Monocyte-derived chicken macrophages exposed to Eimeria tenella sporozoites display reduced susceptibility to invasion by Toxoplasma gondii tachyzoites CURRENT STATUS: POSTED

Background: Both Eimeria (E.) tenella and Toxoplasma (T.) gondii are common apicomplexan parasites in chickens. Host cell invasion by both protozoans includes gliding motility, host cell attachment and active penetration. Chicken macrophages as phagocytic cells participate in the innate host immune response against these two parasites. Methods: In this study, primary chicken monocyte-derived macrophages (MMs) were infected with both pathogens to investigate mutual and host-parasite interactions. MMs cultures were assigned to groups that were infected with E. tenella , T. gondii , or both. In co-infected cultures, MMs were first exposed to E. tenella sporozoites for 2 hours. Afterwards, T. gondii tachyzoite infection was performed. Live-cell imaging was carried out to observe cell invasion and survival of T. gondii by single parasite tracking over a period of 20 hours post infection (hpi). Quantitative analysis for parasite replication was performed at 2, 6, 12 and 24 hpi by real-time PCR (qPCR). Results: We found that during early co-infection T. gondii tachyzoites adhered for more than 4 hours to macrophages. Although they displayed high motility, ability to penetrate the cell membrane of the potential host cell was reduced. qPCR results confirmed that significantly less T. gondii entered E. tenella -activated MMs at 2 hpi, and a reduced proportion of intracellular T. gondii survived and replicated in these cells at 24 hpi. Conclusions: We conclude that E. tenella modulates host cell responses to another apicomplexan agent, T. gondii , reducing active invasion and multiplication in chicken primary macrophages. fluorescence: the nucleus of E. tenella sporozoites; green fluorescence: T. gondii tachyzoites; wide field: single macrophage.

Upon adherence to the host cell membrane apicomplexan parasites start to penetrate into the host cell by forming a so-called moving junction initiated by organelles of the apical complex at the anterior end of the parasites [3]. This moving junction supports the establishment of the parasitophorous vacuole (PV) separating the parasite from the host cell cytoplasma. The adhesive proteins produced by micronemes located in the apical region of the invasive parasite stages allow gliding motility which is essential for the active invasion by T. gondii [4]. In chickens, macrophages play a crucial role in identification and phagocytosis of pathogens including protozoan thus serving as a first line of innate immune defence [5]. However, T. gondii tachyzoites are capable to actively invade macrophages which occurs even faster than phagocytosis displayed by a macrophage [6]. T. gondii tachyzoites are able to replicate in chicken blood monocyte-derived macrophages [7]. In experimental infection of E. tenella in naïve chicken, sporozoites were situated mainly within or next to the lamina propria that was infiltrated with macrophages in response to the infection [8].
Sporozoite-bearing macrophages are able to transport Eimeria sporozoites to the proper site of the intestinal mucosa [9]. However, E. tenella survival and development is rather poor in cultured chicken macrophages [10]. For instance, probiotic bacteria reduced oocyst excretion of E. acervulina in chicken [11] as well as in vitro invasion of E. tenella into Madin-Darby bovine kidney cells [12]. Host immune responses and consequently clinical signs of disease are modulated in co-infected animals. In general, the immune response is dominated by Th1 during protozoan infection whereas Th2 response is typical for helminth infection [13]. Regarding interaction during co-infection by protozoa, it was reported that no competitive effects exist in mixed Eimeria species infection [14]. However, T. gondii replication was increased during co-infection with Trypanosoma in rats [15]. On the other hand, T. gondii supports Plasmodium replication in a rat model [16]. Natural co-occurrence of T. gondii and Eimeria in the same host has been reported. For example, both parasites were found concurrently in blood and organs of a heavily diseased sparrow [17]. Furthermore, wild rabbits tested seropositive for both T. gondii and Eimeria stiedae infection in Scotland [18].
In spite of high seroprevalence rates reported for both T. gondii and E. tenella in chickens, little is known to date about the mutual interplay between these two parasites, particularly during host cell invasion. Experimental in vivo and ex vivo co-infection models in chicken macrophages were established recently and thus suitable tools are now available for further co-infection studies [19,20].
Results demonstrated that the mutual interaction during co-infection modulated both parasite replication as well as the host immune response. In our recent study, macrophage phagocytosis was distinctly altered during in vitro co-infection by T. gondii and E. tenella (unpublished data).
In the current study, we used live-cell imaging of T. gondii and/or E. tenella infected cells to monitor parasite invasion and survival at the single cell-parasite level in both mono-infected and co-infected cultures. The study focuses on the period of early parasite invasion in monocyte-derived chicken macrophages. A previous study showed that asexual stages of T. gondii and Eimeria displayed ultrastructural similarities [21]. Moreover, host cell invasion mechanisms, micronemal adhesins, gliding motility of both T. gondii tachyzoites and Eimeria sporozoites were identified to be affected in vitro by the same inhibitor of protozoan cGMP-dependent protein kinase [22]. Thus, we are particularly interested in whether the considerable capacity of T. gondii to invade and survive in macrophages is affected by host-specific E. tenella infection. Parasite tracking was performed to determine the motility of T. gondii during the invasion phase following co-infection by E. tenella. The life span of individual T. gondii was also monitored by live cell imaging. The animal experiments performed to collect chicken blood samples were approved by the responsible authorities (Landesdirektion Sachsen, Germany, trial registration number V13/10).

Parasites and host cells
Chicken peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood of adult chickens according to the established protocols with slight modifications kindly provided by Dr.
Braukmann, Friedrich-Loeffler-Institute Jena, Germany. Briefly, PBMCs were separated from 2 ml blood by centrifugation (250 x g, 45 min) with Biocoll (density 1.077 g/ml; Biochrom AG, Berlin, Germany). For imaging, cell cultures were assigned to four groups (n = 2 per group). In co-infected group CI, MMs were exposed to 2 × 10 3 E. tenella sporozoites 2 h before T. gondii infection (-2 hpi). Group LPS cultures served as positive controls and were stimulated at the same time with 1 µg/mL lipopolysaccharide (LPS). Cultures of group Tg (monoinfection with T. gondii) and group NC (negative control) were not exposed to LPS stimulation. At 0 hpi cultures of groups CI, LPS, and Tg were infected with 2 × 10 3 T. gondii tachyzoites per well. Group NC remained uninfected. All cultures were observed until 20 hpi. The whole experiment was repeated once biologically.

Study design for experiment 2 (Fig. 1B):
For quantification by qPCR, cell cultures kept in 24-well plates were assigned to five groups (n = 5 cultures/group). At -2 hpi, MMs of group CI (co-infection) and group Et (single infection with E. tenella) were exposed to 2 × 10 5 E. tenella sporozoites per culture. Group LPS cultures were pretreated with T. gondii multiplication was analyzed by a probe-based qPCR detecting the 529-bp repeat element [24]. ITS1 fragment quantification was used to assess the replication of E. tenella by a SYBR Green-based PCR [25]. The relative copy number of E. tenella DNA was implemented by measurement of pSCA-17 plasmid standard dilution as described before [26]. qPCR was conducted in a total volume of 20 µL:

Live cell imaging of T. gondii in MMs
In general, penetration of T. gondii tachyzoites into the MMs started within 2 hpi after tachyzoites were seeded into cultures. CLSM analysis ( Fig. 2A) of group CI cultures showed that T. gondii tachyzoites remained loosely adherent to MMs for more than 4 hours in most cases before they started to actively invade the host cell. During this phase, distinct helical gliding motility of tachyzoites was seen. This untypical behavior of tachyzoites in group CI was observed regardless whether intracellular E. tenella sporozoites were still alive or dead. An additional movie file shows this in more detail (see Additional file 1). In contrast, tachyzoites, which were actively or passively incorporated into MMs after attachment, were seen within minutes in the mono-infected group Tg and the LPS-treated group.
Tachyzoites showed lowest motility in group LPS at most of the captured time points (Fig. 2B). In group CI, significantly higher motility of tachyzoites were observed than in group LPS over the period from 10 min to 120 min post infection (p < 0.05); with the exception of 70 to 110 min. Comparing between groups CI and Tg, tachyzoites of group CI showed significantly higher motility (p < 0.05) during the first 20 min. Average motility values in group CI tended to remain higher than in both groups Tg and LPS with a significant difference (p < 0.05) at 80, 90 and 110 min compared to group Tg.

Parasite Quantification
In experiment 1, the relative intensity (RI) of green fluorescence of 27 to 30 individual intracellular T. gondii tachyzoites which were captured at 3 to 7 hpi by CLSM were anlaysed in all infected groups. RI revealed no significant difference between groups Tg and CI until 4 hpi. Thereafter, RI values remained on a consistently higher level in group CI than in the monoinfected group Tg and in group LPS (Fig. 3A).The RI measured in group CI displayed only slight variation from 3 to 7 hpi, whereas values started to decrease in group LPS and Tg following 4.5 hpi or 5 hpi with a statistically significant difference between groups CI and LPS (p < 0.05) at 4.5 hpi.
In general, less than 45% of traced intracellular tachyzoites of T. gondii were still alive or replicating in MMs at 20 hpi (Fig. 3B). At 20 hpi, the proportion of T. gondii-positive MMs was lower in group CI (27%) than in group Tg (45%) and only slightly higher than in group LPS (21%).
By qPCR, significantly lower DNA copy numbers (p < 0.05) were determined at 2 hpi for T. gondii in group CI and group LPS compared to group Tg (Fig. 3C, left). The number of DNA copies remained on a low level in the two former groups until 12 hpi, followed by a steep increase until 24 hpi in group CI while values remained significantly lower in group LPS (p < 0.05). In group Tg, DNA copy numbers were higher than in the two other groups at 2 hpi, decreased steadily until 12 hpi to a level similar to groups CI and LPS and increased even more distinct than those recorded for group CI thereafter.No statistical difference was observed between group CI and Tg at this time point (p > 0.05). Likewise, no significant difference in DNA copy numbers was detected for E. tenella by comparison of groups Et and CI over the entire observation period of 24 hpi (Fig. 3C, right).

Discussion
After two hours of exposure to E. tenella sporozoites or LPS MMs generally maintained their viability which was demonstrated by negative staining for DRAQ7 (data not shown). A recent study by [20], confirmed earlier findings by [27] that E. tenella sporozoites were mostly located within chicken macrophages at 2 hpi. In the current study, most intracellular E. tenella sporozoites showed red fluorescescence after DRAQ7 staining instead of the expected yellow (YFP) fluorescence within 4 hpi to 6 hpi (data not shown), indicating their death. This CLSM finding corroborates the qPCR results demonstrate that the number of E. tenella declined to lowest numbers at 6 hpi (Fig. 3A, right). Coinfection with T. gondii did not exhibit a significant influence on E. tenella replication when compared to single E. tenella infection. Unfortunately, we could not quantify the E. tenella sporozoite numbers via YFP through time lapsing imaging because of a non-ideal YFP expression (about 80%) in the available parasite strain.
Host cell invasion by T. gondii tachyzoites usually takes only 15 to 20 s [6], 1995). It is assumed that T. gondii tachyzoites and Eimeria sporozoites may invade and traverse several host cells by disrupting the host cell membrane [28]. In our time lapsing study, T. gondii tachyzoites were either floating free in the culture medium (unattached to macrophages) for hours or displayed a rapid entry into the macrophages, in both group LPS and group Tg. Interestingly, adherence of vital tachyzoites was prolonged for more than 4 hours in the co-infection cultures. It was previously demonstrated that most T. gondii tachyzoites remained adherent to murine macrophages that were treated with a phagocytosis inhibitor, Cytochalasin D [29]. Microneme exocytosis is necessary for host-cell entry of both T. gondii [30] and E. tenella [31]. The micronemes appear to be both structurally and functionally conserved between different members of the Apicomplexa [4]. We assumed that E. tenella infection may potentially hamper the entry of T. gondii into host cells by altering recognition of signal receptors or inhibiting phagocytosis.
Rapid invasion and egress are crucial to T. gondii survival and successful replication, thereby minimizing the exposure to destructive reaction by innate protection in a generally hostile extracellular environment [32]. Once inside the host cell, the parasite no longer moves [33]. The intracellular survival of T. gondii depends on the route by which the parasite enters the host cell.
Although T. gondii tachyzoites are phagocytized and internalized through Fc receptor mediation [34], intracellular T. gondii can survive and replicate within their PV by blocking the host macrophage's pathways intended to initiate vacuolar acidification and parasite inactivation [35]. To quantify viable intracellular T. gondii, the intensity of GFP signal expression by the parasite was monitored in this study. It appeared from our observations according to RI values that T. gondii showed better tolerance and survival at the early stage of infection (until 7 hpi) if MMs were previously exposed to E. tenella (Fig. 3A).
Under the conditions of our experimental design (two T. gondii tachyzoites per cell), most infected host cells contained only one parasite at 7 hpi in all infected groups. Compared to group LPS, By CLSM, only slightly high T. gondii-positive cells were monitored compared to group LPS. However, significant high number of total DNA copies of T. gondii was detected in group CI compared to group LPS. Therefore, we conclude that the single cell observation by CLSM may not be completely comparable with quantification of DNA copies in cell cultures due to the different amount of cells considered. Nonetheless, results obtained with both methods indicated a reduced growth of T. gondii in chicken macrophages in a co-infection setting with E. tenella compared to mono-infection.
Macrophages are not only professional phagocytes but also secrete cytokines in response to parasite infection. This innate immune response can be triggered in chicken macrophages by exposure to LPS [36]. A previous study showed that T. gondii blocked LPS-induced production of IL-12 and TNF-alpha in murine bone marrow derived macrophages [37]. For E. tenella, it is known that IL-1β and iNOS expression are significantly enhanced in chicken HTC macrophages by merozoites at 2 hpi [38]. A recent in vitro study demonstrated down-regulation of IL-12 and iNOS in chicken macrophages during simultaneous co-infection by T. gondii and E. tenella [20]. From previous as well as the current data it appears that modulation of innate immunity in chicken differs during mono-and co-infection, which includes cytokine production and macrophage phagocytosis. However, data on concurrent infections with E. tenella and T. gondii are still scarce although both are considered to be common pathogens in poultry and thus deserve more attention.

Conclusions
In summary, life cell imaging by CLSM proved to be a useful tool to evaluate chicken macrophage invasion and / or phagocytosis during mono-and co-infection with two different apicomplexan parasites. It was demonstrated that the mechanisms of T. gondii invasion and survival appear to be altered in E. tenella-exposed macrophages. Further studies into macrophage signaling pathways, particularly modulation of the cytokine response, combined with image analysis and live cell imaging, will help to better understand the function and modulation of the innate immune response during apicomplexan invasion. The animal experiments performed to collect chicken blood samples were approved by the responsible authorities (Landesdirektion Sachsen, Germany, trial registration number V13/10)

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download. Additionalfile1.mp4