Ehrlichia chaffeensis Co-Opts Phagocytic Hemocytes for Systemic Dissemination in the Lone Star Tick, Amblyomma americanum

The immune system of hematophagous arthropods is important for mounting defense responses against invading pathogenic microbes. Ticks are obligate hematophagous arthropods constantly exposed to pathogenic and nonpathogenic microbes from the mammalian host during blood feeding. Pathogen recognition precedes the activation of cellular and humoral components of the immune system, which leads to the killing and elimination of pathogenic microbes from the system [1]. Immune cells, known as hemocytes, play a central role in eliciting cellular and humoral responses, eventually leading to pathogen killing. Hemocytes contribute directly to immune response through phagocytosis, encapsulation, and nodulation. Hemocytes also contribute to the humoral response by producing antimicrobial peptides and lytic enzymes, which indirectly lead to pathogen killing [2, 3].

The recruitment of hemocytes to an infection site is necessary for efficient phagocytosis and subsequent pathogen clearance [4‒6]. Elegant studies in mosquitoes have demonstrated that hemocytes contribute to pathogen survival at the site of midgut infection and subsequent dissemination to other tissues upon acquiring an infected blood meal. Hemocytes have the capability to either promote or hinder pathogen survival, contingent upon the infection site, partly due to the specific attraction of these pathogens to host hemocytes. In mosquitoes, viruses such as Sindbis, Zika, and dengue are able to infect hemocytes [7‒9]. In ticks, Anaplasma phagocytophilum and Rickettsia parkeri also infect the tick’s hemocyte, with the former using hemocyte infection as a vehicle for salivary gland colonization [4, 10]. Plasmodium invasion, a mechanical process, involves the digestion of the midgut peritrophic membrane during midgut infection [11]. The integrity of the midgut epithelium ensures commensal bacteria are protected and, thus, do not induce an immune response. However, damage to the midgut epithelial and basal lamina epithelial integrity, either by Plasmodium invasion or blood feeding, exposes the gut microbes to the epithelial, thus priming the immune system [12]. Plasmodium-induced damage results in the recruitment of hemocytes, specifically granulocytes, to the midgut and the production of hemocyte-specific transcripts [12‒14]. In addition, phagocytic granulocytes have been implicated in mosquito complement recognition of invading Plasmodium ookinetes, an interaction mediated by thioester-containing protein 1 (TEP1) recognition of the parasite surface that is impaired when phagocytes are chemically depleted or overloaded with beads [15, 16]. Together, these studies strengthen the idea that phagocytes play an integral role in the early recognition of ookinetes [16].

Recent studies demonstrated that hemocytes recruited to the midgut during viral infection do not limit viral replication in the midgut; however, they are essential to restrict systemic dissemination [8]. Despite the importance of hemocytes in an immune response against mosquito-borne pathogens, little information exists on their role in tick-pathogen interactions. Ticks can potentially harbor multiple pathogens at the same time. Unlike mosquitoes, immature and mature developmental stages of ticks can transmit tick-borne pathogens (TBPs), including a variety of bacterial, protozoan, and viral pathogens ingested during blood feeding on an infected mammalian host. In addition, some TBPs can also be transovarially transmitted from the female to the eggs. Five hemocyte types are present in ticks based on morphological features, while functional studies have demonstrated phagocytic abilities in some hemocyte types [4]. Granulocytes, predominant cell types, are phagocytic, thus acting as the first line of cellular defense following microbial infection. TBPs can infect hemocytes, specifically granulocytes, to facilitate dissemination to tissue infection, including the salivary gland and ovary. For example, Anaplasma (A) phagocytophilum can infect Ixodes (I) scapularis hemocytes and migrate to infect the salivary gland [10]. Similarly, we recently demonstrated the ability of Rickettsia (R) parkeri to infect Amblyomma (A) maculatum granulocytes in both naturally and laboratory-infected ticks [4]. However, events that preclude hemocyte infection, such as midgut replication, hemocyte recruitment, and subsequent pathogen trafficking through hemocytes during infection, remain a mystery.

In this study, we investigated the role of granulocytic hemocytes in the E. chaffeensis infection and dissemination in A. americanum. We provide evidence that granulocytes are integral to the tick immune response during E. chaffeensis infection. Our results suggest a dual role for phagocytic hemocytes in tissue infection and systemic dissemination, demonstrating that granulocytes are required to reduce tissue infection yet are required for systemic dissemination.

Fully replete Amblyomma americanum nymphs were purchased from the Ecto Services (Henderson, NC, USA) and maintained at 34°C and 65% relative humidity (RH) under 14:10 h L:D photoperiod till needed. Ehrlichia-infected adult ticks were generated as previously described [17]. Fully engorged nymphs were injected with E. chaffeensis (Arkansas strain) using a 32-gauge needle fitted to a Hamilton syringe (Hamilton Company, Franklin, MA, USA). All injected ticks were monitored for 25 h at a temperature of 34°C to remove dead or nonviable nymphs. Viable nymphs were transferred into an incubator maintained at 34°C and 65% RH under 14:10 h L:D photoperiod and monitored until they molted into either adult males or females. Tissues from emerged unfed and partially fed adult ticks were dissected, and RNA was isolated to confirm Ehrlichia infection.

The collection of hemolymph and quantification of hemocytes follows the previous approach with modification to the collection medium [4]. Perfused hemolymph was resuspended in Leibovitz’s L-15 Medium supplemented with 50% BSA on ice. Total hemocytes were quantified using a trypan blue exclusion method. Ten µL of perfused hemolymph was mixed with an equal amount of 0.4% trypan blue, and hemocytes were quantified in a Countess Automated Cell Counter (Invitrogen, Thermo Fisher Scientific Waltham, MA, USA). Differential hemocyte count was estimated by placing 10 μL of hemolymph on the groove of an improved Neubauer chamber. Hemocytes were differentiated morphologically as described previously [4, 18, 19].

Unfed ticks were subjected to injection with 0.2 μL of clodronate liposome (CLD) or control liposomes (LPs) (Standard Macrophage Depletion Kit from Encapsula Nano Sciences LLC, Brentwood, TN, USA) as previously described [4]. To identify an ideal concentration to deplete hemocytes with a minimal impact on tick survival, ticks were injected with different stocks or dilutions of LP (1:2, 1:5) or CLD (1:2, 1:5) in 1X PBS. Additionally, a group of ticks received injections of 1X PBS alone, serving as the injection control. The injected ticks were monitored for 10 days following LP or CLD injection to assess their survival rates. Based on the results obtained from the optimal concentration estimation, a dilution of 1:5 (LP and CLD) was selected for subsequent depletion experiments.

During the partially fed stage, hemolymph was collected by perfusion from CLD- or LP-treated E. chaffeensis-infected ticks. Hemolymph was centrifuged at 500 g for 5 min to separate the hemolymph plasma from the hemocyte component. The cell-free plasma and hemocyte components were maintained on ice until use. Cell-free plasma or hemocytes were injected into uninfected recipient ticks at a final volume of 0.5 μL and were subsequently fed on sheep until the partially fed stage. Partially fed ticks were collected, hemolymph was isolated, and tissues and carcasses were dissected. The effect of hemolymph transfer on total and differential hemocyte population was assessed as described in Hemolymph collection and hemocyte quantification. RNA was isolated from tissues and carcasses for E. chaffeensis quantification as described in RNA extraction, cDNA synthesis, and qRT-PCR.

We utilized in vivo injection of fluorescent-conjugated carboxylated beads to assess and quantify hemocyte phagocytosis, as previously described [4, 16]. To determine hemocyte phagocytosis, we injected ticks with 0.2 μL yellow-green carboxylated-modified microspheres (Thermo Fisher Scientific, Waltham, MA, USA) diluted in PBS into the tick hemolymph. Once the ticks underwent a 4-h recovery period at 22°C and 95% RH, we carefully perfused and processed the hemolymph for imaging. The hemolymph was placed on a cover slide and incubated at room temperature for an hour to promote hemocyte adherence to precisely quantifying phagocytosis. Following this, a 30-min fixation step was undertaken using 4% paraformaldehyde (PFA and a 5-min wash step in 1X PBS. The slides were then mounted and sealed in VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories, catalog number: H-1000) for further image acquisition. Each group underwent processing of 3–5 ticks for the in vivo phagocytosis assay to provide comprehensive data collection. For each tick, three different microscopic fields were carefully selected, each containing 200–500 hemocytes per field of view. This meticulous approach allowed us to determine the proportion of phagocytic hemocytes present.

Quantification of sessile phagocytes in E. chaffeensis-infected tick tissues followed the method described in the previous paragraph. The ticks used for this experiment were infected at the end of the nymphal blood meal as described in the “Tick maintenance and generation of infected ticks” section. Once the hemolymph had been perfused as described above, the ticks were injected with 4% PFA and allowed to fix for 1 h at room temperature. The salivary glands and tissues were dissected in 0.5% Triton X-100 for permeabilization for 30 min before washing in 1X PBS 3 times at 5 min each. The tissues were then mounted and sealed on a microscope slide using a VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories, catalog number: H-1000) for image acquisition and analysis.

Uninfected female A. americanum ticks were restrained with their back on a petri dish, and capillary tubes containing 7 × 10⁸Ehrlichia/μL suspended in sucrose-phosphate-glutamate media (3.76 mm potassium phosphate monobasic, 7.1 mm potassium phosphate dibasic, 4.9 mm potassium glutamate in distilled water) [20] were fitted over their mouthpart. Ticks were placed in an incubator set at 37°C for 24 h for bacteria acquisition. Following infection, ticks were removed, sterilized in 70% ethanol and distilled water and organ dissected for confirmation of infection. Hemocytes and tissues were collected and processed for immunofluorescence assay. Another group of infected ticks was dissected, and the salivary gland, midgut, and carcass were isolated for RNA extraction and Ehrlichia quantification.

Hemocytes from GFP-expressing capillary-fed ticks were incubated on coverslips, while dissected tissues were placed in 24-well plates and fixed in 4% paraformaldehyde diluted in 1X PBS for 1 h at room temperature. Samples were washed 3 times in PBS and permeabilized in 0.5% Triton X-100 for 30 min followed by another wash round in PBS. Coverslips were then mounted and sealed on a microscope slide using a VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories, catalog number: H-1000) for image acquisition and analysis.

Gram-negative and -positive bacteria were injected to access the immune response in hemocyte-depleted ticks. Overnight cultures of Escherichia (E) coli DH5-alpha and Staphylococcus (S) aureus RN4220 maintained at 37°C in LB and TSA media were harvested, centrifuged, and adjusted to OD600 = 0.5 and OD600 = 0.1 in 1X PBS, respectively. E. chaffeensis (Arkansas strain) was recovered, and propagation was as previously described [21]. Briefly, unfed ticks were injected with 0.2 μL of either CLD or LP (1:5) and kept to recover at 22°C and 95% RH. Forty-eight hours after depletion, surviving ticks were injected with 0.2 μL 10⁷E. chaffeensis, E. coli, or S. aureus, using a 33 G removable needle (Hamilton Company, Franklin, MA, USA). Heat-killed S. aureus, E. chaffeensis, and LPS were injected as positive controls. A total of 20 ticks were used per treatment or control group. Ticks were kept for recovery at 22°C and 95% RH and monitored every 24 h for 10-day survival.

The salivary gland, midgut, and carcass were isolated from hemocyte-depleted ticks and ticks that received hemolymph components from hemocyte-depleted ticks. Total RNA was extracted following the Trizol-chloroform separation and isopropanol precipitation method (TRI Reagent, Molecular Research Center, Inc. Montgomery, Cincinnati, OH, USA). RNA pellet was washed in 75% ice-cold ethanol, air-dried, and resuspended in 30 μL nuclease-free water. The final RNA concentration and quality were checked on a nanodrop (Nanodrop One, Thermo Fisher Scientific, Pittsburgh, PA, USA), and RNA was stored at −80°C until needed. Complementary DNA synthesis was synthesized from 1000 ng of isolated RNA as previously described [22]. An absolute quantification method was used to quantify E. chaffeensis. An absolute quantification approach establishes a standard curve of E. chaffeensis 16S rRNA to serve as the reference template for qPCR [23]. The E.chaffeensis 16S rRNA and A.americanumactin gene were initially amplified from laboratory grown E.chaffeensis and A.americanum cDNA (Arkansas strain) using the primers described in online supplementary Table S1 (for all online suppl. material, see https://doi.org/10.1159/000535986) (online suppl. Table S1) [22, 24]. The amplified E. chaffeensis 16S rRNA and A. americanum actin PCR product were purified and serially diluted 10-fold for standard curve preparation. The qPCR reaction mixture (20 μL) consists of approximately 60 ng cDNA template, 0.7 μM of each primer, and 10 μL of SYBR green qPCR master mix. The reaction was performed in a thermal Cycler (CFX96 real-time detection system, Bio-Rad Laboratories) subjected to one cycle each of 50°C for 2 min and 95°C for 2 min, and 45 cycles of 95°C for 15 s and 60°C for 30 s. Each sample was analyzed in triplicate, and the resulting threshold cycle (Ct) values were utilized for determining the copy number using the standard formula.

The microscopic examination of hemolymph collected from A. americanum ticks, co-stained with phalloidin (green) and Hoechst 33342 (blue), revealed five distinct hemocyte populations. These populations exhibited specific characteristics, including spherulocytes (Sp) with a small nucleus-cytoplasmic ratio and a peripherally displaced nucleus. Granulocytes (Gr) also displayed a large nucleus-cytoplasmic ratio and multiple cellular projections. Prohemocytes (Pr) were also present, appearing with a large nucleus-cytoplasmic ratio and smaller than other cell types. Furthermore, we identified oenocytoids (Oe), which exhibited a smaller nuclear-to-cytoplasmic ratio. Lastly, their pyriform shape (Fig. 1a) distinguished plasmatocytes (Pl).

We further assess the impact of hematophagy and Ehrlichia infection on the hemocyte population. We counted total and differential hemocyte populations from the hemolymph of unfed and partially blood-fed ticks. Our results showed hematophagy significantly increased the hemocyte population in uninfected ticks compared to Ehrlichia-infected ticks (Fig. 1b). Although infection seems to stimulate a higher level of basal hemocyte counts, the effects of blood feeding are less prominent in unfed ticks (Fig. 1b). We observed a significant increase in the total hemocyte population when comparing counts between unfed-uninfected ticks and unfed ticks infected with E. chaffeensis (Fig. 1b) (unpaired t test, **p = 0.0095). Specifically, the population of prohemocytes was considerably higher in unfed ticks compared to partially blood-fed ticks, regardless of the infection status. However, unfed Ehrlichia-infected ticks displayed an even higher percentage of prohemocytes when compared to unfed-uninfected ticks (Fig. 1c). Similarly, the prohemocyte population was higher in partially fed ticks infected with E. chaffeensis compared to partially fed uninfected ticks (unpaired t test, *p = 0.0410). In contrast, we observed an opposite trend for the granulocyte population where the percentage of granulocytes was significantly higher in partially fed uninfected and Ehrlichia-infected ticks as compared to unfed ticks (Fig. 1d). As for spherulocytes, plasmatocytes, and oenocytoids, no significant changes were recorded following blood meal or infection (online suppl. Fig. S1A–C). These results suggest that hemocytes respond to host blood meal and, to a lesser extent, pathogen presence through a decline in immature hemocytes and a subsequent increase in granulocyte populations. The increase in the population of granulocytes is likely in response to the overall increase in microbial load that follows a blood meal, as shown in ticks [4] and mosquitoes [25, 26].

Researchers have heavily relied on molecular markers to study the functions of different hemocyte types in arthropod vectors, particularly in understanding their roles in immune functions. Recent studies have demonstrated the functional characterization of phagocytic hemocytes in non-model ticks [4], mosquitoes, and Drosophila through chemical depletion to understand their roles in the immune response [16, 27]. Similarly, in this study, CLD and LP were injected into the uninfected tick hemolymph and examined their effects on the total and differential hemocyte populations (Fig. 2a). To determine the optimal concentration for hemocyte depletion, we injected stock, 1:2, and 1:5 dilutions of CLD and LP and collected hemolymph for hemocyte quantification. Injecting CLD or LP at a 1:5 dilution had no adverse impact on tick survival (Fig. 2b). However, CLD significantly reduced the total hemocyte (Fig. 2c) and granulocyte (Fig. 2d) populations. Interestingly, CLD did not deplete the populations of prohemocytes, oenocytoids, plasmatocytes, and spherulocytes (online suppl. Fig. S2A–D), indicating its specificity in depleting granulocytes. Assessment of hemocyte phagocytosis via in vivo beads phagocytosis assay shows a significant reduction in phagocytic hemocytes as demonstrated by a decrease in the number of hemocytes associated with fluorescent beads (Fig. 2e). Next, we performed injections of CLD or LP (1:5 dilution) into E. chaffeensis-infected ticks and allowed them to blood feed before collecting hemolymph. The quantification of hemocytes showed a significant reduction in the total hemocyte and granulocyte population in the CLD-treated ticks (Fig. 2f, g). Additionally, in vivo phagocytosis analysis confirmed a concurrent decrease in the uptake of fluorescent beads in the CLD-treated ticks (Fig. 2h, i). This result supports using CLD liposome as an effective phagocyte depletion agent in A. americanum ticks.

Ticks harbor diverse microbial communities of pathogenic and nonpathogenic commensals [28]. The innate immune system actively maintains microbial homeostasis by balancing these pathogenic and commensal communities, with hemocytes playing a vital role in this process. With the goal to determine the role of the granulocyte populations in the A. americanum immune response, we infected ticks with E. coli, S. aureus, and E. chaffeensis after CLD treatment. As an injury control, we also included the injection of PBS. Ticks remained unaffected following the injection of PBS (Fig. 3a). CLD treatment significantly impaired tick survival following the S. aureus challenge (Fig. 3b). However, some ticks managed to survive until the end of the experiment. However, phagocyte depletion significantly impaired tick survival against Gram-negative E. coli and E. chaffeensis (Fig. 3c, d), with all ticks dying by the 6th and 8th days postinfection (dpi), respectively. These results underlie the integral role of granulocytic hemocytes as the first line of cellular defense during microbial infection. In addition, it demonstrates the lethality of Gram-negative bacteria in ticks when granulocytic hemocytes are significantly impaired.

Invertebrate hemocytes are distributed into two populations: freely circulating hemocytes found in the hemolymph and sessile populations attached to tissues and the body wall [29, 30]. Several studies have demonstrated that sessile hemocytes play a crucial role in controlling microbial infection in the hemolymph from orally acquired pathogens originating from the midgut epithelium as pathogens disseminate to the hemocoel and other associated tissues [10, 13, 29]. To this end, we assessed the effect of CLD treatment on the number of sessile hemocytes attached to the salivary gland and midgut of E. chaffeensis-infected ticks by quantifying the number of phagocytic hemocytes in these tissues. There was a significant reduction in the proportion of phagocytic hemocytes attached to both the salivary gland and midgut of CLD-treated ticks compared to the LP-injected ticks (Fig. 4a, b). In a parallel experiment, we isolated the salivary gland and midgut from E. chaffeensis-infected ticks 48 h after CLD or LP treatment. Upon quantifying the E. chaffeensis load, no distinction was observed between the CLD- and LP-treated tissues (online suppl. Fig. S23). These data suggest that CLD treatment reduced sessile hemocyte numbers in E. chaffeensis-infected ticks, suggesting their potential immune role, but did not affect pathogen load, implying other factors may control pathogen levels in these tissues.

Tissue infection and subsequent dissemination upon infection are dependent partly on the pathogen’s ability to infect and survive inside the hemocytes [8, 10]. To demonstrate the ability of E. chaffeensis to infect hemocytes and tissues, we exposed uninfected ticks to GFP-expressing E. chaffeensis through capillary feeding and microinjection and allowed the ticks to recover for 24 h. The outcome of this experiment revealed the presence of E. chaffeensis in the cytoplasm of hemocytes, irrespective of the infection route (Fig. 5a). Imaging of tissues from ticks capillary fed with GFP-expressing E. chaffeensis demonstrates the presence of E. chaffeensis in both salivary gland and midgut tissues (Fig. 5b). This further confirms that E. chaffeensis can infect hemocytes and disseminate to all organs after 24 h. Following the establishment of dissemination 24 h post-capillary feeding, an assessment of the role of phagocytic hemocytes in infection was investigated. Uninfected ticks were initially injected with either CLD or LP followed by capillary feeding with GFP-expressing E. chaffeensis. Bacterial density increased in the salivary glands of depleted ticks, while in the midgut, phagocyte depletion led to reduced number of GFP-expressing bacteria compared to the control group. (Fig. 6a, b). Phagocyte depletion causes a significant increase in Ehrlichia load within depleted salivary glands, and a decrease in the depleted midgut, while no difference is observed in the carcass (Fig. 6c–e). These data demonstrate that E. chaffeensis can infect hemocytes and disseminate to various organs, and further investigation showed that phagocytic hemocytes play a crucial role in controlling pathogen density in specific tissues, with significant impacts on bacterial load in the salivary glands and midgut.

Here we decided to inject uninfected ticks with hemocyte or plasma (hemolymph without hemocytes) components collected from E. chaffeensis-infected CLD- or LP-treated ticks (Fig. 7a). In this experiment, we observed a decrease in the total number of hemocytes in ticks that received hemocyte or plasma from CLD-treated ticks (Fig. 7b). Interestingly, injection of hemocyte from CLD-treated ticks significantly increased prohemocyte and decreased granulocyte population in recipient ticks compared to recipients of plasma (Fig. 7c, d). Hemolymph components from neither CLD- nor LP-treated ticks lead to changes in the plasmatocyte and oenocytoid population (online suppl. Fig. S4A, B). Quantifying E. chaffeensis in the respective tissues and carcass of the recipient ticks, we observed varying effects of hemocyte or plasma transfer on bacterial load in tissues compared to carcass. Recipients of hemocyte components from control and depleted ticks exhibited a higher load of E. chaffeensis within the salivary gland (Fig. 7e). However, in the midgut, only individuals who received hemocyte component from LP-treated ticks displayed a higher E. chaffeensis load (Fig. 7e). These results in the salivary gland and tissues demonstrate that phagocytosis is essential to limit pathogen replication inside the tissues. Our results also confirmed that E. chaffeensis could infect A. americanum hemocytes as previously shown (Fig. 5a). Thus, we decided to assess the possibility of systemic dissemination via phagocytes by quantifying E. chaffeensis load in the carcasses of recipient ticks. We observed an opposite outcome when compared to the tissues. Carcasses from ticks that received plasma components from ticks treated with CLD and LP showed increased E. chaffeensis dissemination when compared to ticks that received hemocyte components from CLD- and LP-treated ticks (Fig. 7e). Together, the results presented here highlight the role of phagocytosis in limiting pathogen replication in tissues, raising questions about potential systemic dissemination via phagocytes.

In the current study, we reported the role of granulocytic hemocytes in controlling E. chaffeensis infection in A. americanum. Previous studies from our group and others have shown that these classes of immune cells are essential for their anti-pathogen roles in non-model ticks and mosquitoes [4, 8, 16].

Our findings have identified five morphologically distinct hemocyte types in the hemolymph of A. americanum. Interestingly, the blood meal and, to a lesser extent, the infection caused by E. chaffeensis contribute to changes in the hemocyte population. The hemocyte population increased following a blood meal and E. chaffeensis infection, but the population of granulocytes was significantly higher in infected ticks. This suggests a heightened immune activation in response to infection since granulocytes are professional phagocytic hemocytes. Alternatively, one could posit that the physiological impacts of blood feeding also play a role, including the influence of 20E (20-hydroxyecdysone) post-blood ingestion, which has been linked to hemocyte phenotypes in mosquitoes [31]. Previous studies have demonstrated the use of chemical agents to deplete professional phagocytic hemocytes in A. maculatum [4], mosquitoes, and Drosophila [16, 26]. Partial depletion of phagocytic hemocytes significantly impairs the immune response against commensal and pathogenic microbes in invertebrates. Our study demonstrates that CLD liposomes effectively deplete granulocyte populations in uninfected and E. chaffeensis-infected ticks. Likewise, we observed that ticks with depleted granulocyte populations responded poorly to microbial infection. The most intriguing finding was the complete inability of granulocyte-depleted ticks to survive E. chaffeensis infection. These findings support the earlier reports [4], where granulocyte depletion rendered A. maculatum susceptible to Rickettsia parkeri infection. These findings further strengthen the idea that granulocytes play a crucial role in mounting a cell-mediated immune response against microbial infection in the tick vector.

Due to technical difficulties with tick tissue imaging, we could not quantify the proportion of sessile hemocytes in our tick tissues. However, using in vivo phagocytosis of injected FluoSpheres, we demonstrated that sessile hemocytes are not only present in the salivary gland and midgut tissues but are also phagocytic. The phagocytic ability of the sessile hemocytes reduced following CLD treatment. Surprisingly, depleting sessile hemocytes in E. chaffeensis-infected ticks did not change Ehrlichia load in either the salivary gland or tissues. This could be attributed to the fact that E. chaffeensis was already established in these tissues before hemocyte depletion. However, establishing an infection with capillary-fed fluorescent-tagged bacteria allowed us to establish infection in hemocytes, salivary glands, and midguts of A. americanum ticks 24 h post-feeding. Although pathogen dissemination in tick hemocytes and organs has been previously reported [4, 10, 32], Ehrlichia dissemination in hemocytes and unfed tissues via capillary feeding was reported for the first time. Interestingly, we demonstrated that depleting phagocytes before infection with GFP-expressing bacteria have specific effect on bacterial dissemination and density in the organs. These observations argue tissue-specific role of phagocytic hemocytes in different tick tissues, where they are required to restrict pathogen replication to the midgut, thus limiting systemic dissemination to the salivary gland.

Infection and survival within the hemolymph and hemocyte are required for systemic pathogen dissemination in the tick vector [10]. However, how these pathogens replicate and survive in the hemolymph and hemocytes is yet to be demonstrated. We observed that the transfer of hemolymph plasma and hemocyte components from hemocyte-depleted E. chaffeensis-infected ticks unexpectedly resulted in a drop in total hemocyte and prohemocyte population and a corresponding decline in the granulocyte population. It seems possible that the depletion in the hemocyte population is an outcome of CLD carryover from the hemolymph plasma and hemocyte component in our transfer experiments. It is also likely that receiving hemocytes and hemolymph plasma can induce the proliferation of new hemocytes in the recipient ticks. Another important finding was that when we transferred hemolymph plasma and hemocyte components to recipient ticks, we observed changes in E. chaffeensis load in tissues and carcasses of recipient ticks. Ehrlichia infection and replication in the midgut do not rely on phagocytic granulocytes, but in the salivary gland, the presence of granulocytes is crucial to restrict Ehrlichia replication. However, depletion of phagocytic hemocytes by CLD limits systemic dissemination of Ehrlichia. The single most striking observation to emerge from the transfer experiment was the ability of hemocytes or plasma components from E. chaffeensis-infected ticks to establish infection in naïve recipient ticks with varying degrees of infection. The detection of GFP-expressing E. chaffeensis in the hemocytes further suggests active replication of Ehrlichia in the hemolymph and hemocytes, as was previously demonstrated for R. parkeri [4] and Anaplasma phagocytophilum [10].

In conclusion, this study sheds light on the critical role of granulocytic hemocytes in the immune response against E. chaffeensis infection in A. americanum ticks. E. chaffeensis in tick hemocytes demonstrates the potential for intracellular infection and dissemination within the vector. Additionally, we identified the importance of granulocytes in limiting E. chaffeensis replication in the salivary gland, while their presence is not necessary for midgut infection. Furthermore, our data suggest that E. chaffeensis infection can alter the hemocyte population, leading to changes in the immune response of the tick. These findings highlight the complexity of vector-pathogen interactions and underscore the need for further research to elucidate the precise mechanisms by which tick hemocytes modulate immune responses against understudied TBPs, as with E. chaffeensis.

We thank Dr. Uli Munderloh, University of Minnesota, for the generous gift of the GFP-expressing Ehrlichia chaffeensis strain; Dr. Chris Paddock, CDC, for the generous gift of Ehrlichia chaffeensis (Arkansas strain); and Latoyia Downs for her technical help in culturing E. chaffeensis strains.

Protocols for tick blood feeding were approved by the University of Southern Mississippi’s Institutional Animal Care and Use Committee (USMIACUC protocols #15101501.3 and 17101206.2).

The authors declare that they have no competing interests.

This research was principally supported by the NIH NIAID Awards #R15AI167013; #RO1AI135049. We thank Mississippi INBRE, supported by the NIH-NIGMS (P20GM103476), for using the Imaging Facility. The funders played no role in the study design, data collection, analysis, publication, decision, or manuscript preparation.

Conceptualization: A.A., R.C.S., and S.K.; data curation, investigation, and writing – original draft: A.A., J.H., and S.K.; formal analysis, methodology, and writing – reviewing and editing: A.A., J.H., R.C.S., and S.K.; funding acquisition, project administration, and supervision: S.K.; and resources: R.C.S. and S.K.

All data generated or analyzed during this study are included in this article and its online supplementary material files. Further inquiries can be directed to the corresponding author.