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.