Age and sex matched C57BL/6 wild-type (WT) mice, obtained from Janvier (Cedex, France) were used. All animal care was in accordance with institutional guidelines. Food and water were available ad libitum. Experiments were performed in accordance to German and European legislation.

T. gondii cysts of type II strain ME 49 were harvested from the brains of female NMRI mice infected intra-peritoneally (i.p.) with T. gondii cysts 8–10 months earlier. Brains obtained from infected mice were mechanically homogenized in 1 ml sterile phosphate-buffered saline (PBS). Cyst numbers were counted in a 10 μl brain suspension using a light microscope. Two cysts were administered i.p. in a total volume of 200 μl per mouse as described before (Möhle et al., 2016). Control mice were mock-infected with sterile PBS. The mice were perfused intracardially with 60 ml sterile ice-cold PBS. The brains were removed for further analysis. The acute stage of infection was defined between day 10 and day 14 whereas the chronic stage of infection was starting at day 21. Therefore, to investigate the recruited neutrophil granulocytes in chronic cerebral toxoplasmosis, mice were sacrificed 4 weeks post-infection for further analysis.

Depletion of neutrophils was performed by i.p. administration of anti-Ly6G mAb (clone 1A8, BioXCell). Mice were injected with 500 μg (as described before by Daley et al., 2008; Dunay et al., 2010) of the antibody i.p. on alternate days from days 10 to 23 post-infection. Mice were sacrificed 24 h after the last antibody treatment. Rat IgG2a (BioXCell) was used as a control to mAb.

Brains were homogenized in a buffer containing 1M HEPES (pH 7.3) and 45% glucose and then sieved through a 70 μm strainer. The cell suspension was washed and fractionated on 25–75% Percoll gradient (GE Healthcare). Isolated cells were washed with PBS and used immediately for further experiments. Peripheral blood was obtained from posterior vena cava and lysed with erythrocyte (RBC) lysis buffer (eBioscience). Subsequently, these cells were stained with the desired fluorescent conjugated antibodies (Biswas et al., 2015).

Isolated mononuclear cells were incubated with Zombie NIR™ or Violet™ fixable dye (Biolegend) for live/dead discrimination. Unspecific antibody binding was blocked by incubation with anti-FcγIII/II receptor antibody (clone 93). Thereafter, cells were stained with fluorochrome-conjugated antibodies against cell surface markers: CD45 (30-F11), CD11b (M1/70), Ly6C (HK1.4), MHC I H-2D^b (28-14-8), MHC II I-A/I-E (M5/114.15.2), CD80 (16-10A1), and CD86 (GL1), all purchased from eBioscience, CXCR2 (SA04E1), CXCR4 (L27GF12), and CD64 (X54-5/7.1) from Biolegend, Ly6G (1A8), CD62-L (MEL-14) from BD Bioscience, then washed and fixed in 4% paraformaldehyde. Matched isotype controls were used to assess the level of unspecific binding.

For intracellular cytokine staining, single-cell suspensions (5 × 10⁵ cells/well) were stimulated in 96-well plates in the presence of Toxoplasma lysate antigen (5 μg/ml) and Brefeldin A (10 μg/ml, GolgiPlug, BD Biosciences). After 6 h, cells were incubated with Zombie NIR™ or Violet™ fixable dye and anti-FcγIII/II receptor antibody (clone 93) and surface stained for CD45 (30-F11), CD11b (M1/70), Ly6G (1A8), Ly6C (HK1.4), CRAMP (R-170), washed in FACS buffer [PBS with 1% of fetal calf serum (FCS)] and fixed in 4% paraformaldehyde. Cells were permeabilized using Permeabilization Buffer (Biolegend). To measure the cytokine expression, the staining was performed with the following antibodies: IL-1α (ALF-161), TNF (MP6-XT22), NOS2 (CXNFT), IFN-γ (XMG1.2), IL-1β (NJTEN3), and IL-10 (JES5-16E3) from eBioscience in Permeabilization Buffer (Biolegend) (Biswas et al., 2015).

A total of 100,000 cells was acquired using a flow cytometer (BD FACS Canto II). Data were analyzed using FlowJo software (Version 10 Tree Star). Matched isotype controls were used to assess the level of unspecific binding.

Isolated cells were stained for CD45 (30-F11), CD11b (M1/70), Ly6G (1A8), Ly6C (HK1.4), CXCR4 (L27GF12), and CD62-L (MEL-14) in FACS buffer for 30 min after blocking FcγRs. ROS production was measured by Total ROS Detection Kit (ENZO, 51011), according to the manufacturer's instructions.

RNA and DNA was isolated from the right hemisphere of the infected mice as previously described (Möhle et al., 2016). Following the isolation of the nucleic acids, semi-quantitative PCR was performed to measure the parasite burden and the cytokine gene expression levels in the brain.

Mice were anesthetized with isoflurane and perfused intracardially with saline followed by paraformaldehyde (PFA, 4%) in phosphate buffer (pH 7.4). The brain was removed, post-fixed with PFA overnight, cryoprotected in 30% sucrose, frozen, and 20-μm-thick sections were prepared in a cryostat. The sections were fixed with ethanol, blocked with normal serum and incubated overnight at 4°C with the primary antibody SAG-1 (D61S clone; 1:20, Invitrogen) or PECAM-1/CD31 (MEC13.3 clone; 1:500) and Ly6G (1A8 clone; 1:200). Following the primary antibody staining, lectin staining was performed (Fluorescein labeled Dolichos Biflorus Agglutinin (DBA); 1:500, Vector Laboratories) according to the manufacturer's guidelines. Then, sections were incubated for 2 h at room temperature with secondary antibodies (Alexa Fluor-488, Thermo Fischer Scientific). Controls were carried out by omission of the primary antibodies. Sections were counterstained with Sytox Red Dead Cell Stain (1:20,000, Thermo Fischer Scientific) to visualize the cell nuclei and slides were observed under a confocal laser microscope (LSM510, Carl Zeiss).

Data were analyzed by Mann-Whitney test for two groups or one-way ANOVA for several groups followed by Tukey's post-test with GraphPad Prism 6 (San Diego, CA). In all cases, results were presented as mean ± standard deviation (SD) and were considered significant, with p < 0.05.

Infection of mice with T. gondii induces egress of Ly6C^hi monocytes and Ly6G⁺ neutrophil granulocytes from the BM to the blood. Following our previous characterization of BM derived monocytes in the CNS in 4 weeks T. gondii-infected mice, we analyzed neutrophil dynamics using the same experimental model. CD11b and Ly6C expression were used to identify the inflammatory monocytes (Ly6C^hi), neutrophils (Ly6C^int) and resident monocytes (Ly6C^lo) in the blood (Figures 1A,C). The CD11b⁺ cells were further differentiated based on their Ly6C and Ly6G expression (Figures 1B,D). The neutrophil specific Ab Ly6G (1A8) was applied to distinguish neutrophil granulocytes (Ly6G⁺) from monocytes (Ly6G⁻) (Figures 1B,D). We observed increased percentages of circulating neutrophil granulocytes in peripheral blood of infected mice (30 ± 3.06%) as compared to non-infected controls (15.7 ± 1%; Figures 1B,D).

Alongside the enhanced egress of immune cells in the periphery, cerebral toxoplasmosis leads to the activation of brain resident cells and infiltration of circulating immune cells to the CNS (Möhle et al., 2014; Biswas et al., 2015). Peripheral immune cell influx into the infected brain is characterized by initial invasion of neutrophils, followed by the recruitment of BM-derived monocytes and the lymphocytes. While in non-infected controls the main immune cell population was resting resident microglia (CD45^loCD11b⁺; Figures 1E,F), we observed the entry of CD45^hi myeloid derived population in the brains of T. gondii-infected mice (Figure 1G). The recruited cells included two populations, CD45^hiCD11b⁻ cells (ungated; Figure 1G) and CD45^hiCD11b⁺ cells (upper gate; 10.0 ± 2.1% of the parent population). The CD45^hiCD11b⁺ cells comprised of BM-derived myeloid cells, namely monocytes, neutrophil granulocytes, macrophages and DCs. Upon infection, brain resident activated microglia cells expressed elevated CD45 levels (CD45^intCD11b⁺; 5.0 ± 1.06% of the parent population). The anti-Ly6G (1A8) antibody was applied to distinguish Ly6G⁺ neutrophil granulocytes (Figure 1H; 3.0 ± 1.02% of the CD45^hiCD11b⁺) from monocytes. Next, the total number of mononuclear cells was quantified in the brain during chronic cerebral toxoplasmosis (Figure 1I). Thus, CD45^hiCD11b⁺Ly6G⁺ neutrophil granulocytes (6 × 10³ ± 50) formed a small yet defined population, when compared to the absolute numbers of CD45^intCD11b⁺ microglia (1 × 10⁵ ± 2,000). These data demonstrate that cerebral toxoplasmosis leads to the activation of resident microglia and the recruitment of lymphoid and myeloid immune cells including neutrophil granulocytes.

Neutrophil granulocytes, the earliest immune cells to arrive at the site of infection, can deploy a plethora of mechanisms to attack invading pathogens (Denkers et al., 2012; Bardoel et al., 2014; Perez-de-Puig et al., 2015). To study their localization around T. gondii in the CNS, we performed immunofluorescence analysis of mice brain slices at the acute stage and chronic stage of the infection. We observed that neutrophil granulocytes were not only in the blood vessels, marked by PECAM1, but entered the brain parenchyma (Figure 2A). The recruited neutrophils were in close proximity to the T. gondii tachyzoites in the cerebral cortex during the acute stage infection (Figure 2B). Upon encystation of the T. gondii parasites in the chronic phase, we observed the absence of neutrophils in the vicinity of bradyzoite containing cysts (Figure 2C). These immunofluorescence results indicate the affinity of the neutrophil granulocytes to the infective stage of T. gondii in contrast to the dormant stage during the course of the cerebral toxoplasmosis.

To further characterize neutrophil granulocytes in inflamed brains upon 4 weeks of T. gondii infection, the expression profiles of various cell surface markers were determined by flow cytometry. As control, microglia were selected, because under homeostatic condition neutrophils are not present in the CNS, and immune cells from the same isolate chosen for the comparison. A large percentage of infiltrating neutrophils expressed enhanced levels of MHC I and MHC II (83 ± 1% MHC I; 95 ± 0.5% MHC II) (Figures 3M–P) unlike in the periphery (25 ± 4% MHC I; 3 ± 0.5% MHC II) (Figures 3A–D). According to their function as antigen presenting cells (APCs) of the brain, activated microglia also expressed significant levels of MHC I and MHC II (100% MHC I; 100% MHC II) (Figures 3M–P). We further compared the expression of the co-stimulatory molecules CD80 and CD86 and detected these on small parts of the neutrophil population (15 ± 0.5% CD80; 17 ± 0.5% CD86) and activated microglia (18 ± 1% CD80; 52 ± 1% CD86) (Figures 3Q–T). The phagocytosis mediating receptor CD64 (FcgR1), which was present on a large proportion of activated microglia (89 ± 1% CD64), was only detectable on a small percentage of neutrophils (11 ± 0.5%) (Figures 3U,V). The chemokine receptor CXCR2 was detected on more than half of the neutrophils (65 ± 0.5%). However, only a small percentage of activated microglia expressed CXCR2 (5 ± 0.5%) (Figures 3W,X). The neutrophil granulocytes in the periphery did not express any significant levels of CD80, CD86 and CD64 (Figures 3E–J). However, the entire neutrophil population expressed CXCR2 (Figures 3K,L). Hence, activated microglia and neutrophils express immune modulatory molecules in infected brains indicating their contribution to the establishment of local immunity in response to T. gondii infection.

Several studies have demonstrated that neutrophil granulocytes secrete plenty of immune modulatory molecules including cytokines upon infection (Sturge et al., 2013; Bardoel et al., 2014). To determine their cytokine expression profile, in experimental cerebral toxoplasmosis (4 weeks T. gondii infection), myeloid cells from the infected brain were analyzed by intracellular flow cytometry. We observed that neutrophil granulocytes hardly produced pro-inflammatory cytokines such as IL-1α and TNF (3 ± 1% and 5 ± 0.5%, respectively). On the contrary, microglia served as a considerable source of both cytokines (50 ± 3% and 80 ± 1%, respectively) (Figures 4A,B,E,F). Importantly, 65 ± 0.5% of neutrophil granulocytes synthesized IL-1β (Figures 4C,D). Interestingly, IFN-γ was also produced by the neutrophils (85 ± 0.5%) contrary to activated microglia (Figures 4I,J). IL-10 and iNOS were synthesized by negligible numbers of neutrophil granulocytes (0 ± 0.5% IL-10; 2 ± 0.5% iNOS) and microglial cells (0 ± 0.5% IL-10; 8 ± 1% iNOS) (Figures 4G,H,K,L). Besides, all neutrophils synthesized ROS unlike activated microglia (95 ± 0.5%) (Figures 4M,N). In summary, these data demonstrate that neutrophil granulocytes produce multiple immune modulatory molecules including IFN-γ.

IFN-γ plays a critical role in the host response to cerebral toxoplasmosis. Previous studies defined NK cells, T cells and microglia as sources of IFN-γ during cerebral toxoplasmosis (Gazzinelli et al., 1993; Hunter et al., 1994; Gavrilescu et al., 2004; Kim et al., 2012; Gazzinelli and Sher, 2014). However, the contribution of neutrophil granulocytes to IFN-γ production in the brain has not been elucidated (Suzuki, 2002; Sa et al., 2015). Therefore, we performed flow cytometry from the brains of mice during the acute (2 weeks) as well as the chronic stage (4 weeks) of T. gondii infection. In the acute phase, 83.5 ± 0.5% of neutrophil granulocytes produced IFN-γ (1.5 ^* 10 ^∧ 3 ± 200 cells). This was the case for only 47 ± 0.5% of activated microglia. At this time-point, only negligible numbers of CD11b⁻ lymphocytes (247 ± 50 cells) were detectable in the brain to make a significant contribution (6 ± 0.5%) (Figures 5A,B). However, during the chronic phase of infection enhanced number of lymphocytes infiltrated the brain, which then became the major IFN-γ producers (88 ± 0.5%) (1.5 ^* 10 ^∧ 6 ± 550 cells) (Figures 5C–F). Neutrophils were secondary producers of IFN-γ at the chronic stage of infection (85 ± 4%) (1.4 ^* 10 ^∧ 3 ± 60 cells) (Figures 5C–F). Thus, our results demonstrate that Ly6G⁺ neutrophil granulocytes are representing an important early source of early IFN-γ in the acute phase of cerebral toxoplasmosis.

It is still unclear whether neutrophils contribute to control of cerebral toxoplasmosis. To investigate this, we took advantage of the previously described specific depleting anti-Ly6G mAb (clone 1A8). Following our observation that Ly6G⁺ neutrophils are early IFN-γ producers in the acute phase of cerebral toxoplasmosis, we started the ablation at day 10 post-infection when neutrophil granulocytes began entering the brain. We continued the ablation, injecting Ly6G mAb or control IgG2 mAb every alternate day, as the infection progressed from the acute to chronic stage. Twenty-four hours after the last Ab treatment, mice were sacrificed and the successful depletion of CD11b⁺ Ly6G⁺ monocytes in the blood was confirmed (7.0 ± 0.13 to 0%; Figures 6A,B).

In agreement with this, anti-Ly6G treatment of infected mice also resulted in a significant reduction of neutrophils in the brain. Further characterization revealed a reduction of recruited myeloid cells (24.0 ± 1.5 to 19.0 ± 0.28%) in the brains of anti-Ly6G treated infected mice (Figure 6C, upper panel). This observation was further confirmed with a significant decrease of Ly6G⁺ neutrophils (Figure 6C, middle panel; 3.0 ± 0.5% to 0.6 ± 0.3%). Further investigation revealed reduced recruitment of CD11b⁺Ly6C^hi inflammatory monocytes (Figure 6C, lower panel; 48.0 ± 3.0% to 32.0 ± 1.3%) in the brains of anti-Ly6G treated mice. Moreover, absolute cell numbers of CD45^hi CD11b⁺ myeloid cells (p < 0.05), Ly6G⁺ neutrophils (p < 0.001) and Ly6C^hi monocytes (p < 0.01) were significantly reduced. On the contrary, numbers of T lymphocytes (CD45^hiCD11b⁻CD4⁺ and CD45^hiCD11b⁻CD8⁺) and microglia (CD45^intCD11b⁺) remained unaltered (Figures 6D,E).

Using q and qRT-PCR we investigated whether the ablation of Ly6G⁺ neutrophil granulocytes affected parasite burden and cytokine gene expression levels in brains of anti-Ly6G treated mice. We found that anti-Ly6G treatment was associated with elevated parasite burden (Figure 7A). Correspondingly, we observed a 40 and 60% reduction of pro-inflammatory TNF and IFN-γ, respectively, as compared to IgG treated controls (Figures 7C,E). Furthermore, IL-10 and IL-1β mRNA levels were reduced in anti-Ly6G treated mice (Figure 7B,D). Together, these results demonstrate that increased parasite burden correlates inversely with the reduced levels of TNF, IFN-γ, IL-1 β, and IL-10 in brains of neutrophil-depleted mice with cerebral toxoplasmosis. Hence, the recruitment of neutrophil granulocytes and their early cytokine production appear to promote inflammatory immune responses restricting pathogen load in brains of T. gondii-infected mice.

Depending on the infection model, different subsets of neutrophil granulocytes can be discriminated based on their phenotypic and functional properties (Tsuda et al., 2004; Pillay et al., 2010; Beyrau et al., 2012). However, in the case of cerebral toxoplasmosis, it was largely unclear whether recruited neutrophils represent a heterogeneous or rather homogeneous population in the brain. Therefore, CD45⁺CD11b⁺Ly6C⁺Ly6G⁺ neutrophil granulocytes from 4 weeks T. gondii-infected mice were analyzed by flow cytometry (Figures 8A,B). We found that this population contained cells expressing different levels of CD62-L (Figure 8C). Contrary to CD62-L^lo neutrophils (20 ± 0.5%), a high percentage of CD62-L^hi neutrophils (50 ± 0.5%) expressed CRAMP (Figures 8D,E). On the contrary, only 25 ± 0.5% of CD62-L^hi neutrophils secreted IFN-γ while this was the case for 45 ± 0.5% of CD62-L^lo neutrophils (Figures 8F,G). However, CD62-L^hi and CD62-L^lo neutrophil granulocytes produced similar levels of ROS (90 ± 0.5% vs. 95 ± 0.5%) (Figures 8H,I). These data suggest the phenotypic heterogeneity of brain-derived neutrophil granulocytes further defining CD62-L^lo neutrophils as a source of protective IFN-γ.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.