Fungal and Parasitic Infections

Inherent Oxidative Stress in the Lewis Rat Is Associated with Resistance to Toxoplasmosis

William H. Witola, Chi Yong Kim, Xuejin Zhang
John H. Adams, Editor
DOI: 10.1128/IAI.00289-17

ABSTRACT

The course of Toxoplasma gondii infection in rats closely resembles that in humans. However, compared to the Brown Norway (BN) rat, the Lewis (LEW) rat is extremely resistant to T. gondii infection. Thus, we performed RNA sequencing analysis of the LEW rat versus the BN rat, with or without T. gondii infection, in order to unravel molecular factors directing robust and rapid early T. gondii-killing mechanisms in the LEW rat. We found that compared to the uninfected BN rat, the uninfected LEW rat has inherently higher transcript levels of cytochrome enzymes (Cyp2d3, Cyp2d5, and Cybrd1, which catalyze generation of reactive oxygen species [ROS]), with concomitant higher levels of ROS. Interestingly, despite having higher levels of ROS, the LEW rat had lower transcript levels for antioxidant enzymes (lactoperoxidase, microsomal glutathione S-transferase 2 and 3, glutathione S-transferase peroxidase kappa 1, and glutathione peroxidase) than the BN rat, suggesting that the LEW rat maintains cellular oxidative stress that it tolerates. Corroboratively, we found that scavenging of superoxide anion by Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) decreased the refractoriness of LEW rat peritoneal cells to T. gondii infection, resulting in proliferation of parasites in LEW rat peritoneal cells which, in turn, led to augmented cell death in the infected cells. Together, our results indicate that the LEW rat maintains inherent cellular oxidative stress that contributes to resistance to invading T. gondii, and they thus unveil new avenues for developing therapeutic agents targeting induction of host cell oxidative stress as a mechanism for killing T. gondii.

INTRODUCTION

Toxoplasma gondii is a remarkably successful protozoan capable of infecting virtually all mammalian species, with about one-third of the world human population estimated to be infected. T. gondii infection and clinical outcome vary among species (1), depending on, among others, the immune status and genetic predisposition of the host (2–4). Unlike mice, rats are known to resist the development of clinical toxoplasmosis upon infection with T. gondii (5, 6). The phenomenon of T. gondii infection in rats closely mirrors the clinical progression of the infection in immunocompetent humans (7, 8). This resemblance in the progression of toxoplasmosis between rats and humans warrants the use of rats as quintessential animal models for elucidating T. gondii infection in humans (7, 8).

Among rat strains, variations in resistance/susceptibility to toxoplasmosis have been reported. For instance, compared to the Brown Norway (BN) rat, the Lewis (LEW) rat is extremely resistant to T. gondii infection (9). This refractoriness of the LEW rat to toxoplasmosis has been associated with a rat genomic locus named Toxo1 on chromosome 10 (10). Pursuant to this, two genes called NLRP1 and ALOX1 in the orthologous Toxo1 locus on chromosome 17 in the human genome have been demonstrated to possess alleles linked to susceptibility to human congenital toxoplasmosis (3, 4). The inhibition of intracellular T. gondii growth in LEW rat peritoneal macrophages (10) has been linked to rapid death of both parasites and infected host cells (11). This mode of clearance of parasites in LEW rat cells suggests the involvement of a rapid and vigorous killing response at the site of infection, thus impeding the dissemination of the parasites in the host animal.

Reactive oxygen species (ROS) such as hydrogen peroxide, superoxide radicals, and hydroxyl radicals are highly reactive metabolites of molecular oxygen in mammalian cells (12). Cytochrome P450 (CYP) enzymes catalyze the endogenous oxygenation of organic substrates through the reduction of molecular oxygen in mammalian CYP-dependent microsomal and mitochondrial electron transport chains (13–15). During these enzymatic reactions, ROS are generated if the transfer of oxygen to a substrate is not tightly controlled (16). In healthy cells, production of ROS takes place at a controlled rate because excessive intracellular amounts of ROS can lead to a state called oxidative stress (17). Augmented oxidative stress can be toxic to cells, resulting in oxidative damage of cellular membranes and macromolecules and thus leading to cellular apoptosis and death (18). Generation of ROS has been shown to be upregulated during microbial infection in immune effector cells, including neutrophils, eosinophils, and macrophages, resulting in oxidative stress that is toxic to the invading pathogens (19).

In the present study, we endeavored to perform a global transcriptome analysis of the LEW rat versus the BN rat, with or without T. gondii infection, in order to unravel the molecular mechanisms directing a robust and rapid early innate immune response that mitigates the infection. We provide evidence that the LEW rat has inherent higher expression of cytochrome genes than the BN rat. Because cytochrome enzymes are involved in the generation of intracellular ROS that have been shown to be important in killing intracellular pathogens (19), we investigated whether the inherent high expression of cytochrome genes in the LEW rat contributes to its robust resistance to T. gondii infection. Using in vitro assays, we show that in comparison to those of the BN rat, the LEW rat primary peritoneal cells have augmented ROS levels that are associated with resistance to T. gondii infection.

RESULTS

Progression of T. gondii in LEW versus BN rat peritoneal cells.Part of the freshly isolated peritoneal cells from T. gondii-infected and uninfected LEW and BN rats were maintained in culture and analyzed by fluorescence microscopy to detect yellow fluorescent protein (YFP) fluorescence from T. gondii tachyzoites at 24 h and 48 h postharvest. As expected, in peritoneal cells from the infected BN rats, parasites progressively proliferated over time, while extremely few to no parasites could be observed in the cells derived from the infected LEW rats at both time points (Fig. 1). This was consistent with the previously reported phenotypes of the BN and LEW rats in response to T. gondii infection (9, 10).

FIG 1
FIG 1

In vitro growth of T. gondii parasites in peritoneal cells isolated from BN and LEW rats at 24 h after intraperitoneal infection of the rats. YFP-expressing T. gondii parasites are shown fluorescing green. The images are representative of 4 replicates.

Differentially expressed genes in peritoneal cells of LEW versus BN rats.All RNA samples extracted from the BN and LEW rats and used for RNA sequencing had RNA integrity numbers (RINs) of 9.5/10 and above, as determined with an Agilent Bioanalyzer. The rat reference genome database used was a mixed-strain assembly based on mixed female BN/SsNHsdMCW and male SHR mice, although it apparently did not include any LEW strain. Nevertheless, the alignment metrics showed only negligible differences between the LEW samples and the BN samples (not aligned, 1.3% versus 1.2%; multiply aligned, 9.8% versus 8.9%), such that any strain differences in sequence were minor enough to be ignored overall. The weighted gene correlation network analysis (WGCNA) on 6,942 rat genes that had a one-way analysis of variance (ANOVA) false-discover rate (FDR) (P < 0.2) was run. A liberal cutoff was used to focus on the genes that showed patterns related to the four experimental groups (T. gondii-infected and uninfected LEW and BN rat groups; n = 4 per group) but minimize the problems associated with running WGCNA on genes that showed only a few expression patterns in total. Preprocessing of the combined raw read counts using the R (v 3.3.0) and Bioconductor packages generated reads ranging from 13.1 to 18.3 million per sample (rat) that aligned uniquely within 32,662 rat genes and 8,637 T. gondii genes. About 12,749 rat genes and 890 Toxoplasma genes met the criteria for further analysis using EdgeR's (v 3.14.0) negative binomial generalized linear model with likelihood ratio tests and tagwise dispersion estimates. By ANOVA FDR (P < 0.2), 6,942 genes with moderate evidence for differential expression were selected. Using blockwise modules, this resulted in 55 modules, each of which contained genes clustered based on their expression patterns. Genes that did not fit any of the 55 module patterns were clustered in a separate module. From the modules generated, cytochrome genes (Cyp2d3, Cyp2d5, and Cybrd1) were clustered in one module and were found to be expressed significantly higher in the LEW rats (both infected and uninfected) than in the BN rats (both infected and uninfected) (Table 1). By comparison analysis of the RNA sequence data, among the genes that were significantly (FDR P values of <0.05) differentially expressed, the two cytochrome P450 family genes Cyp2d3 and Cyp2d5 were found to be 464.43-fold and 221.12-fold upregulated, respectively, in the infected LEW rats compared to the infected BN rats (Table 1). Interestingly, in the uninfected rats, Cyp2d3 and Cyp2d5 were 1072.80-fold and 402.16-fold upregulated, respectively, in the LEW compared to the BN rats (Table 1), suggesting that their expression was not induced by infection but was inherently higher in the LEW rat than in the BN rat. Additionally, the cytochrome b reductase 1 gene (Cybrd1) was found to be upregulated (3.64-fold) in the infected LEW rats compared to the infected BN rats. However, unlike for Cyp2d3 and Cyp2d5, Cybrd1 upregulation was augmented by infection of the rats with T. gondii (Table 1). In contrast, LEW rats had lower transcript levels for some antioxidant enzymes than the BN rats (Table 2). These antioxidant enzymes included lactoperoxidase (LPO), microsomal glutathione S-transferase-2 (Mgst2), microsomal glutathione S-transferase-1 (Mgst1), glutathione S-transferase kappa 1 (Gstk1), and glutathione peroxidase (Gpx4). When the infected LEW rat was compared to the uninfected LEW rat, T. gondii infection was found to be associated with the downregulation of the genes for the antioxidant enzymes, while there was no apparent effect in the BN rat (Table 2).

View this table:
TABLE 1

Differentially expressed genes for enzymes that catalyze generation of reactive oxygen species in LEW versus BN ratsa

View this table:
TABLE 2

Differentially expressed genes for antioxidant enzymes in LEW versus BN ratsa

LEW rat peritoneal cells contain inherently higher ROS levels than BN rat peritoneal cells.Because cytochrome enzymes are involved in the generation of intracellular ROS, we endeavored to investigate whether the LEW rat peritoneal cells, which had higher expression of cytochrome enzymes (Cyp2d3, Cyp2d5, and Cybrd1), would also contain higher levels of ROS than the BN rat cells. We measured the relative levels of ROS using the CellROX Deep Red reagent in both T. gondii-infected and uninfected freshly isolated peritoneal cells from the LEW and BN rats. We found that both the infected and uninfected peritoneal cells of the LEW rat contained significantly (P < 0.05) higher levels of ROS than the uninfected or infected BN rat cells (Fig. 2A and C). This corroborated the higher expression levels of cytochrome enzymes in the LEW rat than in the BN rat (Table 1). By quantification of the numbers of infected and uninfected cells in the microscopic images for the cultures in which ROS levels were measured, we found that the LEW rat-derived cultures had fewer than 2% infected peritoneal cells, while the BN rat-derived cultures had about 40% infected peritoneal cells (Fig. 2B and D). This was consistent with the LEW rat's refractoriness to T. gondii infection (Fig. 1).

FIG 2
FIG 2

(A) Relative reactive oxygen species (ROS) levels in rat peritoneal cells cultured for 16 h in vitro with (+) or without (−) T. gondii infection. Fluorescence generated by the reaction of CellROX Deep Red reagent (Molecular Probes) with cellular ROS is shown on the y axis as the mean fluorescence intensity (measured per microscopic field at a magnification of ×20) proportional to the level of ROS in the cells. Lew− and Lew+ represent LEW rat peritoneal cells without and with T. gondii infection, respectively. BN− and BN+ represent Brown Norway rat peritoneal cells without and with T. gondii infection, respectively. (B) Mean percentage of T. gondii-infected cells (black bars) and uninfected cells (gray bars) in cultures of LEW rat (LEW Cells) and BN rat (BN Cells) peritoneal cells cultured with T. gondii tachyzoites at an MOI of 1:1 for 16 h. (C) Representative images depict T. gondii-infected and uninfected LEW and BN peritoneal cells stained with CellROX Deep Red Reagent for detecting reactive oxygen species (red fluorescence) content in cells, captured at a magnification of ×20 at 16 h postinfection. (D) Representative images of LEW and BN peritoneal cells depicting the proportion of infected (with parasites fluorescing green) and uninfected cells at 16 h postinfection. The data shown represent means from three independent experiments with standard error bars, and the points at which data are significantly different (P < 0.05) are shown by asterisks.

Scavenging of ROS decreases LEW rat peritoneal cell resistance to T. gondii infection.We hypothesized that the high level of ROS that we observed in the LEW rat peritoneal cells played a role in resistance to T. gondii infection. Mammalian phagocytic cells are known to produce large amounts of superoxide anion, which is a precursor for other highly reactive ROS such as hydrogen peroxide, hydroxyl radicals, and peroxynitrite (15). Therefore, to investigate whether superoxide anion was playing a role in the LEW rat's refractoriness to T. gondii, we infected LEW and BN rat peritoneal cells in the presence or absence of various concentrations of Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) (a superoxide scavenger) at a multiplicity of infection (MOI) of 1:4 (lower than the MOI used for the assay for Fig. 2 to avoid overgrowth of parasites due to a prolonged culture period) and analyzed the proliferation of intracellular parasites in culture. We observed that while untreated LEW peritoneal cells had extremely few to no observable parasites at the various time points, the MnTBAP-treated LEW peritoneal cells showed a significant (P < 0.05) increase in the number of observable intracellular parasites as well as the number of infected cells, proportional to the concentration of MnTBAP added to the cells (Fig. 3A; see Fig. S1 in the supplemental material). In LEW peritoneal cells treated with 50 and 100 μM MnTBAP, parasites progressively increased in number and peaked at about 56 h postinfection, while in the cells with 150 μM MnTBAP, parasite growth peaked at 18 h postinfection and thereafter declined to the level in the LEW rat peritoneal cells with 50 μM MnTBAP (Fig. 3A). These findings indicated that scavenging of ROS from peritoneal cells by MnTBAP decreased the resistance of LEW cells to T. gondii infection. By measuring ROS levels in uninfected LEW and BN rat peritoneal cells, we found that indeed, MnTBAP treatment decreased the levels of intracellular ROS in a concentration-dependent manner (see Fig. S2 in the supplemental material). As expected, in all the infected BN rat peritoneal cells with or without various concentrations of MnTBAP, parasites progressively proliferated over time (Fig. 4B). Notably, at 18 h postinfection, the amount of parasites in the BN rat peritoneal cells treated with 150 μM MnTBAP was significantly (P < 0.05) higher than that in other cells (Fig. 3B).

FIG 3
FIG 3

Effect of the superoxide scavenger Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) on T. gondii survival and growth in rat peritoneal cells cultured in vitro. Freshly isolated peritoneal cells from LEW (A) and BN (B) rats were cultured without or with various concentrations (50, 100, and 150 μM) of MnTBAP and infected with YFP-expressing T. gondii RH tachyzoites. The YFP fluorescence generated by the parasites in culture was measured at 0 h, 18 h, 32 h, and 56 h postinfection, and the relative mean fluorescence intensities shown on the y axis are proportional to the parasite amount in the cultures. The data shown represent means from three independent experiments with standard error bars, and the points at which data are significantly different (P < 0.05) are depicted by asterisks.

FIG 4
FIG 4

Analysis of the number of tachyzoites per parasitophorous vacuole in T. gondii-infected LEW rat (A) and BN rat (B) peritoneal cells during treatment with or without the superoxide scavenger MnTBAP in vitro. The mean number of parasites per vacuole was determined as an average of parasites in one parasitophorous vacuole for 20 infected cells selected randomly from 10 independent microscopic fields (×20 objective). The data shown represent means from three independent experiments with standard error bars, and the points at which data are significantly different (P < 0.05) are depicted by asterisks. (C) Representative images of infected cells in which parasites per vacuole were counted, containing 1, 2, 3, 4, or 5 tachyzoites (green) of T. gondii.

To ascertain that scavenging of ROS not only increased survival of parasites but also resulted in parasite proliferation in the infected LEW peritoneal cells, we quantified the average number of parasites per parasitophorous vacuole (PV) in the microscopic images from which the data described in Fig. 3 were derived. We found a time-dependent and MnTBAP concentration-dependent increase in the number of parasites in the MnTBAP-treated LEW rat peritoneal cells (Fig. 4A). On the other hand, treatment of BN peritoneal cells with MnTBAP did not have a significant effect on the number of parasites per PV, as both treated and untreated cells allowed a progressive increase in the number of parasites at similar rates (Fig. 4B). Using the WST1 reagent, analysis of the toxic concentrations of MnTBAP in uninfected LEW and BN rat peritoneal cells showed that MnTBAP had 50% inhibitory concentrations (IC50s) of 200 ± 12.2 μM and 215 ± 15.3 μM, respectively. This indicated that the highest concentration (150 μM) of MnTBAP used for scavenging ROS in the cell cultures was below the toxic levels to significantly affect the viability of the cells.

Increase in T. gondii growth in MnTBAP-treated LEW peritoneal cells augments cell death.The resistance phenotype of the LEW rat peritoneal macrophages to T. gondii infection has been associated with the rapid death of both parasites and infected host cells (10, 11). Thus, we endeavored to investigate whether the increase in T. gondii growth in LEW rat peritoneal cells treated with MnTBAP would lead to a higher cell death rate in the LEW than in the BN rat peritoneal cells. Using propidium iodide (PI) staining to detect dead cells, we observed that infected LEW rat peritoneal cells treated with MnTBAP had a higher rate of cell death than the untreated cells (Fig. 5A). The rate of cell death corresponded to the rate of parasite growth in the cells. Comparatively, the infected BN rat peritoneal cells treated with equivalent concentrations of MnTBAP supported a higher parasite growth rate but yet had a lower rate of cell death than observed in the LEW rat peritoneal cells (Fig. 5B). Because we had earlier determined that the concentrations of MnTBAP used in the assays were nontoxic to the cells, the augmentation in cell death could be associated with increased parasite growth in the LEW rat cells. Therefore, from the microscopic images used to derive the data shown in Fig. 5, we randomly selected 50 cells per microscopic field and determined the percentage of cells that both were propidium iodide positive (dead cells) and contained T. gondii parasites intracellularly. Additionally, we also derived the percentage of cells that were positive for propidium iodide but did not contain T. gondii parasites intracellularly. We found that, for the LEW rat cells, the MnTBAP-treated cultures had significantly more dead cells containing intracellular parasites, as well as dead cells without intracellular parasites, than untreated cultures (Fig. 6A). On the other hand, when comparing the MnTBAP-treated and untreated BN peritoneal cells, there was no observable significant difference in the number of dead cells containing intracellular parasites or in the number of dead cells without intracellular parasites (Fig. 6B). Together, these findings suggested that the MnTBAP-induced parasite proliferation in LEW peritoneal cells significantly contributed to host cell death.

FIG 5
FIG 5

Determination of the viability of T. gondii-infected rat peritoneal cells during treatment with the superoxide scavenger MnTBAP in vitro. Freshly isolated peritoneal cells from LEW (A) and BN (B) rats were cultured with various concentrations (0, 50, 100, and 150 μM) of MnTBAP during infection with T. gondii RH tachyzoites. The fluorescence generated by propidium iodide staining of dead cells in the cultures was quantified as relative values from which the background fluorescence of uninfected cells treated with respective MnTBAP concentration was subtracted. Dotted, gray, white, and black bars represent propidium iodide fluorescence intensity from cells treated with 0, 50, 100, and 150 μM MnTBAP, respectively. The data shown represent means from three independent experiments with standard error bars, and the points at which data are significantly different (P < 0.05) are depicted by asterisks.

FIG 6
FIG 6

Quantification of propidium iodide (PI)-positive cells in T. gondii-infected rat peritoneal cells during treatment with or without the superoxide scavenger MnTBAP in vitro. Freshly isolated peritoneal cells from LEW (A) and BN (B) rats were cultured with 0 or 100 μM MnTBAP and infected with YFP-expressing T. gondii RH tachyzoites. At 0 h, 18 h, and 32 h postinfection, the cultures were analyzed by fluorescence microscopy by randomly selecting 50 cells per ×20 microscopic field and deriving the percentage of those with a combination of red fluorescence (PI positive) and green fluorescence (T. gondii infected) (black bars). In the same field, the percentage of cells that were positive for PI but negative for T. gondii infection was also determined (white bars). The data shown represent means from three independent experiments with standard error bars, and the points at which data are significantly different (P < 0.05) are depicted by asterisks.

DISCUSSION

The LEW rat is extremely resistant to T. gondii infection, to the extent that invading parasites are rapidly killed by the host innate immune responses without the generation of anti-T. gondii antibodies in the host (9). This kind of resistance phenotype indicates the involvement of inherently swift and aggressive killing mechanisms. In our attempts to elucidate the molecular mechanisms underlying the robust resistance of the LEW rat to T. gondii infection, we performed a global transcriptome analysis of T. gondii-infected and uninfected LEW rats versus T. gondii-infected and uninfected BN rats. In RNA sequencing, the quality of the RNA samples used is critical, considering that RNA is rapidly digested in the presence of the nearly ubiquitous RNase enzymes. Degraded RNA can result in shorter fragments of RNA that can compromise the sequencing results. Therefore, prior to sequencing, we evaluated the quality of all the RNA samples and used only samples with an RNA integrity number (RIN) of 9.5 or above, on a scale of 1 to 10 with 1 being totally degraded RNA and 10 being intact RNA (20). Previous data from our sequencing facility at the University of Illinois at Urbana-Champaign have demonstrated that RNA samples with a RIN of 7 and above (as determined with an Agilent Bioanalyzer) provide reliable and consistent RNA sequencing results (unpublished data). The RIN is an important tool for validating RNA samples used in gene expression measurement experiments (20).

Our data show that compared to the BN rat, the uninfected LEW rat has inherently higher transcript levels of Cyp2d3, Cyp25, and Cybrd1 that are maintained even during infection with T. gondii. These findings were corroborated by our observation that both the uninfected and infected LEW rat peritoneal cells contained higher levels of ROS than the BN rat peritoneal cells. Cyp2d3 and Cyp2d5 are cytochrome P450 family proteins that catalyze the microsomal and mitochondrial electron transport chains leading to the generation of ROS (including superoxide radical anion, hydroxyl radical, and hydrogen peroxide) in mammalian cells (15). The Cybrd1 gene is a member of the cytochrome b561 gene family that encodes an iron-regulated protein (21). It can catalyze the reduction of ferric iron to form ferrous iron, which reacts with hydrogen peroxide to produce a hydroxyl radical via the Fenton reaction (15). In the normal physiological state, cellular ROS are detoxified by cellular antioxidant enzymes to prevent the development of oxidative stress, which is deleterious to cells (22, 23). Interestingly, in the present study, we found that despite having high expression of cytochrome enzymes, with the associated high levels of ROS, the LEW rat had lower transcript levels of antioxidant enzymes than the BN rat. This implied that the LEW rat maintained cellular microenvironmental oxidative stress which it is able to tolerate. During microbial invasion, immune effector cells, including neutrophils, eosinophils, and macrophages, have been shown to generate an oxidative burst that is toxic to the invading pathogen (19, 24).

Based on our findings it is logical that in the LEW rat cells, the high expression of cytochrome enzymes with concomitantly high ROS levels, but without augmented expression of antioxidant enzymes, creates a toxic cellular environment that rapidly and effectively kills invading T. gondii tachyzoites. Indeed, generation of ROS in infected mammalian cells has been reported to limit intracellular T. gondii growth and survival (25–28), but the involvement of ROS as a critical component for killing T. gondii during or shortly after host cell invasion remains poorly elucidated. Although T. gondii has been shown to express antioxidant enzymes that might protect it from a host cell oxidative burst after invasion (29), it is likely this is not sufficient or comes too late to prevent toxic effects of the inherently high ROS content in LEW rat cells. Despite previous reports that increased generation of nitric oxide (NO) during T. gondii infection plays a role in controlling the infection (5), we did not observe a significant difference in the expression levels of the inducible nitric oxide (iNOS) gene between the infected LEW and BN rats, indicating that neither rat strain had an advantage over the other in the utilization of NO in fighting T. gondii infection.

Further, we found that scavenging of superoxide anion by MnTBAP led to a decrease in the refractoriness of the LEW rat peritoneal cells to T. gondii infection, resulting in establishment of infection and proliferation of the parasites in the cells. Mindful of the fact that the LEW rat peritoneal cells rapidly kill T. gondii, in our experimental setup, we first treated the cells with MnTBAP for 30 min to allow scavenging of ROS to occur prior to inoculating the cultures with T. gondii tachyzoites. MnTBAP acts as a superoxide dismutase mimetic that can rapidly scavenge cellular superoxide anion within a few minutes (30, 31). Superoxide anion has been shown to be produced in large quantities by phagocytic cells and is a precursor for other highly reactive ROS such as hydrogen peroxide, hydroxyl radicals, and peroxynitrite (15). Thus, scavenging superoxide in cells would in turn decrease other intracellular ROS. ROS in phagocytic cells have been shown to induce the recruitment of autophagy components to phagosomes, which may facilitate lysosomal fusion (32, 33). In the case of intracellular T. gondii, lysosomal fusion of the nonfusogenic parasitophorous vacuole leads to killing of the intracellular parasites (34). Consistent with our observation that scavenging ROS augments parasite survival and growth in the LEW rat peritoneal cells, others (11) have demonstrated the involvement of ROS and caspase-1 in a T. gondii killing mechanism that is linked to the Toxo1 locus in the LEW rat.

We found that the decrease in resistance to T. gondii infection associated with scavenging of superoxide in LEW rat cells significantly augmented parasite growth and the rate of infected host cell death. Along with the increase in the death of infected cells, we observed a significant increase in the death of uninfected cells as well, suggesting a bystander host cell killing phenomenon. The killing of bystander noninfected host cells during T. gondii infection has been reported previously and has been associated with cell lytic factors released from dying infected cells as well as the effect of nitric oxide (35). However, in the BN peritoneal cells, even though parasites grew progressively, with a notable increase in the proportion of dying infected cells, the proportion of dead uninfected cells was comparatively lower than those observed in the LEW rat cells. This indicated that the mechanism underlying the resistance of the LEW rat to T. gondii infection is multifaceted, involving the rapid killing of both the invading parasite and the infected cells, thus effectively curtailing the dissemination of the infection. Underscoring this hypothesis, previous studies have found that a highly conserved Toxo1 genomic locus in T. gondii-resistant rats (including the LEW rat), contains genes (NLRP1 and ALOX12) that have been shown to be involved in innate immunity mechanisms for the rapid killing of T. gondii parasites and infected host cells by macrophages (3, 4, 10, 11, 36). Cellular invasion by pathogens activates the NLRP1 inflammasome, which leads to initiation of pyroptosis, a caspase-1-dependent highly inflammatory cell death process often observed during infection with cytosolic pathogens (37), including T. gondii infection (11).

In conclusion, this study provides evidence that the LEW rat cells have inherently high levels of ROS that are involved in the killing of invading T. gondii parasites and thus contribute to the natural resistance of the LEW rat to T. gondii infection. Despite the deleterious effects of high levels of ROS on the normal physiological state of cells, our findings suggest new avenues for developing therapeutic agents by targeting the induction of oxidative stress as a mechanism for controlling T. gondii infections. Indeed an effective veterinary drug in current use called monensin, which has potent activity against coccidian protozoa, has been shown to cause cellular oxidative stress (38), suggesting that its mode of action against protozoa could be dependent on the generation of ROS in cells.

MATERIALS AND METHODS

Culture and purification of T. gondii tachyzoites.Confluent monolayers of human foreskin fibroblasts (HFF) were maintained in Iscove's modified Dulbecco's medium supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, 1% (vol/vol) GlutaMAX, and 1% (vol/vol) penicillin-streptomycin-amphotericin B (Fungizone) (Life Technologies) at 37°C with 5% CO2. T. gondii RH strain parasites were cultured in the HFF cells. To isolate T. gondii tachyzoites, HFF (cultured in T-75 flasks) in which T. gondii had proliferated were scraped off the culture flask and resuspended in 10 ml culture medium, and parasites were extruded by passing the cell suspension twice through a 25-gauge needle. The extruded parasites were isolated from cell debris by passing through a 3-μm filter, followed by washing the filtered parasites three times in sterile phosphate-buffered saline (PBS). The final parasite concentration was determined by counting using a hemocytometer.

Infection of rats and isolation of peritoneal cells.The care and use of rats for experimental procedures in this study were performed following a protocol approved by the University of Illinois at Urbana-Champaign Institutional Animal Care and Use Committee. Four-week-old male BN and LEW rats were purchased from Charles River and allowed to acclimatize for 10 days. Following acclimatization, for each rat strain, four animals in the treatment group were each inoculated intraperitoneally with 3.5 × 106 freshly isolated T. gondii RH strain tachyzoites constitutively expressing cytosolic yellow fluorescent protein (YFP) (39) in 0.5 ml sterile PBS, while the control group of four animals were each inoculated with 0.5 ml of sterile PBS only. The rats were sacrificed at 24 h postinfection by CO2 asphyxiation and peritoneal cells isolated immediately by peritoneal lavage with 20 ml of unsupplemented RPMI medium. The cells were washed three times in RPMI medium and counted with a hemocytometer. Part of the freshly isolated peritoneal cells from T. gondii-infected LEW and BN rats were seeded in 24-well plates at a density of 8.5 × 105/well in supplemented RPMI medium (with 10% fetal bovine serum, 2.05 mM l-glutamine, 1× nonessential amino acids [Sigma], and 1% penicillin-streptomycin [Life Technologies]) and analyzed by fluorescence microscopy to detect YFP fluorescence from T. gondii tachyzoites at 24 h and 48 h postculture.

RNA extraction from rat peritoneal cells.For each rat, 1 × 107 peritoneal cells were used for total RNA extraction using the RNeasy minikit (Qiagen) following the manufacturer's instructions. The extracted RNA was quantified using a Qubit 3.0 fluorometer RNA BR kit (Life Technologies) and the quality determined using an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA samples were stored at −80°C until use.

Library preparation and RNA sequencing.RNA samples from individual animals were submitted for RNA sequencing (4 individual samples per treatment group). RNA library preparation and sequencing were performed at the Roy J. Carver Biotechnology Center's High Throughput Sequencing and Genotyping Unit of the University of Illinois at Urbana-Champaign. Briefly, the RNA sequencing libraries were prepared with Illumina's TruSeq Stranded mRNAseq Sample Prep kit (Illumina) following the manufacturer's instructions. The libraries were quantitated by fluorometry (Qubit), run on a Bioanalyzer for fragment analysis, diluted to 10 nM, and quantified by quantitative PCR (qPCR). The libraries were sequenced from one end of the fragments on a HiSeq2500 using a HiSeq SBS sequencing kit version 4. Trimmomatic (version 0.33) was used to trim any residual adapter content and low-quality bases. Fastq files were generated and demultiplexed with the bcl2fastq v2.17.1.14 conversion software (Illumina). The quality-scores line in fastq files used an ASCII offset of 33 known as Sanger scores. All gene counts were generated using Feature Counts in the Sub-read (version 1.5.0) package. Gene annotations, including Gene Ontology terms, were downloaded from Ensembl release 84 for Rnor_6.0 and ToxoDB.org release 28 for T. gondii GT1. The number of reads mapping to each rat chromosome and to the T. gondii genome were calculated using the Samtools (v. 1.3) idxstats tool.

Measurement of levels of ROS in rat peritoneal cells.Freshly isolated peritoneal cells from BN and LEW rats were seeded in 96-well plates at a density of 3.5 × 105/well in 200 μl RPMI medium supplemented with 10% fetal bovine serum, 2.05 mM l-glutamine, 1× nonessential amino acids (Sigma), and 1% penicillin-streptomycin. For each rat strain's peritoneal cells, triplicate wells were either maintained uninfected or infected with T. gondii RH strain at a multiplicity of infection (MOI) ratio of 1:1 and incubated at 37°C with CO2 and humidity for 16 h. After 16 h, 5 μM (final concentration) CellROX Deep Red reagent (Life Technologies) was added to all wells and the cultures incubated for a further 30 min. The cultures were then washed three times in PBS by carefully aspirating out 150 μl of medium and replacing it with 150 μl of PBS, followed by centrifugation of the plates at 300 × g for 5 min. After the last wash, 150 μl of PBS was added and the cells analyzed by fluorescence microscopy to determine the fluorescence generated by the reaction of the CellROX reagent with ROS. Fluorescence quantification per microscopic field (magnification, ×20) was done using ImageJ version 1.37v software (NIH). From the microscopic images of the cultures, 100 cells were randomly counted per microscopic field and the percentage of infected and uninfected cells calculated.

Determination of effect of MnTBAP on T. gondii growth in LEW and BN rat peritoneal cells.Freshly isolated peritoneal cells from BN and LEW rats were seeded at a density of 8.5 × 105/well in 24-well plates in 1 ml of supplemented RPMI medium. Triplicate wells were treated with a superoxide scavenger, Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), at a final concentration of 0, 50, 100, or 150 μM, incubated for 30 min, and then infected with YFP-expressing T. gondii RH strain at an MOI ratio of 1:4 (1 parasite to 4 cells), followed by incubation at 37°C with 5% CO2 and humidity. At 18 h, 32 h, and 56 h of incubation, the cultures were analyzed by fluorescence microscopy using the fluorescein isothiocyanate (FITC) channel to measure the parasite YFP fluorescence. After each of the 18-h and 32-h time points of analysis, 500 μl of medium was aspirated from each well and replaced with an equivalent volume of fresh medium containing fresh MnTBAP at the respective final concentration. Fluorescence quantification was done using ImageJ version 1.37v software (NIH). The number of tachyzoites per parasitophorous vacuole in T. gondii-infected LEW and BN rat peritoneal cells during treatment with or without the superoxide scavenger MnTBAP was determined from the microscopic field images taken using a ×20 objective.

Cell viability assay using PI.Freshly isolated peritoneal cells from BN and LEW rats were seeded in 96-well plates at a density of 3.5 × 105/well in 200 μl of supplemented RPMI medium. Triplicate wells were treated with MnTBAP at a final concentration of 0, 50, 100, or 150 μM, incubated for 30 min, and then infected with YFP-expressing T. gondii strain RH at an MOI ratio of 1:4 (1 parasite to 4 cells), followed by incubation at 37°C with 5% CO2 and humidity. Cells treated with MnTBAP but without T. gondii infection were also cultured. For each category of cultures, three plates were set for reading at time points of 0 h, 18 h, and 32 h postinfection. For the plates designated for reading at 0 h postinfection, immediately after inoculating the parasites into the cells, the plates were centrifuged to sediment the cells, and then 150 μl of medium was removed from each well and replaced with an equal volume of fresh medium containing propidium iodide (PI) at a final concentration of 20 μg/ml, followed by incubation in darkness at 37°C for 5 min. The cells were washed three times with PBS by aspirating out 150 μl of the medium and replacing it with 150 μl of PBS, followed by centrifugation of the plates to sediment the cells. After the last wash, 150 μl of PBS was added and the wells analyzed by fluorescence microscopy using the Texas Red channel to detect PI fluorescence and the FITC channel to detect YFP-expressing T. gondii. The plates designated for reading at 18 h and 32 h postinfection were processed and analyzed in the same way at the respective time points. Fluorescence quantification was done using ImageJ version 1.37v software (NIH). The relative fluorescence was derived by subtracting the quantified fluorescence of the uninfected cultures from that of the infected cultures. In microscopic images taken at 0 h, 18 h, and 32 h postinfection, 50 cells were randomly counted per ×20 microscopic field, and the percentage of cells with a combination of red fluorescence (PI positive) and green fluorescence (T. gondii infected), as well as the percentage of cells that were PI positive but negative for T. gondii infection, was calculated.

Cell viability assay using cell proliferation reagent WST-1.Freshly isolated peritoneal cells from BN and LEW rats were seeded in 96-well plates at a density of 3.5 × 105/well in 200 μl of supplemented RPMI medium (without red phenol). Triplicate wells were treated with MnTBAP at final concentrations of 0, 25, 50, 75, 100, 125, 150, 175, 200, 250, and 300 μM and incubated at 37°C with 5% CO2 and humidity. After 24 h of culture, a colorimetric assay using the cell proliferation reagent WST-1 (Roche) for the quantification of cell viability was performed on the cultures by adding 20 μl of the WST-1 reagent to each well. After mixing, the plates were wrapped in aluminum foil and incubated for 1 h at 37 C with 5% CO2. After 1 h of incubation, 150 μl of the medium from each well was transferred to a new 96-well plate, and quantification of the formazan dye produced by metabolically active cells was read as absorbance at a wavelength of 420 nm using a scanning multiwell spectrophotometer (Spectra Max 250; Molecular Devices). Three independent assays were performed, the dose-response curves of the means from triplicate assays were generated using GraphPad Prism software, and the IC50s were derived.

Data analysis.For RNA sequencing data, the combined raw read counts were subjected to preprocessing and analysis using the R (v 3.3.0) and Bioconductor packages (40, 41). Analysis was done using EdgeR's (v 3.14.0) negative binomial generalized linear model with likelihood ratio tests and tagwise dispersion estimates (42–44). The equivalent of a one-way ANOVA across the four groups was calculated for each gene, along with the four logical pairwise comparisons between the groups. Multiple-hypothesis test correction was done using the false-discovery rate method (45). To assess the complex patterns of gene expression across the four treatment groups, a weighted gene correlation network analysis (WGCNA v 1.51) using the trimmed mean of M-values (TMM)-normalized log2 counts per million (CPM) values was performed (46, 47). Heat maps were made separately for rat and Toxoplasma genes with ANOVA FDR (P < 0.05) using TMM-normalized log2 CPM values that were scaled to have a mean of zero and a standard deviation of one. All statistical analyses on the sequencing data were done on log2 scale values and then back translated from log2 fold change (FC) to regular FC. For in vitro assays, data analyses were performed using the two-tailed Student's t test. P values of 0.05 or less were considered significant.

Accession number(s).The entire series of RNA sequencing data obtained in this study has been submitted to the GEO repository and assigned accession number GSE100203.

ACKNOWLEDGMENTS

This study was funded in part by the University of Illinois at Urbana-Champaign.

We are grateful to Alvaro G. Hernandez, Jenny Drnevich Zadeh, and Jessica (Kirkpatrick) Holmes of the Roy J. Carver Biotechnology Center's High Throughput Sequencing and Genotyping Unit of the University of Illinois at Urbana-Champaign for help with RNA sequencing and analysis of the results.

FOOTNOTES

    • Received 24 April 2017.
    • Returned for modification 10 June 2017.
    • Accepted 17 July 2017.
    • Accepted manuscript posted online 24 July 2017.

  • Supplemental material for this article may be found at https://doi.org/10.1128/IAI.00289-17.

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KEYWORDS

Brown Norway rat
cytochrome enzymes
Lewis rat
Toxoplasma gondii resistance
innate immunity
oxidative stress

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