Phagocyte-mediated innate immune reactions against the apicomplexan parasite Eimeria bovis

INTRODUCTION

Eimeriosis in cattle is an important enteric protozoan parasitosis causing economic losses and severe clinical disease in calves (daugschies et al., 1998). Unlike most other eimerian species in cattle or in rodent models, the life cycle of E. bovis includes the development of macromeronts (up to 250 µm) within an endothelial host cell (Hammond et al., 1964). This rather long lasting process (14-18 days) requires enlargement and re-organisation of the host cell, involving, e. g., host cell cytoskeletal elements (Hermosilla et al., 2008; lutz et al., 2011). Once the parasite begins growth and proliferation within the parasitophorous vacuole (PV) it must acquire nutrients from the host cell as reported for other intracellular apicomplexans (Behrendt et al., 2008; Taubert et al., 2011). Furthermore, given that endothelial cells generally represent a highly reactive cell type possessing a broad range of effector mechanisms to initiate pathogen elimination, E. bovis has to trigger a complex modulation of the host cell transcriptome and proteome to ensure its successful development (Taubert et al., 2010; Lutz et al., 2011). Interactions of E. bovis-infected endothelium with leucocytes were shown on the level of PBMC (Taubert et al., 2007) and PMN adhesion and seem to rely on infection-induced up-regulation of distinct adhesion molecules (Taubert et al., 2006; Hermosilla et al., 2006). However, at least the PMN adhesion appears to be contained in its magnitude by the parasites.

So far, early innate immune response of ruminant hosts against Eimeria spp. have hardly been investigated, although these reactions may be crucial for the outcome of a primary infection with respect to the severity of the disease and effective induction of adaptive immunity. The first line of defense against invading pathogens is represented by professional phagocytes, such as macrophages, monocytes, dendritic cells and polymorphonuclear neutrophils (PMN). Main effector mechasnims of phagocytes are the killing of pathogens and the production of immunomodulatory molecules, such as cytokines or chemokines, thereby initiating acquired immune responses. Classical PMN-conducted killing involves phagocytosis. In addition, the formation of neutrophil extracellular traps (NETs) has been recently identified as a further effector mechanism of PMN-mediated pathogen killing. NETs act effectively against bacteria and fungi (Brinkmann et al., 2004; Brinkmann and Zychlinsky, 2007; Fuchs et al., 2007) and may represent a common mechanism to eliminate invading pathogens.

For successful infection, E. bovis sporozoites have to traverse the mucosal layer of the ileum to reach the lymphatic capillaries for infection of the adequate host cells, lymphatic endothelial cells. In consequence, E. bovissporozoites should be exposed to the interstitial fluid and to the lymph and should be recognized as potential targets by phagocytes such as PMN and monocytes/macrophages. In the case of Eimeria infections, PMN show distinct infiltration of parasitized gut tissue and accumulate very early at the site of meront formation in infected rodents (Mesfin et al., 1978; Blagburn and todd, 1984) and in E. bovis-infected calves (Friend and Stockdale, 1980). The importance of PMN in Eimeria infections is further underlined by the observation, that PMN-depleted SCID mice significantly produce more E. papillata oocysts after primary infection than control mice (Schito and Barta, 1997).

There is previous evidence that PMN for instance can directly interact with Eimeria parasite stages. PMN have been shown to actively lyse E. falciformis sporozoites in the presence of antibodies and complement (Bekthi et al., 1992). We have recently reported that bovine PMN interact with E. bovis sporozoites (Behrendt et al., 2008) and are capable to eliminate sporozoites in vitro. Moreover, bovine PMN were identified as an in vitro source of several pro-inflammatory cytokines (Il-6, Il-12, TNF-α), chemokines (MCP-1, GRO-α, Il-8, IP-10) and iNOS when exposed to E. bovis sporozoites or merozoite I antigens (Behrendt et al., 2008). The key role of PMN in E. bovis control was further underlined by in vitro and ex vivo data showing enhanced phagocytic and oxcidative burst activities of PMN either exposed to sporozoites in vitro or derived from E. bovis-infected calves (Behrendt et al., 2008).

Hardly anything, however, is known on early role of macrophages or monocytes against the enteropathogen E. bovis. Friend and Stockdale (1980) demonstrated macrophages in degenerating macromeronts of E. bovis-infected calves. Nonetheless, Hughes et al. (1987) even described macromeront formation in cultured bovine monocytes. Mucosal macrophage infiltration was reported in E. tenella- and E. acervulina-infected chickens and in E. separate-infected rats (Trout and Lillehoi, 1993; Vervelde et al., 1996; Shi et al., 2000). A biphasic increase of large mononuclear cells was observed in the peripheral blood of E. nieschulzi-infected rats and E. maxima-infected chickens (Rose et al., 1979). In vitro analyses suggested avian and murine macrophages isolated from immune animals as potent phagocytes of Eimeria sporozoites (Rose, 1974; Rose and Lee, 1977; Bekthi and Pery, 1989), although elimination of the parasites appeared to depend on the presence of immune serum and complement (Bekthi and Pery, 1989).

Additional effector functions utilized by macrophages/monocytes are the release of oxidative radicals and the production of immunomodulatory molecules, such as cytokines or chemokines, in order to attract other leukocytes to the site of infection, initiating, thereby, acquired immune responses. Avian macrophages isolated from E. tenella- and E. maxima-infected animals showed enhanced Il-1 and TNF-α production (Byrnes et al., 1993). The latter was further found increased in a macrophage cell line co-cultured with E. tenella stages (Zhang et al., 1995). Microarray analyses on avian macrophages, which had been previously exposed to sporozoites of different Eimeria subspecies showed parasite-induced effects on the synthesis of various cytokines and chemokines, which were partially subspecies specific (Dalloul et al., 2007). However, for the bovine system detailed data concerning macrophage/monocyte actions in coccidiosis are still lacking. In order to characterize early macrophage-mediated, innate immune reactions against E. bovis, we analysed in vitro interactions between macrophages and sporozoites. We showed that macrophages phagocytise sporozoites under serumfree conditions and in the presence of immune serum, whilst monocytes failed to do so (Taubert et al., 2009). Additionally, bovine macrophages were identified as an in vitro-source of several critical cytokines and chemokines upon exposure to E. bovis-sporozoites and parasite-antigens (Taubert et al., 2009). Moreover, the potential role of monocytes and macrophages in parasitic control in vivo was clearly underlined by the demonstration of macrophage mucosal infiltration in E. bovis-infected calves and by ex vivo data demonstrating enhanced phagocytosis and oxidative burst activities in monocytes derived from E. bovis-infected calves throughout the coccidiosis infection.

The current study was conducted to characterize early innate reactions of bovine PMN, macrophages/monocytes against E. bovis with respect of phagocytosis, ROS production and NETs

MATERIALS AND METHODS

Calves

Holstein Friesian calves were purchased from a local farmer at the age of 2 weeks, treated with Baycox® (Bayer) and Halocur® (Intervet) in the second week after birth, assessed for parasitic infections, and when found parasite free, maintained under parasite-free conditions in autoclaved stainless metabolic steel cages (Woetho) until experimental E. bovis infection. Calves were fed with milk substitute (Hemo Mischfutter) and commercial concentrate pellets (raiffeisen). Water and sterilized hay were given ad libitum.

Parasite maintenance

The E. bovis strain H used in the current study was maintained by passages in Holstein Friesian calves. For the production of oocysts, calves were infected at the age of 10 weeks with 5 x 10⁴ sporulated oocysts each. Excreted oocysts within the faeces were then isolated beginning 18 days p. i. according to Hermosilla et al. (2002). Sporulation was achieved by the incubation in a 2% (w/v) potassium dichromate (Sigma) solution at room temperature (RT). Sporulated oocysts were stored in this solution at 4 °C until further use.

Sporozoites were excysted from sporulated oocysts as previously described (Hermosilla et al., 2002) and free sporozoites were collected and suspended at concentrations of 10⁶ /ml in complete endothelial cell growth medium (ECGM, PromoCell). For in vitroE. bovis-infections bovine umbilical vein endothelial cells (BUVEC, Taubert et al., 2006), Grown to confluence, were infected with freshly isolated sporozoites (10⁶ sporozoites/75 cm² tissue culture flasks). Culture medium (ECGM, PromoCell) was changed 24 h p. i. and thereafter every second day. From day 18 p. i. onwards, E. bovis merozoites I were harvested from BUVEC cultures as previously described (Hermosilla et al., 2002).

Host cells

BUVEC were isolated according to Taubert et al. (2006). Briefly, umbilical veins were isolated from umbilical cords of calves born by sectio caesarea and kept at 4° c in 0.9 % HBSS-HEPES buffer (w/v, pH 7.4, Gibco) supplemented with 1 % penicillin (v/v, 500 U/ml) and streptomycin (v/v, 500 mg/ ml, Sigma) until use. Under sterile conditions one end of the umbilical cord veins was clamped shut and 0.025 % collagenase type II (w/v, Worthington Biochemicals Corporation) in Puck's saline A solution (PSA, Gibco) was infused into the lumen. After clamping the remaining open end of the umbilical veins, they were incubated at 37° c and 5 % CO2 atmosphere for 20 min. Thereafter umbilical veins were gently massaged, unclamped and the resulting collagenase solutions were each collected in 50 ml plastic tubes (Nunc) containing 1 ml FCS (Gibco) to inactivate collagenase. The umbilical veins lumens were washed two times with RPMI 1640 medium (Gibco). Washes were pooled, centrifuged (400 x g, 10 min), resuspended in complete ECGM, plated in 75 cm2 plastic tissue culture flasks (Greiner) and incubated at 37° c and 5 % CO2. BUVEC were fed with complete ECGM medium one day after isolation and, thereafter, every 2-3 days. They were used for infection after 1-2 passages in vitro.

Infections, bleedings and necropsies of experimental animals

Calves (n = 3); group 1 = primary infection, group 2 = challenge infection) were infected orally with 5 x 10⁴ sporulated e. bovis oocysts. Challenge infection was performed on day 40 after primary infection. Non-infected calves (n = 3, group 3) were used as negative controls. Shedding of oocysts was determined from day 18 p. i. onwards by daily faecal examination (McMaster technique). For the determination of oxidative burst and phagocytic activities of monocytes and PMN blood samples were drawn from e. bovis experimentally infected calves on days -1, 1, 5, 7 13, 15, 18, 20, 22 and 25 p. i. by puncture of the jugular vein.

Calves were necropsied on days 26 after primary infection or challenge infection. Tissue samples of the jejunum, ileum, caecum and colon and associated lymph nodes (Lnn. jejunales, Lnn. ileocaecales and lnn. colici) were excised for immediate fixation (4% formaldehyde in phosphate-buffered saline, 24 h) and embedded in paraffin.

Immunohistochemistry

Cross-sections of formalin-fixed tissues (5 µm) were deparaffinied according to standard histological procedures. Endogenous peroxidase was inactivated in 0.5% H202 (30 min, RT, Roth). Samples were washed for 5 min in trisbuffered saline (TBS) and treated with protease (protease type 24, 5 min, 37 °c, Sigma). Protease activity was then stopped by dipping the slides in 4 °c TBS. tissue samples were then probed with monoclonal mouse anti-human monocyte/macrophage-specific antibodies (1:1000, 60 min, 37 °c, humidity chamber, MAC387, Serotec), which cross-react with bovine cells (Gutierrez et al., 1999). After rinsing three times in TBS (5 min), samples were incubated in sheep anti-mouse IgG conjugated with peroxidase (1:50, 30 min, 37 °c, humidity chamber, NA 931, Amersham). After three washings in TBS (5 min), reactions were visualized by adding substrate (0.048 g DAB, Fluka, and 800 µl 3% H202 in 80 ml imidazole buffer, 3-5 min). After rinsing three times in TBS (5 min) and once in Aqua dest (5 min), the tissue samples were counterstained for 15 s in Papanicolaou solution (1:10, Merck), washed in tap water (5 min), dehydrated according to standard procedures and mounted in Aquatex® (Merck). Immunostained macrophages present in the gut mucosa were counted in 10 randomly selected vision fields (200 x magnification) per sample.

Detection of the ex vivo phagocytic and oxidative burst activities of monocytes and PMN

Phagocytic and oxidative burst activities were determined by using Phagotest® and Phagoburst® kits (ORPEGEN-Pharma), according to Taubert et al. (2009). All tests were performed in duplicates. Four ml of heparinized blood were mixed with 36 distilled water (40 s, shaking) to lyse erythrocytes, supplemented with 10x Hank's buffer (Gibco) and pelleted (10 min, 400 x g). After washing (10 ml PBS/EDTA, 10 min, 400 x g) cells were transferred to V-shaped microtitre plates (2 x 105 cells/well, Nunc) and centrifuged (4 °c, 200 x g, 7 min).

For ex vivo quantification of phagocytic activity cells were suspended in 100 µl ice-cold autologous plasma. After addition of 10 µl FItc-labelled Escherichia coli preopsonized with human serum (provided with the commercial kit), cells were incubated for 10 min at 37 °c (shaking water bath) or on ice, the quenching of surface-bound bacteria, fixation and permeabilisation of cells was performed according to the manufacturer's instructions.

For ex vivo quantification of the inducible oxidative burst activity, cells were suspended in 100 µl ice-cold PBS, supplemented with either 10 µl non-labelled E. coli, phorbol-12-myristate 13 acetate solution (PMA 8.1 µM, ORPEGEN-Pharma; =positive control) or PBS (=negative control) and incubated at 37 °c (shaking water bath). After 10 min, 10 µl dihydrorhodamine 123 substrate solution was added and cells were then incubated for further 20 min (37 °c, shaking water bath). After transferring the plates onto ice, cells were fixed and permeabilised according to the manufacturer's instructions.

In both assays PBS/EDTA were then added to the wells (4 °c, 5 min) to recover plastic-adherent cells. Cells were counterstained with 'DNA solution' (provided with the kits) and analysed by flow cytometry (FCM; FACScalibur, BD Biosciences).

Isolation and cultivation of bovine PMN, monocytes and macrophages

For PMN isolation, calves were bled by puncture of the jugular vein and blood was collected in 50 ml plastic tubes (Nunc) containing 0.1 heparin (Sigma) as anticoagulant. Heparinised blood was centrifuged in a discontinuous Percoll (Amersham) gradient according to Hjorth et al. (1981) to yield a PMN fraction of > 97% purity. PMN were washed twice with medium (RPMI 1640) to remove Percoll and resuspended in medium (RPMI 1640).

For both monocytes and macrophages PBMC had to be isolated in advance. therefore, 18 ml of blood, substituted with 2 ml 3.8% citric acid, were mixed with 17 ml of 0.9% NaCl and applied on the top of 12 ml Ficoll-paque (density = 1.007 g/l, Biochrom) in 50 ml centrifugation tubes (Nunc). After centrifugation (45 min, 400 x g) the lymphocyte/monocyte layer was collected and the cells were washed three times (10 min, 400 x g, 4 °c) in RPMI 1640 medium (Gibco). Using trypan blue (Sigma) exclusion test, viable cells were counted in a Neubauer haemocytometer chamber.

Bovine monocytes were isolated as previously described by Goddeeris et al. (1986). If not stated elsewhere, we used monocytes of infected animals. In brief, 7.5 x 10⁷ PBMC were allowed to adhere (1 h, 37 °c), thereafter dried and incubated in autologous plasma (1 h, 37 °c, thereafter washed twice with RPMI 1640/1% penicillin/1% streptomycin, all Sigma). Non-adhering PBMC were removed and monocytes were washed with pre-warmed RPMI 1640/1% penicillin/1% streptomycin. Monocytes were detached (5-10 min in10 mM EDTA in Mg²⁺- and Ca ²⁺-free Hank's solution, RT), washed (10 min, 400 x g, 4 °c) and resuspended in 4 °c RPMI 1640/1% penicillin/1% streptomycin. The cells were kept on ice until further use and counted in a neubauer haemocytometer chamber.

Bovine macrophages were prepared according to Jungi et al. (1996). If not stated differently, we used macrophages of infected animals. PBMC were sealed in Teflon bags (20 ml, 5 x 10⁶ PBMc/ml) as described by Jungi et al. (1996) and cultured for 7-8 days at 37 °c in a humidified atmosphere of 5% CO₂ . The medium was Iscove's modified Dulbecco's Medium (IMDM Glutamax®, Sigma) containing 100 Iu/ml penicillin, 100 µg/ml streptomycin, 1% (v/v) non-essential amino acids for minimal essential medium (MEM, Gibco), 0.4% (v/v) vitamin solution for MEM (Gibco), 1 mM sodium pyruvate (Gibco), 2.5 µM amphotericin B (Gibco), 50 mM 2-mercaptoethanol (Gibco) and 20% FCS (Biowest). From the cell mixture, macrophages were purified by selective adherence to microtitre plate wells for 4 h as previously described by Jungi et al. (1996).

Scanning electron microscopy

Bovine PMN were incubated with freshly isolated E. bovis sporozoites at a ratio of 10:1 for 2, 3 and 4 h on poly-_L-lysine pre-coated coverslips. After incubation, cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 15 min and afterwards washed with 0.1% M cacodylate buffer for 15 min and afterwards washed with 0.1% cacodylate buffer. The cells were then post-fixed in 1% osmium tetroxide in 0.1% cacodylate buffer, washed three times in distilled water, dehydrated in ascending ethanol concentrations, critical point dried with CO₂ and sputtered with gold. Specimens were examined using a Phillips XL20 scanning electron microscope.

Co-culture of bovine PMN and Eimeria bovis sporozoites for NET-formation and -quantification

To test for sporozoite-induced NET-formation, 10⁵ sporozoites (vital or heat inactivated at 60 °c for 30 min) were added. To test a sporozoite-homogenate for its ability to induce net-formation, sporozoites underwent three freeze and thaw cycles (freezing in liquid nitrogen for 1 min and complete thawing at 37 °c) and subsequent sonification (15 min, 50 kHz). The amount of homogenate per well corresponded to 10⁵E. bovis sporozoites.

For DNase treatment 90 u DNase I (Roche Diagnostics) per well were added at the start of incubation. Inhibition assays were performed using 5 µM diphenylene iodonium (DPI) or 10% neonatal FCS throughout the incubation period.

NETs were quantified after staining extracellular DNA with sytox orange (Invitrogen) according to others (Martinelli et al., 2004; Lippolis et al., 2006). Samples were stained by Sytox Orange (Invitrogen) at a final concentration of 1 µM for 10 min. They were analysed by a fluorometric reader (Ascent Fluoroskan, Labsystems) using an excitation wavelength of 530 nm and detecting at 590 nm. Results were always confirmed by microscopical observations.

Figure 1. Interactions of PMN with Eimeria bovis sporozoites illustrated by SEM analyses. Bovine PMN were exposed to E. bovis sporozoites for 4 h in the presence of immune serum in vitro. Interactions were illustrated by SEM analyses ranging from PMN surface-derived compact protrusions towards the sporozoite (a) or leading to parasite uptake by two activated PMN (b).

Phagocyte-mediated innate immune reactions against the apicomplexan parasite Eimeria bovis - Image 1

Real-time Pcr for the relative quantification of IFN-γ, iL-12, IL-6, TNF-α, CXCL1, CXCL8, CXCL10, CCL2, COX-2, iNOS and GAPDH cDNAs

The relative quantification of IFN-γ, Il-12, Il-6, TNF-α, CXCL1, CXCL8, CXCL10, CCL2, iNOS and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene transcripts was done by real-time PCR applying TaqMan® probes (Applied Biosystems). The sequences of primers (MWG Biotech) and probes (Eurogentec) are published elsewhere (Taubert et al., 2009). Probes were labeled at the 5'-end with a reporter dye FAM (6-carboxyfluorescein) and at the 3'-end with the quencher dye TAMRA (6-carboxytetramethylrhodamine). PCR amplification was performed employing an automated fluorometer (ABI PRISM ^TM® 5700 Sequence Detection System, applied Biosystems) using 96-well plates. Samples were analyzed according to Taubert et al. (2009) and semi-quantitative analyses used the comparative ct method according to the instructions of the ABI PRISM ^TM® 5700 sequence detector manufacturer and reported as n-fold differences in comparison to the respective medium control (after normalizing the samples referring to their corresponding housekeeping gene GAPDH).

RESULTS

Macrophages infiltrate eimeria bovis infected intestinal mucosa and accumulate in lymph nodes

E. bovis-infected calves showed significantly more macrophages in the gut than naïve animals. The significant increase in macrophage numbers was apparent in both primary and challenge infected animals (both p > 0.01). In challenged calves macrophage counts significantly exceeded those of primary infected animals (p > 0.01). Macrophage infiltration occurred in all gut samples tested in comparable proportions. In the late phase of primary infection (26 days p. i.), enhanced accumulation of macrophages in associated lymph nodes was found only for Lnn. jejunales. In contrast, challenge infection caused an increase of macrophage numbers in all associated lymph nodes investigated (e. g. Lnn. jejunales, Lnn. ileocaecales, Lnn. colici).

Monocytes and PMN display enhanced oxidative burst and phagocytic activities during eimeria bovis infection

Data generated on day -1 p. i., which reflect the situation of non-infected animals, revealed low phagocytic and oxidative activities, whilst a biphasic upregulation of the phagocytic and oxidative burst activities of monocytes and PMN were observed when compared to the negative control. oxidative burst activity for both phagocytes was enhanced already 1 day p. i. and a second peak was detected at 13-18 days p. i. Highest values occurred on day 15 p. i. when, by means, more than 50% of monocytes and PMN showed increased oxidative burst activity.

Macrophages and PMN phagocytize Eimeria bovis sporozoites in vitro

Macrophages and PMN co-incubated with sporozoites were found loaded with whole parasites after 4 h (Fig. 1 and 2). Extracellular sporozoites appeared fully vital and active. Elimination of sporozoites from the medium increased significantly (p > 0.05) with increasing phagocyte-sporozoite-ratios. The data were confirmed by flow cytometry analyses using CFSE-stained sporozoites. This staining does not affect the parasite viability as previously demonstrated by Hermosilla et al. (2008). As observed microscopically, the sporozoites^CFSE accumulated in the macrophages and PMN irrespective of heat inactivation of the sporozoites.

Figure 2. Macrophage-mediated elimination of CFSE-stained eimeria bovis sporozoites in vitro. Bovine macrophages were exposed to viable CFSE-stained e. bovis sporozoites for 4 h in the absence of immune serum: (a) uptake of CFSE-stained sporozoites by bovine macrophages illustrated by phase contrast and (b) fluorescence microscopy

Phagocyte-mediated innate immune reactions against the apicomplexan parasite Eimeria bovis - Image 2

Exposure of phagocytes to Eimeria bovis sporozoites or meroizoite i antigen (EbAg) leads to differential upregulation of immunoregulatory molecule gene transcription

Co-culture of macrophages with sporozoites and stimulation with merozoite I antigen (EbAg) induced distinct levels of cytokine and chemokine gene transcription. Transcription of genes encoding for IFN-γ, Il-12, TNF-α, Il- 6, CXCL1, CXCL8, CXCL10 and COX-2 was up-regulated after sporozoite encounter. In contrast, EbAg merely induced the gene transcription of IL-6 and Il-12 and failed to up-regulate IFN-γ and TNF-α. In PMN only CXCL1, CXCL8, CXCL10 and TNF-α were found enhanced. Monocytes reacted upon exposure to E. bovis sporozoites by enhanced IFN-γ gene transcription. TNF-α, Il-6, CCL2, CXCL8, COX-2 and iNOS gene transcripts were induced rather weakly. No upregulation of IL-12 and CXCL1 mrna was detected in monocytes.

Effective sporozoite-induced net formation is dependent on the parasite viability/integrity

Experiments performed with either viable, dead or crushed sporozoites implicated that E. bovis-induced NET formation is dependent on the sporozoite viability and/or integrity. Thus, homogenized and heat-inactivated sporozoites only slightly, but nevertheless significantly, enhanced the DNA-related fluorescence in comparison to parasite-free controls (p > 0.01 and p > 0.005, respectively). Stimulation led to much stronger reactions (p > 0.01). Stimulation with PMA induced much weaker signals of NETs than viable sporozoites, but led to stronger fluorescence intensities when compared to dead or homogenized parasites. Parasite-induced NETs prevent sporozoites from invading host endothelial cells.

DISCUSSION

Early innate immune reactions of professional phagocytes (PMN, macrophages, monocytes) against cattle Eimeria spp. have scarcely been investigated so far, although, the first encounter between parasite and the innate part of the immune system should be decisive for the subsequent outcome of the infection. In this work we have focused on monocyte-, macrophage- and PMN-mediated immune reactions against E. bovisin vivo, ex vivo and in vitro. We found enhanced general phagocytic and oxidative burst activities of PMN and monocytes obtained from calves experiencing experimental E. bovis infection. Macrophages were shown to accumulate in the gut mucosa of E. bovis infected animals. Direct exposure of macrophages to E. bovis sporozoites in vitro resulted in the elimination of the parasite from the medium and upregulated transcription of genes encoding for various immunoregulatory molecules. These results suggest macrophages as anti-parasitic effector cells and active mediators of immune response against E. bovis.

Macrophage infiltration depends on adequate chemotactic signals. PMN, which are generally accepted as the earliest leukocytes to be involved in inflammatory processes (Burgos et al., 2011), have recently been identified as an early source of chemokines upon encounter with E. bovis sporozoites (Behrendt et al., 2008), including TNF-α and CCL3, which are of relevance with respect to macrophage infiltration and activation.

The observation of additional macrophage accumulation in the gut tissue of challenged calves in combination with the sporozoite opsonizing efficacy of immune serum emphasizes the in vivo relevance of these phagocytes in abrogating E. bovis challenge infections. All these observations are in agreement with reports on avian and murine Eimeria infections which also show enhanced in vitro anti-sporozoite phagocytosis of macrophages isolated from previously immune animals (Rose, 1974; Rose and Lee, 1977; Bekthi and Pery, 1989) and increased macrophage-mediated antibody dependent cytotoxicity (Bekthi and Pery, 1989).

Monocytes and PMN of E. bovis infected calves exhibited biphasic increased, general phagocytic and oxidative burst activities coinciding with periods of time when E. bovis stages most probably are not yet or no longer situated intracellular and, consequently, should be accessible for professional phagocytes, such as PMN and monocytes (Behrendt et al., 2008; Taubert et al., 2009). It is noteworthy, that the proportions of monocytes involved in these reactions are far lower than those of PMN (Behrendt et al., 2008). However, in in vitro experiments bovine monocytes failed to effectively phagocytize heat-inactivated sporozoites, although the fact, that sporozoite uptake was increased by supplementation of immune serum, argues for the potentially ability of monocytes for antibody-dependent phagocytosis. Furthermore, monocytes were identified as a source of IFN-γ and TNF-α, i. e., molecules involved in macrophage and PMN activation. In addition, monocytes may attract NK cells and actively initiate adaptive immune reactions in E. bovis infected animals as they showed enhanced gene transcription of CXCL10 after stimulation with ebag, a chemokine which acts mainly on NK cells (Muller et al., 2001; Lande et al., 2003) and T cells (Taub et al., 1993).

Primary bovine macrophages, like PMN, phagocytized sporozoites even at serum-free conditions, indicating their ability to fight efficiently against these parasitic stages in the first encounter. The sporozoite uptake occurred irrespective of the viability of the parasite, as heat-inactivated sporozoites and viable ones were both phagocytized. Thus, active invasion by the sporozoite cannot be excluded it appears of minor role. Nonetheless, Hughes et al. (1987) reported on development of E. bovis sporozoites into macromeronts in a macrophage-like cell line. In our current experiments we could not observe development of sporozoites, neither in a permanent bovine macrophage cell line (BoMac, unpublished data) nor in primary bovine macrophages, but the cells were only incubated for up to 8 days.

Bovine macrophages reacted upon exposure to viable sporozoites by upregulation of INF-γ and IL-12 mRNAs and consequently can play an active role in the activation of NK cells (Subauste et al., 1992; Trinchieri, 1995, 1998a,b; Biron et al., 1999) and in the transition of innate to adaptive immune reactions as these two cytokines are well recognized to trigger Th1 associated immune responses. In fact, Th1 dominated responses have recently been reported for E. bovis infected calves during the prepatency (Taubert et al., 2008). Similar situations are well known in other Eimeria infections (Rose et al., 1989, 1991a,b; Smith and Hayday, 2000; Shi et al., 2001) and seem to be a key feature of control (Ovington and Smith, 1992).

NETs were firstly described by Brinkmann et al. (2004) who showed that activated PMN can form sticky extracellular traps capable of binding and killing Gram-positive and –negative bacteria. By now, NETs are not only described to be involved in defense against bacteria, but also against fungi (Urban et al., 2006) and apicomplexan parasites such as Plasmodium falciparum (Baker et al., 2008) and E. bovis (Behrendt et al., 2010). Assembly and activation of the NADPH oxidase complex, resulting in the production of reactive oxygen species (ROS), is an essential step in the process of NET formation (Brinkmann and Zychlinsky, 2007; Fuchs et al., 2007). Furthermore, antimicrobial extracellular trap formation is seemingly not unique for PMN, but is also described as effector mechanism for mast cells (von Köckritz-Blickwede et al., 2008).

PMN derived NETs firmly attached to E. bovis sporozoites and SEM analyses rather suggested immobilization of the parasites, which, in contrast to extracellular bacteria and fungi, may have a preventive effect on host cell invasion. Thus, we could show for the first time that pre-incubation of sporozoites with PMN clearly affects the sporozoite infectivity causing approximately 65% reduction of infection rates (Behrendt et al., 2010). As supplementation with DNase abolished this effect, it appears convincing that NETs hampers sporozoites of E. bovis from host cell invasion. Overall, NETs may not kill sporozoites directly, but might have detrimental effects on successful E. bovis establishment by immobilizing the parasite in order to abrogate the parasite replication and to facilitate subsequent phagocytosis by other phagocytes. In consequence, sporozoite-induced net formation should also play an important role in the in vivo situation (Behrendt et al., 2010).

Signals and corresponding receptors for NET-activation are still not known. In the case of unopsonized bacteria, pattern recognition receptors (PRR), such as Toll-like receptors (TLR) or dectin are discussed (Urban et al., 2006). Also intracellular tlrs may be involved since several parasitic protozoans are sensed by TLRmolecules (Gazzinelli and Denkers, 2006). We recently described for the first time the presence of mRNA transcripts for TLR1, TLR2, TLR4, TLR6, TLR7, TLR9 and TLR10 in bovine PMN (Conejeros et al., 2011). Additionally, zymosan, a dectin-1/TLR2 ligand, induced ROS in a CD11b-, but not dectin-1-dependent in activated bovine PMN (conejeros et al., 2011). Therefore, in the bovine system not only tlrs but also CD11b could be considered as PMN specific PRR in the activation of NADPH oxidase, the production of ROS which could lead to NET formation but further investigation in the signaling pathway is required.

The current data emphasize the role of PMN, macrophages and monocytes in E. bovis induced early innate immune reactions. Enhanced phagocytic and oxidative burst activities and increased accumulation of macrophages in the gut mucosa of E. bovis infected calves indicate in vivo relevance of these cells. In vitro analyses of PMN showed E. bovis net-formation and macrophage antibody-dependent and –independent phagocytosis of sporozoites and point at the parasite induced gene transcription of immunoregulatory molecules, that influence both the chemotaxis of leukocytes of the innate and adaptive immune system and the development of Th1 dominated immune response. Taken together, our results strongly suggest that phagocytemediated innate immune reactions play a key role in the early host immune response to E. bovis infections in calves.

Acknowledgements

The authors acknowledge funding by the German Research Foundation (DFG; projects TA 291/1-2 and He3663/2-1). We also thank Brigitte Hofmann and Birgit Reinhardt for their technical assistance in cell culture.

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This paper was presented at the World Buiatrics Congress 2012, Lisbon, Portugal from 3 to 8 June 2012. Engormix thanks for this huge contribution.

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