Abstract
Ticks secrete various anti-coagulatory, anti-vasoconstrictory, anti-inflammatory, and anti-platelet aggregation factors in their saliva at the bite site during feeding to evade host immunological surveillance and responses. For the first time, we report successful isolation of exosomes (small membrane-bound extracellular signaling vesicles) from saliva and salivary glands of partially fed or unfed ixodid ticks. Our data showed a novel role of these in vivo exosomes in the inhibition of wound healing via downregulation of C-X-C motif chemokine ligand 12 (CXCL12) and upregulation of interleukin-8 (IL-8). Cryo-electron microscopy (cryo-EM) analysis revealed that tick saliva and salivary glands are composed of heterogeneous populations of in vivo exosomes with sizes ranging from 30 to 200 nm. Enriched amounts of tick CD63 ortholog protein and heat shock protein 70 (HSP70) were evident in these exosomes. Treatment of human skin keratinocytes (HaCaT cells) with exosomes derived from tick saliva/salivary glands or ISE6 cells dramatically delayed cell migration, wound healing, and repair process. Wound healing is a highly dynamic process with several individualized processes including secretion of cytokines. Cytokine array profiling followed by immunoblotting and quantitative-PCR analysis revealed that HaCaT cells treated with exosomes derived from tick saliva/salivary glands or ISE6 cells showed enhanced IL-8 levels and reduced CXCL12 loads. Inhibition of IL-8 or CXCL12 further delayed exosome-mediated cell migration, wound healing, and repair process, suggesting a skin barrier protection role for these chemokines at the tick bite site. In contrast, exogenous treatment of CXCL12 protein completely restored this delay and enhanced the repair process. Taken together, our study provides novel insights on how tick salivary exosomes secreted in saliva can delay wound healing at the bite site to facilitate successful blood feeding.
Highlights
Ticks and tick-borne diseases are of major concern due to medical and veterinary health importance (Nuttall et al, 2000; Neelakanta and Sultana, 2015; de la Fuente, 2018)
Our Cryo-electron microscopy (cryo-EM) analysis showed the presence of purified tick saliva or salivary glandderived in vivo exosomes with the size ranges of 30–200 nm in diameter (Figure 1A) that are similar to exosomes isolated
We have found that both tick saliva and salivary gland-derived exosomes were heterogeneous in the population with sizes from 30 to 200 nm in diameter (Figure 1A and Supplementary Figure S1A), suggesting differential cargo being transported via these extracellular vesicles (EVs)
Summary
Ticks and tick-borne diseases are of major concern due to medical and veterinary health importance (Nuttall et al, 2000; Neelakanta and Sultana, 2015; de la Fuente, 2018). Several studies have shown that during blood feeding, ticks transmit a variety of pathogens that mainly includes bacteria from rickettsial family and Lyme disease agent and viruses such as tick-borne encephalitis virus (TBEV) that belongs to Flaviviridae family (Wikel and Whelen, 1986; Labuda et al, 1996; Nuttall et al, 2000; Nuttall and Labuda, 2004; Neelakanta et al, 2010; Sultana et al, 2010; Leitner et al, 2011; Wikel, 2018; Zhou et al, 2018). Some of these tick salivary factors include the inhibitors of the itch and/or pain or in combination of both, anticoagulants, immunomodulators, vasodilators (that dilate blood vessels), and antiplatelet molecules (that can avoid platelet-mediated killing mechanisms) (Wikel and Whelen, 1986; Labuda et al, 1996; Wikel, 1996a; Bowman et al, 1997; Nuttall and Labuda, 2003, 2004; Bernard et al, 2015; Kotal et al, 2015; Lewis et al, 2015; Scholl et al, 2016; Glatz et al, 2017; Vora et al, 2017; Bakshi et al, 2018; Nuttall, 2019)
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