Ticks, Ixodes scapularis, Feed Repeatedly on White-Footed Mice despite Strong Inflammatory Response: An Expanding Paradigm for Understanding Tick-Host Interactions

Abstract

Ticks transmit infectious agents including bacteria, viruses and protozoa. However, their transmission may be compromised by host resistance to repeated tick feeding. Increasing host resistance to repeated tick bites is well known in laboratory animals, including intense inflammation at the bite sites. However, it is not known whether this also occurs in wild rodents such as white-footed mice, Peromyscus leucopus, and other wildlife, or if it occurs at all. According to the "host immune incompetence" hypothesis, if these mice do not have a strong inflammatory response, they would not reject repeated tick bites by Ixodes scapularis. To test this hypothesis, histopathological studies were done comparing dermal inflammation in P. leucopus versus guinea pigs, Cavia porcellus, repeatedly infested with I. scapularis. In P. leucopus, the immune cell composition was like that seen in laboratory mouse models, with some differences. However, there was a broad sessile lesion with intact dermal architecture, likely enabling the ticks to continue feeding unimpeded. In contrast, in C. porcellus, there was a relatively similar mixed cellular profile, but there also was a large, leukocyte-filled cavitary lesion and scab-like hyperkeratotic changes to the epidermal layer, along with itching and apparent pain. Ticks attached to sensitized C. porcellus fed poorly or were dislodged, presumably due to the weakened anchoring of the tick's mouthparts cemented in the heavily inflamed and disintegrating dermal tissues. This is the first time that the architecture of the skin lesions has been recognized as a major factor in understanding tick-host tolerance versus tick bite rejection. These findings broadly strengthen previous work done on lab animal models but also help explain why I. scapularis can repeatedly parasitize white-footed mice, supporting the "immune evasion theory" but cannot repeatedly parasitize other, non-permissive hosts such as guinea pigs.

Keywords: Ixodes scapularis; Peromyscus leucopus; T-cells; inflammation; macrophages; sessile.

Figure 1

Photomicrographs showing histopathology of mouse (Peromyscus leucopus) skin before and after attachment of nymphal tick, Ixodes scapularis. First tick challenge. Stain hematoxylin and eosin (H&E). (A) Normal skin. Bar = 100 µM. (B) Skin 6 h after attachment showing accumulations of neutrophils and a few eosinophils, indicating an early focal inflammatory response. Bar = 50 µM. Black arrow indicates tick capitulum; red arrow indicates invading leukocytes; asterisk indicates hair follicle.

Figure 2

Photomicrographs showing histopathology of mouse (Peromyscus leucopus) skin on day 1 of first tick challenge (D1) showing an increasing inflammatory dermal response and hyperplasia. Stain H&E. (A) Skin with developing lesion surrounding tick mouthparts. Bar = 100 µM. (B) Enlargement showing detail of lesion adjacent to tick mouthparts. Bar = 50 µM. Black arrows indicate tick mouthparts.

Figure 3

Photomicrographs showing histopathology of mouse (Peromyscus leucopus) skin on day 2 of first tick challenge (D2) showing an increasing inflammatory dermal response with widespread mixed leukocytic infiltrates, serocellular crusting, dermal hyperplasia, and hyperkeratosis. Stain H&E. (A) Skin with expanding lesion surrounding tick mouthparts. Bar = 100 µM. (B) Enlargement showing detail of lesion adjacent to tick mouthparts. Arrow = tick mouthparts. Bar = 50 µM. Black arrows indicate tick mouthparts.

Figure 4

Photomicrographs showing histopathology of mouse (Peromyscus leucopus) skin on day 3 of first tick challenge (D3). The inflammatory response was like day 2. See text for details. (A) Skin area of tick-bite showing the sessile lesion, spreading out over a broad area of the tissues near the tick mouthparts, dilated blood vessels, extravasated erythrocytes, and an intense mixed but predominantly neutrophilic inflammation with some eosinophils. Stain H&E. Bar = 100 µM. (B) Higher magnification image showing detail of tick-bite lesion with neutrophils and few eosinophils (asterisks) invading tissues. Stain H&E. Bar = 50 µM. Photomicrographs showing histopathology of mouse (Peromyscus leucopus) skin on day 3 of first tick challenge (D3) showing an increasing inflammatory dermal response with widespread mixed leukocytic infiltrates, vascular disruption, and extravasated blood. (C–G) High magnification image showing detail of the highly focal area (box) with numerous leukocytic infiltrates adjacent to the tick-bite lesion (black arrow indicating fragments of tick hypostome). (C) Stain H&E. Bar = 50 µM. (D–G) Immunohistochemical markers for different leukocyte cell types, staining brown. (D) CD3 identifying numerous T-lymphocytes (dark brown) concentrated in the tick-bite lesion (box). (E) Eosinophil major basic protein identifying few eosinophils in or near the tick-bite lesion (box). (F) IBA1 identifying numerous macrophages in the tick-bite lesion (box) and surrounding dermal tissues. (G) Myeloperoxidase identifying few neutrophils concentrated in the tick-bite lesion (box). Bars = 50 µM.

Figure 5

Photomicrographs showing histopathology of mouse (Peromyscus leucopus) skin from ear collected on day 4 of first tick challenge (D4). Acute inflammatory response with granulomatous dermatitis and hemorrhage. See text for details. (A) Low magnification image showing tick mouthparts, skin with tick-bite lesion, pronounced hyperplasia, and extensive hemorrhage. Stain H&E. Bar = 100 µM. (B) Higher magnification image showing detail of tick-bite lesion adjacent to tick mouthparts showing infusion of mixed leukocytes, including some eosinophils, macrophages, and other polymorphonuclear cell types (e.g., mast cell, black arrow). Stain H&E. Bar = 50 µM. (C) Detail of tick-bite lesion with Luna stain specific for eosinophils. Note greatly diluted blood vessels. Bar = 50 µM. Photomicrographs showing histopathology of mouse (Peromyscus leucopus) skin on day 4 of first tick challenge (D4). (D–H) Immunohistochemical (IHC) markers for different leukocyte cell types (brown stain) in or near the tick-bite lesion (box). (D) H&E stained section for comparison with IHC-stained sections following. (E) CD3 identifying numerous T-lymphocytes (dark brown) concentrated in the tick-bite lesion. (F) Eosinophil major basic protein identifying few eosinophils in or near the tick-bite lesion. (G) IBA1 identifying numerous macrophages in the tick-bite lesion and surrounding dermal tissues. (H) Myeloperoxidase; neutrophils absent from the tick-bite lesion. Bars = 50 µM.

Figure 6

Photomicrographs showing histopathology of mouse (Peromyscus leucopus) skin from the ear collected on day 1 of third tick challenge (D1). Early onset of acute inflammatory response with infiltrating neutrophils and some eosinophils. Stain H&E. See text for details. (A) Low magnification image showing the vicinity of the bite lesion. The tissues show a severe focally diffuse subacute dermatitis. Bar = 100 µM. (B) Detail of tick bite lesion adjacent to tick mouthparts. The tissues show a severe focally diffuse subacute dermatitis with mixed neutrophilic, mastocytic, and histiocytic infiltrates but relatively few eosinophils (black arrows). There is focal epidermal hyperplasia with serocellular crusting. There is focal epidermal hyperplasia with serocellular crusting. Bar = 50 µM.

Figure 7

Photomicrographs showing histopathology of mouse (Peromyscus leucopus) skin from the ear collected on day 2 of third tick challenge (D2). The inflammatory response is more intense, widespread, with mixed leukocytic infiltrates, serocellular crusting, and dermal hyperplasia. The lesion appears sessile, without cavitation. Stain H&E. See text for details. (A) Low magnification image showing the tissues near the tick mouthparts (black arrow), forming a sessile lesion. There is a severe focally diffuse subacute dermatitis with numerous leukocytes. Black arrow indicates edge tick mouthparts. Bar = 100 µM. (B) Higher magnification images of area of the tick-bite lesion. In addition to the numerous infiltrating neutrophils and some eosinophils, there is increasing edema, along with focal muscle breakdown and other tissue necrosis. Bar = 50 µM.

Figure 8

Photomicrographs showing high magnification image illustrating the histopathology of mouse (Peromyscus leucopus) skin on day 3 of the third tick challenge (D3). See text for details. (A) Dermal region adjacent to the tick mouthparts (black arrow) showing the intense, acute dermatitis forming an extensive sessile lesion, substantial leukocytic infiltrates, increasing hyperplasia, and hyperkeratosis. Bar = 100 µM. (B) Higher magnification images of area the tick attachment site, showing the focal lesion with extravasated red blood cells and leukocytic infiltrates. Bar = 50 µM. (C) Higher magnification image of same region showing representative eosinophils (black arrows). Bar = 20 µM. (D–I) Photomicrographs showing high magnification images illustrating the histopathology of mouse (Peromyscus leucopus) skin on day 3 of the third tick challenge (D3). (D) Section of mouse skin selected stained with H&E for comparison with immunohistochemical (IHC) markers. Bar = 50 µM. (E–I) IHC markers (brown stain) for different leukocyte cell types, in or near the tick-bite lesion (box). (E) CD3 identifying T-lymphocytes, dispersed within the focal area of the tick-bite lesion. (F) Eosinophil major basic protein for eosinophils; very few eosinophils were detected in or near the tick-bite lesion. (G) IBA1 identifying numerous macrophages in the tick-bite lesion and surrounding dermal tissues. (H) Myeloperoxidase showing numerous neutrophils in the tick-bite lesion. (I) Mmcp-8 for basophils; no basophils were found. Bars = 50 µM.

Figure 9

Photomicrographs showing high magnification image illustrating the histopathology of mouse (Peromyscus leucopus) skin on day 4 of the third tick challenge (D4). The skin near the tick-bite shows an extensive, predominantly granulomatous mixed leukocytic dermatitis. Stain H&E in (A,B). (A) Low magnification image shows the tick-bite lesion filled with masses of mixed leukocytic cells, extensive hyperplasia, and serocellular crusting. Black arrow indicates hair follicle; red asterisk denotes intense granulomatous area. Red arrow indicates vicinity of tick bite. The lesion is sessile, no cavity formation evident. Bar = 100 µM. (B) Higher magnification image of the tick-bite lesion. There are many macrophages and abundant histiocytes interspersed with numerous neutrophils and occasional eosinophils. There is widespread hemorrhaging, diluted and ruptured blood vessels, and tissue necrosis. Bar = 50 µM. (C–H) Photomicrographs illustrating the histopathology of mouse (Peromyscus leucopus) skin on day 4 of the third tick challenge (D4). The skin near the tick-bite shows an extensive, predominantly granulomatous mixed leukocytic dermatitis. (C) Section of mouse skin selected stained with H&E for comparison with immunohistochemical (IHC) markers (box). Bar = 50 µM. (D–H) IHC markers (brown stain) for different leukocyte cell types, in or near the tick-bite lesion. (D) CD3 identifying T-lymphocytes, dispersed within the focal area of the tick-bite lesion. (E) Eosinophil major basic protein for eosinophils; very few eosinophils were detected in or near the tick-bite lesion. (F) IBA1 identifying numerous macrophages in the tick-bite lesion and surrounding dermal tissues. (G) Myeloperoxidase showing numerous neutrophils in the tick-bite lesion. (H) mMCP-8 for basophils. Arrows indicate few, scattered basophils, mostly in small blood vessels. Bars = 50 µM. See Figure 10 for a higher magnification of the IHC assay.

Figure 10

Photomicrographs illustrating the results of an immunohistochemical (IHC) assay for basophils in mouse (Peromyscus leucopus) skin on day 4 of the third tick challenge (D4). (A,B) Two separate sections of tick-bite lesion showing basophils (arrows). Most of the IHC-positive cells occur within the lumens of small blood vessels (triangular arrows); a few are scattered in the dermis (small arrows). Bar = 20 µM.

Figure 11

Photomicrographs showing a feeding Ixodes scapularis nymphs attached to skin of a mouse (Peromyscus leucopus) on day 3 of the third tick challenge (D3) showing the normal internal body tissues. Stain H&E. (A) Low magnification image showing the entire tick body. Bar = 100 µM. (B) Higher magnification image showing the tick’s anterior body region. Bar = 50 µM. (C) High magnification image showing the interior of the tick’s body tissues. The midgut epithelial cells have expanded greatly with masses of hematin accumulating after hemoglobin digestion. The epidermis is enlarged, indicating cuticle growth consistent with the blood engorging tick body. Bar = 20 µM.

Figure 12

Photomicrographs showing the histopathology of guinea pig (Cavia porcellus) skin on day 3 of first tick challenge (D3) near the tick mouthparts. There is hyperplasia and keratosis in the epidermal layer next to the tick’s mouthparts. There is leukocytic infiltration indicating an early inflammatory response. Stain H&E. (A) Low magnification image showing the skin region adjacent to the tick mouthparts. Bar = 100 µM. (B) High magnification image of the skin tissues next to the tick mouthparts. Bar = 50 µM. (C) Section of guinea pig skin stained with H&E for comparison with Immunohistochemical (IHC) markers (box). Section shows tissue necrosis and cavitation in the focal area of the tick-bite lesion. Bar = 50 µM. (D–G) IHC markers (brown stain) for different leukocyte cell types, in or near the tick-bite lesion. (E) CD3 identifying T-lymphocytes, concentrated around margins of the tick-bite lesion. (F) Eosinophil major basic protein for eosinophils; none were detected in or near the tick-bite lesion. (G) IBA1 identifying numerous macrophages throughout the tick-bite lesion and surrounding dermal tissues. (H) Myeloperoxidase showing numerous heterophils in the tick-bite lesion. Bars = 50 µM.

Figure 13

Photomicrographs showing the histopathology of guinea pig (Cavia porcellus) skin on day 3 of third tick challenge (D3) near the tick bite. Stain H&E. See text for details. (A) Low magnification image showing the tissues adjacent to the tick-bite. The section shows a severe, focally diffuse inflammation with numerous neutrophils, eosinophils, macrophages, and mononuclear cells, also with a dense serocellular crust, very severe epidermal hyperplasia and hyperkeratosis. There is a very large cavity lesion in the center. Bar = 100 µM. (B) High magnification image showing region of the tick-bite lesion, with masses of mixed, infiltrating leukocytes, severe epidermal hyperplasia, hyperkeratosis, a broad cavity lesion, breakdown of dermal collagen, and extensive hemorrhage. Bar = 50 µM. (C) Very high magnification image showing masses of neutrophils and eosinophils in the tick-bite lesion, especially concentrated in the central cavity. Insets either side of image C indicate eosin-staining degranulating leukocytes, presumably eosinophils. Bar = 20 µM. (D–H) Immunohistochemical markers (brown stain) for different leukocyte cell types, in or near the tick-bite lesion. (E) CD3 identifying T-lymphocytes, concentrated around margins of the tick-bite lesion. (F) Eosinophil major basic protein for eosinophils; few eosinophils were recognized by this stain in or near the tick-bite lesion. (G) IBA1 identifying numerous macrophages throughout the tick-bite lesion and surrounding dermal tissues. (H) Myeloperoxidase showing numerous neutrophils in the tick-bite lesion. The tick had fallen off. Bars = 50 µM.

Figure 14

Photomicrographs comparing the histopathology of the mouse (Peromyscus leucopus) versus guinea pig (Cavia porcellus) tick-bite dermal lesion on day 3 of the third tick challenge. Stain H&E. (A) Mouse dermal region adjacent to the tick mouthparts (black arrow) showing an acute dermatitis forming a broad sessile lesion, substantial leukocytic infiltrates hyperplasia, and keratosis. Arrow indicates tick. Bar = 100 µM. (B) Guinea pig dermal lesion showing an acute dermatitis forming a large focal cavity lesion, with numerous heterophils, macrophages, histiocytes and mononuclear cells, dense serocellular crusting, severe hyperplasia, and hyperkeratosis. The scab-like keratotic layer has separated from the underlying dermis. Tick had fallen off. Bar = 100 µM.