Inflammation is characterized by complex interactions between innate and adaptive immunity . Pro-inflammatory cytokines and chemokines recruit immune cells to the site of tick feeding. Tick salivary proteins then mitigate the secretion of cytokines by immune cells, thereby, diminishing inflammation [1, 2, 4]. Despite significant progress in the past decades, how ectoparasites, such as ticks, regulate host innate immune signaling during transmission of the rickettsial agent A. phagocytophilum to the mammalian host remains mostly elusive. In this study, we demonstrate that I. scapularis saliva has the ability to inhibit cytokine secretion by murine and human immune cells. These findings are supported by our results showing that extracellular and cytosolic stimulation of macrophages with PAMPs can be inhibited by I. scapularis saliva. We also performed experiments with A. phagocytophilum and demonstrated that similar mitigation effects occur in macrophages. The implications for these findings are wide in scope as ticks, mosquitoes, biting flies, fleas and blood-feeding bugs have also evolved similar strategies for modulating host defenses .
We reported an effect of tick saliva on TLR and NLR signaling in macrophages. Although the effect of tick saliva was previously demonstrated during TLR stimulation of dendritic cells [29, 30, 48–50], whether I. scapularis saliva affects the response to stimulation of murine macrophages and human peripheral blood mononuclear cells had not been determined. Macrophages and peripheral blood mononuclear cells are important because these immune cells respond to A. phagocytophilum infection [33, 51]. To our knowledge, we describe for the first time that secretion of IL-6 and IL-12p40 after stimulation with the Nod2 ligand MDP was diminished in macrophages during treatment with tick saliva. Nod2 has emerged as a critical regulator for immunity and inflammation since it activates canonical and non-canonical NF-κB signaling, mitogen-activated protein kinases, cytokines, chemokines and antimicrobial reactive oxygen species .
Previously, we showed that A. phagocytophilum is partially recognized by the NLRC4 inflammasome , a protein scaffold that regulates the secretion of IL-1β and IL-18 . We also demonstrated that mice deficient in caspase-1 and asc, essential components of the inflammasome, were more susceptible than wild-type animals to A. phagocytophilum infection. These findings were due to the absence of IL-18 secretion and reduced interferon (IFN)-γ levels in the peripheral blood. It is unclear how I. scapularis saliva regulates IL-1β secretion by macrophages during A. phagocytophilum stimulation. However, it is possible that multiple salivary proteins regulate IL-1β secretion during hematophagy. Ticks have large genomes and carry many gene paralogs . These gene paralogs may act redundantly to provide inhibition of immune protein scaffolds in the mammalian host. Two earlier articles provided experimental support for this hypothesis. Ramachandra and Wikel showed that salivary gland extracts from the tick Dermacentor andersoni reduced IL-1 levels during the early phases of tick feeding , whereas Fuchsberger et al., 1995 determined that human IL-1β secretion was mitigated when treated with LPS and salivary gland extracts from partially fed adult female Rhipicephalus appendiculatus.
In addition, A. phagocytophilum may need redundant mechanisms of innate immune recognition to trigger IL-1β secretion. Secretion of IL-1β requires NF-κB activation to generate pro-IL-1β [56, 57]. Dumler and colleagues demonstrated that A. phagocytophilum triggers TLR2 activation during immune cell stimulation . TLR activation is known to initiate NF-κB signaling in immune cells . More recently, our group participated in a study showing that receptor interacting protein-2 (RIP2) affects A. phagocytophilum infection in mice . RIP2 is an adaptor molecule for the innate immune receptors Nod1/2, which also regulates NF-κB signaling . Finally, assembly of a multi-protein complex coined “inflammasome” is critical for IL-1β secretion [56, 57]. We previously demonstrated that the inflammasome is critical for immunity against A. phagocytophilum infection . Taken together, our findings reinforce the notion that A. phagocytophilum immunity is multi-factorial, and suggests a holistic inhibitory effect of tick saliva on innate immunity. This is important because a pathogen such as A. phagocytophilum may need multiple layers of immune evasion during transmission. Therefore, the holistic properties of tick saliva may be a major strategy of host immune evasion during pathogen transmission.
The above hypothesis is supported by several lines of evidence. Post-genomic approaches show that A. phagocytophilum actively modulates gene expression in ticks [62, 63]. Recently, the P11 salivary protein was shown to be required for A. phagocytophilum migration from hemocytes to the salivary glands in ticks . Another salivary gland protein named SALP16 was deemed important for A. phagocytophilum survival within the tick vector . A. phagocytophilum alters the monomeric/filamentous (G/F) actin ratio leading to the translocation of phosphorylated/G-actin to the nucleus . This event affects salp16 gene transcription in association with the RNA polymerase II (RNAPII) and the TATA-binding protein. Fikrig and colleagues have also demonstrated that A. phagocytophilum appears to increase the ability of I. scapularis to survive in cold temperatures by up-regulating an antifreeze glycoprotein  and α1, 3- fucosyltransferases, which are important for pathogen colonization . On the other hand, the Janus kinase (JAK)-signaling transducer activator of transcription (STAT) pathway seems to be important for the restriction of A. phagocytophilum infection in ticks. Clearly, further studies are necessary to determine the contribution of salivary proteins to A. phagocytophilum pathogenesis and immunity.
Elucidating the underlying effect of tick saliva in vivo should be considered a high-priority in tick research. Several experiments show that findings obtained in vitro may sometimes differ from those occurring in vivo, and mRNA and protein levels do not necessarily correlate well . In vivo characterization of tick salivary proteins, however, is not a trivial task. The presence of multiple paralogues in the I. scapularis genome  make the use of siRNA or dsRNA technology challenging because of known off-target effects . Another technical limitation is the lack of strategies to introduce and/or delete genes in ticks. Thereby, we were unable to generate knock-out, knock-in or transgenic I. scapularis. The development of this technology would enable researchers to characterize tick salivary proteins and clarify underlying events at the vector-pathogen-host interface. Despite all these technical restrictions, we posit that I. scapularis saliva may inhibit cytokine secretion during rickettsial transmission. We look forward with reasonable confidence that our findings may be used as a prelude for future in-vivo experimentation.