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\begin{document}

\title{Receptor for advanced glycation end-product (RAGE) modulates
inflammation during feeding of tick, Haemaphysalis longicor}



\author[1]{* Anisuzzaman}%
\author[1]{Md. Abdul Alim}%
\author[2]{Makoto Matsubyashi}%
\author[1]{Md. Shahadat Hossain}%
\author[1]{Sharmin Shahid Labony}%
\author[3]{Ireen Sultana Shanta}%
\author[1]{Md. Haydar Ali}%
\author[4]{Yasuhiko Yamamoto}%
\author[5]{Takeshi Hatta}%
\author[5]{Naotoshi Tsuji}%
\affil[1]{Bangladesh Agricultural University}%
\affil[2]{Osaka Koritsu Daigaku Jui Gakubu Juigaku Kenkyuka}%
\affil[3]{International Centre for Diarrhoeal Disease Research Bangladesh}%
\affil[4]{Kanazawa Daigaku Daigakuin Iyaku Hokengaku Sogo Kenkyuka Iyaku Hoken Gakuiki Igakurui}%
\affil[5]{Kitasato Daigaku Igakubu}%


\vspace{-1em}



  
  \date{June 14, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{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, \emph{Haemaphysalis longicornis} using
\emph{RAGE\textsuperscript{-/-}} 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
\emph{RAGE\textsuperscript{-/-}} 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 3 \textsuperscript{rd}
infestations number of inflammatory cells reached to the highest level
(350.3\selectlanguage{ngerman}±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
\emph{RAGE\textsuperscript{-/-}} mice. RAGE was highly expressed in the
3 \textsuperscript{rd} infestation in wt mice. In the 3
\textsuperscript{rd} 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 in \emph{RAGE}
\textsuperscript{-/-} mice. Also, peripheral eosinophil counts
significantly increased in wt but not in \emph{RAGE}
\textsuperscript{-/-} mice. Taken together, our study revealed that
RAGE-mediated inflammation and eosinophils played crucial roles in the
tick induced inflammatory reactions.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Receptor for advanced glycation end-product (RAGE) modulates
inflammation during feeding of tick, \emph{Haemaphysalis longicornis} in
mice}

\textbf{Anisuzzaman\textsuperscript{1*}, Md. Abdul
Alim\textsuperscript{1}, Makoto Matsubyashi\textsuperscript{2}, Md.
Shahadat Hossain\textsuperscript{1}, Sharmin Shahid
Labony\textsuperscript{1}, Ireen Sultana Shanta\textsuperscript{3}, Md.
Haydar Ali\textsuperscript{1,4}, Yasuhiko Yamamoto\textsuperscript{5},
Takeshi Hatta\textsuperscript{6, 7}, Naotoshi Tsuji\textsuperscript{6,
7}}

\textsuperscript{1}Department of Parasitology, Faculty of Veterinary
Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

\textsuperscript{2}Department of Veterinary Immunology, Graduate School
of Veterinary Sciences, Osaka Metropolitan University, Izumisano, Japan.

\textsuperscript{3}International Centre for Diarrhoeal Disease Research:
Dhaka, Bangladesh

\textsuperscript{4}Department of Pathology and Parasitology, Faculty of
Veterinary and Animal Science, Hajee Mohammad Danesh Science and
Technology University (HSTU), Dinajpur, Bangladesh

\textsuperscript{5}Department of Biochemistry and Molecular Vascular
Biology, Kanazawa University Graduate School of Medical Sciences,
Kanazawa, Japan

\textsuperscript{6}Department of Parasitology and Tropical Medicine,
Kitasato University School of Medicine, 1-15-1 Kitasato, Minami,
Sagamihara, Kanagawa 252-0374, Japan.

\textsuperscript{7}Laboratory of Parasitic Diseases, National Institute
of Animal Health, National Agricultural and Food Research Organization,
Tsukuba, Ibaraki 305-0856, Japan

\textsuperscript{\textbf{*}}\textbf{Corresponding authors}

\textbf{Anisuzzaman} \emph{:} zaman.a@bau.edu.bd

\textbf{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,\emph{Haemaphysalis longicornis} using
\emph{RAGE\textsuperscript{-/-}} 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
\emph{RAGE\textsuperscript{-/-}} 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 3\textsuperscript{rd}
infestations number of inflammatory cells reached to the highest level
(350.3\selectlanguage{ngerman}±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
\emph{RAGE\textsuperscript{-/-}} mice. RAGE was highly expressed in the
3\textsuperscript{rd} infestation in wt mice. In the
3\textsuperscript{rd} 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 in\emph{RAGE}
\textsuperscript{-/-} mice. Also, peripheral eosinophil counts
significantly increased in wt but not in\emph{RAGE}
\textsuperscript{-/-} mice. Taken together, our study revealed that
RAGE-mediated inflammation and eosinophils played crucial roles in the
tick induced inflammatory reactions.

\textbf{KEYWORDS:} Ticks, RAGE, alarmin, innate lymphoid cells,
eosinophils

\textbf{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.

\textbf{MATERIALS AND METHODS}

\textbf{Ticks}

We propagated parthenogenetic Okayama strains of \emph{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.

\textbf{RAGE\textsuperscript{-/-} mice}

RAGE deficient (\emph{RAGE} \textsuperscript{-/-}) mice were developed
and maintained as described elsewhere (12) .

\textbf{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: \emph{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 (

\textbf{Feeding of ticks on mice}

We allowed two adult \emph{H. longicornis} ticks on the shaven back of
each tick-naïve or previously tick infested C57BL6 mice or
RAGE\textsuperscript{-/-} 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 \emph{ad libitum} . The blood feeding
pattern of ticks was observed and recorded.

\textbf{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.

\textbf{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 \selectlanguage{greek}μ\selectlanguage{english}m)
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) .

\textbf{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\%
H\textsubscript{2}O\textsubscript{2} and blocked with 5\% skimmed milk.
They were then incubated overnight at 4 \selectlanguage{ngerman}°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\selectlanguage{english} 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(r) 488 goat anti-mouse
IgG (H+L), Invitrogen). Slides were mounted with
VECTASHIELD\textsuperscript{(r)} mounting medium containing DAPI (Vector
Laboratories) and examined under a fluorescent microscope (Leica).

\textbf{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
~\textless{} 0.05 was considered as statistically significant.

\textbf{RESULTS}

\textbf{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).

\textbf{3.2} \textbf{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).

\textbf{3.3 Degranulation of mast cells}

To detect mast cells at the attachment sites of ticks, we stained the
tissue sections with toluidine blue. Toluidine blue staining revealed
that there was no significant difference in the presence of mast cells
in the tick bite site at different feeding periods (day 0 to day 5) of
the primary infestations. In the primary infestation, very few
degranulated mast cells were found in the surrounding tissues of tick
attachment. Also, no significant increase in the infiltration and
degranulation of mast cells was noticed in the secondary and tertiary
infestations (Supplementary Figure 1).

\textbf{3.4 Roles of RAGE in the feeding process of hard ticks}

To evaluate the impact of RAGE-ligands axis on the tick bite induced
inflammation, ticks were allowed to feed on wt
or\emph{RAGE\textsuperscript{-/-}} mice. We observed that infiltration
of inflammatory cells gradually increased in the subsequent infestations
in wt mice but it was not evident in \emph{RAGE\textsuperscript{-/-}}
mice, indicating RAGE plays a vital role in the development of
pathological changes induced during tick bite (Figure 1A). Counting of
the inflammatory cells per focus by two blind investigators revealed
that the number of inflammatory cells significantly (p\textless{}0.001)
increased at the site of attachment of ticks in the different feeding
phases in wt mice but not in \emph{RAGE} -/- mice. However, in the
3\textsuperscript{rd} infestations, cellular infiltration reached to the
highest level (350.3+-16.8 cells/focus) in wt mice, whereas in
the\emph{RAGE\textsuperscript{-/-}} mice it was almost comparable to the
cellular infiltration detected in fresh skin (Figure 1B).

\textbf{3.5 RAGE is upregulated during blood feeding processes by ticks}

Since inflammations were more pronounced during feeding on wt mice,
therefore, we planned to assess the expression of RAGE at the site of
attachment of ticks. Tissues were collected and analyzed to determine
expression of RAGE using fluorescence. We detected very high-level
RAGE-specific green fluorescence at the biting site of ticks in wt mice
(Figure 2), which was almost absent in non-infested normal mouse skin
and in previously tick bitten \emph{RAGE\textsuperscript{-/-}} mouse
skin, suggesting RAGE is highly expressed at the site of attachment of
ticks.

\textbf{3.6 DAMPs are highly upregulated during tick feeding}

Since RAGE is highly expressed during feeding process of ticks,
therefore, we planned to detect the expression of RAGE ligands at the
site of tick attachment. By immuno-histochemical study, we detected
S100A8- and S100B-specific reactions at the biting sites of ticks on wt
mice, whereas expression of these molecules were minimum even at the
tertiary infestations in \emph{RAGE\textsuperscript{-/-}} mice (Figure
3), suggesting the possible roles of RAGE-ligand axis in eliciting tick
bite induced inflammation.

\textbf{3.7 Infiltration of ILC2s and neutrophils at the site of
attachment of ticks}

Infiltrations of macrophages and basophils had been detected at the site
of attachment of ticks (6,14). However, ILC2s are abundant in skin and
are responsible to provide innate immunity against parasitic infections
(15,16). Therefore, we planned to detect responsiveness of ILC2s at the
tick bitten areas. For this, we collected skin samples from the sites
tick attachment or non-infested wt or
\emph{RAGE\textsuperscript{-/-}}mice and subjected to
immune-histochemical analysis using anti-CD44. Our study revealed
massive infiltration of CD44\textsuperscript{+} cells at the tick bitten
lesion. Infiltration of the cells increased in the subsequent
infestations and the highest infiltrations were detected during the
tertiary infestations. However, accumulation of CD44\textsuperscript{+}
cells was minimum during the feeding process of ticks on
\emph{RAGE\textsuperscript{-/-}} mice, indicating the important role of
RAGE on the trafficking of ILC2s at the attachment sites (Figure 4).
Furthermore, infiltrations of neutrophils were detected by
immune-histochemistry using anti-MPO. Very few anti-MPO-positive cells
were found both in wt and \emph{RAGE\textsuperscript{-/-}} mice skin
(Figure 5), indicating that neutrophils has no significant roles in the
tick-inflicted injuries in skin.

\textbf{3.8 Systemic eosinophilia}

Excessive infiltrations of eosinophils in the lesions instigated us to
determine whether tick bites induce systemic eosinophilia or not. For
this, ticks were allowed to feed on wt or
\emph{RAGE\textsuperscript{-/-}} mice and examination of peripheral
blood in a blind revealed that peripheral eosinophils count increased
rapidly in wt, but not in\emph{RAGE\textsuperscript{-/-}} mice even in
the primary infestation. At day 0 (prior to the attachment of ticks)
eosinophil count was 0.6\% which increased \textasciitilde{}3 times
within 24 h and reached up to the pick (8 times increase compared to day
0) at the rapid feeding phase (day 5) prior to spontaneous detachment of
tick from the host following full engorgement. In the tertiary
infestation, at day 0, eosinophil count was 2.6\% but at day 5 post
attachment eosinophil count reached up to \textasciitilde{}7\% (Figure
5). We did not find any significant change in the count of other cells
of blood.

\textbf{DISCUSSION}

All blood feeding arthropods, including ticks induce local inflammation
at the site of biting and elicits infiltration of innate immune cells,
predominantly eosinophils, attempting to thwart feeding. Ticks imbibe
blood over a long periods of time gradually from the blood pool created
by multifaceted mechanical and bio-chemical processes (3,6,17). During
development of a blood pool, a tick causes extensive tissue damage, and
dead or devitalized tissues release intracellular proteins, termed as
DAMPs/allarmins. These alarmins are then recognized by the PRR and
induced sterile inflammations (18). Here, we report that RAGE-alarmins
axis regulates the migration of eosinophils and ILC2s at the site of
attachment of ticks.

Initially, we showed that blood pools developed during the primary
infestations were minimally infiltrated by the inflammatory cells. In
fact, ticks secret a lot of molecules from the salivary glands and also
regurgitate from midgut with saliva into the blood pool to prevent
inflammation and keep blood in a fluid state throughout the feeding
periods (9). In a previous study, Anisuzzaman et al. (6) isolated and
characterized a pluripotent molecule, named longistatin, from the
salivary glands of a hard tick, \emph{H. longicornis} , which
simultaneously prevents blood coagulation and inflammation. Furthermore,
factor Xa (FXa) inhibitors (e.g., tick anticoagulant peptide {[}TAP{]},
from \emph{Ornithodoros} \emph{moubata} , Salp14 from \emph{Ixolaris
scapularis} and FXaI from \emph{Ornithodoros savignyi} ), tissue factor
pathway inhibitor (e.g., Ixolaris from \emph{I. scapularis} ), direct
thrombin inhibitor (e.g., microphilin from \emph{Ripicephalus microplus}
, savignin from \emph{O} . \emph{savignyi,} ornithodorin from\emph{O.
moubata} , madanin 1 and 2 from \emph{H. longicornis} and variengin from
\emph{Amblyomma variegatum} ), complement inhibitors (e.g., OMC from
\emph{O. moubata} , Isac and Salf 20 from \emph{I. scapularis} and IRAC
1 and 2 from \emph{Ixodes ricinus} ), T cell inhibitors (e.g., Salp 15
and siolostatin L from \emph{I. scapularis}and Iris from \emph{I.
ricinus} ) and B cell inhibitors have been identified and characterized
(9).

The immunogenic properties of some of these molecules have been
demonstrated. In the present study, we found that the number of
inflammatory cells gradually increased due to repeated infestations, and
inflammatory changes reached to the maximum level at the secondary and
tertiary infestations. It is presumed that due to repeated exposure to
the ticks, immune responses are triggered targeting the vital bio-active
molecules secreted by ticks; thus, provides a band of protection to the
mammalian hosts against tick infestation.

During the third infestation RAGE and its ligands were highly expressed.
RAGE is constitutively present in lung, intestine and skin as these
organs are highly exposed to infectious agents or invading pathogens
(19,20). Previously, we showed that tick's salivary molecules,
particularly longistatin, bind with RAGE (6). Longistatin is also
immunogenic (21); thus, we presume that antibodies develop against
RAGE-binding key feeding related salivary molecules due to repeated
feeding. Taken together, RAGE inhibitors are sequestrated and ultimately
RAGE is upregulated in the subsequent repeated feeding. Alarmins or RAGE
ligands such as S100 proteins are mostly intracellular proteins and they
are released following cellular damage. Here, we observed that S100A8
and S100B are highly expressed following sterile inflammation induced by
tick. S100A8 and S100B are EF-hand Ca\textsuperscript{2+}-binding
proteins belonging to the S100 family. They are constitutively expressed
by keratinocytes and immunocytes (e.g., neutrophils, macrophage and
eosinophils), and keratinocytes are the most important source of S100
proteins, including S100A8/A9 and S100B.

S100A8 has both intracellular and extracellular functions. S100A8 is
intensely expressed in various inflammatory conditions, particularly in
trauma and infections, and inflammations accelerate S100A8 protein
secretion. They participate in the innate immunity. It has been proved
that S100A8 activate RAGE signaling and induces various inflammatory
processes such as psoriasis, rheumatoid arthritis (RA), systemic lupus
erythromatosus (SLE), asthma, food allergies, severe glomerulonephritis,
diabetic nephropathy, juvenile dermatomyositis etc., resulting severe
damages. On the other hand, S100B protein is constitutively present in
the epidermis and dermis, especially, in sweat glands, Langerhans'
cells, melanocytes, Schwann cells, sensory corpuscles, eosinophils, and
play significant roles in various skin diseases, including vitiligo,
melanoma and psoriasis (22,23). S100 proteins and other DAMPs
particularly bind with RAGE and play key roles in the progression of
inflammatory processes. Blockage of RAGE, but not TLR4, inhibits S100
mediated trafficking of macrophages and leukocytes (24,25). S100A8/A9 is
becoming a more sensitive biomarker than routine inflammation indices,
including C-reactive proteins (CRP) to predict prognosis and therapeutic
progresses. Sequestrations of S100 proteins by tasquinimod (an oral
quinolone-3-carboxamide) or blockage of secretions of soluble S100
proteins ameliorate severe inflammatory conditions in diabetes, SLE,
multiple sclerosis and RA. In addition, blockage of RAGE with small
molecules (e.g., paquinimod) suppresses diabetes-related cardiovascular
diseases (CVDs). In a mice/tick-feeding model, the ticks' salivary
glands derived molecule, longistatin, has been shown to block RAGE, but
not TLR4. Longistatin mitigates tick induced inflammatory responses (6).

During this study, we detected huge accumulation of ILC2 at the site of
attachment of ticks in wt, but not in
\emph{RAGE\textsuperscript{-/-}}mice, indicating RAGE receptor plays
critical roles in the tick bite dermatitis and injuries. Based on
developmental biology, phenotype, and signaling, until now, ILCs have
been classified into five sub groups such as~NK
cells,~ILC1s,~ILC2s,~ILC3s, and lymphoid tissue inducer (LTi) cells, of
which ILC2s are vital for the lethality following haemorrhagic shock
(HS), trauma (Ref) and parasitic infections and helps in repair of
tissue damage (26) . They are abundant in tissues of the skin
(27,28),~lung, liver, and gut~(29,30) and are characterised by the
production of IL-4,~IL-5, and~IL-13, the signature type 2 cytokines and
considered as the innate counterparts of~Th2 cells. In fact,
immunological characteristic of ILC2s is of type 2 immunity, and deals
with indigestible oversized pathogens, such as expulsion of helminths
and other large organisms (31). We detected ILC2s using anti-CD44, as
ILC2s produce~CD44, but not~CD161 in mice (32). ILC2s is instrumental
for repairing damaged tissues by facilitating differentiation of
epithelial cells for tissue repair (29).~ It has been reported that
ILC2s are recruited to the damaged~dermis~in both mice and humans
(33).~During blood feeding by ticks, damage in the skin extends up to
the dermis and ILC2s take part in the tissue repair (6).

We found that repeated tick infestations gradually increased systemic
circulating eosinophils. Eosinophils are multifunctional leucocytes,
which govern the pathogenesis of multiple inflammatory condition,
including allergic diseases, tissue injuries, helminth infections and
ectoparasitic attacks. Eosinophils express several PRRs such as TLR1--5,
TLR7, TLR9, NOD1, NOD2, Dectin-1 and RAGE. Eosinophils are produced by
and become mature in the bone marrow, and finally released into blood
circulation. In peripheral blood, they constitute 1--3\% of the
circulating white blood cells (WBL), and are recruited to various
tissues. Granules of eosinophils contain four toxic proteins namely
major basic protein (MBP), eosinophil cationic protein (ECP),
eosinophil-derived neurotoxin (EDN) and eosinophil peroxidase (EPO).
Moreover, they release reactive oxygen species (ROS), leukotrienes,
prostaglandins and several cytokines and chemokines. These molecules
cause epithelial damage, smooth muscle constriction, increased vascular
permeability and recruitment of inflammatory cells (34). RAGE is one of
the most prominent DAMP sensors by responding to a range of signals
derived from dying or damaged cells (35). Alarmins induce degranulation
and oxidative burst, and serve as a chemoattractant and an eosinophil
survival factor.

\textbf{CONCLUSIONS}

Taken together, we proved that inflammatory reactions and number of
circulating eosinophils gradually increased in the repeated tick
infestations in wt mice, which were tightly governed by RAGE. RAGE also
dictated trafficking of eosinophils and ILC2s in the tick mediated
insults at the site of attachment of ticks.

\textbf{ACKNOWLEDGEMENTS}

This work was supported by a postdoctoral fellowship for foreign
researchers (to A) provided by the Japan Society for the Promotion of
Science (JSPS). This work was also supported by JSPS KAKENHI Grant
Numbers 20F20093, 20H03001 and the JSPS Core-to-Core Program
(JSJSCCB20200010).

\textbf{AUTHOR CONTRIBUTIONS}

Conceptualization by A, MAA and NT; data generation by A, MM, MAA, and
TH; A, MSH, SSL, ISS wrote draft; A, TH, MSH, MHA, and ISS analyzed
data; A, TH, MAA, YY and NT revised manuscript.

\textbf{DATA AVAILABILITY STATEMENT}

The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request.

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\textbf{Figure legends}

\textbf{Figure 1 Roles of RAGE in modulating tick-bite induced
inflammation at different phases of tick infestations. (A) Histological
feature of the tick-bitten areas or normal (control) skin.} Ticks were
allowed to feed on C57BL/6 or \emph{RAGE} \textsuperscript{-/-} mice,
and the same mice were used for repeated tick infestations. Mice were
euthanized after repletion of tick in each infestation (n=3 for each
feeding phase). Skin from the attachment site of ticks was collected and
preserved in 4\% paraformaldehyde added with 0.1\% gluteraldehyde
overnight at 4 °C under gentle shacking. Thin (5 \selectlanguage{greek}μ\selectlanguage{english}m) sections were
prepared and subjected to H\&E\textbf{. (B) Comparative features of
cellular infiltration in tick-bitten skins.} Ticks were allowed to feed
on C57BL/6 or \emph{RAGE} \textsuperscript{-/-} mice in the same manner
and subjected to histopathological analysis. Inflammatory cells per
focus were counted by two independent blind investigators. **,
P\textless{}0.001; NS, non-significant.

\textbf{Figure 2 Upregulation of RAGE in tick bite areas.} Ticks were
allowed to feed on C57BL/6 or \emph{RAGE} \textsuperscript{-/-} mice,
and the same mice were used for repeated tick infestations. Mice were
euthanized after repletion of tick in the tertiary infestation (n=3).
Skin from the attachment site of ticks was collected and preserved in
4\% paraformaldehyde added with 0.1\% gluteraldehyde overnight at 4 \selectlanguage{ngerman}°C
under gentle shacking. Thin (5 \selectlanguage{greek}μ\selectlanguage{english}m) sections were prepared and subjected
to immunofluorescence using anti-RAGE as mentioned in the methodology
section.

\textbf{Figure 3 Detection of RAGE ligands at the tick bite areas.}Ticks
were allowed to feed on C57BL/6 or\emph{RAGE} \textsuperscript{-/-}
mice, and the same mice were used for repeated tick infestations. Mice
were euthanized after repletion of tick in the tertiary infestation
(n=3). Skin from the attachment site of ticks was collected and
preserved in 4\% paraformaldehyde added with 0.1\% gluteraldehyde
overnight at 4 \selectlanguage{ngerman}°C under gentle shacking. Thin (5 \selectlanguage{greek}μ\selectlanguage{english}m) sections were
prepared and subjected to immunohistochemistry using anti-S100A/8 or
anti-S100B as mentioned in the methodology section.

\textbf{Figure 4 RAGE dictates ILC2s during ticks' feeding.} Ticks were
allowed to feed on C57BL/6 or \emph{RAGE} \textsuperscript{-/-} mice,
and the same mice were used for repeated tick infestations. Mice were
euthanized after repletion of tick in the tertiary infestation (n=3).
Skin from the attachment site of ticks was collected and preserved in
4\% paraformaldehyde added with 0.1\% gluteraldehyde overnight at 4 \selectlanguage{ngerman}°C
under gentle shacking. Thin (5 \selectlanguage{greek}μ\selectlanguage{english}m) sections were prepared and subjected
to immunohistochemistry using anti-CD44 or anti-MPO as mentioned in the
methodology section.

\textbf{Figure 5 Tick-bite induced eosinophilia is governed by
RAGE.}Ticks were allowed to feed on C57BL/6 or\emph{RAGE}
\textsuperscript{-/-} mice, and the same mice were used for repeated
tick infestations. Blood were collected different feeding phase at
different time points (day 0-day 5) from tail tip and examined.

\textbf{Hosted file}

\verb`Figures_Anisuzzaman et al.pptx`  available at \url{https://authorea.com/users/595353/articles/674141-receptor-for-advanced-glycation-end-product-rage-modulates-inflammation-during-feeding-of-tick-haemaphysalis-longicor}

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