Abstract
Ticks are notorious blood-sucking ectoparasites affecting both humans and animals, and serve as a unique vector of various deadly diseases. Here, we have shown the roles of the receptor for advanced glycation end-products (RAGE) during repeated infestations by the tick,Haemaphysalis longicornis using RAGE-/- mice. In primary infestation, a large blood pool developed which was flooded with numerous RBC, especially during the rapid feeding phase of the tick both in wild type (wt) and RAGE-/- mice. Very few inflammatory cells were detected around the zones of hemorrhage in the primary infestations. However, number of inflammatory cells gradually increased in the subsequent tick infestations and at the 3rd infestations number of inflammatory cells reached to the highest level (350.3±16.8 cells/focus), and the site of attachment was totally occupied by the inflammatory cells in wild type (wt) mice whereas very few cells were detected at the ticks’ biting sites in RAGE-/- mice. RAGE was highly expressed in the 3rd infestation in wt mice. In the 3rd infestation, infiltration of innate lymphoid cells type 2 (ILC2s), expression of S100A8 and S100B significantly increased at the biting sites of ticks in wt, but not inRAGE -/- mice. Also, peripheral eosinophil counts significantly increased in wt but not inRAGE -/- mice. Taken together, our study revealed that RAGE-mediated inflammation and eosinophils played crucial roles in the tick induced inflammatory reactions.
KEYWORDS: Ticks, RAGE, alarmin, innate lymphoid cells, eosinophils
INTRODUCTION
Ticks (Arachnida: Acari) serve as a unique vector of various deadly diseases, such as Lyme disease, tick-borne encephalitis, Rocky Mountain spotted fever, severe fever with thrombocytopenia syndrome (SFTS), babesiosis, theileriosis and anaplasmosis through hematophagy (1,2). Ticks remain attached to hosts for long times firmly embedding their barbed mouthparts, which are adapted for lacerating tissues. Unlike hematophagous insects, ticks cannot canulate individual blood vessels, rather they lacerate tissues and vascular beds with their specialized mouthparts and establish a feeding lesion in the skin, known as a blood pool, from which they feed blood and exudate until repletion. Development of a blood pool is a prerequisite to the feeding biology of ticks. Tick-bite mediated extensive tissue injury is almost similar to trauma induced sterile inflammation. During experimental infestation in dogs, rabbits and mice, we observed that size of the blood pool and feeding success of ticks largely depend on the types of infestations. In primary infestation, blood pool is relatively larger and ticks feed on more successfully in terms of post engorgements body weight gained and repletion time needed than those parameters observed in secondary or tertiary infestations (3,4).
Inflammation plays vital role for host defense against attacking pathogens. In response to an infection, a cascade of signals first activates innate immune systems consisting of neutrophils, macrophages, eosinophils or mast cells which phagocytoses infectious agents or kills them by releasing chemical mediators (5,6). Microbially induced inflammation is elicited by structural moieties found on or released by microorganisms, which are known as pathogen-associated molecular patterns (PAMPs). PAMPs for example lipopolysaccharide (LPS, from Gram-negative bacteria), lipoteichoic acid (from Gram-positive bacteria), peptidoglycan (in most bacteria), bacterial DNA, viral DNA/RNA, and mannans (from yeast cells) are mainly recognized by Toll-like receptors (TLRs), retinoid acid-inducible gene I (RIG I)-like receptors (RLRs), AIM2 like receptors (ALRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and lead to inflammatory response (7). Similar to microbial inflammation, sterile inflammation also induces pro-inflammatory cytokines and chemokines production, and adhesion molecule expression leading to recruitment of cells of innate immune system. Sterile injury causes variable degrees of tissue injury leading to release of S100 proteins/calgranulins, high mobility group box 1 protein (HMGBi or amphoterin), hit shock protein (e.g., HSP70), SPARC (secreted protein acidic and rich in cysteine) and spillage of self-DNA which are commonly known as damage-associated molecular patterns (DAMPs) or alarmins. DAMPs are chiefly recognized by the DAMPs prototypic receptors RAGE (receptor for advanced glycation end product). RAGE, a pattern recognition receptor (PRR), can recognize diverse groups of structurally unrelated molecules through recognition of their three-dimensional structures rather than specific amino acid sequences. Some DAMPs are also recognized by extracellular TLRs (8).
Ticks remain attached to hosts usually up to a week for full engorgement. Therefore, during feeding, ticks need to minimize several host defense mechanisms such as coagulation, inflammation, immune responses, pain and itching responses, which are engaged to prevent an invading pathogen. Ticks have been shown to secret several peptidases and inhibitors, which play crucial roles to block coagulations and prevent cellular infiltrations at the site of attachment (9) . In addition, eosinophils play vital roles in tick induced inflammatory reactions in mammalian hosts (6) . However, trafficking of eosinophils and other innate immune cells at the site of attachment of ticks is still unknown. Furthermore, during the primary tick infestations, a big blood pool develops, and ticks become engorged rapidly than during the secondary or tertiary infestations. Several researches have shown that an immune response to salivary glands’ molecules released during ticks’ feeding elicits protective immunity and thwart feeding (3,6,9). Mammalian hosts had been shown to acquire resistance to ticks following repeated and even after in primary tick infestation, manifested by lower post engorgement ticks’ weights, prolonged repletion time, very few engorged ticks or the death of the ticks in the subsequent infestations (10). Here, we have elegantly drawn a complete picture of cellular infiltration during different phases of tick infestations and we also show the roles of RAGE receptor in providing a band of protection against tick infestations.
MATERIALS AND METHODS
Ticks
We propagated parthenogenetic Okayama strains of Haemaphysalis longicornis at the Laboratory of Parasitic Diseases, the National Institute of Animal Health (NIAH), Tsukuba, Japan, by feeding on the ear of tick-naïve, SPF Japanese White rabbits according to methods as described previously (11). Briefly, ticks were allowed to feed on clipped ears of rabbits providing support by ear bags and a head collar. Ticks were collected after detachment following full engorgement.
RAGE-/- mice
RAGE deficient (RAGE -/-) mice were developed and maintained as described elsewhere (12) .
Laboratory animals used
All animals used in this study were acclimatized to the experimental conditions for two weeks prior to the commencement of the experiment. The protocol for the care and use of Laboratory Animals was approved by the Committee of the Ethics of Animal Experiments of the National Institute of Animal Health (NIAH) (Permit Numbers: 09-017, 09-018, 10-008, 10-010, 11-027 ). All methods were performed in accordance with the relevant guidelines and regulations approved by the NIAH. The study is reported in accordance with ARRIVE guidelines (
Feeding of ticks on mice
We allowed two adult H. longicornis ticks on the shaven back of each tick-naïve or previously tick infested C57BL6 mice or RAGE-/- mice by giving support with a mouse-back-cell of about one centimeter in height prepared from a 14 ml Falcon tube. Between two subsequent feedings at least two weeks’ interval were kept for would healing. The mice were reared in the same experimental conditions suppling feed and water ad libitum . The blood feeding pattern of ticks was observed and recorded.
Hematology
We collected blood from mice at different feeding periods (day 0 to day 5) from tail tip and prepared blood smears. Differential leucocytes count (DLC) was performed by staining the smears with Leishman’s stain.
Euthanasia, tissue collection and histopathology
The tick infested mice were euthanized by injecting sodium pentobarbital at the day 1-day 5 and gross appearances of the blood pools were carefully examined. Skin from the attachment site of ticks was collected, and preserved in 4% paraformaldehyde added with 0.1% glutaraldehyde overnight at 4 °C under gentle shaking. Thin (5 μm) tissue sections were prepared and subjected to hematoxylin and eosin (H&E), toluidine blue and direct fast scarlet red (DFS) staining following the standard protocol (13) .
Immunohistochemistry and Immunofluorescence
Immunohistochemistry was performed as previously described (11). Briefly, mouse skin sections were deparaffinized and rehydrated with graded series of alcohol. Inactivation of endogenous peroxidase with 1% H2O2 and blocked with 5% skimmed milk. They were then incubated overnight at 4 °C with commercially available antibodies to myeloperoxidase (MPO), and CD44. Slides were rinsed thoroughly with ice-cool PBS and subsequently reacted with the biotin-conjugated secondary antibody and the substrate 3´, 3´diaminobenzidine tetrahydrochloride (Fast™ DAB set, Sigma, St. Luis, Mo, USA). After color development, the slides were dehydrated with graded series of alcohol washes, cleared in xylene and then covered with cover slips and examined under a microscope (Leica Microsystem, Wetzlar, Germany).
For immunofluorescent staining, sections were treated with anti-RAGE antibody and bound antibodies were detected using green fluorescent-labeled secondary antibody (Alexa Flour® 488 goat anti-mouse IgG (H+L), Invitrogen). Slides were mounted with VECTASHIELD® mounting medium containing DAPI (Vector Laboratories) and examined under a fluorescent microscope (Leica).
Statistical analysis
Data were presented as mean ± SEM. For the comparison infiltration of inflammatory cells of multiple groups, 1-way ANOVA followed by post-hoc Bonferroni’s analysis was used. For direct comparisons, unpaired 2-tailed Student’s t  test was employed. A value of at least P  < 0.05 was considered as statistically significant.
RESULTS
Blood pool is flooded with RBC, especially at the rapid feeding phase
During our study, we observed that at the initial phase (24 h of attachment) the ticks attached their hypostome into the skin making a notch in the epidermis. There were very minimum tissue damages and cellular responses into the surrounding tissues of the invading mouthparts obvious hemorrhagic changes. At 48 h of feeding, tissue damage was almost similar but accumulation of reactive cells increased and the hemorrhagic changes were evident. At 72 h of feeding, cellular infiltration increased but hemorrhagic changes were almost same to the level of 48 h of feeding. At 96 h post attachment, a large blood pool developed which was flooded with numerous RBC. Very few inflammatory cells were detected around the zones of hemorrhage in the primary infestations. However, number of inflammatory cells gradually increased in the subsequent tick infestations, and at the tertiary infestations, the site of attachment was totally occupied by the inflammatory cells (Supplementary Figure 1).
3.2 Eosinophil, the Trojan horse, present in tick-bite sites
To demonstrate the eosinophils, present in the tick-bite sites, we stained the histological sections with DFS. In the primary infestations, we found very few eosinophils up to day 4 of feeding. At day5 of attachment, eosinophil infiltration increased a little bit at the periphery of the blood pool. Notably, the blood pool was almost free from eosinophils. However, in the subsequent infestations, infiltration of eosinophils significantly increased and reached to the maximum level in the tertiary infestations (Supplementary Figure 1).