CD4⁺ T cells are trigger and target of the glucocorticoid response that prevents lethal immunopathology in toxoplasma infection

GC receptor (GR) signaling in T cells is required for host survival during acute T. gondii infection

Although able to control infection, WT mice inoculated with nonlethal T. gondii strains undergo transient weight loss and display a hunched and scruffy appearance suggestive of a GC-mediated stress response. To determine whether toxoplasma infection triggers GC production, we measured corticosterone by ELISA in the sera of C57BL/6 mice challenged i.p. with cysts of the ME49 strain while simultaneously assaying IL-12, IFN-γ, IL-10, and IL-27. Serum GC levels increased sixfold during acute infection with kinetics that closely resembled those determined for the antiinflammatory (IL-10 and IL-27) as opposed to proinflammatory (IL-12 and IFN-γ) cytokines (Fig. 1 A). Thus, whereas IL-12 (p40 and p70) and IFN-γ reached peak levels on days 5 and 6 after infection, respectively, GCs together with IL-10 and IL-27 displayed minor increases at these time points and did not peak until day 8.

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Figure 1.

T. gondii infection elicits a GC response, and the lack of GR signaling in T cells results in acute mortality of toxoplasma-infected mice. (A) C57BL/6 mice were infected i.p. with an average of 15 ME49 cysts, and serum corticosterone, IFN-γ, IL-12p70 and p40, IL-27p28, and IL-10 levels were measured on the days indicated. Symbols represent mean ± SEM of the ELISA values for the individual animals (n = 3–12) at each time point pooled from three independent experiments. (B) Survival of homozygote GR^lck-Cre, heterozygote GR^fl/+lck-Cre, and littermate control animals after infection. The survival curves shown are from one representative of 10 experiments performed, two of which included GR^fl/+lck-Cre mice. (C) Parasite burdens in PECs and spleen on day 8 after infection as determined by plaque assay. Bars represent mean ± SEM number of PFU per organ (n = 3–5 mice). (D) Weight loss and serum levels of AST and CK in infected animals. Results shown are means ± SEM for values for the individual mice (n = 4–5). No distinguishing histopathological changes were detected in lung, heart, liver, and kidney at this day 8 time point. Data presented in C–E are representative of two experiments performed. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Because GCs promote IL-10 production by T cells (Barrat et al., 2002) and dampen IFN-γ production by Th1 lymphocytes (Franchimont et al., 2000; Liberman et al., 2007), we asked whether GCs exert a similar regulatory role during T. gondii infection by acting on T cells. To this end, we infected mice that selectively lack GR expression in T cells (GR^lck-Cre; Mittelstadt et al., 2012). In contrast to littermate control animals, GR^lck-Cre mice rapidly succumbed during the acute phase of infection with similar kinetics to those previously described for both IL-10^−/− and IL-27R^−/− T. gondii–infected animals (Fig. 1 B). Interestingly, mice heterozygous for GR deletion (GR^fl/+lck-Cre) displayed the same mortality as homozygous-deficient animals (Fig. 1 B), consistent with prior findings (Wüst et al., 2008; Mittelstadt et al., 2012), arguing for a threshold requirement in GR signaling in T cells. Together these results demonstrate that T. gondii infection triggers GC production and that this response plays a critical and nonredundant T cell–dependent host-protective function.

T. gondii–infected GR^lck-Cre mice display evidence of enhanced tissue pathology despite unaltered parasite burdens

To determine whether the increased mortality in T. gondii–exposed GR^lck-Cre mice is caused by an ineffective immune response, we measured parasite burdens on day 8 after infection. Despite their distinct survival outcomes, GR^lck-Cre and control animals displayed indistinguishable frequencies of infected cells (not depicted), as well as total numbers of parasites in peritoneum (the site of infection; peritoneum exudate cells [PECs]) and spleen (Fig. 1 C). Instead, the rapid mortality of infected GR^lck-Cre (and heterozygous GR^fl/+lck-Cre) mice was preceded by enhanced weight loss and increased levels of serum aspartate aminotransferase (AST) and creatine kinase (CK), major biomarkers for hepatic dysfunction and muscle damage, respectively (Fig. 1 D).

GR^lck-Cre mice infected with T. gondii exhibit a cytokine expression profile distinct from IL-10^−/− and IL-27R^−/− animals

The phenotype observed for GR^lck-Cre animals was reminiscent of that previously described in T. gondii–infected IL-10^−/− and IL-27R^−/− mice, which display increased levels of serum IL-12p40 and IFN-γ as a result of their deficiencies in IL-10 production. Because in our model GR deletion is restricted to T cells, an effect on innate cytokine production by APCs seemed unlikely. Nevertheless, lack of GR expression in T cells could indirectly cause increased IL-12p40 if GC signaling is required for IL-10 production by CD4⁺ T lymphocytes. However, levels of IL-12p40 or p70 and IL-27p28 in serum and peritoneum were indistinguishable between control and GR^lck-Cre animals (Fig. 2, A–C), a finding inconsistent with either altered APC function or defective IL-10 production.

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Figure 2.

T. gondii–infected GR^lck-Cre mice display enhanced IFN-γ, TNF, and IL-10 production, despite normal levels of innate cytokines and CD4⁺ T cell numbers. (A–F) WT and GR^lck-Cre littermates were infected i.p. with 15 ME49 cysts, and serum IL-12p40 (A), IL-12p70 (B), IL-27p28 (C), IFN-γ (D), TNF (E), and IL-10 (F) were measured on the days indicated as well as cytokine levels in peritoneal exudates on day 8 or 4 in the case of IL-12p70 (bar graphs). Each symbol or bar represents mean ± SEM of values for individual animals (n = 3–22). (G and H) Kinetics of total numbers of CD4⁺ and CD8⁺ T cells in PECs (G) or spleen (H) in T. gondii–infected animals. Bars represent mean ± SEM of values for individual animals (n = 3–28 per time point). Data presented are pooled from 10 independent experiments performed. *, P < 0.05; ***, P < 0.001.

In contrast to IL-12 and IL-27, dramatic increases in IFN-γ levels were observed in the same infected GR^lck-Cre mice. Although the initial phase of the response (before day 5) was similar to that seen in WT animals, on days 7–8 after infection, when GC levels peak and IFN-γ levels are decreasing in WT mice, GR^lck-Cre animals exhibited an uncontrolled increase in the cytokine (Fig. 2 D). Interestingly, infected GR^lck-Cre animals also displayed a dramatic elevation in TNF and IL-10 (Fig. 2, E and F), a phenotype clearly distinct from that of infected IL-10– or IL-27R–deficient mice. Moreover, the delayed onset and combined overexpression of IFN-γ, TNF, and IL-10, which are known Th1 products, support the concept that the defective host resistance in GR^lck-Cre mice results from a perturbation of the adaptive, rather than innate, immune system.

Th1 cells from T. gondii–infected GR^lck-Cre mice display hyperactivity in vivo but not in vitro

The increased production of IFN-γ, TNF, and IL-10 in GR^lck-Cre mice could result from either the expansion of activated T cells or the enhanced responsiveness of cells within an unchanged pool size. Although the first hypothesis is consistent with the known role of GC in T cell apoptosis (Sionov et al., 2006), no significant increases were observed in the total number of CD4⁺ and CD8⁺ T lymphocytes (Fig. 2, G and H) in GR^lck-Cre versus control mice during acute infection. In addition, no differences were detected in the frequencies of Ki-67⁺ versus Annexin-V⁺ T cells in the two mouse strains (not depicted).

To test the second possibility, spleen cells isolated from GR^lck-Cre and WT mice on day 8 after infection were stimulated in vitro with either anti-CD3 mAb or the MHC class II–restricted parasite peptide AS15 (Grover et al., 2012). No differences were observed in IFN-γ or IL-10 responses (not depicted). Moreover, frequencies of IFN-γ⁺–producing cells, which at this time point are restricted to the CD4⁺ CD44⁺ T-bet⁺ Foxp3⁻ population (Jankovic et al., 2007), were comparable between GR^lck-Cre and WT animals (Fig. 3, A–C).

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Figure 3.

GCs regulate the magnitude of the Th1 response in vivo in T. gondii–infected mice. (A) WT and GR^lck-Cre mice were infected i.p. with 15 ME49 cysts, and frequencies of IFN-γ⁺ CD4⁺ CD44⁺ T cells were determined on day 8 in splenocytes stimulated in vitro by plate-bound anti-CD3 mAb. Bars represent mean ± SEM of values for individual animals (n = 5–11) determined by ICS. (B–E) Bulk splenocytes, described in A, were stimulated in vitro with AS15 peptide alone (B and C) or in the presence of Dex (D and E), and frequencies of IFN-γ⁺ and TNF⁺ cells were assayed by ICS. (B and D) The dot plots shown were gated on CD44⁺ CD4⁺ T cells. (C) Bars represent mean ± SEM of values determined from cultures of individual animals (n = 5) shown in B. In E, each symbol indicates the ratio of cytokine MFI for IFN-γ⁺ TNF⁺ CD4⁺ T cells determined by ICS in parallel cultures from individual mice (n = 5) stimulated with AS15 peptide in the presence versus absence of Dex (shown in D and B). (F) Numbers of AS15 tetramer⁺ CD4⁺ T cells in spleen on day 8 after infection. Bars represent mean ± SEM of values for individual animals (n = 13–16). (G and H) Day 8 toxoplasma-infected mice were injected i.v. with AS15 peptide, and 6 h later, frequencies of IFN-γ⁺ tetramer⁺ cells were determined by ICS in splenocytes prepared from individual mice. The dot plots shown were gated on CD44⁺ CD4⁺ T cells (G), and bars represent mean ± SEM of values for individual mice (n = 6; H). (I and J) Uninfected WT and day 8 infected mice were injected i.p. with BFA and ICS was performed. (I) The representative dot plots shown are gated on CD44⁺ CD4⁺ T lymphocytes. (J) Bars represent mean ± SEM of frequencies of IFN-γ⁺ CD44⁺ CD4⁺ T cells for individual mice (n = 5), whereas symbols indicate IFN-γ MFI values for CD44⁺ CD4⁺ T cells from one mouse. The experiments shown in A–J are representative of two to four performed. (K) Ifng, Tnf, and Il10 mRNA expression in CD4⁺ CD44⁺ T cells purified from peritonea of day 8 infected mice (n = 3–5). Bars represent mean ± SEM of cytokine expression relative to Hprt from three independent experiments. AU, arbitrary units. *, P < 0.05; **, P < 0.01.

Although CD4⁺ T lymphocytes from both groups displayed indistinguishable cytokine responsiveness in vitro, their behavior in vivo was clearly distinct. Whereas the T. gondii–specific CD4⁺ T population recognized by the AS15 tetramer (Grover et al., 2012) expanded equally in GR^lck-Cre and control mice (Fig. 3 F), after injection of AS15 peptide i.v. on day 8 of infection, significantly higher frequencies of Th1 cells producing IFN-γ were detected in GR^lck-Cre animals (Fig. 3, G and H). To extend this in vivo analysis to a broader range of T cell specificities, we next analyzed infected mice injected i.p. with brefeldin A (BFA). We found that CD44⁺ T-bet⁺ Foxp3⁻ cells in PECs represent 79% versus 80% of CD4 T lymphocytes in control and GR^lck-Cre-infected animals, respectively. Nevertheless, the frequency of IFN-γ–producing cells was significantly higher in GR^lck-Cre mice as was their overall IFN-γ mean fluorescence intensity (MFI; Fig. 3, I and J). In addition, increased levels of Ifng and Tnf as well as Il10 mRNA were detected by RT-PCR in FACS-sorted PEC CD4⁺ CD44⁺ T lymphocytes from GR^lck-Cre versus control animals (Fig. 3 K). However, no differences in expression of the co-stimulatory molecules CD28, CD69, ICOS, and GITR, as well as cytokine receptors CD25, CD119, and CD127 were detected in the same populations (not depicted).

Together these experiments demonstrated that despite similar antigen (Ag) load and comparable innate cytokine milieu, Th1 effectors in T. gondii–infected GR^lck-Cre animals respond more vigorously to Ag stimulation than the equivalent cell population in WT mice. However, when removed from GC pressure by in vitro culture, cells from both groups exhibited equal responsiveness. When GC pressure was reintroduced in vitro by addition of dexamethasone (Dex), cytokine production by Th1 cells from infected WT, but not GR^lck-Cre, mice was suppressed (not depicted). Interestingly, in WT cultures stimulated with AS15 peptide in the presence of Dex, the frequency of responding CD4⁺ T cells remained unchanged (Fig. 3, B vs. D), but their MFIs for IFN-γ⁺ and TNF⁺ were reduced (Fig. 3 E).

CD4 T lymphocytes drive mortality in T. gondii–infected GR^lck-Cre mice

The above experiments suggest that Th1 cells are a major target of GC-mediated suppression during toxoplasma infection. In support of this hypothesis, anti-CD4, but not anti-CD8, mAb treatment attenuated acute mortality in GR^lck-Cre mice (Fig. 4 A). Importantly, CD4 T cell depletion also prevented the increase in pathological markers observed in the latter group (Fig. 4 B). As predicted (Jankovic et al., 2007), although serum IL-12p40 levels were similarly increased, the systemic IFN-γ and IL-10 responses in sera of CD4-depleted T. gondii–infected mice were reduced to a comparable level in both GR^lck-Cre and WT animals (Fig. 4 C). To directly test the pathological role of CD4⁺ T lymphocytes lacking GR, we reconstituted RAG KO mice with either WT or GR^lck-Cre CD4⁺ T cells from naive mice. In agreement with the depletion experiments, all RAG KO animals that received GR^lck-Cre CD4⁺ T lymphocytes succumbed to infection, whereas the majority of recipients of WT cells survived (Fig. 4 D). Moreover, serum levels of IFN-γ, alanine aminotransferase (ALT), and AST were significantly higher in mice reconstituted with GR^lck-Cre versus WT cells (IFN-γ: 11.6 ± 1.5 vs. 7.2 ± 1.0; ALT: 353 ± 73 vs. 162 ± 28; AST: 662 ± 122 vs. 310 ± 46).

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Figure 4.

Mortality in T. gondii–infected GR^lck-Cre mice is dependent on CD4⁺ T cells that also drive the induction of the GC response. (A–C) T. gondii–infected mice were treated i.p. with anti-CD4, anti-CD8, or control mAb on days −1, 3, 5, 8, and 12 (n = 3–5 mice). Survival was monitored daily (A), and serum levels of AST and CK (B) and IL-12p40, IFN-γ, and IL-10 (C) were measured on day 8. Bars represent mean ± SEM of values for individual animals (n = 3–12). (D) Survival of T. gondii–infected RAG KO mice (n = 4) and those adoptively transferred with naive GR^lck-Cre (n = 6) or WT (n = 9) CD4⁺ T cells. (E) Serum corticosterone levels in animals described in A. The results shown in A–E are representative of three experiments performed. (F–H) Serum corticosterone levels in naive and toxoplasma-infected mice deficient in the genes indicated (F), WT animals treated with anti-TNF and anti–IFN-γ Ab alone or in combination with anti–IL-6 Ab (G), and RAG^−/− and OT-II RAG^−/− mice (H). Bars represent mean ± SEM of the ELISA values for individual mice (n = 4–10) pooled from two independent experiments. (I) Corticosterone levels in uninfected and 8-d T. gondii–infected RAG^−/− animals, one group of which received naive WT CD4⁺ T cells. Bars represent mean ± SEM of ELISA values for individual mice (n = 5–8) from one representative out of three performed. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

The GC response in T. gondii infection is triggered by CD4 T lymphocytes

The above results demonstrated that the GC response induced during the acute phase of infection plays an essential host-protective role by modulating the responsiveness of CD4 T lymphocytes. Because the interaction between the endocrine and immune system is known to be bidirectional, we next tested whether depletion of CD4⁺ T cells might influence GC production during toxoplasma infection. Unexpectedly, serum GC levels were dramatically reduced in infected mice depleted of CD4⁺, but not CD8⁺, T cells (Fig. 4 E). In contrast, GC responses were not compromised in toxoplasma-infected mice lacking the signaling molecules MyD88, IFN-γR, or IL-27R and cytokines TNF, IL-12p40, or IL-10 that are associated with both Th1 responses and have been previously implicated in driving GC responses (Fig. 4 F). Moreover, antibody (Ab) neutralization of IFN-γ, TNF, and IL-6 failed to inhibit the GC response in infected WT mice (Fig. 4 G).

Consistent with the depletion studies, RAG^−/− mice failed to display significant increases in GC levels after toxoplasma infection (Fig. 4 H). In addition, serum GC levels remain unchanged in infected OT-II Tg RAG^−/− mice that possess only OVA-specific CD4⁺ T cells (Fig. 4 H). However, RAG-deficient mice reconstituted with polyclonal WT CD4⁺ T cells showed a full restoration of GC levels, confirming that CD4 T lymphocytes recognizing parasite and/or self-Ags are necessary and sufficient to drive the GC response during acute T. gondii infection (Fig. 4 I).

GCs have multiple and sometimes opposing effects on T lymphocytes. They promote apoptosis but can also induce IL-7R expression and inhibit synthesis of some while enhancing production of other cytokines (Ashwell et al., 2000). In GR^lck-Cre mice, the GR is deleted at the stage of double-negative thymocytes. As a consequence of thymic selection in the absence of GC influence, GR^lck-Cre mice display blunted responses after immunization with soluble Ag (Mittelstadt et al., 2012). One would therefore predict that GR^lck-Cre animals would be unable to control T. gondii infection. However, GR^lck-Cre mice mount a highly effective Th1 immune response against the parasite and, indeed, are hyperresponsive. Thus, challenge with an antigenically complex intracellular pathogen, and the strong proinflammatory response that it induces, appears to completely reverse the immunocompromised state of GR^lck-Cre mice. The nonbiased overproduction in GR^lck-Cre mice of IFN-γ, TNF, and IL-10, three distinct cytokines secreted by Th1 cells, suggests that in T. gondii infection GCs function as a general damper of TCR signaling.

Based on these observations, we propose that the same phenomenon of decreased Ag threshold for TCR activation in the absence of GC signaling, which during thymocyte maturation drives the appearance of hyporeactive T cells, paradoxically, also underlies the hyperreactivity of Th1 cells in vivo during T. gondii infection. Although it is as yet unclear whether GR is a component of the TCR complex (Löwenberg et al., 2007), the blunted TCR response occurring in WT Th1 cells is consistent with the known inhibitory effects of GCs on AP-1, NF-κB, and NFAT (Ashwell et al., 2000). Superimposed on this mechanism may be additional suppressive effects of GCs on the STAT4 and/or T-bet signaling pathways (Franchimont et al., 2000; Liberman et al., 2007).

Although the induction of GCs has been previously observed in a variety of viral, bacterial, and parasitic infections, the signals responsible for triggering this response are poorly defined. Cytokines, and in particular IL-1, have been shown to be potent inducers of GC production, arguing for a strong association with the innate immune system (Sternberg, 2006). Nevertheless, the IL-1 pathway is not involved in the T. gondii system described here as infected MyD88^−/− mice display an uncompromised GC response. Instead, our depletion and adoptive transfer experiments clearly implicate CD4 T lymphocytes themselves as drivers of GC production. Although CD4⁺ T cell–derived cytokines are obvious candidate mediators, our current findings argue against the role of IFN-γ, TNF, and IL-10. Nevertheless, the involvement of other soluble T cell products has not been ruled out. Alternatively, as a stress response, GC production in T. gondii infection could be the indirect result of CD4⁺ T cell–mediated tissue damage (Jamieson et al., 2010).

The findings reported here establish GC signaling as a fourth major pathway (along with IL-10, IL-27, and lipoxin production) for dampening the Th1 response during T. gondii infection to protect the host against lethal immunopathology. Such a multitargeted regulatory mechanism may be necessary to adequately brake the Th1 pathway, which also incorporates a positive feedback-loop through IFN-γ production (Lugo-Villarino et al., 2003; Schulz et al., 2009). In turn, each of these circuits represents a potential target for exploitation by the parasite in enhancing its virulence and/or promoting its own survival (Butcher et al., 2005; Bradley and Sibley, 2007). In this regard, it is of interest that the virulence of avian influenza virus has been associated with induction of the endogenous GC response (Tian et al., 2012).