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, H. longicornis , which
simultaneously prevents blood coagulation and inflammation. Furthermore,
factor Xa (FXa) inhibitors (e.g., tick anticoagulant peptide [TAP],
from Ornithodoros moubata , Salp14 from Ixolaris
scapularis and FXaI from Ornithodoros savignyi ), tissue factor
pathway inhibitor (e.g., Ixolaris from I. scapularis ), direct
thrombin inhibitor (e.g., microphilin from Ripicephalus
microplus , savignin from O . savignyi, ornithodorin fromO. moubata , madanin 1 and 2 from H. longicornis and
variengin from Amblyomma variegatum ), complement inhibitors
(e.g., OMC from O. moubata , Isac and Salf 20 from I.
scapularis and IRAC 1 and 2 from Ixodes ricinus ), T cell
inhibitors (e.g., Salp 15 and siolostatin L from I. scapularisand Iris from 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 Ca2+-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 RAGE-/-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.
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.