A prime example of intracellular bacteria regulating apoptosis is Anaplasma phagocytophilum, a tick-transmitted rickettsial agent that causes human granulocytic anaplasmosis. In vitro, A. phagocytophilum utilizes several mechanisms to inhibit apoptosis in different species of tick cells. For example, besides interfering with endoplasmic reticulum (ER) and the unfolded protein response in Ixodes scapularis ISE6 cells, A. phagocytophilum also downregulates expression of a series of kinases, including phosphoenolpyruvate carboxykinase (PEPCK), mitogen-activated protein kinase (MKK), and apoptosis signal-regulating kinase 1 (ASK1) (Ayllón et al., 2013; Villar et al., 2015). In addition, in I. ricinus IRE/CTVM20 tick cells, transcriptome analysis and flow cytometry revealed that infection with A. phagocytophilum not only regulates JAK and anti-apoptotic factors gene expression but also inhibits the intrinsic apoptosis pathway (Alberdi et al., 2016b; Alberdi et al., 2016a). In vivo, A. phagocytophilum displays a tissue-specific response to mediate cell apoptosis in tick nymphs (Ayllón et al., 2015). It achieves this by targeting the JAK/STAT pathway and decreasing FAS expression in midguts, while reducing porin (voltage-dependent anion-selective channel) expression to inhibit cytochrome c (one of the mitochondrial proteins associated with apoptosis) release from mitochondria in salivary glands via intrinsic apoptosis. Interestingly, tick salivary glands serve as an essential organ for A. phagocytophilum colonization, and have a possible role in activating extrinsic apoptosis to limit bacterial infection. Indeed, mitochondria, caspases, and pro/anti-apoptotic molecules are key players associated with apoptosis during pathogen infection. Similar to its manipulation of the arthropod host, A. phagocytophilum also modulates activity of the above “players” to inhibit apoptosis for their advantage in vertebrate hosts (Scaife et al., 2003; Borjesson et al., 2005; Choi et al., 2005; Ge and Rikihisa, 2006; Niu et al., 2010).

Another very similar system is exploited by Rickettsia rickettsii (agent of Rocky Mountain spotted fever). According to the proteome of Rhipicephalus microplus BME26 cells, R. rickettsii may hamper apoptosis via inhibition of caspase-3 activity, thus favoring bacterial growth and proliferation (Martins et al., 2020). Apart from bacterial pathogens, tick-borne viruses have long received much attention. The I. ricinus IRE/CTVM20 tick cell transcriptome exerts different gene expression patterns relative to infection with A. phagocytophilum after tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) infection, such as raising cytochrome c expression. Interestingly, some apoptosis-related genes, including caspase and hsp70, are expressed differently in flavivirus and intracellular bacterial infections. Whether flaviviruses could benefit from inhibiting tick apoptosis as does A. phagocytophilum, is however unconfirmed (Mansfield et al., 2017).

Although a critical strategy of intracellular pathogens is induction of vertebrate host-cell apoptosis under certain circumstances, the pro-apoptotic response of ticks to tick-borne pathogens is much less defined. Recently, we have shown that Rickettsia parkeri, a tick-transmitted spotted fever group rickettsia, is able to activate mitochondria-dependent apoptosis to promote its infection of and replication in tick cells (Wang X-R. et al., 2021). By employing cell types from different tick species, we demonstrated that R. parkeri initiation of apoptosis was a conserved response and required intracellular rickettsial replication. Considering that apoptosis is initiated via a series of stressors within a cell, the different growth status (exposure, colonization, invasion, and infection) of pathogens in the host might cause different stimuli, thus playing a diverse role in apoptosis. Indeed, R. parkeri exerts pro-and anti-apoptotic activities during different infection phases, which also has been observed in another spotted fever group rickettsia, R. rickettsii (Clifton et al., 1998). However, unlike in the arthropod host, R. parkeri failed to induce apoptosis in vertebrate host cells at the same infection phase (Wang X-R. et al., 2021). Unsurprisingly, pro-apoptotic activity in one cell type or host species may not be the same as in another cell type or host species. Another example of a pathogen that induces different responses in mammalian and tick hosts is Hazara virus, a tick-borne segmented negative-sense RNA virus closely related to Crimean-Congo hemorrhagic fever virus (CCHFV), but which causes less severe disease. Cleavage of virus nucleocapsid (N) protein by caspase-3 results in apoptosis in mammalian cells but fails to activate apoptosis in tick cells. The use of two different strategies to modulate apoptosis in the respective hosts by members of the genus Nairovirus could directly affect the infection outcome (Fuller et al., 2019). Obviously, a flexible strategy is important for the successful colonization of vectors and hosts by those pathogens in a broad range of host cell types and host species. However, how intracellular pathogens execute this flexible strategy is largely unexplored in their arthropod hosts.

The most well-studied mechanisms used by tick-borne bacteria to manipulate the autophagic pathway come from the Anaplasmataceae in their interactions with mammalian cells. Anaplasma phagocytophilum secretes the effector protein Ats-1, which interacts with Beclin1 to recruit autophagosomes to the A. phagocytophilum vacuole, supplying nutrients to support pathogen growth (Niu et al., 2012). A similar but distinct process to induce autophagy and trafficking of autophagosomes to the pathogen-containing vacuole is employed by Ehrlichia chaffeensis (causative agent of human ehrlichiosis) via its effector Etf-1, which interacts with Rab5, Beclin1 and the autophagy-initiating class III phosphatidylinositol 3-kinase complex (Lin et al., 2016). Both A. phagocytophilum and E. chaffeensis also prevent their vacuoles fusing with the lysosome (Niu et al., 2008; Lin et al., 2016; Lina et al., 2017); this is achieved by E. chaffeensis through modulation of the Wnt signaling pathway to inhibit autolysosome formation (Lina et al., 2017).

Infection with a range of spotted fever group rickettsiae (R. conorii, R. japonica, R. montanensis, R. parkeri and R. rickettsii) results in autophagy induction in mammalian host cells (Uchiyama et al., 2012; Engström et al., 2019; Sahni et al., 2020). Pathogenic rickettsiae appear to be capable of evading this immune response, whilst non-pathogenic species lack this ability (Uchiyama et al., 2012; Engström et al., 2019). Infection of human umbilical vein endothelial cells with R. rickettsii or R. conorii results in mTOR activation, potentially as a mechanism by which these rickettsiae limit anti-microbial autophagy (Sahni et al., 2020), whilst Rickettsia parkeri is able to evade autophagy by employing outer membrane protein B (OmpB) to prevent the ubiquitination of surface proteins and their subsequent recognition by autophagic receptors in both human microvascular endothelial cells and mouse bone-marrow-derived macrophages (Engström et al., 2019). Even in the same cell type, different Rickettsia species utilize contrasting strategies to evade autophagy. For example, R. australis induces autophagy to aid successful invasion of mouse bone-marrow-derived macrophages (Bechelli et al., 2019), resulting in the inhibition of inflammatory cytokine secretion to favor bacterial survival (Bechelli et al., 2021). Due to the broad range of rickettsial pathogens and different host cell types studied, we are just unveiling the tip of the iceberg regarding the complex interactions between these pathogens and their hosts. Although we can use the situation in vertebrates as a basis for predicting what might occur in ticks, we cannot assume that the strategies employed by pathogens to infect mammals can be generalized to their persistence in arthropods (as shown above with apoptosis), and so further investigation into how pathogens interact with tick autophagy are warranted.

Despite impressive progress being made on how pathogens “tamper with” the tick immune system, including revealing antibacterial and antiviral pathways and identifying molecular effectors and cells (Fogaça et al., 2021), unlike the well-known arthropod-pathogen systems (Buchon et al., 2014), tick-pathogen interaction is a newly emerging field that is far from completely understood. This is largely due to the tick’s own complex development and the diversity of its transmitted pathogens. Firstly, in vitro study depends on tick cell lines, which are relatively fragile compared to more common cell types, and have more intensive culture requirements (Munderloh and Kurtti, 1989; Bell-Sakyi, 1991; Mattila et al., 2007; Bell-Sakyi et al., 2009). Additionally, most tick cell lines were derived from embryos, however, their tissue(s) of origin are unconfirmed. Different cell types might possess different characteristics and ontogenies, resulting in unique properties (Mattila et al., 2007; Wang et al., 2020). In vivo studies on ticks are more challenging because of their unique life cycle, which is influenced by an array of elements, including species, host feeding preference, different ecological and geographic factors in nature, and strict maintenance requirements in the laboratory (Sonenshine and Roe, 2013; Jia et al., 2020). Besides some medically and veterinary important species, most ticks are a blind-spot due to insufficient data on genomic information, let alone the interactions with potential pathogens that they may harbor. Genomic data is only available for a limited number of tick species including Ixodes scapularis, Ixodes ricinus, Ixodes persulcatus, Haemaphysalis longicornis, Dermacentor silvarum, Hyalomma asiaticum, Rhipicephalus sanguineus, and Rhipicephalus microplus (Gulia-Nuss et al., 2016; Cramaro et al., 2017; Jia et al., 2020). The most critical difficulty is pathogens themselves, as many different factors such as species/strain pathogenicity, difficulty in laboratory maintenance, etc. may alter the observed results. For example, even different strains of the same pathogen, R. rickettsii, exhibits exclusive manners in different cell types or organs (Lehman et al., 2018). Thus, pathogens initiate a critical step for tick immune response, directing more complex communication than simple inhibition or activation. Studies to address these difficulties would pave the way for future research centered on the tick immune system ( Figure 2 ).

It is unquestionable that PCD acts as one piece of the puzzle for tick innate immunity, and more work needs to be done to gain more clues to solve a tick’s “Jigsaw puzzles”. This would include investigating the network of other immune pathways as well as the cross-talk between apoptosis and autophagy under certain conditions (Fairlie et al., 2020). Paradigms in vertebrate hosts utilize these communications to enhance the recognition and destruction of intracellular pathogens (Hua et al., 2019; Van Opdenbosch and Lamkanfi, 2019), and we expect that immune responses in ticks behave similarly. Interestingly, tick-borne intracellular pathogens also make use of effective communications in their vertebrate hosts to tip the scales in their favor. For example, as well as inducing autophagy, both the A. phagocytophilum effector Ats-1 and the E. chaffeensis Etf-1 are also translocated into the host mitochondria to inhibit apoptosis initiation (Niu et al., 2010; Liu et al., 2012). Another A. phagocytophilum effector AptA induces autophagy and the ubiquitin-proteasome system, whilst reducing the efficiency of apoptosis (Ma et al., 2021). There is also interplay between autophagy and inflammatory pathways during both Ehrlichia and Rickettsia infection (Tominello et al., 2019; Bechelli et al., 2021), and autophagy induction is balanced by signaling of MyD88 (a downstream adaptor for many pattern recognition receptors) during ehrlichial infection (Kader et al., 2017).

As with other arthropods, crosstalk within the tick innate immune system associated with the response to pathogen infection has also been explored in recent decades (Capelli-Peixoto et al., 2017; Fogaça et al., 2021). Although antibacterial and antiviral pathways, including JAK-STAT (Janus kinase/signal transducer and activator of transcription), Toll, IMD (Immune Deficiency) and RNA interference (RNAi), possess a certain specificity, they are also capable of collaboration under certain conditions. For example, ticks utilize the IMD pathway in response to infection with B. burgdorferi, A. phagocytophilum, or A. marginale (Shaw et al., 2017; McClure Carroll et al., 2019; Kurokawa et al., 2020). The downstream pathways include NF-kB/Relish and Jun N-terminal kinase (JNK) (Ramphul et al., 2015; Chowdhury et al., 2020; Tafesh-Edwards and Eleftherianos, 2020), which are involved in viral-induced apoptosis and have been well characterized in insects. It is possible that bacteria induce a similar response in ticks. However, the components of these pathways in ticks are highly divergent from vertebrate and insect systems, and whether those mechanisms also apply in ticks needs further investigation. Towards this end, identification of the apoptosis and autophagy components of ticks by employing comparative genomics would be a significant step. Utilizing tick and other arthropod genome sequences, the homologues to apoptosis/autophagy-related genes of known function should be confirmed (Wang et al., 2020).

To understand the biological processes involved in apoptosis/autophagy in response to pathogens, multi-omics (including genetics, epigenetics, transcriptomics, proteomics, metabolomics, and cellomics) would be valuable approaches (Hasin et al., 2017; Chu et al., 2021). For example, combining genomic and transcriptome data can reveal apoptosis-related genes and their roles in pathogen infection. Using metabolomics and proteomics, proteins involved in the apoptosis pathway and their connection to molecular changes in metabolic pathways can also be identified. Single-cell/nucleus omics also can determine functional molecules of each cell and specific tick cell subtypes in response to pathogens. Finally, combining properly analyzed approaches and apoptosis assay measurements, the study of apoptosis in tick-pathogen interactions would be significantly enhanced. Genetic tools for editing microorganisms, including mutagenesis and CRISPR as well as RNAi, facilitate the growing body of research in host-pathogen interactions (Durvasula et al., 1997; Billmyre et al., 2013; Wilke and Marrelli, 2015; Vo et al., 2021). As well as using the multi-omics methods outlined above, further work to characterize interactions between pathogens and tick autophagy could employ transgenic reporter bacteria or viruses to visualize responses to autophagy. In addition, with the use of directed mutants or random mutant libraries, the underlying mechanisms used by pathogens to influence autophagy and apoptosis pathways could be revealed, as well as identification of specific factors essential for bacterial invasion and replication in their tick vectors ( Figure 2 ).