Interleukin-17A-Deficient Mice Are Highly Susceptible to Toxoplasma gondii Infection Due to Excessively Induced T. gondii HSP70 and Interferon Gamma Production
ABSTRACT
Interleukin17A (IL-17A) is known to be involved in the host defense against pathogens and the pathogenesis of autoimmune diseases. Previously, we showed that excessive amounts of interferon gamma (IFN-γ) play an important role in the pathogenesis of the lethal effects of Toxoplasma gondii by inducing anaphylactic responses. In the study described in this report, we examined the effects of IL-17A deficiency on murine host defense against oral T. gondii infection. IL-17A-deficient C57BL/6 (B6) mice exhibited higher rates of mortality than wild-type (WT) mice during the acute phase of T. gondii infection. CD4+ T cells in the mesenteric lymph nodes (mLNs) and ileum of T. gondii-infected IL-17A-deficient mice produced higher levels of IFN-γ than did those of WT mice. In addition, the level of T. gondii HSP70 (T.g.HSP70) expression was also significantly increased in the ileum, mLNs, liver, and spleen of infected IL-17A-deficient mice compared with that in WT mice. These elevated levels of expression of T.g.HSP70 and IFN-γ in infected IL-17A-deficient mice were presumably linked to the IL-17A defect since they decreased to WT levels after treatment with recombinant IL-17A. Furthermore, IL-17A-deficient mice were highly susceptible to the anaphylactic effect of T.g.HSP70, and the survival of IL-17A-deficient mice during the acute phase was improved by treatment with an anti-T.g.HSP70 monoclonal antibody. These results suggest that IL-17A plays an important role in host survival against T. gondii infection by protecting the host from an anaphylactic reaction via the downregulation of T.g.HSP70 and IFN-γ production.
INTRODUCTION
Toxoplasma gondii is an obligate intracellular protozoan that causes the parasitic infectious disease toxoplasmosis in humans and animals worldwide (1). In intermediate hosts, such as humans and mice, infected orally (p.o.), encysted T. gondii bradyzoites or sporozoites in sporulated oocysts undergo stage conversion into rapidly dividing tachyzoites, responsible for acute toxoplasmosis in immunocompromised individuals and fetuses infected during pregnancy (1, 2).
Interferon gamma (IFN-γ) is essential in the induction and maintenance of host protective immunity against T. gondii infection (2, 3), and IFN-γ-deficient mice with either a susceptible background (C57BL/6 [B6] mice) or a resistant background (BALB/c mice) succumb 9 to 10 days after p.o. T. gondii infection, with markedly increased parasite loads being found in multiple organs (4).
Interleukin-17A (IL-17A) is a proinflammatory cytokine which is produced by Th17 cells, a subset of CD4+ T cells, NK cells (5), and other types of cells, contributing to various inflammatory responses (6–9). Among the IL-17 family members (IL-17A to IL17F), IL-17A and IL-17F share 50% amino acid sequence homology and bind to the same heterodimer receptor, consisting of IL-17 receptor A (IL-17RA) and IL-17RC (10). Although it is thought that they likely possess similar biological activities, they perform some distinct functions (10). IL-17A (also referred to as IL-17) is known to play important functions in the development of autoimmune diseases, allergic diseases, and tumors through the induction of proinflammatory and neutrophil-mobilizing chemokines (10).
IL-17A is involved in the host defense against many pathogens, including bacteria and fungi, by inducing the development and recruitment of neutrophils and inducing antimicrobial peptides (11–13). IL-17A is also shown to be important for protective immunity against T. gondii by recruiting neutrophils to the infected sites (14). However, another report indicated that the IL-17A during chronic T. gondii infection instead promotes neuroinflammation (6). Furthermore, IL-17R-deficient mice show reduced ileitis with diminished neutrophil recruitment and inflammation and prolonged survival (15), although another report indicated that IL-17A is not involved in the intestinal inflammation induced by p.o. T. gondii infection (16). Therefore, the roles of IL-17 in protective immunity against T. gondii remain elusive.
Previously, we showed that Th1 cells are preferentially differentiated in T. gondii-infected mice (17). In these mice, Th17 cell differentiation is suppressed, consistent with the observation that Th17 cell differentiation and Th1 cell differentiation are mutually suppressive (18).
T. gondii HSP70 (T.g.HSP70) is a tachyzoite-specific virulent molecule (19–23). The level of T.g.HSP70 expression greatly increases just before host death (21), and this molecule has deleterious effects on disease development and host defense mechanisms by (i) inducing a platelet-activating factor (PAF)-mediated lethal anaphylactic reaction in the T. gondii-infected host (24–26); (ii) downregulating nitric oxide (NO) production, which is an important parasiticidal mechanism (21, 22); and (iii) enhancing the production of anti-HSP70 autoantibody from B1 cells, which accelerates pathogen growth (27, 28).
The T.g.HSP70-induced lethal anaphylactic reaction is an IFN-γ-dependent reaction because the reaction is not observed in IFN-γ-deficient mice and is transferable to IFN-γ-deficient mice by splenocytes but not by serum (i.e., immunoglobulins) from infected wild-type (WT) mice (24). T.g.HSP70 induces a large amount of IFN-γ in T. gondii-infected WT mice, and IFN-γ-dependent PAF activation is involved in this reaction since (i) the level of PAF acetylhydrolase increases in WT mice but not in IFN-γ-deficient mice during the anaphylactic reaction and (ii) WT mice are rescued from anaphylaxis by treatment with the PAF receptor antagonist (24, 25).
In the present study, we analyzed the roles of IL-17 in T. gondii-induced protective immunity by using IL-17A-deficient B6 mice and show that IL-17A deficiency renders the mice susceptible to T. gondii during acute-phase infection. Interestingly, the T. gondii burden in the mesenteric lymph nodes (mLNs) and ileum is not significantly influenced by the IL-17A deficiency, but IFN-γ and T.g.HSP70 production is highly upregulated in T. gondii-infected IL-17A-deficient mice. Based on the observed elevations in the levels of T.g.HSP70 and IFN-γ expression, we addressed the induction of T.g.HSP70-stimulated lethal anaphylaxis in T. gondii-infected IL-17A-deficient mice and the effect of anti-T.g.HSP70 antibody on the acute-phase survival of IL-17A-deficient mice. Our results suggest that IL-17A is important for the protection against T. gondii not because it inhibits pathogen growth but because it suppresses the anaphylactic reaction induced by IFN-γ and T.g.HSP70.
RESULTS
Influence of IL-17A deficiency on survival of T. gondii-infected mice.
Previous results of the influence of an IL-17R deficiency on the survival of T. gondii-infected B6 mice have been controversial, despite the use of the same 76k parasitic strain (14, 15). Thus, the effects of a deficiency in IL-17A on murine survival from infection with the Fukaya strain of T. gondii were examined with IL-17A-deficient B6 mice. While all WT mice infected with 10 T. gondii cysts survived for over 2 weeks, the survival of IL-17A-deficient mice infected with same number of cysts began decreasing on day 10, with the final survival rate being 50% at 2 weeks (P < 0.01 versus WT mice) (Fig. 1A). When WT mice were infected with 30 cysts, the survival rate of these mice (60%) also remained higher than that of IL-17A-deficient mice (<30%) (P < 0.05) (Fig. 1B). Thus, IL-17A was shown to participate in host susceptibility to acute-phase T. gondii infection.
FIG 1
T. gondii load in mLNs and ileum during acute phase of infection.
The T. gondii loads in the mLNs and ileum of WT and IL-17A-deficient B6 mice infected with 10 or 30 T. gondii cysts were compared at 1 week postinfection (p.i.) by measuring the relative level of expression of mRNA for the T. gondii B1 molecule. The differences in the T. gondii burdens in the mLNs between IL-17A-deficient and WT mice did not reach statistical significance (P = 0.17 and 0.49 when the mice were infected with 10 or 30 cysts, respectively), while the loads in IL-17A-deficient mice tended to be slightly higher than those in WT mice (Fig. 2A). Similarly, the T. gondii burden in the ileum was not statistically significantly different between groups (P = 0.58 and 0.53 when the mice were infected with 10 or 30 cysts, respectively) (Fig. 2B). These results suggest that the susceptibility of IL-17A-deficient mice during the acute phase of infection is not simply due to an increase in the T. gondii load in these organ tissues.
FIG 2
Moreover, the T. gondii cyst number and the relative levels of expression of mRNA for the T. gondii B1 and bradyzoite-specific BAG1 molecules in the brains of the surviving mice were not influenced by the IL-17A defect during the chronic phase of infection (at 6 weeks p.i.) (data not shown).
Cytokine expression by mLN cells during acute phase of infection.
IFN-γ is crucial for host protective immunity against T. gondii infection since IFN-γ-mediated immune responses limit the proliferation of tachyzoites and accelerate conversion to the bradyzoite stage in immunocompetent hosts (2, 3). However, we previously revealed that the increased IFN-γ production mediated by T.g.HSP70 stimulation induces lethal anaphylactic responses in T. gondii-infected hosts (24). Accordingly, the influences of an IL-17A deficiency on IFN-γ expression were examined in T. gondii-infected mice by measuring the levels of expression of IFN-γ, IL-4, and IL-17 (as representative Th1, Th2, and Th17 cytokines, respectively) by CD4+ cells. Intracellular cytokine staining of mLN cells at 1 week p.i. revealed that CD4+ T cells from T. gondii-infected IL-17A-deficient mice produced IFN-γ more prominently than those from WT mice (mean number of IFN-γ-producing cells ± standard deviation [SD] in IL-17A-deficient and WT mice, 10.8 ± 2.2 and 5.5 ± 1.2, respectively; these values were significantly different [P < 0.05]) (Fig. 3A). The levels of IFN-γ expression by the CD4− cell population, which included CD8+, NK, NKT, γδ T, and other cells, were similar between the groups (Fig. 3A). In contrast, the influence of an IL-17A deficiency on IL-4 expression was minor (Fig. 3B). A low level of IL-17 expression was observed in WT mice but not in IL-17A-deficient mice (Fig. 3C). Thus, an IL-17A deficiency markedly upregulated IFN-γ expression by the mLN cells of T. gondii-infected mice. Comparable results were observed with the splenocytes of infected mice as well (data not shown).
FIG 3
Effects of IL-17A deficiency in the ileum during acute phase of infection.
IL-17A has been shown to be involved in the recruitment of neutrophils to infection sites (10). Since the ileum is the primary infection site for T. gondii cysts after p.o. infection, the effects of an IL-17A deficiency on neutrophil recruitment to ileal tissue were examined. Histopathological analysis of ileal mucosa infected by T. gondii revealed inflammation with various degrees of epithelial damage, inflammatory infiltrates, and goblet cell depletion in WT mice (Fig. 4A and B). The same degree of mucosal damage was also found in IL-17A-deficient mice (Fig. 4G and H). However, such cellular changes were never noted in either WT or IL-17A-deficient mice not infected with T. gondii (Fig. 4E, F, K, and L). The T. gondii SAG1 antigen, indicative of tachyzoites, was largely detected in the cytoplasm of mononuclear cells infiltrating the lamina propria of the ileal mucosa in both WT and IL-17A-deficient mice (Fig. 4C and I). Occasionally, T. gondii tachyzoites extracellularly discharged from infected cells were also found. On the other hand, neutrophil infiltration of the ileal mucosa was evident in WT mice but not IL-17A-deficient mice (Fig. 4D and J). These histopathological findings may suggest a possible role of IL-17A in neutrophil-derived protection in the ileum during the acute phase of infection.
FIG 4
Regarding Th cytokines, the level of expression of IFN-γ mRNA in the ileum of T. gondii-infected IL-17A-deficient mice was significantly higher than that in the ileum of infected WT mice (Fig. 5A). The level of expression of IL-4 mRNA was also higher in IL-17A-deficient mice when they were infected with 30 cysts, albeit to a lesser degree (Fig. 5B).
FIG 5
Next, the level of T.g.HSP70 expression in the ileum of infected mice was measured to examine the relationship with IL-17A-deficient mouse susceptibility during the acute phase of infection. While the T. gondii loads in the ileum, assessed by determination of the level of B1 mRNA expression, were not different between WT and IL-17A-deficient mice (Fig. 2B), the level of expression of T.g.HSP70 mRNA was significantly higher in IL-17A-deficient mice than in WT mice when they were infected with 30 T. gondii cysts (P < 0.01) (Fig. 6A and B). Both the ATP-binding and C-terminal sites of T.g.HSP70 mRNA were more highly expressed in T. gondii-infected IL-17A-deficient mice than in infected WT mice, and neither was expressed in uninfected animals (Fig. 6A and B). Thus, it appears that an IL-17A deficiency participates in T.g.HSP70 expression during the acute phase of infection.
FIG 6
Effect of rmIL-17A treatment.
To clarify the relevance of an IL-17A deficiency in T.g.HSP70 expression, IL-17A-deficient mice were treated with recombinant mouse IL-17A (rmIL-17A) from 2 days before infection to 6 days p.i. and then sacrificed at 7 days p.i., i.e., 1 day before the rate of survival began to decrease (Fig. 1B). Following the treatment, the ileal expression of mRNA for T.g.HSP70 (both the ATP-binding and C-terminal domains) decreased to that in infected WT mice (P < 0.05) (Fig. 7A and E). Similar findings were observed in the mLNs (Fig. 7B and F), liver (Fig. 7C and G), and spleen (Fig. 7D and H) of T. gondii-infected IL-17A-deficient mice. Treatment with rmIL-17A had no marked effect on T.g.HSP70 production in infected WT mice (Fig. 7). Neither the ATP-binding nor the C-terminal domain of T.g.HSP70 was detected in uninfected IL-17A-deficient or WT mice (data not shown).
FIG 7
Treatment with rmIL-17A also decreased the excess IFN-γ production in the ileum of infected IL-17A-deficient mice to levels approximating those in infected WT mice (P < 0.05) (Fig. 8A). Comparable results were observed in the mLNs (Fig. 8B), liver (Fig. 8C), and spleen (Fig. 8D). The influence of rmIL-17A treatment was not remarkable in infected WT mice (Fig. 8). The difference in the levels of IFN-γ production between uninfected and infected mice was significant (P < 0.001) for both IL-17A-deficient and WT mice.
FIG 8
These results indicate that an IL-17A deficiency is linked to increases in the levels of T.g.HSP70 and IFN-γ expression during the acute phase of infection.
Anaphylactic reaction in T. gondii-infected IL-17A-deficient mice.
T.g.HSP70 is considered a danger signal because the level of expression of T.g.HSP70 greatly increases just 1 to 2 days before host death (21) and induces an IFN-γ-dependent, PAF-mediated, lethal anaphylactic reaction in infected hosts (24, 25). Since T.g.HSP70 expression was more prominent in T. gondii-infected IL-17A-deficient mice than in infected WT mice, the occurrence of an anaphylactic reaction in the two groups was examined. As expected, both IL-17A-deficient and WT mice began exhibiting clinical signs of an anaphylactic reaction 1 h after administration of recombinant T.g.HSP70 (rT.g.HSP70). While the rectal body temperature of both IL-17A-deficient and WT mice declined similarly, the hematocrit (Ht) increase was more prominent in IL-17A-deficient mice (Fig. 9A). The peripheral platelet number relative to the red blood cell (RBC) number was significantly decreased in both IL-17A-deficient and WT mice (Fig. 9B). Clinical signs were scored according to criteria reported previously (26). The average scores ± SDs for the WT and IL-17A-deficient mice were 5.81 ± 0.69 and 6.46 ± 0.66, respectively, which were significantly different (P < 0.05) (Fig. 9C). After rT.g.HSP70 administration, both IL-17A-deficient and WT mice produced markedly more IFN-γ: IFN-γ production in the liver and spleen was highly upregulated (P < 0.01) in T. gondii-infected WT and IL-17A-deficient mice compared to that in mice infected alone (Fig. 9D).
FIG 9
Thus, rT.g.HSP70 stimulation induced a severe anaphylactic reaction in IL-17A-deficient mice, implicating the participation of a T.g.HSP70-stimulated anaphylactic reaction in the susceptibility of IL-17A-deficient mice during the acute phase of T. gondii infection.
Effect of anti-T.g.HSP70 antibody on survival of IL-17A-deficient mice.
To confirm the participation of T.g.HSP70 in the acute-phase susceptibility of IL-17A-deficient mice after T. gondii infection, IL-17A-deficient mice infected with 30 T. gondii cysts were treated or not treated with an anti-T.g.HSP70 monoclonal antibody (MAb; TgNCR A5) at days 5 to 11 p.i. Without anti-T.g.HSP70 MAb treatment, IL-17A-deficient mice succumbed earlier than WT mice after T. gondii infection (P < 0.02), similar to the results presented in Fig. 1B. Treatment with the anti-T.g.HSP70 MAb prolonged the survival of IL-17A-deficient mice, and although the difference between IL-17A-deficient mice treated or not treated with the MAb did not reach statistical significance (P = 0.07), antibody-treated IL-17A-deficient mice were as resistant as WT mice (P = 0.35) (Fig. 10). These results also support the participation of T.g.HSP70 in the acute-phase susceptibility of IL-17A-deficient mice after T. gondii infection.
FIG 10
DISCUSSION
We report here that an IL-17A deficiency may increase the susceptibility of B6 mice to the acute phase of T. gondii infection. An IL-17A deficiency participated in the enhanced upregulation of T.g.HSP70 and IFN-γ expression in the ileum and other tissues, which induced a severe anaphylactic reaction in the hosts. Th1 polarization in T. gondii-infected hosts is enhanced by an IL-17A deficiency, and overproduction of IFN-γ plays a crucial role in the T.g.HSP70-induced anaphylactic reaction through the mechanism of PAF generation enhancement (24, 25).
When B6 mice were infected with either 10 or 30 cysts of the T. gondii Fukaya strain, the rate of mortality of IL-17A-deficient mice was higher than that of WT mice. However, the higher susceptibility of IL-17A-deficient mice could not be simply explained by the T. gondii burden in the organs, because the T. gondii loads in the mLNs and ileum were not statistically significantly different between IL-17A-deficient and WT mice during the acute phase of infection. This was confirmed by measuring the level of T. gondii-specific B1 mRNA expression and histological detection of T. gondii SAG1, although the level of SAG1 staining was rather reduced in IL-17A-deicient mice compared to WT mice. These results are consistent with those reported by Muñoz et al. (16), who analyzed the T. gondii load at day 8 after p.o. infection with 100 cysts of T. gondii ME49.
Previously, Kelly et al. (14) reported an increased susceptibility of IL-17R-deficient B6 mice, indicating the importance of IL-17R-mediated signaling for the host defense during the acute phase of infection. These results are consistent with our results. Although they reported that the T. gondii burden measured at 1 and 2 weeks p.i. in the organs (spleen, gut, liver, and brain) of IL-17R-deficient mice was higher than that in WT mice, we could not detect a significant increase in the T. gondii load in IL-17A-deficient mice. This is probably explained by the redundant activity of the remaining IL-17F in IL-17A-deficient mice, because the IL-17F function is also inhibited in IL-17R-deficient mice. In support of this idea, we previously showed that either IL-17A or IL-17F is enough to protect the host from Staphylococcus aureus infection (12). In contrast, Guiton et al. (15) reported the prolonged survival of IL-17RA-deficient B6 mice compared with that of WT mice and concluded that the IL-17R signaling was deleterious in T. gondii infection. Despite our different results on the survival of infected mice, we agree that IL-17A contributes to the pathogenesis of T. gondii infection. The reason why our results are different from those of Guiton et al. (15) is not known at present, but it is possible that IL-17F and/or other IL-17 family members that use IL-17RA play an important role in the T. gondii-induced pathology, although this cannot explain the discrepancy with the results reported by Kelly et al. (14). Other possibilities are the difference in the T. gondii strains used and also differences in the genetic backgrounds of the mice or the intestinal commensal microbiota, although all mice were kept under specific-pathogen-free conditions. Further examinations are needed to explain the apparent discrepancy.
The current study showed that T.g.HSP70 expression is highly upregulated at 1 week p.i. in the ileum and other organs of IL-17A-deficient mice. T.g.HSP70 is a tachyzoite-specific virulence molecule whose expression increases rapidly just 1 to 2 days before the death of the host (21) and whose systemic detection has been suggested to associate with parasite death mediated by host immune responses (23). As mortality in IL-17A-deficient mice p.o. infected with 30 cysts commenced on day 8 p.i., the increased level of T.g.HSP70 expression on day 7 p.i. was considered to coincide well with this survival pattern. Because HSP70 release occurs both through physiological secretion mechanisms and during cell death (29), its extracellular levels do not necessarily depend on the T. gondii load but, rather, depend on the efficiency of the secretory system of eukaryotic cells. Our data suggest that IL-17A deficiency may cause an increase in the level of T.g.HSP70 secretion from infected cells during the acute phase of infection.
Histopathological analyses suggest the participation of IL-17A in the recruitment of neutrophils to the ileum during the acute phase of infection. Thus, the level of T.g.HSP70 expression is inversely correlated to neutrophil activity in tissues or organs, suggesting the involvement of neutrophils in the regulation of T.g.HSP70 secretion. However, the precise mechanisms that exist between them remain to be analyzed.
Preferential Th1 polarization was observed in CD4+ T cells from the mLNs and spleens of T. gondii-infected mice. This polarization was more prominent in T. gondii-infected IL-17A-deficient mice than in WT mice. We also showed that the level of IFN-γ expression in the ileum of T. gondii-infected IL-17A-deficient mice was higher than that in the ileum of WT mice. Consistent with our observations, Guiton et al. (15) reported a higher concentration of IFN-γ in the culture supernatants of splenocytes from IL-17RA-deficient mice than in those of splenocytes from WT mice upon cocultivation with T. gondii extract, although the supernatants might have included IFN-γ produced by cells other than Th1 cells, such as CD8+ T or NK cells. Muñoz et al. (16) showed the marked expression of IFN-γ in the ileum and in the supernatant of ileal biopsy specimens from infected IL-17A-deficient mice as well, although the expression levels were similar between IL-17A-deficient and WT mice. Thus, our results and others' results suggest that an IL-17A deficiency excessively increases the level of IFN-γ expression during the acute phase of T. gondii infection, although the enhancing effect of IL-17A deficiency may not be detected under some experimental conditions.
The markedly upregulated expression of T.g.HSP70 and IFN-γ in the ileum and other organs of T. gondii-infected IL-17A-deficient mice prompted us to analyze the occurrence of the T.g.HSP70-stimulated anaphylactic reaction (24, 25). Administration of rT.g.HSP70 apparently induced a severe anaphylactic reaction in infected IL-17A-deficient mice, and the reaction was more prominent than that in WT mice. These findings suggest that a T.g.HSP70-stimulated severe anaphylactic reaction is a part of the cause of mortality in IL-17A-deficient mice during the acute phase of T. gondii infection. It is well-known that T.g.HSP70 functions as an agonist for Toll-like receptor 2 (TLR2) and TLR4 (30–32), and PAF generation in the T.g.HSP70-induced anaphylactic reaction is caused by the activation of cytosolic phospholipase A2 (cPLA2), involving the TLR4–myeloid differentiation factor 88 (MyD88)–mitogen-activated protein kinase (MAPK) signaling pathway (25). However, the IL-17A-induced transcription factors and collaborating molecules that regulate T.g.HSP70 expression remain to be analyzed in a future study.
Lastly, it is known that p.o. T. gondii infection causes intestinal inflammation and massive necrosis in the ileum of susceptible B6 mice and ensuing death (33, 34). T. gondii-induced damage of the villi and mucosal cells in the ileum of B6 mice is not due to tissue destruction by tachyzoites but, rather, is mediated by the overproduction of proinflammatory mediators from CD4+ T cells, such as IFN-γ, tumor necrosis factor, and NO (33–36). Thus, we have shown here that T. gondii-infected IL-17A-deficient mice succumb earlier than WT mice due to the anaphylactic reaction against T.g.HSP70, which is overproduced together with IFN-γ in IL-17A-deficient mice, suggesting that IL-17A plays a beneficial role in the pathogenesis of toxoplasmosis by suppressing Th1 polarization and T.g.HSP70 production. Also, the potential use of anti-T.g.HSP70 antibodies against acute toxoplasmosis has been suggested. These observations may provide a cue to treat toxoplasmosis in humans together with basic therapy to block the folic acid synthesis of this microbe.
MATERIALS AND METHODS
Mice and T. gondii strain.
IL-17A-deficient mice (37) with a B6 background were maintained in the animal centers of Chiba University and Shinshu University under specific-pathogen-free conditions. WT female B6 mice were purchased from SLC (Hamamatsu, Japan). Both the IL-17A-deficient and WT mice were used at the age of 8 weeks. Cysts of an avirulent T. gondii Fukaya strain were obtained from the brains of chronically infected mice as previously described (38). Mice were p.o. infected with 10 or 30 T. gondii cysts suspended in 500 μl of phosphate-buffered saline (PBS) by using a needle with a round head. All animal procedures used in this study complied with guidelines set by the Animal Care and Use Committees of Chiba and Shinshu Universities.
Real-time PCR.
During the acute phase of infection (at 1 week postinfection [p.i.]), total RNA was isolated from the ileum, mLNs, liver, or spleen of both WT and IL-17A-deficient B6 mice and transcribed to cDNA by reverse transcription (RT), and then the cDNA was used for real-time quantitative PCR using a TaqMan PCR system (Applied Biosystems, Tokyo, Japan).
The T. gondii abundance in the mLNs and ileum of WT and IL-17A-deficient B6 mice was determined by measurement of the relative level of expression of mRNA for the T. gondii B1 molecule, which is specific for both tachyzoites and bradyzoites, by using forward primer 5′-AAC GGG CGA GTA GCA CCT G-3′, reverse primer 5′-TGG GTC TAC GTC GAT GGC AT-3′, and 6-carboxyfluorescein (FAM)-labeled probe 5′-ATA GAG AGT ACT GGA ACG TC-3′.
The level of ATP-binding or C-terminal T.g.HSP70 mRNA expression was evaluated by using forward primer 5′-CAG GTG CAG GAT TTG CTT CT-3′, reverse primer 5′-TTG GTC GGG ATC GTT GTG T-3′, and FAM-labeled probe 5′-CCA AGC TGA TTG AAA G-3′ or forward primer 5′-CAC GTA CGC GGA CAA CCA-3′, reverse primer 5′-CGC ACG CTC ACC TTC GT-3′, and FAM-labeled probe 5′-CAG GAG TGC TGA TTC A-3′, respectively.
All primer/probe sets mentioned above and those specific for murine IFN-γ, IL-4, or IL-17 were purchased from Applied Biosystems. We normalized each set of samples using the difference in the number of threshold cycles (ΔCT) between the sample gene (CTsample) and the GAPDH glyceraldehyde-3-phosphate dehydrogenase) internal control gene (CTGAPDH) as follows: ΔCT = CTsample − CTGAPDH. The calibrator sample was the sample with the highest ΔCT (ΔCTcalibrator) in each set. The relative scale of mRNA measurement is represented on the y axes of the relevant figures by the expression of log-transformed 2−ΔΔCT, where ΔΔCT = ΔCTsample − ΔCTcalibrator. Each reaction was done in triplicate.
Flow cytometry analysis.
Mesenteric lymph node cells were isolated from WT or IL-17-deficient B6 mice infected or not infected with 30 T. gondii cysts at 1 week p.i. and stimulated with 25 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, Tokyo, Japan) plus 1 μg/ml ionomycin (Sigma-Aldrich) for 6 h in the presence of GolgiStop protein transport inhibitor (BD Biosciences), which was added for the last 4 h. Then, they were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (clone GK1.5) monoclonal antibody (MAb), fixed, and permeabilized using Cytofix/Cytoperm fixation and permeabilization solution (BD Biosciences), followed by intracellular staining with phycoerythrin (PE)-conjugated anti-IFN-γ (clone XMG1.2), IL-4 (clone 11B11), IL-17A (clone TC11-18H10), or an isotype control IgG MAb at 4°C for 30 min. The MAbs were purchased from BD Biosciences or BioLegend (BioLegend Japan, Tokyo, Japan). After washing, the level of cytokine expression was analyzed by use of a FACSCanto II flow cytometer (BD Biosciences).
Treatment of mice with recombinant mouse IL-17A.
IL-17A-deficient B6 mice infected with 30 T. gondii cysts were treated or not treated intraperitoneally (i.p.) with recombinant mouse IL-17A (rmIL-17A; R&D Systems, Minneapolis, MN, USA) at a dose of 0.5 μg/mouse every other day from 2 days before infection to 6 days p.i. and sacrificed at 7 days p.i. The concentration of rmIL-17A applied was from a previous report (39).
Immunohistochemistry.
Ileal tissues were removed from mice infected or not infected with 30 T. gondii cysts at 1 week p.i., fixed in 20% buffered formalin for 48 h, and then embedded in paraffin. Serial sections with a 3-μm thickness were prepared and stained with hematoxylin and eosin (H&E) and alcian blue–periodic acid-Schiff (AB-PAS) stains. Immunohistochemistry for T. gondii SAG1 antigen and neutrophils was carried out on the remaining tissue sections with anti-T. gondii SAG1 antigen (TP3 antigen; HyTest, Turku, Finland) diluted 1:100 and antimyeloperoxidase (catalog number ab9535; Abcam, Cambridge, UK) diluted 1:50, respectively. Before immunostaining, antigen retrieval was performed by microwaving tissue sections in 10 mM citrate buffer, pH 6.0, for 30 min for the anti-T. gondii SAG1 antigen or in 10 mM Tris-HCl buffer, pH 8.0, containing 1 mM EDTA for 30 min for antimyeloperoxidase. For secondary antibodies, Histofine simple stain Max-Po (M) and Histofine simple stain Max-Po (R) (Nichirei Bioscience, Tokyo, Japan) were used for anti-T. gondii SAG1 antigen and antimyeloperoxidase, respectively. Peroxidase activity was visualized with a diaminobenzidine-hydrogen peroxide solution. Negative controls were performed by omitting the primary antibodies from the procedure, and no specific staining was seen.
Induction of PAF-mediated anaphylactic reaction by T. gondii HSP70.
The method used for the induction of a PAF-mediated anaphylactic reaction by T.g.HSP70 in T. gondii-infected WT mice but not in IFN-γ-deficient mice was previously reported (24, 25). Recombinant T.g.HSP70 (rT.g.HSP70) was prepared as previously described (40). Lipopolysaccharide (LPS) in rT.g.HSP70 was undetectable (<0.5 endotoxin units in 1 mg/ml rT.g.HSP70, determined by use of an E-Toxate kit [Sigma, St. Louis, MO, USA]). According to dose-response analysis (24), 100 μg of rT.g.HSP70 was i.p. injected into WT or IL-17-deficient B6 mice at 1 week p.i. with 10 T. gondii cysts. The survival of the mice, their rectal body temperature, and clinical signs of an anaphylactic reaction were monitored every 30 min after rT.g.HSP70 injection as described previously (24, 26). Peripheral blood samples were collected from the tails of mice before or 3 h after rT.g.HSP70 injection, smeared on glass slides, and stained by the Giemsa method. The numbers of platelets in peripheral blood were counted under a microscope at a magnification of ×200. Results are shown as the ratio of the platelet number to 1,000 red blood cells (RBC) according to the Fonio method (41). The mice were carefully monitored and sacrificed before the endpoint to avoid unnecessary suffering.
Treatment of IL-17A-deficient B6 mice with anti-T.g.HSP70 monoclonal antibody.
Hybridoma clones which produce MAbs specific for T.g.HSP70 were generated, and an anti-T.g.HSP70 MAb (TgNCR A5, IgG1) was purified as previously described (27, 42). IL-17A-deficient B6 mice infected with 30 T. gondii cysts were intraperitoneally immunized with 100 μg TgNCR A5 MAb daily at days 5 to 11 p.i.
Statistics.
Statistically significant differences in the survival experiment were analyzed by the Kaplan-Meier method (log-rank test). Other statistically significant differences between groups were determined by Student's t test. A P value of <0.05 was taken to indicate statistical significance.
ACKNOWLEDGMENTS
We thank Akemi Sakamoto (Biomedical Research Center, Chiba University) for discussions.
We declare that we have no conflict of interest.
This work was supported by JSPS K grant numbers JP21590462 and JP25460512.
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Information & Contributors
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Published In
Infection and Immunity
Volume 85 • Number 12 • December 2017
eLocator: 10.1128/iai.00399-17
Editor: John H. Adams, University of South Florida
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© 2017 American Society for Microbiology. All Rights Reserved .
History
Received: 5 June 2017
Returned for modification: 17 July 2017
Accepted: 1 September 2017
Published online: 17 November 2017
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John H. Adams
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University of South Florida
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Interleukin-17A-Deficient Mice Are Highly Susceptible to Toxoplasma gondii Infection Due to Excessively Induced T. gondii HSP70 and Interferon Gamma Production. Infect Immun
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