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The Journal of Immunology, 2006, 176: 7263-7271.
Copyright © 2006 by The American Association of Immunologists

Tyk2 Negatively Regulates Adaptive Th1 Immunity by Mediating IL-10 Signaling and Promoting IFN-{gamma}-Dependent IL-10 Reactivation1

Michael H. Shaw*, Gordon J. Freeman{dagger}, Mark F. Scott*, Barbara A. Fox{ddagger}, David J. Bzik{ddagger}, Yasmine Belkaid§ and George S. Yap2,*

* Department of Molecular Microbiology and Immunology, Brown University, Providence, RI 02906; {dagger} Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215; {ddagger} Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, NH 03756; and § Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 02892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The Jak, Tyk2, is activated in response to IL-12 and IFN-{alpha}beta and promotes IFN-{gamma} production by Th1-type CD4 cells. Mice deficient in Tyk2 function have been previously shown to be resistant to autoimmune arthritis and septic shock but are acutely susceptible to opportunistic pathogens such as Toxoplasma gondii. In this study, we show that Tyk2, in addition to mediating the biological effects of IL-12 and IFN-{alpha}beta, is an important regulator for the signaling and expression of the immunosuppressive cytokine IL-10. In the absence of Tyk2, Ag-reactive CD4 cells exhibit impaired IL-10 synthesis following rechallenge of T. gondii vaccine-primed mice. The impaired IL-10 reactivation leads to unopposed antimicrobial effector mechanisms which results in a paradoxically superior protection of immune Tyk2–/– mice against virulent T. gondii challenge. We further demonstrate that Tyk2 indirectly controls CD4 IL-10 reactivation by signaling for maximal IFN-{gamma} secretion. The unexpected role of IFN-{gamma} in mediating IL-10 reactivation by Th1 cells provides compelling evidence that conditions driving Th1 responses establish a negative feedback loop, which will ultimately lead to its autoregulation. Thus, Tyk2 can be viewed as a dual-function Jak, mediating both pro and anti-inflammatory cytokine responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The mammalian Jak family of nonreceptor tyrosine kinases consists of four members: Jak1, Jak2, Jak3, and Tyk2. Jak kinases are activated following interactions between cytokines and their cognate receptors on the cell surface. The activated Jak kinases, in turn, tyrosine phosphorylate latent cytoplasmic transcription factors known as Stats (1), which promote gene transcription. The critical role of the Jak-Stat signaling pathways in development is underscored by the phenotype of the mice genetically deficient in the various Jak kinases. Deletion of Jak1 and Jak2 gene results in perinatal and fetal lethality, respectively, whereas Jak3-deficient mice are viable but are lacking NK and B cells and have reduced numbers of T lymphocytes (2). Tyk2-deficient mice, unlike other Jak kinase mutants, are developmentally normal but have diminished signaling in response to IFN-{alpha}beta and IL-12 and exhibit partial susceptibility to certain viruses (3, 4). Further characterization of the Tyk2–/– mice revealed a critical role of Tyk2 in mediating not only LPS-induced endotoxin shock (5, 6), but also allergic airway hypersensitivity (7). In addition, our laboratory has identified a naturally occurring mutation in the Tyk2 gene of the B10.Q/J strain of mice, which are spontaneously susceptible to Toxoplasma gondii and yet highly refractory to collagen-induced arthritis (8, 9, 10). Thus, Tyk2 likely plays a regulatory role broadly impacting innate and adaptive phases of both Th1- and Th2-mediated immune responses.

Immunologically naive Tyk2-null mice are susceptible to T. gondii and Leishmania major infection, presumably because of impaired Th1 responses resulting from blunted IL-12-induced IFN-{gamma} production by NK and T lymphocytes (8, 9, 11). Th1 effector cytokines (IFN-{gamma} and TNF-{alpha}) provide critical protection against other intracellular microbial pathogens including Mycobacteria, Salmonella, and Listeria (12). Despite the beneficial effects of Th1-associated cytokines during infection, an unchecked proinflammatory cytokine response can cause collateral tissue damage. Therefore, the host must actively regulate the intensity and duration of the immune response to maximize pathogen clearance while minimizing nonspecific host injury. It is now evident that endogenous IL-10 plays a central role in striking a balance between host protection and pathology (13). The absence of IL-10 invariably results in enhanced pathogen clearance (14, 15, 16, 17), but can lead to shock-like lethality, in the case of T. gondii, (15) or to the loss of concomitant immunity against L. major (16). Thus, the paradigm emerging from these studies is that the dynamic balance between locally produced activating (TNF-{alpha}, IFN-{gamma}) and deactivating (IL-10) cytokines will dictate the outcome of an active infection. Nonetheless, one issue yet to be addressed is how this balance is initiated and established. Moreover, there is presently (18, 19, 20, 21) an increasing body of evidence from both mouse and human models demonstrating that both IFN-{gamma} and IL-10 originate from the same CD4 Th1-type effector cell. What determines the sequence of production and the intensity of these two antagonistic lymphokine responses during an adaptive Th1 response remains unknown.

In our studies of protective immunity in Tyk2-deficient animals immunized with attenuated T. gondii parasites, we observed that vaccine-primed Th1 cells responded to in vitro Ag restimulation by rapid synthesis of IFN-{gamma}, whereas IL-10 secretion required additional Tyk2-regulated signal(s) provided by in vivo re-exposure to pathogen at the effector site. This requirement is linked to the role of Tyk2 in IL-12 induction of IFN-{gamma}, but is unrelated to either the IL-10 or IFN-{alpha}beta signaling pathways. Importantly, an intact IL-12/IFN-{gamma} signaling cascade is essential only for the reactivation of IL-10 expression during secondary infection but appears dispensable for the initial programming of CD4 IL-10 competence upon acute vaccine exposure. We propose that the induction of IL-10 reactivation by IFN-{gamma} during an effector/memory Th1 response represents a classical negative feedback loop, which initializes and contributes to the establishment of a balance between activating and deactivating cytokines at the site of infection. This novel regulatory circuit may provide the host an adaptive mechanism for maximizing pathogen clearance while minimizing tissue collateral damage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and infection

C57BL/6, IFN-{gamma}R–/–, IL-10–/– and TCRbeta–/– mice were purchased from The Jackson Laboratory. Tyk2-, IL-12p35-, Stat4-, IFN-{alpha}betaR-, Stat1-, and T-bet-deficient mice were maintained and housed under specific pathogen-free conditions at Brown University. Sex- and age-matched mice were used in all experiments.

Mice were vaccinated with attenuated live carbamoyl phosphate synthase (CPS)3 tachyzoites (22). The vaccination consisted of a single i.p. dose of either 106 or 104 of the CPS strain of parasites that had been irradiated (15 krad) from a 137Cs source. One month after vaccination, mice were challenged with varying doses of the virulent RH strain of parasites as indicated. Tachyzoites of both the CPS and RH strains of T. gondii were harvested from infected monolayers of human foreskin fibroblasts.

In vivo Ab treatment

To evaluate the role of IFN-{gamma} and IL-10 in reactivation of IL-10 secretion, vaccinated mice were injected i.p. with 0.5 mg of anti-IFN-{gamma} (XMG-6 (23)), anti-IL-10R (1B1.3A (18)), or control mAb (GL113) at the time of parasite challenge. For evaluating the role of IL-10 during CPS priming, anti-IL-10R mAb was administered on days –1 and 3. To assess the role of ICOS ligand (ICOSL) and B7-2 interaction for IL-10 reactivation, anti-ICOS ligand Ab (0.5 mg/mouse) (16F.7E5 (24)) or anti-B7-2 Ab (0.5 mg/mouse) (GL-1; BioExpress) was administered twice at 6 h before and at the time of parasite challenge.

Cell cultures and cytokine assays

Peritoneal exudate cells (PECs) and splenocytes were harvested from immune or immune-challenged mice at the indicated times. Single-cell suspensions (PEC: 1 x 106/ml and spleen: 4 x 106/ml) were prepared from individual peritoneum and spleen from each mouse following secondary infection. Initially, RH parasites were use for reinfection, but CPS were used subsequently to minimize parasite replication. Cell suspensions were cultured in complete RPMI 1640 (Invitrogen Life Technologies) with live CPS tachyzoites (at 1/10 the concentration of the PECs and splenocytes) for either 24 or 72 h as indicated. Supernatants were removed for cytokine assays. IL-12p40, IFN-{gamma}, and IL-10 were detected using commercial ELISA sets from BD Biosciences. Where indicated, 20 µg/ml blocking anti-CD4 mAb (GK1.5, rat IgG2b) or anti-CD8 mAb (2.43, rat IgG2b) (25) was added to the cultures.

Purification and adoptive transfer of bone marrow and CD4+ cells

CD4+ cells were purified from donor spleens by using CD4-positive selection microbeads (Miltenyi Biotec). The purity (≥90%) of the isolated CD4 cells was determined by flow cytometry before transfer into TCRbeta-null recipients (2 x 106 cells/mouse). CD4-reconstituted mice were also vaccinated with (106) CPS at the same time as reconstitution. One month after vaccination, reconstituted mice were challenged with the RH strain of parasites. For peritoneal CD4+ cell isolation, pooled PECs from immune challenged WT mice were separated into either CD4-enriched or CD4-depleted fractions (purity was assessed by flow cytometry) and cultured in the absence of parasite Ag for 24 h.

Flow cytometry

For intracellular cytokine analysis, PECs and splenocytes from vaccinated animals were cultured in RPMI 1640 complete in the presence or absence of live CPS tachyzoites for 24 h, with the addition of brefeldin A (10 µg/ml) during the last 4 h. Fc receptors were blocked with excess anti-Fc (2.4G2) before surface staining with direct conjugated mAbs: FITC-conjugated anti-CD3 (clone 145-2C11), PE-conjugated anti-NK1.1 (clone PK136) and PerCP-conjugated anti-CD4 (clone H129.19) (BD Biosciences). Following surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm and Perm/Wash (BD Biosciences) according to the manufacturer’s instructions. For staining of intracellular cytokine, allophycocyanin-conjugated anti-IFN-{gamma} (clone XMG1.2; BD Biosciences) was added to permeabilized cells (30 min on ice) followed by washing once with PBS. For detection of ICOSL surface expression, PECs were surface stained with FITC-conjugated anti-B220 (clone RA3-6B2), PerCP-conjugated CD11b (clone M1/70) (BD Biosciences), PE-conjugated anti-ICOSL (clone HK5.3), PE-conjugated anti-ICOS (clone 7E.17G9), and allophycocyanin- conjugated anti-CD11c (clone N418) (eBioscience). Flow cytometry analysis was performed with a FACScan (BD Biosciences) on 100,000 cells. Data analysis was performed using CellQuest software.

In vitro assays for cytokine signaling/responsiveness

Cell extracts were prepared from immune PECs or thioglycolate-elicited macrophages. Cells were either lysed directly (immune PECs) or serum starved (thioglycolate macrophages) for 4 h followed by stimulation, with or without IL-10 (BD Biosciences), at the indicated concentrations for 15 min before lysis with radioimmunoprecipitation assay buffer. Total cell lysate (5–10 µg) was immunoblotted for phospho-Stat3, Stat3, phospho-Stat1, and Stat1 (Santa Cruz Biotechnology) or IFN-inducible GTPase (IGTP; BD Biosciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Vaccination overcomes innate susceptibility of Tyk2-null mice to T. gondii infection

We have previously demonstrated that mice with either a spontaneous or a targeted deletion of the Tyk2 gene are acutely susceptible to infection with T. gondii because of an impaired IL-12-induced IFN-{gamma} response (8, 9). Nevertheless, studies by us and other investigators indicate that Tyk2-deficient mice have the potential to develop an adaptive Th1 immune response (10, 11). To evaluate the protective function of this Tyk2-independent adaptive response, wild-type (WT) and Tyk2–/– mice were vaccinated i.p. with a high dose (106) of the radiation-attenuated auxotrophic strain (CPS) of T. gondii. One month following vaccination, mice were challenged i.p. with 200 virulent (RH) strain T. gondii. Vaccinated Tyk2–/– mice, similar to immunized WT controls, were completely resistant to challenge (Fig. 1A), indicative of a functionally protective type I immune response. Consistent with this conclusion, similar frequencies and numbers of IFN-{gamma}-producing CD4+ and CD8+ T lymphocytes were observed in WT and Tyk2–/– PECs (Fig. 1A). Thus, in vaccinated Tyk2-null hosts, the frequency of IFN-{gamma}-secreting effector cells found at the site of challenge appears sufficient for protection against lethal RH challenge.


Figure 1
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  		FIGURE 1. Vaccinated Tyk2-deficient mice develop functionally protective Th1 immunity. A (left panel), Survival of naive or 106 CPS-immunized WT and Tyk2–/– mice following i.p. challenge with 200 RH strain T. gondii (right panel): single-cell analysis of IFN-{gamma} secretion by CD4+ and CD8+ lymphocytes from PEC derived from 106 CPS-immunized WT and Tyk2–/– mice following 24 h in vitro restimulation with live CPS tachyzoites. B (left panel), Single-cell analysis of IFN-{gamma} secretion by CD4+ and CD8+ lymphocytes from PEC derived from 104 CPS-immunized WT and Tyk2–/– mice following 24 h in vitro restimulation with live CPS tachyzoites. The FACS dot plots shown are gated on TCRbeta+. Survival of naive, 104, or 106 CPS-vaccinated WT (middle panel) and Tyk2–/– (right panel) mice following i.p. challenge with 200 RH tachyzoites. All data shown are representative of at least three experiments with n = 3 mice per group and all mice were analyzed individually for flow cytometric analysis.

 
Low-dose vaccination reveals the development of a paradoxically superior protective immunity in Tyk2–/– mice

The high numbers (106) of parasites used for vaccination could be masking the Tyk2 effect by promoting TCR-driven IL-12-autonomous IFN-{gamma} production (26). To test this possibility, a lower dose (104) of CPS vaccine was used. Indeed, low-dose vaccination resulted in lower frequencies of peritoneal IFN-{gamma}-producing T cells in Tyk2–/– relative to WT mice (Fig. 1B, left panel). We next tested whether low-dose vaccination led to a loss of protective immunity in vaccinated Tyk2–/– mice. Contrary to our expectation, low-dose CPS-vaccinated Tyk2–/– animals remain completely protected (Fig. 1B, right panel). Paradoxically, low-dose vaccinated WT mice are now susceptible to lethal RH challenge (Fig. 1B, middle panel). The susceptibility of low-dose vaccinated WT mice is associated with uncontrolled systemic parasite growth (data not shown) and indicates that, in the case of WT mice, a vaccine dose of 104 is ineffective or suboptimal. Consistent with the greater protection observed in low-dose immunized Tyk2-deficient animals, challenge with 106 RH tachyzoites revealed a consistent delay in the time of death only in high-dose immunized Tyk2-mutant, but not WT mice (data not shown). Taken together, the data presented above indicate that, following vaccination, Tyk2-deficient hosts exhibit a higher level of protective immunity. This observation is surprising and paradoxical, given the obvious impairment in their IFN-{gamma} response in the peritoneal effector site. These data imply that Tyk2 signaling, independent of its role in promoting IFN-{gamma}-producing T cells, negatively regulates host resistance against secondary RH infection.

Tyk2 is critical for optimal IL-10-mediated signaling and suppressor function

The unexpected resistance phenotype of vaccinated Tyk2-deficient animals led us to hypothesize that the absence of Tyk2 relieves a suppressive pathway, allowing unopposed signaling by the low amounts of IFN-{gamma} produced locally. Among the cytokines that have been shown to activate Tyk2 (2), IL-10 is of particular interest because it can down-regulate IFN-{gamma} production by both CD4+ and NK cells and antagonize effector functions of IFN-{gamma}-activated macrophages (13). Although earlier studies of Tyk2-knockout mice indicated a nonessential role for Tyk2 in IL-10-induced Stat1 and Stat3 activation (3, 4), we decided to re-examine this question by careful comparison of the responses of WT and Tyk2–/– thioglycolate-elicited peritoneal macrophages to graded doses of IL-10. The tyrosine phosphorylation of both Stat1 and Stat3 proteins following IL-10 stimulation was attenuated by Tyk2 deficiency (Fig. 2A). For instance, ~100-fold higher concentration of IL-10 was required to achieve a similar intensity of Stat3 activation in Tyk2-null macrophages, suggesting an important role of Tyk2 in optimal IL-10 signaling.


Figure 2
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  		FIGURE 2. Tyk2–/– macrophages have impaired IL-10-mediated signaling and function. A, Thioglycolate-elicited macrophages from both WT and Tyk2–/– mice were serum starved for 4 h before a 15-min stimulation with graded concentrations of rIL-10. Cell extracts were used for direct immunoblotting of phospho-Stat1 and phospho-Stat3. Protein loading was controlled by reprobing with Abs specific for Stat1, Stat3, and p38. B–D, Thioglycolate-elicited macrophages from both WT and Tyk2–/– mice were preincubated with rIFN-{gamma}, as indicated, in the presence or absence of increasing concentrations of rIL-10. Nitrite accumulation in culture was measured colorimetrically by the Griess reaction. Percent inhibition was calculated by subtracting from 100% the ratio of NO made in the presence of IL-10 vs the absence of IL-10. All experiments were performed with pooled thioglycolate-elicited macrophages from a minimum of n = 3 age- and sex-matched mice per genotypic group. All data shown are representative of at least three experiments.

 
A caveat of the above experiments is the decreased expression of Stats in Tyk2-null macrophages (4, 6), prompting us to examine whether IL-10-mediated biological function was in fact impaired in Tyk2–/– cells. Because inhibition and killing of T. gondii has been shown to involve NO (27), IL-10 suppression of IFN-{gamma}-induced NO synthesis (28) in Tyk2-null macrophages was examined. Initial characterization indicated that NO production by Tyk2-null macrophages following IFN-{gamma} stimulation is similar to that of WT macrophages (Fig. 2B). Next, the ability of increasing doses of IL-10 to inhibit NO production by WT and Tyk2–/– macrophages was compared at three different concentrations of activating cytokine. At a low dose of IFN-{gamma} (0.8 ng/ml; Fig. 2C), IL-10 suppressed NO synthesis by WT and Tyk2-deficient cells with equal efficiency. As the concentration of IFN-{gamma} was increased, the effect of Tyk2 on IL-10-mediated suppression was revealed. At an intermediate dose of IFN-{gamma} (Fig. 2D), Tyk2-deficient macrophages remained IL-10 responsive but required higher doses of IL-10 to achieve the same levels of inhibition observed in WT cells. Strikingly, at high concentrations of IFN-{gamma}, the inhibitory affect of IL-10 on Tyk2–/– cells is completely abolished (Fig. 2E). Overall, the data presented above (Fig. 2) indicate clearly that Tyk2 is necessary for optimal IL-10-induced Stat activation. Furthermore, the extent to which Tyk2 deficiency impacts IL-10 immunosuppression appears to be conditioned by the strength of the opposing signal delivered by IFN-{gamma}. Thus, it is likely that pathogen clearance by peritoneal effector cells will be influenced by the timing and the relative concentrations of IFN-{gamma} and IL-10 produced locally.

Tyk2 regulates the "reactivation" of CD4-dependent IL-10 secretion during secondary infection

To measure the local production of IL-10 and IFN-{gamma}, an ex vivo culture system using PECs derived from vaccinated hosts following secondary infection was used. Cytokine concentrations were determined in the conditioned medium after in vitro restimulation of PECs from either RH-challenged or unchallenged immune mice. As expected, IFN-{gamma} secretion by vaccinated WT PECs was observed following in vitro CPS restimulation (Fig. 3A). Surprisingly, in vitro parasite restimulation failed to induce IL-10 production from immune WT PECs. IL-10 secretion by WT PECs, in contrast to IFN-{gamma} synthesis, was only evident following in vivo parasite challenge (Fig. 3A). This IL-10 production is not observed in PEC cultures of challenged naive mice (data not shown) and, therefore, represents an adaptive response induced by CPS vaccination. Thus, peritoneal effector cells are differentially poised for IFN-{gamma} and IL-10 secretion, the latter requiring a reactivation signal provided by in vivo pathogen re-exposure.


Figure 3
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  		FIGURE 3. CD4 IL-10 reactivation requires Tyk2-mediated signaling. A, PECs were harvested from day 1 RH-challenged (+) and unchallenged (–) immune WT mice. PECs were then cultured in vitro in the presence or the absence of live CPS tachyzoites for 24 h. The culture supernatants were assayed for the presence of IFN-{gamma} and IL-10 by ELISA. B and C, PECs from RH-challenged or unchallenged immune WT, Tyk2–/–, and IL-10–/– mice were isolated and restimulated in vitro with live CPS tachyzoites for 72 h. The cultured supernatants were then assayed for IFN-{gamma} (B) or IL-10 (C). D, PECs from immune WT and Tyk2–/– host were harvested at the indicated days after receiving i.p. injection of 106 CPS. PECs were cultured for 24 h in the presence of live CPS tachyzoites, and the cultured supernatants were then assayed for IL-10. E (left panel), PECs from immune WT mice were harvested day 1 following i.p. injection of 200 RH parasites. PECs were restimulated for 24 h with live CPS tachyzoites in the presence or absence of CD4 blocking mAb (right panel). Pooled PECs from challenged immune WT mice were magnetically separated into CD4-enriched (>90% purity) and CD4-depleted (<1% contaminating CD4 cells) fractions and cultured in the absence of Ag for 24 h. The culture supernatants were then assayed for IL-10 and IFN-{gamma} by ELISA. F, PECs were derived from individual immune WT and Tyk2–/– mice following in vivo infection with or without live 200 RH tachyzoites for 24 h. Whole-cell lysates were analyzed for IGTP induction by Western blot analysis. Protein loading was controlled by reprobing the blot with mouse beta-actin. All experiments were performed with a minimum of n = 3 age- and sex-matched mice per genotypic group. All data shown are representative of at least three experiments; with the exception of E (right panel), all mice were individually analyzed for cytokine production or protein expression.

 
We next compared the PEC production of IFN-{gamma} and IL-10 during secondary infection by vaccinated WT, Tyk2–/– as well as IL-10–/– hosts, the latter serving as a baseline for detecting an IL-10 recall response. As shown in Fig. 3B, immune Tyk2–/– PECs secreted 50% less IFN-{gamma} relative to WT levels, regardless of in vivo parasite rechallenge. Unexpectedly, secondary infection of vaccinated Tyk2–/– hosts failed to induce a detectable IL-10 response (Fig. 3C). The lack of IL-10 production by Tyk2–/– PECs at all time points examined following reinfection implies a general impairment, rather than simply a delay in local IL-10 secretion (Fig. 3D). Thus, in addition to parasite re-exposure in vivo, IL-10 reactivation in peritoneal effector cells requires Tyk2-dependent signaling. The defect in IL-10 reactivation observed in Tyk2-–/– hosts cannot be readily explained by a deficit in cell recruitment, because total peritoneal cell counts and CD4 T cell numbers were not different following challenge of Tyk2-sufficient and Tyk2-deficient hosts (data not shown). Furthermore, the IL-10 recall response was not abrogated by pertussis toxin treatment (data not shown).

Tyk2 may regulate the reactivation of IL-10 secretion by either T cells or non-T cells, such as macrophages and/or dendritic cells (DCs), following infection. Based on a previous report using Toxoplasma as model Ag, it was demonstrated that IL-10-producing cells are predominantly IFN-{gamma}+CD4+ cells (19). We attempted to stain intracellular IL-10 synthesis by peritoneal CD4 lymphocytes directly ex vivo following challenge, but due to poor detection sensitivity, we were unable to ascertain, at the single-cell level, the exact source of IL-10 during reinfection. To further assess the contribution of CD4+ cells to the peritoneal IL-10 production in our vaccination and challenge model, PECs were cultured in the presence of either CD4 or CD8 blocking mAbs. The presence of anti-CD4 mAb, but not CD8 mAb (data not shown), significantly reduced IL-10 and IFN-{gamma} secretion by WT PECs (Fig. 3E, left panel). Thus, during rechallenge of immunized mice, the reactivation of IL-10 expression probably occurs within Toxoplasma-responsive Th1 CD4+ lymphocytes previously described (19). To formally demonstrate a direct role of vaccine-primed CD4 cells in IL-10 secretion, peritoneal cells from immune-challenged WT hosts were separated into CD4-enriched (>90% CD4+ TCRbeta+) and CD4-depleted fractions and their IL-10 secretion was assayed. As Fig. 3E (right panel) shows, IL-10 production was observed only in cultures containing the CD4-enriched fraction, indicating that CD4 cells are the major source of IL-10 during parasite challenge.

The unexpected absence of IL-10 in the peritoneum of vaccinated Tyk2–/– mice further favors our hypothesis that enhanced immunity is related to unopposed activation of effector cells by the low amounts of IFN-{gamma} produced locally. To address whether the reduced IFN-{gamma} production by Tyk2–/– PECs (Fig. 3B) is capable of promoting IFN-{gamma}-dependent antimicrobial mechanism(s), the expression of IGTP was examined (29). As shown in Fig. 3F, similar levels of IGTP induction was observed between Tyk2–/– and WT PECs following in vivo challenge, confirming the unopposed induction of a critical IFN-{gamma}-regulated microbicidal mechanism in Tyk2-deficient cells. Overall, the results suggest that the enhanced protective immunity observed in vaccinated Tyk2-null hosts can be explained by a more productive activation of effector cells due to impaired IL-10 secretion and function.

IL-12-induced IFN-{gamma} is required for the reactivation of peritoneal IL-10 secretion during secondary challenge

The impaired reactivation of CD4 IL-10 secretion following secondary infection of Tyk2-null animals may be associated with either impaired IL-12 or IFN-{alpha}beta signaling (3, 4, 8). To distinguish between these two cytokines, peritoneal IL-10 levels from immune and immune-challenged IL-12p35- and IFN-{alpha}betaR-null mice were determined. As Fig. 4A demonstrates, PECs lacking IFN-{alpha}beta signaling exhibited normal IL-10 secretion following in vivo parasite challenge, whereas PECs from immune-challenged IL-12p35–/– mice exhibited depressed levels of peritoneal IL-10 secretion similar to that of Tyk2-null cells (Fig. 4B), indicating a stringent requirement for IL-12 in mediating the reactivation of CD4 IL-10 secretion at the local effector site.


Figure 4
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  		FIGURE 4. IL-12-induced IFN-{gamma} is required for IL-10 reactivation. A and B, CPS-immunized Tyk2–/–, IFN-{alpha}betaR–/–, and 129SvEv control mice (A) or CPS-immunized Tyk2–/–, IL-12p35–/–, IL-10–/–, and WT mice (B) were i.p. challenged with 200 RH parasites. PECs were harvested at the indicated days following parasite challenge and cultured in vitro with live CPS tachyzoites for 24 h. The culture supernatants were then assayed for IL-10. C and D, PECs were harvested from CPS-immunized WT, IL-12p35 ––, Tyk2–/–, and Stat4–/– mice (C) or CPS-immunized WT, IFN-{gamma}R–/–, Stat1–/–, and T-bet–/– mice (D) that were either unchallenged or challenged with 200 RH parasites. PECs were harvested 1 day following parasite challenge and cultured in vitro with live CPS tachyzoites for 24 h. The cultured supernatants were then assayed for IL-10. All experiments were performed with a minimum of n = 3 age- and sex-matched mice per genotypic group. All data shown are representative of at least three experiments, with all mice analyzed individually for cytokine production.

 
IL-12 might promote the reactivation of CD4 IL-10 production directly or indirectly via the induction of IFN-{gamma}. To differentiate between the direct effects of IL-12 vs IFN-{gamma}, mice genetically deficient in either IL-12 or IFN-{gamma} signaling components were used in our vaccination/challenge model. Following Ag challenge, IL-12p35–/–, Tyk2–/–, and Stat4–/– PECs exhibited severe impairment in their IL-10 production (Fig. 4C). Nevertheless, similarly striking reductions were observed in IFN-{gamma}R-, Stat1- and T-bet-deficient PECs (Fig. 4D). Thus, local production of IFN-{gamma} provides an essential signal for CD4 IL-10 reactivation during an in vivo recall response.

T cell-derived IFN-{gamma} acts on accessory cells to promote IL-10 reactivation in preexisting resident effector CD4 cells

To confirm the role of IFN-{gamma}, immune WT mice were treated with anti-IFN-{gamma} mAb during challenge. As shown in Fig. 5A, in vivo neutralization of IFN-{gamma}, but not IL-10, during secondary infection significantly reduced the amount of IL-10 secreted by the PECs as compared with control-treated PECs. To better characterize how Tyk2-dependent IFN-{gamma} is reactivating IL-10 production by CD4+ cells, we sought to elucidate the source and the cellular target(s) of IFN-{gamma} during secondary infection. To identify the cellular source of IFN-{gamma}, TCRbeta-null mice were reconstituted with purified CD4+ cells derived from either WT or Tyk2-deficient mice. Thirty days after CPS vaccination of T cell reconstituted mice, IL-10 reactivation was assayed after RH challenge. As shown in Fig. 5B, vaccinated TCRbeta-null mice reconstituted with Tyk2–/– CD4+ cells, in contrast to recipients of WT CD4 cells, displayed an impaired IL-10 recall response. The blunted IL-10 reactivation in Tyk2–/– CD4-reconstituted mice is associated with impaired IFN-{gamma} secretion (data not shown), suggesting that CD4 T cells are the critical source of IFN-{gamma} necessary to reactivate IL-10 secretion. To address the potential role of IFN-{gamma} derived from non-CD4 cells in IL-10 reactivation, mice doubly deficient in TCRbeta and Tyk2 were also used as recipients. Selective Tyk2 deficiency in the non-CD4 compartment does not result in diminished IL-10 production (Fig. 5B).


Figure 5
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  		FIGURE 5. T cell-derived IFN-{gamma} acts on accessory cells to promote ICOSL-mediated IL-10 reactivation. A, CPS-immunized WT mice were treated i.p. with either anti-IFN-{gamma} or anti-IL-10R mAb at the time of 106 CPS rechallenge. Day 1 after reinfection, PECs were harvested and cultured for 24 h in the presence of live CPS tachyzoites and subsequently assayed for IL-10. B, Purified WT or Tyk2-deficient CD4 cells were adoptively transferred into either TCRbeta–/– or TCRbeta–/–Tyk2–/– (DKO) recipient mice, which were then vaccinated with 106 CPS on the day of CD4 cell transfer. One month following reconstitution/vaccination, the mice were challenged i.p. with 106 CPS. PECs were harvested 1 day after challenge and cultured for 24 h in the presence of live CPS tachyzoites. The culture supernatants were assayed for IL-10. C, WT bone marrow (B.M.) cells were adoptively transferred into irradiated WT and IFN-{gamma}R–/– recipient mice, which were then vaccinated with 106 CPS at 8 wk after transfer. One month after vaccination, mice were challenged i.p. with 106 CPS. PECs were harvested 1 day after challenge and cultured for 24 h in the presence live CPS tachyzoites. The culture supernatants were assayed for IL-10. D, Purified WT and IFN-{gamma}R-deficient CD4 cells were adoptively transferred into TCRbeta-null recipient mice, which were then vaccinated with 106 CPS on the day of CD4 cell transfer. One month following reconstitution/vaccination, mice were challenged i.p. with 106 CPS. PECs were harvested 1 day after challenge and cultured for 24 h in the presence of live CPS tachyzoites. The culture supernatants were assayed for IL-10. E, Immune WT host were injected twice i.p. with either anti-ICOSL mAb or isotype control Ab. PECs were harvested 24 h after challenge and cultured in vitro with live CPS tachyzoites for 24 h. The culture supernatants were then assayed for IL-10 and IFN-{gamma}. All experiments were performed with a minimum of n = 3 mice per group. All data shown are representative of at least three experiments, except for C, which was performed twice, with all mice analyzed individually for cytokine production.

 
CD4-derived IFN-{gamma} probably acts on either APCs or T cells themselves to reactivate IL-10 production. Consistent with these possibilities, transfer of WT bone marrow into lethally irradiated IFN-{gamma}R-deficient recipient mice was sufficient to rescue CD4 IL-10 reactivation (Fig. 5C). To delineate whether Th1-type CD4 cells are themselves responding to IFN-{gamma} for IL-10 reactivation, purified CD4+ T cells derived from either WT or IFN-{gamma}R–/– mice were adoptively transferred into TCRbeta-deficient animals. Following secondary infection, IFN-{gamma}R–/– CD4 cell-reconstituted animals displayed normal PEC IL-10 production (Fig. 5D), indicating that IL-10 production by CD4 cells does not require IFN-{gamma} responsiveness by CD4 T cells themselves. Rather, T cell-derived IFN-{gamma} is acting in a paracrine fashion on accessory cells which, in turn, signal local CD4+ cells to reactivate expression of IL-10.

IL-10 reactivation by IFN-{gamma} is dependent on ICOSL costimulation

We next hypothesized that IL-10 reactivation by IFN-{gamma} may be mediated indirectly by inducing the expression of costimulatory molecule(s), on APCs, which promote T cell IL-10 production. Among the B7 family of costimulatory molecules, ICOSL costimulation has been demonstrated to be required for IL-10 expression by activated T cells (30, 31). To explore the role, if any, of ICOS costimulation in the reactivation of CD4 IL-10 expression, WT mice were pretreated with either anti-ICOSL or control mAb. As shown in Fig. 5E, in vivo neutralization of ICOSL resulted in a major (>50%) reduction in peritoneal secretion of IL-10, demonstrating that ICOSL plays a critical costimulatory function in IL-10 reactivation. Thus, IFN-{gamma} may indirectly induce CD4 IL-10 reactivation by promoting accessory cell expression of costimulatory molecules such as ICOSL.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have previously shown that the impaired development of type I immune response exhibited by Tyk2-null mice renders them highly susceptible to Toxoplasma infection (8, 9). However, in the present study, we demonstrated that vaccinated Tyk2–/– mice, similar to WT animals, are capable of mounting a protective type I immune response against lethal RH challenge. Interestingly, our data now indicate that Tyk2-null mice, following vaccination, express heightened levels of protection against lethal parasite challenge. The superior immunity exhibited by vaccinated Tyk2–/– mice seems to be related to a higher "net" effector function mediated by a partially defective type I cytokine response not subject to IL-10-mediated suppression. In addition to documenting the role of Tyk2 in IL-10 signaling and function, the most surprising and significant discovery of our study is that, during secondary exposure to the pathogen, a proinflammatory cytokine cascade involving IL-12 induction of IFN-{gamma} provides an obligate signal for the re-expression of the anti-inflammatory cytokine IL-10 at the immune effector site. Furthermore, we demonstrate that the reactivation of IL-10 expression is critical for tonic regulation of adaptive type I immunity and is ultimately essential for the survival of the immunized host.

The initial characterization of the Tyk2-deficient mice (3, 4) identified an essential role of Tyk2 that is restricted to the IL-12 and IFN-{alpha}beta signaling pathways. The results presented here suggest that the functional role of Tyk2 can now be further extended to the IL-10 signaling cascade. In contrast to previous reports where apparently normal Stat1 and Stat3 tyrosine phosphorylation was detected in high-dose (~100 ng/ml) IL-10-stimulated Tyk2-null cells, we detected a general defect in IL-10-induced Stat3 and Stat1 activation in Tyk2-deficient macrophages. The discrepancy between our data and the earlier studies may stem from the different cell types used to assay IL-10 signaling. In support of our data showing defective IL-10 signaling in Tyk2-null macrophages, IL-10-mediated suppression of IFN-{gamma}-induced NO production is also affected by Tyk2 deficiency. Together with the finding that vaccinated mutant mice do develop an adaptive Th1 response and exhibit superior protective immunity, the IL-10 signaling defect in Tyk2–/– macrophages argues that a genetic or functional impairment in Tyk2, whether identified in human subjects, may lead to a more subtle or benign form of immune deficiency, as compared with the susceptibility phenotype associated with IFN-{gamma}R or IL-12R mutations (12). A further implication is that it may be difficult to predict the net effect of pharmacologic inhibition of Tyk2 on autoimmune or inflammatory diseases, given its role in cellular responses to proinflammatory (including IL-12, IL-23) as well as anti-inflammatory cytokines (IL-10 and type I IFNs). Consistent with an anti-inflammatory role of Tyk2 is the observation that contact hypersensitivity responses are paradoxically enhanced in Tyk2-deficient mice despite decreased levels IL-12 and IFN-{gamma} at the dermal reaction site (32).

During infections with intracellular pathogens, including Mycobacteria, Toxoplasma, Leishmania, and certain viruses, IFN-{gamma} derived from activated Th1 cells is critical for host resistance by mediating the activation of effector cells and promoting inflammatory responses. Nevertheless, overproduction of the potent antimicrobial Th1 cytokines IFN-{gamma} and TNF-{alpha} can lead to collateral tissue damage. Endogenous mechanisms involving suppressive cytokines such as IL-10 and TGF-beta, however, can prevent host immunopathology by curtailing proinflammatory cytokine production and action. Interestingly, both activating (IFN-{gamma}) and deactivating (IL-10) cytokines have been shown to originate from the same pathogen-reactive CD4 T lymphocyte (33). The potential for antagonism between IFN-{gamma} and IL-10 raises the question of whether the two cytokines are secreted simultaneously or sequentially following antigenic stimulation. We now demonstrate that immune effector lymphocytes are poised for rapid IFN-{gamma} secretion, whereas reactivation of IL-10 synthesis by peritoneal CD4+ T cells requires an additional signal(s) delivered by the reintroduction of pathogen to the effector site in vivo. Our observation suggests that during secondary infection IFN-{gamma} production likely precedes IL-10 synthesis, allowing efficient activation of effector cells before they become targets for IL-10-mediated regulation. This differential regulation of pro- and anti-inflammatory lymphokine secretion may be a general feature of IFN-{gamma}+IL-10+CD4+ effector cells responding to various intracellular pathogens (18, 19, 20).

The most surprising finding of the present study is that the immunoregulatory interactions between IFN-{gamma} and IL-10 activities extend beyond their well-documented stereotypically reciprocal antagonism (13). We now demonstrate, for the first time, that in vivo IFN-{gamma} production by vaccinated animals provides an inductive signal for the reactivation of effector site, but not splenic CD4 IL-10 secretion. The effector site-specific requirement for IFN-{gamma} to reactivate IL-10 production is conceptually appealing, suggesting that IFN-{gamma} is functioning not only as an effector cytokine facilitating parasite clearance but also as a mediator for the induction of the suppressive cytokine IL-10 from primed lymphocytes.

It is interesting to note that the requirement for an intact IL-12/IFN-{gamma} signaling cascade for IL-10 production by CD4 cell is only evident during the secondary reactivation response, but not during vaccine priming (M. Shaw, unpublished results). This observation argues that the dependence of IL-10 reactivation on IL-12/IFN-{gamma} signaling does not reflect a general programming function for IL-10 competence (as previously shown for human Th1 lines) (34), but rather represents an inductive event leading to the re-expression of the cytokine locus in presumably IL-10-committed effector Th1 cells. Consistent with this interpretation, in vivo neutralization of IFN-{gamma} activity only during the rechallenge phase was sufficient to compromise IL-10 production. However, once IL-10 has been reactivated in vivo, further stimulation by IFN-{gamma} produced ex vivo appears unnecessary for IL-10 secretion (M. Shaw, unpublished results). Although the picture of how IL-10 reactivation occurs is only beginning to emerge (see discussion below), even far less is known about what commitment signals program IL-10 competence in developing Th1 cells. Nonetheless, the concomitant increase in IFN-{gamma} and IL-10 observed following IL-10 neutralization (data not shown) indicates that IL-10 signaling per se is not required for the differentiation of IL-10-producing Th1 cells and further supports the notion that IL-10 originates from pathogen-reactive Th1 cells (19, 33), whose development is antagonized or inhibited by IL-10.

In Fig. 6, we depict a model for how IFN-{gamma} promotes CD4 IL-10 reactivation in situ. Upon Ag re-exposure, IL-12 promotes IFN-{gamma} secretion from parasite-reactive CD4+ lymphocytes present at the effector site. T cell-derived IFN-{gamma} (Fig. 5B) then acts in a paracrine fashion on non-T cells (Fig. 5, C and D), which in turn reactivates IL-10 expression by local Th1 CD4+ cells via the ICOS and related costimulatory pathways. In our T. gondii vaccination and rechallenge experiments, B cells appear to be the principal APC subset targeted by IFN-{gamma}. During secondary challenge, ICOSL expression on B cells, but not on DCs and macrophages, was severely reduced in the absence of IFN-{gamma} signaling (M. Shaw, unpublished data). Regardless of which ICOSL-expressing APC subset acts as the key mediator of IFN-{gamma}-induced IL-10 reactivation, the negative feedback immune regulatory circuit described here provides a general mechanism for down-regulating adaptive Th1 responses directed toward microbial as well as tissue Ags. Recently, a crucial, but unexplained, requirement for IFN-{gamma} production by adaptive regulatory T (Treg) cells in mediating allograft accommodation in a well-established model of IL-10-dependent transplantation tolerance has been reported (35). Our model predicts that impairment of adaptive Treg IFN-{gamma} production will disrupt APC expression of costimulatory molecules that promote Treg IL-10 expression, resulting in unopposed IFN-{gamma} secretion and rejection of the allograft.


Figure 6
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  		FIGURE 6. Proposed model for the role of Tyk2 in IL-12-induced IFN-{gamma}-dependent CD4 IL-10 reactivation. Schematic representation of the proposed negative feedback circuit regulating local Th1 immunity (the sites of Tyk2 action are highlighted using Roman numerals I and II): Upon Ag re-exposure, IL-12 via Tyk2 signaling induces high level IFN-{gamma} production by effector Th1 cells, which act on peritoneal accessory cells and promote ICOSL expression on this subset of APCs. ICOS-ICOSL interaction costimulates parasite-reactive Th1 cells for IL-10 secretion. The resulting IL-10 then down-modulates the ongoing adaptive Th1 response by dampening IL-12 secretion and antagonizing IFN-{gamma} signaling as denoted by the dashed lines. Also depicted on the far right end is the role of Tyk2 in mediating IL-10R signaling and IL-10 inhibition of IFN-{gamma}-induced antimicrobial responses in effector cells. We propose that, in the absence of Tyk2 function, a low level of IFN-{gamma} is produced sufficient to activate macrophage antiparasitic mechanisms because of (I) impaired IL-10 reactivation and (II) inefficient IL-10-mediated signaling and suppression.

 
Together with previous reports of innate susceptibility of Tyk2-deficient hosts to infection, our current results showing the paradoxical enhancement of protective immunity following vaccination, in the same Tyk2-null background, illustrate that the immunoregulatory function of Tyk2 Jak is "antagonistically pleiotropic." Before the development of an adaptive Th1 response, the function of Tyk2 in IL-12 signaling is the principal determinant of host survival. In contrast, within the context of pre-established Th1 immunity, the roles of Tyk2 in promoting IL-10 signaling and reactivation emerge as the dominant factor. An apparent incongruity in the phenotype of vaccinated Tyk2–/– mice is the presence of an intact IFN-{gamma}-induced antimicrobial effector activity while IFN-{gamma}-dependent IL-10 reactivation appears to be abrogated. This contradiction can be resolved by postulating a higher threshold requirement of the latter immune regulatory function for IFN-{gamma}. A hierarchical regulation of IFN-{gamma} effector vs immunomodulatory functions could provide the host a triggering mechanism to reactivate IL-10 once a pathological level of IFN-{gamma} is reached.


   Acknowledgments
 
We thank S. Hieny, A. Sher, and M. Kaplan for Ab reagents and mice, and M. Tessmer for cell separation. We are grateful to D. Jankovic, P. Gil, C. Biron, and D. Wilson for their critical review of the manuscript, and to U. VonAndrian and L. Ivashkiv for advice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported National Institutes of Health Grant AI 50618 (to G.S.Y.) and Grants CA84500 and AI39671 (to G. F.). Back

2 Address correspondence and reprint requests to Dr. George S. Yap, Department of Molecular Microbiology and Immunology, Division of Biology and Medicine, Box GB-6, Brown University, Providence, RI 02912. E-mail address: George_Yap{at}Brown.edu Back

3 Abbreviations used in this paper: CPS, carbamoyl phosphate synthase, PEC, peritoneal exudates cells, ICOSL, ICOS ligand; WT, wild type; IGTP, IFN-inducible GTPase; Treg, regulatory. Back

Received for publication January 17, 2006. Accepted for publication March 28, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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