit later events in the chemotactic cascade and also effector functions of neutrophils. Against this backdrop, the present study shows leukocytes such as neutrophils and pro-inflammatory geneTick-Host InterfaceFigure 5. Histopathology of Ixodes scapularis nymphal bite sites at 1, 3, 6, and 12 hrs PI. Skin biopsies were fixed in formaldehyde followed by decalcification prior to paraffin embedding. Five micron sections were stained with hematoxylin and eosin, as described in the methods section. The arrowhead marks a marginating neutrophil at 6 hrs PI 1000x, while the arrow marks 1326631 areas of putative myositis/muscle necrosis at 12 hrs PI 100x. doi:10.1371/journal.pone.0047301.gtranscription was initiated before 3 hours post-infestation. Thus despite the impressive arsenal of inhibitory tick salivary proteins, the host is able to mount a surprisingly timely immune response.Studies in mice with labeled neutrophils (enhanced GFP expression under the control of the lysozyme M promoter) have shown that neutrophils migrate into sites of sterile cutaneous injuryTick-Host Interfaceas soon as 20 minutes post-injury. Neutrophil numbers then increased rapidly for 2 hrs when a plateau.