IL-6 Mediates the Susceptibility of Hypermorphs to Toxoplasma gondii

This information is current as Jonathan S. Silver, Jason S. Stumhofer, Sara Passos, of September 29, 2021. Matthias Ernst and Christopher A. Hunter J Immunol 2011; 187:350-360; Prepublished online 23 May 2011; doi: 10.4049/jimmunol.1004144

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-6 Mediates the Susceptibility of Glycoprotein 130 Hypermorphs to Toxoplasma gondii

Jonathan S. Silver,* Jason S. Stumhofer,* Sara Passos,*,1 Matthias Ernst,† and Christopher A. Hunter*

IL-6 and IL-27 are closely related that play critical but distinct roles during infection with Toxoplasma gondii. Thus, IL-6 is required for the development of protective immunity to this pathogen, whereas IL-27 is required to limit infection-induced pathology. Paradoxically, these factors both signal through gp130, but little is known about how the signals downstream of gp130 are integrated to coordinate the immune response to infection. To better understand these events, gp130 Y757F mice that have a mutation in gp130 at the binding site for suppressor of signaling 3, a critical negative regulator of gp130 signaling, were infected with T. gondii. These mutant mice were acutely susceptible to this challenge, characterized by an early defect in the production of IL-12 and IFN-g and increased parasite burdens. Consistent with the reduced IL-12 levels, IL-6, but not other Downloaded from gp130 cytokines, was a potent antagonist of IL-12 production by gp130 Y757F and dendritic cells in vitro. Moreover, in gp130 Y757F mice, blocking IL-6 in vivo, or administration of rIL-12, during infection restored IFN-g production and pro- tective immunity. Collectively, these studies highlight that a failure to abbreviate IL-6–mediated gp130 signaling results in a pro- found anti-inflammatory signal that blocks the generation of protective immunity to T. gondii. The Journal of Immunology, 2011, 187: 350–360. http://www.jimmunol.org/ he transmembrane gp130 is a signal-transducing prominent regulators of STAT signaling. These are in- component of the receptors used by several closely re- duced in response to several cytokines that use the STAT-signaling T lated cytokines that include IL-6 and IL-27, as well as IL- pathway, and among the SOCS family members, SOCS3 plays 11, , LIF, cardiotrophin-like cytokine, cytokine-like a critical role in specifically limiting gp130-mediated signaling. factor 1, and ciliary neurotrophic factor (1). Binding of IL-6/ This inhibitory effect has been associated with the ability of gp130 family members to their specific receptor a-chains allows SOCS3 to bind to gp130 at the residue in position 757 as for high-affinity interactions with gp130 and activation of JAKs well as its ability to act as a ubiquitin ligase, targeting the asso- (JAK1, JAK2, and Tyk2), which ultimately results in recruitment ciated receptor complex for degradation (3–7). of STAT1 and STAT3. Following , the STATs The significance of SOCS3 in limiting gp130 signaling is il- by guest on September 29, 2021 dimerize and translocate to the nucleus where they can act as lustrated by the work of two groups that generated gp130 mutant transcription factors. There are several mechanisms in place that mice: gp130 Y757F mice have a mutation in the binding site of limit gp130-mediated Jak/STAT signaling; these include dissoci- SOCS3, whereas gp130 Y759F mice express the human gp130 ation of JAKs from the receptor, endocytosis of the receptor receptor with a similar mutation (8, 9). In both cases, activation complex, and nuclear export and dephosphorylation of activated of STAT3 is sustained in response to stimulation with gp130- STAT molecules (2). However, members of the suppressor of signaling cytokines, which leads to aberrant pathology. For ex- cytokine signaling (SOCS) family are regarded as the most ample, the gp130 Y757F mice develop gastric adenoma and he- matopoietic defects, driven by IL-11 and IL-6, respectively (10, 10–13). In contrast, gp130 Y759F mice develop an autoimmune *Department of Pathobiology, School of Veterinary Medicine, University of Penn- arthritis driven by STAT3-mediated overproduction of IL-7 (14, sylvania, Philadelphia, PA 19104; and †Ludwig Institute for Cancer Research, Mel- bourne, Victoria, Australia 3052 15). Consistent with the phenotype of the gp130 mutant mice, 1Current address: Servic¸o de Imunologia Hospital Universita´rio, Salvador, Bahia, multiple studies have highlighted the role of this receptor and Brazil. its corresponding cytokines in mediating the inflammation asso- Received for publication December 23, 2010. Accepted for publication April 16, ciated with cancer and autoimmunity (16–18). However, despite 2011. the body of work highlighting the importance of regulated This work was supported by Ludwig Institute for Cancer Research, the state of gp130-mediated signaling in inflammation associated with cancer Pennsylvania, National Institutes of Health Grant AI42334, and National Institutes and autoimmunity, very little is understood about how control of Health Grant AI 084882. J.S. Silver is supported by National Institutes of Health T32 Training Grant AI007532, the state of Pennsylvania, and the Penn Center for of gp130-mediated signaling impacts infection-induced inflam- Molecular Studies in Digestive and Diseases. mation. Address correspondence and reprint requests to Dr. Christopher A. Hunter, Depart- Toxoplasma gondii is an obligate intracellular parasite that is an ment of Pathobiology, School of Veterinary Medicine, Hill Pavilion, Room 313, 380 important opportunistic infection in immunocompromised indi- South University Avenue, University of Pennsylvania, Philadelphia, PA 19104-6008. E-mail address: [email protected] viduals, especially those with defects in function (19, 20). The online version of this article contains supplemental material. Challenge with this organism induces a highly polarized Th1 re- Abbreviations used in this article: BFA, brefeldin A; DC, ; KO, knock- sponse characterized by production of IL-12 by macrophages, out; MHC II, MHC class II; PEC, peritoneal exudate cell; SHP2, Src homology neutrophils, and dendritic cells (DCs), which leads to IFN-g region 2 domain-containing phosphatase 2; SOCS, suppressor of cytokine signaling; production by NK cells as well as CD4+ and CD8+ T cells (21, STAg, soluble toxoplasma Ag; WT, wild-type. 22). IFN-g is the major mediator of resistance to T. gondii and Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 induces a variety of antimicrobial mechanisms that allow the host www.jimmunol.org/cgi/doi/10.4049/jimmunol.1004144 The Journal of Immunology 351 to control this infection (23). Relevant to this work, the cytokines Preparation of bone marrow-derived macrophages and IL-6 and IL-27, which both signal through gp130, have important isolation of primary DC roles during experimental toxoplasmosis. Thus, mice deficient in Bone marrow from naive WT and gp130 Y757F littermates was harvested, IL-6 develop high cyst burdens and succumb to a severe en- and single-cell suspensions were cultured at a density of 4 3 105 cells/petri cephalitis associated with a failure to control parasite replication dish for 7 d. DMEM was supplemented with HEPES, penicillin, strepto- (24, 25). In addition, mice that lack gp130 specifically in astro- mycin, sodium pyruvate, and cell-free supernatant from cultures of L929 cytes are more susceptible to infection because of uncontrolled fibroblasts. Media and L929 supernatant were replenished at day 4. For isolation of primary DC, and lymph nodes were pooled from parasite replication, resulting in pathology in the brain (26). Thus, two to three 6- to 8-wk-old naive WT and gp130 Y757F mice. Single-cell IL-6 and gp130 are essential for the development of a protective suspensions were lysed in 0.86% NH4Cl and run on Lympholyte gradients immune response that allows the host to control parasite replica- to enrich for live cells. Remaining cell suspensions were then used to + tion. In contrast, mice that lack WSX-1, the a-chain of the purify CD11c DCs using a MACS column (Milltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. A total of IL-27R, control this parasite effectively but develop lethal T cell- 7.5 3 104 purified DCs were cultured per well in 200 ml complete RPMI mediated immunopathology, indicating that IL-27 plays a critical 1640 medium on a 96-well plate and activated with LPS (100 ng/ml), role in limiting infection-induced inflammation and pathology (27, IFN-g (100 U/ml), IL-6 (50 ng/ml), or IL-27 (100 ng/ml; Amgen, Thou- 28). Taken together, these studies highlight the diverse signals sand Oaks, CA) for 24 h before harvesting supernatants for ELISA. through gp130 that are required to coordinate a balanced immune For intracellular cytokine staining, macrophages or DC were stimulated in the presence of media or with LPS (100 ng/ml; Sigma-Aldrich, St. Louis, response that allows the host to control T. gondii while limiting the MO), IFN-g (100 U/ml; R&D Systems, Minneapolis, MN), IL-6 (50 ng/ml; development of immune pathology. These findings are also rele- eBioscience, San Diego, CA), and brefeldin A (BFA) for 8–12 h before vant to other infectious models, where IL-6 and IL-27 contribute being fixed with 4% paraformaldehyde, permeabilized with 0.5% saponin Downloaded from to the control of pathogen replication or limit pathology, re- in flow cytometry buffer, and stained intracellularly for IL-12p40 (eBio- science). spectively (29). However, little is known about the factors that coordinate the balance between the pro- and anti-inflammatory T cell transfers signals of gp130 during infection. For T cell transfers into RAG2/2 mice, spleens and lymph nodes were To better understand how gp130-mediated signaling coor- pooled from two to three naive WT or gp130 Y757F mice. Single-cell dinates the immune response against a pathogen, gp130 Y757F suspensions were lysed with 0.86% NH4Cl and run on a Lympholyte http://www.jimmunol.org/ mice were infected with T. gondii. Unlike wild-type (WT) lit- (Cedarlane Laboratories, Burlington, ON, Canada) gradient to enrich for termate controls, mutant mice succumbed to this challenge be- live cells. The remaining cells were then run over a T cell purification column (R&D Systems) according to the manufacturer’s protocol to purify cause of an inability to control parasite replication. Susceptibility CD3+ T cells. Eight to 10 million total CD4+ and CD8+ T cells were was associated with defective production of IL-12 and IFN-g by transferred into RAG knockout (KO) mice 3 d before being infected i.p. DC and NK cells, respectively, which correlated with the ability with T. gondii. Mice were sacrificed at day 8 postinfection for analysis of of IL-6 to block the production of IL-12 by gp130 Y757F DC parasite burden and cytokine production. and macrophages. Accordingly, administration of IL-6 blocking Intracellular staining for phosphorylated STAT1 and STAT3

Abs or rIL-12p70 during infection restored the production of + +

CD4 T cells were purified from WT or gp130 Y757F mice with a CD4 by guest on September 29, 2021 IFN-g and decreased parasite burdens in gp130 Y757F mice. isolation (Milltenyi Biotec). Purified CD4+ T cells (1 3 106) were Collectively, these studies highlight that a failure to abbreviate incubated with IL-6 for 5, 30, 60, or 180 min. Cells were then fixed for 10 IL-6–mediated gp130 signaling results in a profound anti- min at 37˚C with 2% paraformaldehyde. After being fixed, cells were made inflammatory signal that blocks the generation of protective im- permeable for 30 min on ice with 90% methanol and then were stained for munity to T. gondii. CD4 and phosphorylated STAT1 and STAT3. In vitro differentiation of IL-10–producing CD4+ T cells was done as described previously (31). Materials and Methods Flow cytometry Abs Mice and parasites FITC DX5 (Dx5), Alexa Fluor 488 phospho-STAT3 (pS727, clone 49/p- ), Alexa Fluor 647 phospho-STAT1 (pY701, clone 4a), PE IL-17 gp130 Y757F mice on a C57BL/6 and C57BL/6/129 background were (TC11-18H10), and PerCP Cy5.5 CD4 (RM4-5) were purchased from provided by Dr. M. Ernst (Ludwig Institute for Cancer Research, Mel- BD Biosciences (San Jose, CA). FITC anti-MHC class II (MHC II) IA/IE bourne, VIC, Australia) and have been described previously (8). RAG mice (M5/114.15.2), PerCP-Cy5.5 CD11b (M1/70), Pe-Cy7 CD11c (N418), are maintained in-house. Mice were housed and bred in specific pathogen- allophycocyanin CD62L (IM7), allophycocyanin IFN-g (XMG1.2), allo- free facilities in the Department of Pathobiology at the University of phycocyanin IL-10 (JES5 16E3), and allophycocyanin Alexa Fluor 750 Pennsylvania in accordance with institutional guidelines. The Me49 strain anti-CD3 (17A2) were purchased from eBioscience. PE anti-CD126 (IL- of T. gondii was prepared from chronically infected CBA/ca mice, and 6Ra–chain, 255821) and allophycocyanin anti-CD130 (gp130, 125623) experimental animals were infected i.p. with 20 cysts. For IL-6 blockade, were purchased from R&D Systems. PE-Cy7 CD62L (MEL-14), Pacific mice were treated i.p. with 100 mg anti–IL-6–blocking Abs (BioXCell, Blue anti-CD3 (17A2), Pacific Blue anti-CD19 (6D5), and Pacific Blue Lebanon, NH) 1 d before infection and at days 2 and 5 postinfection. For anti-NK1.1 (PK136) were purchased from BioLegend (San Diego, CA) 250 ng, recombinant murine IL-12p70 (PeproTech, Rocky Hill, NJ) was given i.p. on the day of infection as well as days 1–3 postinfection. Soluble Statistical analysis toxoplasma Ag (STAg) was prepared from tachyzoites of the RH strain as previously described and used at a concentration of 10 mg/ml for Ag- An unpaired Student t test or one-way ANOVA was used to determine , specific recalls. For histology, , lungs, and brains were collected significant differences, and p 0.05 was considered significant. from mice, fixed in 10% formalin, embedded in paraffin, sectioned, and stained with H&E. For measurement of parasite burden, the 35-fold re- Results petitive T. gondii B1 was amplified by real-time PCR with SYBR gp130 Y757F mice are susceptible to T. gondii Green PCR Master Mix (Applied Biosystems, Foster City, CA) in an AB7500 fast real-time PCR machine (Applied Biosystems) using pre- To address the role of gp130 signaling in the regulation of the viously published conditions (30). To assess parasite burden by cytospin immune response to infection, gp130 Y757F mice were challenged count, single-cell suspensions were prepared from PECs, and 1 3 105 cells with the Me49 strain of T. gondii. Although WT littermate controls in 200 ml RPMI 1640 were spun onto glass slides at 500 rpm for 5 min. survived to the chronic phase of infection, gp130 Y757F mice Slides were stained with Diff-Quick (Merck, Darmstadt, Germany) and mounted in CytoSeal (Edmund Scientifics, Tonawanda, NY). The per- succumbed by day 12 (Fig. 1A). Microscopic analysis of perito- centage of infected cells was calculated based on counts of at least 250 neal exudate cells (PECs) at day 8 postinfection revealed that, as total cells. expected, WT mice controlled parasite replication with ,5% of 352 Gp130 Y757F MICE ARE SUSCEPTIBLE TO T. GONDII

FIGURE 1. gp130 Y757F mice are susceptible to infection with T. gondii. A, Survival of WT (n = 15) and gp130 Y757F mice (n = 12) infected i.p. with 20 cysts of the Me49 strain of T. gondii. B, Per- centage of T. gondii-infected PECs at day 8 post- Downloaded from infection in WT (n = 14) and gp130 Y757F (n = 12) mice. **p , 0.001. Enumeration of parasite DNA by real-time PCR in livers (C) and brains (D)of infected WT (n = 10) and gp130 Y757F (n = 14) mice at day 8 postinfection. **p , 0.001. Histo- pathology of lungs from naive and infected WT (E, G) and gp130 Y757F (F, H) mice, analyzed by http://www.jimmunol.org/ staining with H&E at day 8 postinfection. E–H, Original magnification 310. by guest on September 29, 2021

PECs infected. In contrast, gp130 Y757F mice had .35% of 12p40 production were also observed when whole splenocytes peritoneal cells infected at this time point (Fig. 1B). Consistent from mice infected for 3 d were stimulated with STAg (Fig. 2B), with these data, real-time PCR to quantify parasite DNA at the whereas splenocytes from mice infected for 8 d produced equiv- same time point revealed increased parasite burdens in the liver alent levels of IL-12p40 (data not shown). Moreover, analysis of and brain of gp130 Y757F mice (Fig. 1C,1D). This high parasite the PECs at day 4 postinfection revealed that, although 4–8% of burden was accompanied by the presence of overt inflammation in WT DCs produce IL-12p40 postinfection, a smaller percentage of multiple peripheral tissues, in particular the lungs (Fig. 1E–H). gp130 Y757F DCs stained positive for intracellular IL-12p40, and this difference is reflected in the total number of IL-12p40+ DCs gp130 Y757F mice have an early defect in the ability to (Fig. 2C,2D). Despite the difference in the number of IL-12+ DC, produce IL-12 the expression of MHC II (Fig. 2E) is not different between DC in The ability of IL-12 to promote the production of IFN-g is critical WT and Y757F mice. Taken together, these data demonstrate that for the control of T. gondii, and defects in this pathway lead to gp130 Y757F mice infected with T. gondii have an early de- heightened susceptibility to this infection (21, 22). Therefore, to ficiency in the production of IL-12, but this does not correlate with determine whether dysregulated gp130-mediated signaling resul- differences in overall activation. ted in alterations in the production of IL-12, a kinetic analysis of the levels of this cytokine was performed. In uninfected mice, Gp130 Y757F mice have lower levels of IFN-g early after there were low basal amounts of serum IL-12p40 (data not T. gondii infection, despite normal T cell activation shown), but infection of WT mice resulted in increased circulating The early production of IL-12 following challenge with T. gondii levels of this cytokine by days 3 and 8 postinfection (Fig. 2A). In drives the secretion of IFN-g by NK and T cells, which is required contrast, gp130 Y757F mice had significantly lower circulating to control this infection (21). To determine whether the early levels of this cytokine at day 3 postinfection, but by day 8, levels defects in IL-12 levels observed in gp130 Y757F mice affected the were comparable to WT mice (Fig. 2A). These differences in IL- expression of IFN-g, a kinetic analysis of this cytokine was The Journal of Immunology 353 Downloaded from FIGURE 2. gp130 Y757F mice have an early defect in the ability to produce IL-12 during infection with T. gondii. A, Kinetics of IL-12p40 production in the serum of infected WT (n = 8) or gp130 Y757F mice (n = 10), as measured by ELISA. **p , 0.001. B, ELISA of IFN-g levels in the supernatants of splenocytes isolated from WT and gp130 Y757F mice that were incubated with STAg for 48 h. Data are representative of three independent experiments. **p , 0.001. C, Flow cytometry of intracellular IL-12p40 in DC isolated from PECs of infected WT or gp130 Y757F mice at day 4 postinfection; cells were incubated with brefeldin A for 6 h before staining. D, Total number of IL-12+ DC in the PECs at day 4 postinfection in WT or gp130 Y757F mice, calculated from percentages based on flow cytometry. *p , 0.01. E, Representative histrogram of MHC II expression on DC from the PEC of WT (solid

line) or gp130 Y757F (dotted line) mice at day 4 postinfection. Data are representative of two independent experiments. http://www.jimmunol.org/ performed. In naive WT and gp130 Y757F mice, there were low To determine the functional consequence of the early defect in basal levels of circulating IFN-g (data not shown), but by day 3 IFN-g production in gp130 Y757F mice, real-time PCR was used postinfection, WT mice had ∼7 ng/ml IFN-g circulating in the to quantify the expression of the IFN-g–inducible p47 GTPases serum, whereas gp130 Y757F mice had significantly lower levels IGTP, Lrg47, and IGIP, which are required for resistance to of this cytokine (Fig. 3A). However, by day 8 postinfection, WT T. gondii (32–34). Relative to the levels in naive mice, infection of and gp130 Y757F mice had equivalent levels of IFN-g (Fig. 3A). WT mice induced a 10-to 20-fold increase in expression of IGTP, Stimulation of whole splenocytes from mice infected for 3 d with Lrg47, and IGIP in the livers from WT mice by day 3 post-

STAg mirrored the defect in circulating IFN-g levels, and cells infection (Fig. 3C). In contrast, the levels of these GTPases in by guest on September 29, 2021 from WT controls produced significantly more than gp130 Y757F gp130 Y757F mice only increased by 1- to 2-fold (Fig. 3C). These (Fig. 3B). data indicate that the gp130 Y757F mice have an early defect in

FIGURE 3. gp130 Y757F mice have lower levels of IFN-g early after T. gondii infection. A, Kinetics of IFN-g production in the serum of infected WT (n = 10) or gp130 Y757F mice (n = 11), as measured by ELISA. Data are pooled from four independent experiments. ***p , 0.001. B, At day 3 postinfection, whole splenocytes from WT and Y757F mice were stimulated with STAg for 48 h and assayed for IFN-g. Data are representative of four independent experiments. **p , 0.01. C, Real-time PCR of total cellular RNA isolated from the livers of WT and Y757F mice at day 3 postinfection to detect IGTP, Lrg 47, and IGIP mRNA. Data are representative of two independent experiments. D, Flow cytometry of IL-17A versus IFN-g from splenocytes isolated from WT or gp130 Y757F mice at day 4 postinfection. Plots are gated on NK1.1+CD32 cells; cells were incubated with PMA, ionomycin, and BFA for 4 h before being stained. Total numbers of of IFN-g+ (E) and IL-17+ (F) splenic NK cells from WT (n = 5) and Y757F mice (n =7) at day 4 postinfection. Data are pooled from two independent experiments. *p , 0.05. 354 Gp130 Y757F MICE ARE SUSCEPTIBLE TO T. GONDII the production of IFN-g and reduced expression of antimicrobial a potent antagonist of IL-12 production, which can be promoted by effector molecules downstream of this cytokine. IL-6 and IL-27 (31, 40–45). Indeed, experiments assessing the Because NK cells are an innate source of IFN-g and IL-17 during activation and phosphorylation of STAT molecules in response acute toxoplasmosis (35, 36), studies were performed to assess to IL-6 (Fig. 5A) and IL-27 (Supplemental Fig. 1) (28) on T cells whether the gp130 Y757F NK cells have a defect in the ability revealed that responsiveness to both cytokines was enhanced in to produce these cytokines. WT and gp130 Y757F mice were gp130 Y757F cells compared with WT cells, as measured by infected, and at day 4 postinfection, splenocytes were stimulated levels of intracellular phospho-STAT3 and STAT1 (data not ex vivo with PMA/ionomycin and BFA for 4 h before being shown). In addition, naive CD4+ T cells from gp130 Y757F mice stained for intracellular IFN-g and IL-17. As shown in Fig. 3D and produce significantly higher levels of IL-10 when activated in the 3E, gp130 Y757F mice have fewer IFN-g+ NK cells at this time presence of TGF-b and IL-6 or IL-27 (Fig. 5B). In vivo, although point, but consistent with the ability of IL-6 to drive the production IL-10 was not detected in the serum of naive mice of either ge- of IL-17 by NK cells (37), gp130 Y757F mice have significantly notype (data not shown), by 8 d postinfection, serum IL-10 levels more IL-17+ NK cells (Fig. 3D,3F). By day 8 postchallenge, the in gp130 Y757F mice were almost twice those detected in WT NK cell response was diminished, and few of these cells produced mice (Fig. 5C). Moreover, when splenocytes from infected gp130 IFN-g or IL-17 (data not shown), consistent with reports that the Y757F mice were restimulated ex vivo with anti-CD3, there was NK cell response to T. gondii peaks 4–5 d postinfection (37–39). a significant increase in the production of IL-10 compared with To assess whether the altered signaling through gp130 also af- WT cells (Fig. 5D). There was also a 2- to 4-fold increase in the fected the T cell compartment, studies were performed to compare percentage of IL-10+CD4+ T cells as revealed by intracellular the T cell responses in WT and gp130 Y757F mice. At days 4 and 8 cytokine staining, and this was reflected in the total number of Downloaded from postinfection, there were equivalent percentages and total numbers splenic IL-10+CD4+ T cells (Fig. 5E,5F). Thus, the production of of activated CD44highCD62LlowCD4+ T cells in the spleens of the potent anti-inflammatory cytokine IL-10 is augmented in infected WT and gp130 Y757F mice (Fig. 4A,4B) and similar gp130 Y757F mice during toxoplasmosis. results were observed for CD8+ T cells (data not shown). Single- Previous studies demonstrated that CD4+ T cells are the pri- cell analysis revealed that there were similar percentages and total mary source of host-protective IL-10 during toxoplasmosis (46). + + numbers of IFN-g CD4 T cells in WT and gp130 Y757F mice at Therefore, to determine whether the enhanced production of IL-10 http://www.jimmunol.org/ days 4 and 8 postchallenge (Fig. 4C,4D). In addition, stimulation of by gp130 Y757F T cells contributed to the increased susceptibility splenocytes from infected mice with anti-CD3 at day 8 post- to infection, experiments were performed using a RAG transfer infection show equivalent production of IFN-g between WT and system. In these experiments, 10 million CD4+ and CD8+ T cells gp130 Y757F mice (Fig. 4E). These data suggest that the T cell isolated from spleens and lymph nodes of naive WT and gp130 response is normal in gp130 Y757F mice and that the diminished Y757F mice were transferred into RAG2 KO mice 1 d prior to early production of IFN-g is due to an NK cell defect. infection. At day 8 postinfection, mice were sacrificed and ana- lyzed for parasite burden and cytokine production. Although gp130 Y757F T cells produce augmented levels of IL-10 in infected RAG2 KO mice have high parasite burdens at this time response to IL-6 and IL-27 but are still protective during point, mice that received WT or gp130 Y757F T cells had a sig- by guest on September 29, 2021 toxoplasmosis nificant reduction in the percentage of infected PECs (Fig. 5G). One possible explanation for the early defect in IL-12 levels during In addition, there were equivalent percentages of IFN-g+CD4+ infection of gp130 Y757F mice is increased expression of IL-10, T cells in splenocytes of recipient mice (Fig. 5H), and the

FIGURE 4. Infection-induced T cell activation is normal in Y757F mice. A, Flow cytometry of splenocytes from WT or gp130 Y757F mice at day 8 postinfection showing expression of CD62L and CD44. Gated on CD3+CD4+ cells. B, The number of CD44hiCD62LlowCD4+ T cells was enumerated in naive WT and gp130 Y757F mice and at days 4 and 8 postinfection. n $ 5 mice per time point. C, Flow cytometry of IFN-g in splenocytes isolated from WT or gp130 Y757F mice at day 8 postinfection. Gated on CD3+CD4+ events; cells were incubated with PMA, ionomycin, and BFA for 4 h before being stained. D, Enumeration of IFN-g+CD4+ T cells in naive or infected WT or gp130 Y757F mice. n $ 5 mice per time point. E, ELISA of IFN-g levels from cultures of splenocytes isolated at day 8 postinfection and incubated with STAg for 48 h. Data are representative of four independent experiments. The Journal of Immunology 355 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. Gp130 Y757F T cells produce augmented levels of IL-10 in response to IL-6 and IL-27. Purified CD4+ T cells from naive WT and Y757F mice were stimulated in vitro with IL-6 (A) for various time points before being fixed, permeabilized, and stained for intracellular phosphorylated STAT3. Data are pooled from five independent experiments. *p , 0.01. B, Purified CD4+ T cells from naive WT or gp130 Y757F mice were activated with anti- CD3 and anti-CD28 for 48 h in the presence of TGF-b with IL-6 or IL-27. IL-10 levels in culture supernatants were determined by ELISA. Data are representative of four independent experiments with similar results. **p , 0.001. C, IL-10 levels in the serum of infected WT (5) or gp130 Y757F (8) mice were determined by ELISA at day 8 postinfection. *p , 0.01. Data are pooled from two independent experiments. D, At day 8 postinfection, whole splenocytes from WT and Y757F mice were stimulated with media or anti-CD3 for 48 h and assayed for IL-10 by ELISA. Data are representative of three independent experiments. *p , 0.01. E, Flow cytometry of IL-10 from splenocytes isolated from WT or gp130 Y757F mice at day 8 postinfection. Gated on CD3+CD4+ events, cells were incubated with PMA, ionomycin, and BFA for 4 h before being stained. F, The total number of IL-10+CD4+ T cells was calculated in WT (n = 5) and Y757F mice (n = 6) at 8 d postinfection. Data are pooled from two independent experiments. *p , 0.01. G, Ten million splenic CD4+ and CD8+ T cells were isolated from WT and Y757F mice and transferred into RAG KO mice. The recipient RAG mice that received T cells from WT (n = 5) or gp130 Y757F mice (n = 5) were infected with T. gondii, and at 8 d postinfection, the percentage of infected PECs was enumerated. **p , 0.001. H, Flow cytometry of IFN-g from splenocytes isolated from RAG KO mice that received WT or gp130 Y757F mice at day 8 postinfection. 356 Gp130 Y757F MICE ARE SUSCEPTIBLE TO T. GONDII transferred T cells produced equivalent levels of IFN-g upon ex asses whether the production of IL-12 by these cells was also vivo STAg (data not shown) and anti-CD3 (Fig. 5I) restimulation. affected by IL-6. In response to LPS and IFN-g, WT bone Similarly to infected gp130 Y757F mice, transferred gp130 marrow-derived macrophages produced .20 ng/ml IL-12p40, Y757F T cells produced significantly more IL-10 during infection and addition of IL-6 reduced the production of IL-12p40 by WT compared with WT T cells (Fig. 5J), but these mice were able to macrophages by ∼25%, whereas blocking IL-6 during activation appropriately control the infection based on parasite burden in the had no effect on IL-12p40 levels (Fig. 6F). While LPS and IFN-g– PECs (Fig. 5G). Taken together, these experiments demonstrate activated macrophages produce significantly less IL-12p40 than that the overproduction of IL-10 is intrinsic to the gp130 Y757F WT controls, the addition of exogenous IL-6 almost completely T cells and can be driven by IL-6 and IL-27 but that this pheno- inhibits the expression of this cytokine (Fig. 6F). In contrast, type does not account for the increased susceptibility of the gp130 blocking IL-6 during activation restored production of IL-12p40 to Y757F mice. WT levels (Fig. 6F). Moreover, the addition of other gp130- signaling cytokines had no effect on the production of IL-12p40 IL-6 blocks the production of IL-12 by gp130 Y757F APCs by gp130 Y757F macrophages (Fig. 6F,6G). Collectively, these Taken together, these experiments suggest that the ability of gp130 data demonstrate that the observed defect in IL-12 production Y757F T cells to protect during challenge with T. gondii is not during toxoplasmosis in gp130 Y757F mice could be recapitulated compromised and indicate that the susceptibility of gp130 Y757F in vitro and that sustained signaling through gp130 by IL-6, but mice to T. gondii is due to an intrinsic defect in the innate immune not other gp130-signaling cytokines, blocks proinflammatory response. Previous work demonstrated that in the absence of responses by macrophages and DCs.

SOCS3, IL-6 acquired the ability to block LPS-mediated IL-12 Downloaded from production by macrophages, providing a potential mechanism IL-6 blockade or addition of exogenous IL-12 restores underlying the IL-12 defect in gp130 Y757F mice (47–49). To protective immunity to T. gondii in gp130 Y757F mice assess whether IL-6 could be acting directly on innate APCs The data described above demonstrate that IL-6, but not other during infection, macrophages and DCs in the PECs were ana- gp130-signaling cytokines, has an enhanced ability to directly lyzed by flow cytometry for the IL-6Ra–chain and gp130 at day 4 block IL-12 production by gp130 Y757F APCs and that ∼50% of

postinfection. In both cell types, the expression of gp130 was DCs and macrophages express both chains of the IL-6R complex http://www.jimmunol.org/ ubiquitous, and all cells expressed this receptor (Fig. 6A), whereas during infection (Fig. 6A–G). Although infection of WT and ∼50% of each population expressed the IL-6Ra–chain (Fig. 6A). gp130 Y757F mice results in similar levels of circulating IL-6 There were no differences in the expression of either chain of the (∼200–600 pg/ml), experiments were performed to assess the receptor between cells from either naive or infected WT and role of endogenous IL-6 in the susceptibility of gp130 Y757F gp130 Y757F mice. Strikingly, significantly more MHC II- mice to T. gondii. In these studies, gp130 Y757F mice were treated positive PEC DCs expressed the IL-6Ra–chain than those that with anti–IL-6–blocking Ab or rat IgG and sacrificed at day 8 were MHC II negative (Fig. 6B,6C), and similar trends were postchallenge to assess cytokine production, parasite burden, and observed for splenic DC. These data suggest that IL-6 has the pathology. As expected, WT littermate controls survived the acute ability to act directly on a subset of macrophages and activated DC infection and effectively controlled parasite burden, whereas by guest on September 29, 2021 during the acute challenge to T. gondii. infected gp130 Y757F mice that received rat IgG were susceptible To directly test the effect of IL-6 on the production of IL-12 by to challenge and had high parasite burdens (Fig. 7A,7B). In accessory cells, purified splenic DC from WTor gp130 Y757F mice contrast, gp130 Y757F mice that were treated with anti–IL-6 were cultured for 24 h in the presence of LPS or LPS and IFN-g survived the acute infection and had significantly lower parasite with or without IL-6. As shown in Fig. 6D, WT DCs secreted IL- burdens than controls (Fig. 7A,7B). Consistent with our in vitro 12p40 after stimulation with LPS, and this response was enhanced experiments, blocking IL-6 resulted in a significant increase in in the presence of IFN-g, but the addition of IL-6 to these cultures circulating levels of IL-12p40 and IFN-g at day 3 (Fig. 7C,7D). In had no effect on IL-12 production. In contrast, DCs from gp130 addition to significantly reducing parasite burdens in infected Y757F mice produced equivalent levels of IL-12p40 in response gp130 Y757F mice, anti–IL-6 treatment resulted in decreased to LPS alone, but when these cells were activated in the presence infection-induced lung pathology (Fig. 7E–G). Taken together, of LPS and IFN-g (Fig. 6D), there was a reduced IL-12p40 pro- these studies demonstrate that, in the absence of SOCS3-mediated duction compared with WT DCs, and the addition of IL-6 to these regulation of gp130-mediated signals, IL-6 signaling is a key cultures almost completely blocked secretion of IL-12p40 (Fig. factor that antagonizes the initiation of protective immunity to 6D,6E). Although WT and gp130 Y757F DCs produced equiva- T. gondii. lent levels of IL-6 (data not shown), the addition of anti–IL-6– Although blockade of IL-6 during infection resulted in signifi- blocking Abs had no effect on the production of IL-12 by WT DCs cantly decreased parasite burdens, the mechanism underlying these (data not shown) but resulted in a significant increase in the results remained unclear. Therefore, to address whether the ca- production of IL-12p40 by gp130 Y757F DC (Fig. 6E). In- pacity of IL-6 to block IL-12 production was the primary defect in terestingly, when purified splenic DCs from gp130 Y757F mice the gp130 Y757F mice, these mice were treated with rIL-12p70 for were stimulated with LPS and IFN-g in combination with IL-6, 4 d, starting at the time of infection. Similar to the effects of IL-6 IL-11, IL-27, or oncostatin M, only IL-6 had the capacity to block blockade, the administration of rIL-12p70 to gp130 Y757F mice IL-12p40 production (Fig. 6E). resulted in a significant reduction in parasite burden, and these mice Macrophages are also an innate source of IL-12 during toxo- survived the acute infection (Fig. 7A,7B) associated with reduced plasmosis (50, 51), and similar experiments were performed to infection-induced lung pathology (Fig. 7E,7F,7H). The addition

Gated on CD3+CD4+ events; cells were incubated with PMA, ionomycin, and BFA for 4 h before being stained. I, At 8 d postinfection, whole splenocytes from recipient RAG KO mice were stimulated with anti-CD3 for 48 h and assayed for IFN-g by ELISA. J, At 8 d postinfection, whole splenocytes from recipient RAG KO mice were stimulated with anti-CD3 for 48 h and assayed for IL-10 by ELISA. Data are representative of three independent experiments with similar results. *p , 0.01. The Journal of Immunology 357 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. IL-6 blocks the production of IL-12 by gp130 Y757F APCs. A, Flow cytometry of IL-6Ra and gp130 expression on PECs from WT mice at day 4 postinfection. Gated on CD3/CD19/NK1.1neg, CD11c, or CD11b+ cells. Representative FACS plot (B) and enumeration (C) of the percentage of MHC II-negative or MHC II-positive DCs that express the IL-6Ra. Data are representative of two independent experiments with similar results. D, Purified splenic DC from naive WT and Y757F mice were stimulated in vitro with LPS, IFN-g, and IL-6 for 24 h before IL-12p40 was measured in culture supernatants by ELISA. E, Purified splenic DC from naive gp130 Y757F mice were stimulated in vitro with LPS, IFN-g and IL-6, IL-11, IL-27, oncostatin M, or anti–IL-6 Abs for 24 h before IL-12p40 was measured in culture supernatants by ELISA; n = 3 samples/condition. Data are representative of three independent experiments. F, Bone marrow-derived macrophages from WT and Y757F mice were stimulated in vitro with LPS, IFN-g and IL-6, or anti–IL-6 Abs for 24 h before IL-12p40 was measured in culture supernatants by ELISA. G, Bone marrow-derived macrophages from gp130 Y757F mice were stimulated in vitro with LPS, IFN-g and IL-6, IL-10, IL-11, IL-27, or oncostatin M for 24 h before IL-12p40 was measured in culture supernatants by ELISA. *p , 0.01, **p , 0.001. of exogenous IL-12p70 also restored IFN-g to WT levels (Fig. a major inhibitor of IL-12 production was initially unexpected, 7D), consistent with the idea that the early IFN-g defect is and there were several possible explanations for this observation. a consequence of the reduced production of IL-12. Collectively, Recent studies have highlighted the importance of IL-6 and IL-27 these results suggest that the ability of IL-6 to block IL-12 pro- signaling in promoting the generation of IL-10–producing CD4+ duction in gp130 Y757F mice prevents the development of pro- T cells that are essential for limiting infection-induced pathology tective immunity to T. gondii. (31, 40, 42–46, 52, 53). One scenario for the increased suscepti- bility of the gp130 Y757F mice was that enhanced gp130 sig- Discussion naling observed in T cells from gp130 Y757F mice explained the Previous studies have identified a critical role for IL-6 in resistance elevated IL-10 levels in these mice, which in turn antagonized the to T. gondii (24, 25), and although the mechanism by which IL-6 early production of IL-12. This hypothesis is consistent with promotes resistance to this pathogen remains unclear, these find- previous work, which established that CD4+ T cells are the pri- ings are consistent with its role as a proinflammatory factor. mary source of host-protective IL-10 during toxoplasmosis (46). Consequently, the finding in gp130 Y757F mice that IL-6 becomes However, the studies in which mutant T cells transferred into RAG 358 Gp130 Y757F MICE ARE SUSCEPTIBLE TO T. GONDII

FIGURE 7. Anti–IL-6 treatment, or administra- tion of IL-12p70, rescues gp130 Y757F mice. A, Survival of T. gondii-infected WT (n = 5) or gp130 Y757F mice that were treated with rat IgG (n = 12), anti–IL-6 Abs (n = 9), or rIL-12p70 (n = 6). B, Percentage of PECs infected in WT (n = 5) or gp130 Y757F mice that were treated with rat IgG (n = 5), anti–IL-6 Abs (n = 8), or rIL-12p70 (n = 5). C,At Downloaded from day 3 postinfection, WT (n = 5) or gp130 Y757F mice treated with rat IgG (n = 7) or anti–IL-6– blocking Abs (n = 8) were bled, and the levels of circulating IL-12p40 were determined by ELISA. **p , 0.01. D, At day 3 postinfection, WT (n =5) or gp130 Y757F mice treated with rat IgG (n = 7),

anti–IL-6–blocking Abs (n = 8), or rIL-12p70 (n = http://www.jimmunol.org/ 5) were bled, and the levels of circulating IFN-g were determined by ELISA. **p , 0.01. E–H, Histopathology of lungs from an infected WT or gp130 Y757F mouse that was treated with rat IgG, anti–IL-6, or rIL-12p70 and stained with H&E. E–H, Original magnification 310. by guest on September 29, 2021

mice were able to provide protection against T. gondii indicated Regardless of any intrinsic effects of the Y757F mutation on that susceptibility of the gp130 Y757F mice was due to an innate T cell function, our studies revealed that IL-6 was a potent inhibitor defect that affected the production of IL-12. of IL-12 production by various and DC populations IL-12 is an important factor that promotes NK and T cell ex- from these mice. In contrast, other gp130 family cytokines did not pansion, differentiation, and production of IFN-g during numerous display this activity. Notably, IL-27Ra–chain expression was not intracellular infections including T. gondii (54). Although the detected on APCs, and we did not see any effects of IL-27 on the IL-12 levels in gp130 Y757F mice eventually recover, previous production of IL-12 or IL-10 by gp130 Y757F DCs or macro- studies have demonstrated that production of IL-12 during the first phages. Nonetheless, the basis for this defect was initially unclear 3 d of infection is the most critical for host resistance (38, 50). as the Y757F mutation prevents SOCS3 binding to gp130 but also Paradoxically, although the gp130 Y757F mice have an early abrogates Src homology region 2 domain-containing phosphatase defect in the production of IFN-g by NK cells associated with 2 (SHP2)/Erk signaling (56). However, gp130 Y757F and SOCS3- lower levels of IL-12, at later time points, there are normal deficient cells (which have hyper-SHP2/Erk signaling) have sim- numbers of IFN-g+ T cells. This compensatory response may be ilar defects in the ability to produce IL-12 (48, 57), indicating that a consequence of the increased Ag load at later time points that the inability to activate SHP2/Erk does not explain this phenotype. leads to heightened T cell responses in gp130 Y757F mice. Al- Rather, although SOCS3 normally abbreviates the duration of ternatively, SOCS3 has been shown to negatively regulate the IL-6–mediated STAT3 activation, in its absence, IL-6 generates proliferation of activated T cells (55), and the inability of SOCS3 a prolonged STAT3 signal, similar to that induced by IL-10, to limit gp130 signaling in mutant mice might result in increased a potent antagonist of accessory cell inflammatory responses. proliferation of activated T cells. In support of this idea, both Notably, when the and receptors, which also gp130 Y757F mice and gp130 Y759 mice, which have an anal- activate STAT3, were engineered to yield sustained signaling ogous mutation, accumulate activated T cells with age (J. Silver, profiles, they also generated a similar anti-inflammatory outcome unpublished observations) (15). (57). Thus, the studies presented in this paper indicate that, in the The Journal of Immunology 359 gp130 Y757F mice challenged with T. gondii, the IL-6 that is 4. De Souza, D., L. J. Fabri, A. Nash, D. J. Hilton, N. A. Nicola, and M. Baca. 2002. produced becomes an efficient antagonist of IL-12 production that SH2 domains from suppressor of cytokine signaling-3 and protein tyrosine phos- phatase SHP-2 have similar binding specificities. Biochemistry 41: 9229–9236. is essential to control this parasite. 5. Nicholson, S. E., D. De Souza, L. J. Fabri, J. Corbin, T. A. Willson, J. G. Zhang, The observation that IL-6 drives the hematopoietic defects A. Silva, M. Asimakis, A. Farley, A. D. Nash, et al. 2000. Suppressor of cytokine signaling-3 preferentially binds to the SHP-2-binding site on the shared cytokine observed in aged gp130 Y757F mice (10, 10–13) indicates that receptor subunit gp130. Proc. Natl. Acad. Sci. USA 97: 6493–6498. 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