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).