TPL2 Kinase Is a Crucial Signaling Factor and Mediator of NKT Effector Cytokine Expression in Immune-Mediated Liver Injury

Unlike the majority of secondary immune organs, the liver is typified by an overrepresentation of cellular components of the innate immunity such as Kupffer, dendritic, NK, and NKT cells endowed with Ag recognition and regulation of immunogenic versus tolerogenic functions. Disruption of immunological balance as a result of viral infection, autoimmune reactions, excess alcohol consumption, or metabolic disease leads to exaggerated and uncontrolled inflammation, hepatocyte death, and eventually, permanent loss of organ function (1). Emerging evidence underscores a central role for NKT cells in orchestrating immune homeostasis and disease pathogenesis in the liver (2).

NKT cells are a heterogeneous group of nonconventional T lymphocytes that are found with highest frequency in the liver (3). Invariant NKT (iNKT) cells, the prototypical NKT cell subtype (also referred as type I NKT), express a TCR comprising a semi-invariant α-chain (Vα14-Jα18 in mice; Vα24-Jα18 in humans) and a restricted β-chain repertoire (Vβ8.2, Vβ2, or Vβ7 in mice; Vβ11 in humans) that, unlike conventional T lymphocytes, recognize self- or foreign glycolipid Ags presented by the nonclassical MHC class I molecule CD1d (4). Studies using a mouse model of fulminant hepatitis induced by Con A have revealed a major pathogenic role for iNKT cells in liver injury. Thus, CD1d^−/− and Jα18^−/− mice, which both lack iNKT cells, are resistant to Con A–induced pathology (5, 6). Further studies have shown that upon activation, iNKT cells produce copious amounts of immunoregulatory molecules, including the Th1 cytokine IFN-γ and the Th2 cytokine IL-4 (4, 7), which endow them with the capacity to direct the licensing of other immune cell types and thus orchestrate inflammatory immune reactions in the liver (4). Remarkably, the intracellular signaling pathways that mediate the pathogenic functions of iNKT cells remain poorly defined.

TPL2 is an MAP3 kinase with an obligatory role in signal transduction on the MEK/ERK axis downstream of receptors involved in innate immunity, including Toll-like (8) and TNF family receptors (9), thereby affecting the production of mediators of inflammation such as TNF, IL-6, cyclooxygenase-2, and TIMP-1 (10-12). As a result, TPL2 ablation in mice ameliorates the severity of various inflammatory pathologies including LPS-induced endotoxic shock (10), inflammatory bowel disease (13), and the onset and progression of experimental autoimmune encephalomyelitis (14, 15). In contrast, TPL2 ablation exacerbates the inflammatory response to intracellular pathogens and bronchoalveolar allergens (16, 17), highlighting cell type– and stimulus-dependent roles of TPL2 in the immune system. In this study, we have addressed the role of TPL2 in immune-mediated liver pathology in the mouse and uncovered a novel obligatory role for this kinase in CD3⁺NK1.1⁺ iNKT signal transduction induced by lipid Ags and in the regulation of the pathogenic effects of iNKT cells.

Tpl2^+/+ and tpl2^−/− mice (C57BL/6 background) are described in Ref. 10, and C57BL/6-CD1d^−/− mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Tpl2^+/− animals were used to generate experimental groups of different tpl2 genotypes, which were cohoused and maintained in identical conditions prior to treatments. To induce immune-mediated hepatitis, 8- to 10-wk-old female mice were injected i.v. with Con A (Sigma-Aldrich) at a dose of 10 mg/kg body weight and were euthanized at the indicated time points after Con A treatment. For the assessment of the effects of kinase inhibition, a TPL2 inhibitor (Calbiochem) dissolved in DMSO was administered i.p. at a dose of 250 μg/mouse 15 h and 30 min before Con A treatment, whereas the control animals received equal amount of DMSO. To induce NKT cell–mediated liver injury, mice received 100 μg/kg α-galactocylceramide (αGalCer, KRN7000; Enzo Life Sciences) for the indicated time points. Control mice of all study groups received PBS as control. Experimental procedures were approved by the Veterinary Department of the Prefecture of Crete (renewed license number 6161/28–03-2014).

For histopathology, liver tissue sections were excised at various time points, fixed overnight in 10% neutral formalin solution, and embedded in paraffin. Sections of 5 μM were prepared, placed on glass lesions, and stained with H&E to assess liver injury. To assess the extent of apoptotic areas, liver tissue was stained for activated caspase-3. Inactivation of endogenous peroxidase was achieved by incubating the deparaffinized sections with hydrogen peroxide/methanol. Ag retrieval was performed by using 10 mM sodium citrate buffer (pH 6). Sections were blocked using an Avidin/Biotin Kit (Vector Laboratories), followed by addition of 20% pig serum in PBS. A primary Ab was used against caspase-3 (CS9664; Cell Signaling Technology) at a 1:200 dilution in PBS, followed by overnight incubation at 4°C. Sections were washed and further incubated with biotinylated swine anti-rabbit at 1:200 (DakoCytomation) for 1 h and washed, and immunohistochemistry was completed with streptavidin biotin–peroxidase complex incubation (Vector Laboratories) for 45 min. Caspase-3–positive cells were visualized by 3,3′-diaminobenzidine tetrahydrochloride, and sections were hematoxylin counterstained. Morphometric image analysis was performed using Leica QWin software.

To detect serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity, serum was collected from peripheral blood via cardiac puncture at the indicated time periods postinjection under isoflurane anesthesia. Serum samples were stored at −20°C until ready to be used. Levels of AST and ALT were assayed by standard enzymatic procedures using an automatic biochemical analyzer Olympus AU5400 (Medicon) in the Laboratory of Clinical Biochemistry, University Hospital of Heraklion (Crete, Greece).

Hepatic lymphocytes were isolated using the Percoll gradient as described previously (18). In brief, following anesthesia with isoflurane inhalation (Baxter), livers were perfused with PBS and excised carefully. Livers were chopped into small pieces and incubated for digestion in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 0.05% collagenase/dispace (Roche) and 0.01% trypsin inhibitor (Life Technologies) for 30 min at 37°C on a rotary shaker. The digested materials were filtered through 40-μm nylon cell strainer and pelleted to enrich lymphocytes. Cell pellet containing liver mononuclear cells (LMNCs) was collected and washed in RPMI 1640 medium. The cell suspension was gently overlaid by a 33% Percoll (Sigma-Aldrich) gradient containing 100 U/ml heparin and centrifuged for 30 min at 800 × g. LMNCs were collected from the cell pellet, followed by lysis of erythrocytes using RBC lysis buffer containing 155 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM Na₂EDTA. LMNCs were washed twice in PBS and resuspended in PBS supplemented with 5% FBS. Splenic or thymic cells were isolated by pressing the spleen or thymus through a 40-μm nylon strainer and were resuspended in PBS, followed by erythrocyte lysis using RBC lysis buffer. Cell yields and viability were measured with trypan blue staining.

For flow cytometric analysis, cells were resuspended in 100 μl FACS buffer (PBS containing 5% FBS). For extracellular markers, single-cell suspensions of LMNCs were stained with specific fluorescently conjugated Abs for 30 min at 4°C in the dark. The following Abs were used from eBioscience: CD3 (clone 145-2C11), NK1.1 (clone PK136), CD69 (clone H1.2F3), CD4 (clone RM4-5), CD8 (clone 53-6.7), Gr1 (clone; RB6-8C5), CD11b (clone; M1/70), CD11c (clone N418), CD19 (clone MB19-1), CD24 (clone 30-F1), CD44 (clone IM7), and IL-4 (clone BVD6-24G2). For tetramer staining, LMNCs (2 × 10⁶ cells) were preincubated for 10 min with anti-CD16/32 for blocking of FcRs and labeled with appropriate dilutions of PE-conjugated CD1d^PBS57-tetramer (a gift from Drs. M. Verykokakis and B. L. Kee, University of Chicago, Chicago, IL) and TCRβ-APC (H57-597; purchased from eBioscience) for 45 min at 4°C in the dark. Cells were washed and resuspended in PBS/5% FBS. For IL-4 intracellular staining, DN32.D3 cells were treated with 200 ng/ml αGalCer in the presence or absence of TPL2 inhibitor. Secretion of cytokines was blocked by either momensin or brefeldin A protein transport inhibitors (eBioscience). Cells were stained with cell surface markers and then fixed and permeabilized with eBioscience fixation and permeabilization buffers, according to the manufacturer’s intracellular staining protocol. Flow cytometric analysis was performed and acquired on a FACSCalibur (BD Biosciences), and data were analyzed using the FlowJo software (Tree Star).

The TCRβ⁺CD1d tetramer⁺ NKT cell population and CD11c⁺ dendritic cells (CD11c⁺ DCs) were sorted from LMNCs of tpl2^+/+ and tpl2^−/− mice using the high-speed cell sorter MoFlo (DakoCytomation). Tpl2^+/+ and tpl2^−/−CD11c⁺ DCs (purity > 90%) were treated with αGalCer to the final concentration 100 ng/ml for 18 h. Tpl2^+/+ and tpl2^−/− αGalCer–pulsed DCs were cocultured with purified tpl2^+/+ and tpl2^−/− NKT cells (purity > 95%) in all possible combinations at ratio 1:2 for 24 h. Coculture supernatants were collected and analyzed for IL-4 and IFN-γ secretion.

LMNCs were isolated from tpl2^+/+ and tpl2^−/− mice as described above. NKT cells were enriched from hepatic MNCs by magnetic cell sorting (130-096-513; Miltenyi Biotec). In brief, CD3-positive cells were enriched by negative selection (MACS), according to the manufacturer’s protocol. CD3-enriched cells were stained with NK1.1 APC mAb and incubated with anti-APC microbeads, and NK1.1-positive cells were enriched by positive magnetic cell sorting according to the manufacturer’s recommendations. Approximately 90% of the magnetic cell sorting–purified cells were CD3 and NK1.1 positive. Purified tpl2^+/+ or tpl2^−/− NKT cells (0.5 × 10⁶) were injected i.v. into CD1d-deficient mice, respectively. Con A was injected at a dose of 10 mg/kg, mice were then euthanized 8 h postinjection, and their sera and livers were analyzed. Liver injury was assessed by measuring serum levels of ALT/AST transaminases.

Primary murine LMNCs and splenocytes were cultured in RPMI 1640 supplemented with 10% FBS, 1% nonessential amino acids, 1% penicillin-streptomycin, 1% sodium pyruvate, and 50 μM 2-ME (all purchased by Life Technologies) and treated with 100 ng/ml with αGalCer at the indicated time points. The Va14⁺ CD1d-specific NKT hybridoma cell line DN32.D3 was a gift from Dr. A. Bendelac (University of Chicago, Chicago, IL). DN32.D3 cells were maintained in hybridoma medium consisted of RPMI 1640 supplemented with EHAA (Sigma-Aldrich), 10% FBS, 1% penicillin-streptomycin, 1% l-glutamine (Sigma-Aldrich), gentamicin (Sigma-Aldrich), and 50 μM 2-ME (Life Technologies). Cells were treated with DMSO (Applichem) as vehicle control, 5 μM TPL2 kinase inhibitor (Calbiochem, Merck-Millipore), 5 μM UO126 (Calbiochem, Merck-Millipore), or 0.5 μM wortmannin (Calbiochem, Merck-Millipore) 30 min before stimulation with 200 ng/ml αGalCer for various time points. In another set of experiments, cells were treated with 10 μg/ml purified anti-mouse IL-4 or/and 10 μg/ml purified anti-mouse IFN-γ and were stimulated or not with 200 ng/ml αGalCer for further analysis.

Following treatment, cells or liver tissues were lysed in radioimmunoprecipitation assay buffer, containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitors (Sigma-Aldrich), 100 mM Na₃VO₄ (Sigma-Aldrich), and 1 mM NaF (Sigma-Aldrich). For Western blot analysis, levels of total proteins were determined using the bicinchoninic acid assay (Thermo-Scientific). For fractionation of cytoplasmic and nuclear protein extracts, cells were lysed in buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA, and 10% Nonidet P-40) supplemented with protease inhibitors and 100 mM Na₃VO₄ and 1 mM NaF. Lysates were rotated on a platform for 15 min at 4°C, and the cytosolic fraction was purified by centrifugation at 13,000 rpm at 4°C for 4 min. The pellet of nuclei was resuspended in buffer B (20 mM HEPES [pH 7.9], 0.4 M NaCl, 0.1 mM EDTA, 10% glycerol, protease inhibitors, 100 mM Na₃VO₄, and 1 mM NaF), and extracts were incubated on ice for 30 min. The nuclear fraction was purified by centrifugation at 13,000 rpm at 4°C for 4 min. Cytoplasmic and nuclear protein concentration was measured using the Bio-Rad DC protein assay kit. An amount of 30 μg/sample was loaded and subjected to SDS-PAGE and subsequently transferred to a nitrocellulose membrane (Whatman) following blocking with 5% nonfat milk in TBS-T before incubation with primary Ab. The following Abs were used for immunoblotting: p-ERK1/2 (CS4370; Cell Signaling Technology), pp38 (CS4511; Cell Signaling Technology), pJNK1/2 (CS4668; Cell Signaling Technology), pAKT (CS4060; Cell Signaling Technology), pGSK3β-Ser9 (CS5558; Cell Signaling Technology), pSTAT1 (CS9171; Cell Signaling Technology), pSTAT3 (CS9131; Cell Signaling Technology), pSTAT6 (CS9361; Cell Signaling Technology), ERK1/2 (CS4695; Cell Signaling Technology), p38 (CS8690; Cell Signaling Technology), JNK1/2 (CS9258; Cell Signaling Technology), AKT (CS2920; Cell Signaling Technology), STAT1 (610115, BD Transduction Laboratories.), STAT3 (CS9132; Cell Signaling Technology), STAT6 (CS9362; Cell Signaling Technology), NFATc1 (CS5861; Cell Signaling Technology), Sp1 (PEP-2; Santa Cruz Biotechnology), TPL2 (M-20; Santa Cruz Biotechnology), IκBα (CS4814; Cell Signaling Technology), p-IκB kinase (IKK)α/β (CS2697; Cell Signaling Technology), IKKβ (CS8943; Cell Signaling Technology), and β-actin (clone X-4; Millipore). Secondary HRP-conjugated Abs were purchased by Sigma-Aldrich and used at a concentration of 1:20,000. ECL method (PerkinElmer Life Sciences) was used for signal development.

Cytokine concentration in serum and cell culture supernatants was determined by ELISA at the indicated time points using ELISA kits for mouse IL-4, IFN-γ, TNF, IL-6, IL-2, and IL-12 (all purchased by eBioscience), according to the manufacturer’s instructions. Colorimetric reactions were stopped by the addition of 1 N HCl, and the optical absorbance at 450 and 570 nm was determined using a microplate absorbance reader (Model 680 Microplate Reader; Bio-Rad).

RNA was isolated from NKT hybridoma cell line DN32.D3 or liver tissue using TRIzol (Invitrogen) and Nucleospin RNA kit (MACKEREY-NAGEL), respectively. Total RNA was quantified with a Nanodrop Spectrophotometer. A High-Capacity cDNA Reverse Transcription kit was used to synthesize cDNA from 500 ng RNA, according to the manufacturer’s protocol using the High-Capacity cDNA Archive kit (Applied Biosystems). Applied Biosystems TaqMan Universal PCR Mastermix and TaqMan gene expression probes for mouse IL-4 (Mm00445259_m1, FAM labeled), IFN-γ (Mm01168134_m1, FAM-labeled), GATA-3 (Mm00484683_m1, FAM labeled), MAF (Mm02581355_s1, FAM-labeled), T-bet (Mm00450960_m1, FAM-labeled), Fas ligand (Mm00438864_m1, FAM-labeled), JunB (Mm04243546_s1, FAM-labeled), CXCL1 (Mm04207460 _m1, FAM-labeled), CXCL2 (Mm00436450_m1), CCL3 (Mm00441259_g1, FAM-labeled), and β-actin (ACTB; Mm00607939_s1) as endogenous control (VIC-labeled) were obtained from Applied Biosystems and used on an Applied Biosystems ViiA Real-Time PCR Instrument. All assays were run in duplicate on an Applied Biosystems ViiA Real-Time PCR system, according to the manufacturer’s instructions, and the mean value was used for the analysis. mRNA levels were expressed as relative quantification (RQ) values, which were calculated as RQ = 2^(−ΔΔCt), where ΔCt is (Ct [gene of interest] − Ct [housekeeping gene]).

Data are expressed as the mean values for all mice treated similarly. Error bars represent ± SEM. The statistical significance of differences between two groups was determined using a two-tailed Student t test or the nonparametric Mann–Whitney U test where appropriate or one-way ANOVA for comparison of three or more groups, followed by Tukey’s post hoc test. The p values <0.05 were considered to be statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001). All analyses were performed using GraphPad PRISM version 5.00 (Graph Pad Software, San Diego, CA).

The physiological role of TPL2 in immune-mediated liver injury was explored using an established mouse model of fulminant hepatitis-like pathology induced by Con A (19). Wild-type (WT; tpl2^+/+) and TPL2-deficient (tpl2^−/−) mice were administered Con A i.v., and disease severity was monitored over time. WT animals progressively developed congestive livers characterized by increased blood accumulation (Fig. 1A), histological manifestations of extensive tissue damage (Fig. 1B), and elevated expression of cleaved caspase-3 (Fig. 1C, 1D). Strikingly, tpl2^−/− mice exposed to Con A displayed reduced macroscopic and histological features of liver injury (Fig. 1A–D). The serum levels of ALT and AST, which represent biochemical markers of hepatic injury (Fig. 1E) and of the proinflammatory cytokines TNF-α and IL-6 (Supplemental Fig. 1A) as markers of systemic effects, were also significantly reduced in tpl2^−/− compared with WT mice treated with Con A. Con A also led to accumulation of TPL2 mRNA and protein levels in the liver of WT animals (Fig. 1F, 1G).

To determine whether the catalytic activity of TPL2 is required for the observed effects, WT mice were treated with a TPL2 kinase inhibitor prior to Con A administration. This treatment resulted in reduced hepatic congestion and significantly lower serum ALT/AST levels compared with animals receiving Con A alone (Fig. 1H, 1I). We conclude that TPL2 mediates pathogenic signals in Con A–induced liver injury, which require intact TPL2 kinase activity.

The pathogenesis of Con A–induced hepatitis critically depends on T and NKT cells (5, 19). To dissect the cellular mechanisms underlying the aforementioned pathogenic TPL2 effect, we analyzed T cell content and activation status in LMNCs isolated from WT and tpl2^−/− mice during disease progression. This analysis demonstrated progressive accumulation of CD3⁺ and CD4⁺ T (Fig. 2) in Con A–treated WT mice, which was similar to that observed in tpl2^−/− animals. Simultaneous assessment of the early activation marker CD69 also showed absence of significant differences in the activation status of CD3⁺ and CD4⁺ lymphocytes between strains (Fig. 2), indicating that conventional T cells are not the primary target of the TPL2 effect on Con A–induced liver injury.

However, detailed examination of flow cytometry data revealed a discernible variation between strains in a CD3⁺CD69⁺ subpopulation bearing intermediate CD3 expression levels, a feature of NKT cells (Fig. 2A). Because the vast majority of hepatic NKT cells in C57BL/6 mice express both CD3 and NK1.1 (CD161), a marker of NK cells (4), and CD3⁺NK1.1⁺ NKT cells confer pathogenic effects on Con A–induced liver injury (5), we analyzed tpl2^+/+ and tpl2^−/− LMNCs for CD3 and NK1.1 expression by flow cytometry. The results revealed progressive accumulation of CD3⁺NK1.1⁺ NKT cells in WT mice exposed to Con A that was attenuated in tpl2^−/− animals (Fig. 3A, 3B). Moreover, TPL2-deficient liver NKT cells expressed reduced CD69 levels compared with WT equivalents both in untreated and Con A–treated mice (Fig. 3A). CD4⁺ NKT cells displayed the same pattern of accumulation and CD69 expression as CD3⁺ NKT cells in tpl2^−/− mice (data not shown). In contrast, the percentage of CD3⁻NK1.1⁺ NK cells remained unaffected by the TPL2 deficiency (Fig. 3A). Treatment of WT mice with TPL2 kinase inhibitor prior to Con A administration also resulted in reduced accumulation of NKT but not NK cells (Fig. 3C, 3D).

LMNCs were further analyzed using CD1d-tetramers loaded with the prototypic iNKT TCR–specific glycolipid ligand αGalCer, which identify the subset of CD1d-restricted T cells expressing the invariant Vα14-Jα18 TCR. Previous studies have shown that NKT cell stimulation results in a transient downregulation of cell surface TCR that may provide a mechanism to avoid undesirable consequences of chronic NKT cell activation, including overproduction of effector cytokines (20). In line with these reported observations, Con A administration in WT animals resulted in a transient reduction in the number of LMNCs stained double positive for CD1d^tet and TCRβ⁺, which, however, was largely reversed by 8 h of treatment. In contrast, tpl2^−/− mice exposed to Con A showed a more marked and prolonged reduction in the number of CD1d^tet/TCRβ⁺ LMNCs (Supplemental Fig. 2). Assessment of CD69 expression of CD1d^tetTCRβ⁺ gated cells demonstrated reduced activation of TPL2-deficient liver iNKT cells both in untreated and Con A–treated mice (Supplemental Fig. 2).

IFN-γ and IL-4 produced by NKT cells have been reported to play important roles in Con A–induced liver pathology by mediating hepatocyte killing (6, 21, 22) and neutrophil infiltration (23), respectively. Serum levels of IL-4 and IFN-γ (Fig. 3E), accumulation of Gr1^highCD11b^high-expressing inflammatory cells in the liver (Supplemental Fig. 3A), and hepatic mRNA expression of neutrophil and monocyte chemokines such as CXCL1, CXCL2, and CCL3 (MIP1-α) (Supplemental Fig. 3B) were attenuated in Con A–treated tpl2^−/− mice. A TPL2 kinase inhibitor mirrored the effects of TPL2 ablation on Con A–induced neutrophil recruitment in vivo (Supplemental Fig. 3C).

To substantiate a role for TPL2 in mediating the pathogenic effects of Con A through CD3⁺NK1.1⁺ NKT cells, we performed adoptive transfer of WT or TPL2-deficient NKT cells into CD1d^−/− recipients (Fig. 4A), which lack NKT cells and are protected from Con A–induced fulminant hepatitis (Fig. 4B) (5). In line with previous reports (5), the partial reconstitution of the NKT cell pool in CD1d^−/− mice partly restored their responsiveness to Con A, evidenced by elevated serum ALT/AST levels (Fig. 4B) (5). In comparison, the adoptive transfer of TPL2-deficient NKT cells led to significantly reduced response of CD1d^−/− mice to Con A (Fig. 4B) with decreased levels of IL-4 and IFN-γ in the serum (Fig. 4C) and reduced numbers of infiltrating neutrophils in their livers (Fig. 4D, 4E).

NKT cells comprise two main subpopulations with opposing roles in immune-driven liver diseases, type I (iNKT) and type II NKT cells. The former confer pathogenic effects during Con A–induced hepatitis which are antagonized by type II NKT cells (24). The observation that TPL2 operates within the CD3⁺NK1.1⁺ NKT cell compartment to mediate liver injury (Fig. 4) prompted us to explore more specifically the in vivo role of TPL2 in iNKT cell function by using the prototypic iNKT TCR–specific glycolipid ligand αGalCer (25, 26). Mice immunized with αGalCer undergo mild hepatitis as reflected by the progressive increase in plasma ALT/AST, reaching maximal levels between 16 and 24 h after administration (Fig. 5A) (27). In comparison, mice lacking TPL2 were found to possess 2- to 3-fold lower serum ALT/AST levels following αGalCer administration (Fig. 5A).

iNKT cells respond to αGalCer by producing copious amounts of IL-4, IL-6, and TNF-α, followed by IFN-γ, which orchestrate the licensing and activation of other immune cells (3, 4, 7). TPL2-deficient mice exposed to αGalCer displayed significantly reduced serum levels of these cytokines (Fig. 5B). Congruent with this defect, the number of neutrophils (Fig. 5C) and the activation of liver CD3⁺, CD4⁺, and CD19⁺ cells (Fig. 5D) were attenuated in tpl2^−/− compared with WT mice challenged with this iNKT cell ligand. In vitro stimulation of TPL2-deficient splenocytes and LMNCs with αGalCer (Fig. 6A) also led to significant reduction in IL-4 and IFN-γ production compared with WT cultures (Fig. 6B).

αGalCer is presented to iNKT TCR by CD1d expressed on DCs. The diminished response of tpl2^−/− mice, splenocytes and LMNCs to αGalCer could thus be attributed to iNKT cell-autonomous mechanisms, defects in αGalCer presentation by DCs or both. To dissect cell-specific roles of TPL2 in this effect, DCs from WT and tpl2^−/− mice were exposed to αGalCer in vitro and then cocultured with tpl2^+/+ and tpl2^−/− NKT cells in all possible combinations. The results showed that cultures of tpl2^−/− NKT cells in combination with either tpl2^+/+ or tpl2^−/− DCs released lower levels of IL-4 and IFN-γ than cultures of tpl2^+/+ NKT cells (Fig. 6C). CD1d expression on DCs did not differ between strains and similar levels of IL-12 were produced by tpl2^+/+ and tpl2^−/− DCs pulsed with αGalCer in vitro when cocultured with tpl2^+/+ and tpl2^−/− NKT cells, respectively (Fig. 6D, 6E) and in αGalCer-administered tpl2^+/+ and tpl2^−/− mice in vivo (Fig. 6F). Collectively, these data demonstrate that the expression of TPL2 in iNKT cells modulates their effector function.

iNKT cells develop in the thymus through a developmental program distinct from conventional T cells (28). The earliest iNKT cell population to emerge (developmental stage 0) expresses CD24 and binds glycolipid Ag-loaded CD1d tetramer (CD1d tet⁺). In stage 1, CD24 is downregulated and the cells enter into a proliferative phase during which they up-regulate CD44 and rapidly enter stage 2. The final maturation step, stage 3, is defined by the upregulation of NK1.1 (4, 28). We examined the impact of TPL2 ablation on iNKT cell development by performing flow cytometric analysis of thymocytes isolated from WT and tpl2^−/− mice and gated in CD1d^tet+ for stage 0 and in CD24^loCD1d^tet+ for stages 1, 2, and 3. As shown in Fig. 7A and 7B, the differentiation process and numbers of mature iNKT cells in the thymus did not differ between strains. Moreover, the activation status (CD69⁺) of the thymic CD3⁺NK1.1⁺ pool was similar in WT and tpl2^−/− mice (Fig. 7C). Therefore, TPL2 does not influence iNKT cell development in the thymus. Interestingly, the tpl2 mRNA expression levels in thymic NKT cells were found to be dramatically reduced compared with liver iNKT cells (Fig. 7D), indicating that TPL2 may typify late stages of immune cell maturation, a possibility that warrants further investigation.

To gain insight into the mechanism by which TPL2 influences pathogenic iNKT cell functions, we have explored αGalCer-induced signal transduction in the mouse NKT hybridoma cell line DN32.D3 engineered to express CD1d (29). αGalCer-stimulated DN32.D3 cells express IL-2 and upregulate genes (Il4, Il10, and Ifng) encoding cytokines that define classic iNKT cells (30). Treatment of DN32.D3 cells with TPL2 kinase inhibitor prior to αGalCer stimulation led to a dramatic reduction in secreted IL-2 (Fig. 8A) in the absence of an effect on cell viability (data not shown). This treatment also led to reduced IL-4 secretion (Fig. 8A) and il4 mRNA expression (Fig. 8B) and intracellular IL-4 protein levels (Fig. 8C). Similar results were obtained for IFN-γ expression (Fig. 8A, 8B), mirroring the effects of TPL2 ablation on αGalCer responses in vivo and in primary cultures (Figs. 5, 6).

On the basis of these findings, we proceeded to identify αGalCer-induced signaling pathways affected by TPL2 kinase inhibition and define their functional roles in IL-4 and IFN-γ expression. We have found that αGalCer treatment leads to phosphorylation of ERK1/2, Akt, and its downstream target GSK3β but not of JNK, p38, or IKK/IκBα, which was attenuated by TPL2 kinase inhibition (Fig. 8D, Supplemental Fig. 4A). Reduced phosphorylation of ERK1/2, Akt, and GSK3β was also observed in αGalCer-treated primary tpl2^−/− compared with tpl2^+/+ splenocyte cultures (Fig. 8G), supporting the results of the inhibitor studies in DN32.D3 NKT hybridoma cells.

Akt is activated downstream of PI3K and ERK is directly phosphorylated by MEK. To explore the impact of the MEK-ERK and PI3K-Akt signaling pathways on IL-4 and IFN-γ expression, DN32.D3 cells were exposed to the MEK inhibitor UO126 or the PI3K inhibitor wortmannin prior to αGalCer stimulation. Inhibition of either MEK or PI3K reduced but did not eliminate secreted IL-4 compared with cells treated with αGalCer alone. This observation coupled with the partial effect of TPL2 inhibitor on αGalCer-mediated IL-4 synthesis (Fig. 8E, upper panel) indicates the contribution of TPL2-independent pathways in IL-4 regulation. In contrast, αGalCer-induced IFN-γ production was abolished by UO126 and TPL2 kinase inhibitor but remained unaffected by wortmannin (Fig. 8E, lower panel). Under these conditions of inhibitor treatment, UO126 attenuated αGalCer-mediated ERK but not Akt phosphorylation, and wortmannin inhibited Akt and its downstream target GSK3β but not ERK activation (Fig. 8D), excluding a cross-talk between these signaling pathways downstream of TPL2.

IL-4 and IFN-γ are known to activate the transcription factors STAT6 and STAT1, respectively. Exposure of DN32.D3 cells to αGalCer also led to robust phosphorylation of STAT1 and STAT6 but not STAT3, which was abolished upon neutralization of IFN-γ and IL-4 respectively (Supplemental Fig. 4B–D). Congruent with the effects of kinase inhibitors on IL-4 synthesis (Fig. 8E), treatment of DN32.D3 cells with UO126, wortmannin, or the TPL2 inhibitor attenuated the αGalCer-mediated induction of STAT6 phosphorylation (Fig. 8F, Supplemental Fig. 4B). In contrast, αGalCer-induced STAT1 phosphorylation was inhibited by treatment with UO126 or TPL2 kinase inhibitor but not wortmannin (Fig. 8F, Supplemental Fig. 4B). These data implicate both the TPL2/Akt and TPL2/ERK pathways in IL-4 regulation and identify TPL2/ERK as the signaling axis required for IFN-γ synthesis in iNKT cells.

Transcriptional activation of the IL-4 gene in T cells is orchestrated by GATA3-mediated chromatin remodeling, which enables binding of c-Maf and NFAT:AP1 to the il4 promoter. NFAT is activated by TPL2 overexpression (31), but whether it is a physiological target of TPL2 remains unknown. JunB is a component of the NFAT:AP-1 transcriptional complex that regulates il4 transactivation in mouse T cells (32, 33), is upregulated in αGalCer-stimulated iNKT cells, and required for the transcription of the IFN-γ gene (34). On the basis of these premises, we analyzed the impact of inhibition of TPL2 kinase activity on αGalCer-induced transcription factor engagement in DN32.D3 cells. The results showed that TPL2 is required for αGalCer-mediated upregulation of JunB (Fig. 9A) and the nuclear translocation of NFAT (Fig. 9B) but not of GATA3 and c-Maf (data not shown).

ERK has been implicated in upregulation of JunB in response to CD30 ligation (35) and the PI3K-Akt signaling pathway inhibits GSK3β, the protein kinase that phosphorylates NFAT and promotes its export from the nucleus (36). Because TPL2 is required for the activation of both ERK and Akt signals in αGalCer-stimulated iNKT cells (Fig. 8D, 8G), we analyzed the effect of UO126 and wortmannin on JunB upregulation and NFAT localization, respectively. As shown in Fig. 9A, inhibition of the MEK/ERK axis leads to a profound reduction in αGalCer-induced junB mRNA levels, similar to the effect of TPL2 inhibitor treatment. Immunoblot analysis of nuclear protein extracts isolated from αGalCer-stimulated DN32.D3 cells revealed a dramatic reduction in NFAT levels following TPL2 or wortmannin inhibitor (Fig. 9B) but not UO126 administration (data not shown). We conclude that the ifng and il4-regulating transcription factors JunB and NFAT are targets of the TPL2/ERK and TPL2/Akt signaling pathways, respectively (Fig. 9C).

Immune-mediated hepatitis represents a major health burden and cause of mortality worldwide (37), reflecting the need for improved understanding of the mechanisms underlying disease pathogenesis and development of evidence-based therapeutic strategies. Previous studies have revealed opposing roles for NKT subtypes in the regulation of immunogenic versus tolerogenic outcomes and identified iNKT cells as orchestrators of the initiation and maintenance of hepatic inflammation underpinning liver damage. However, the intracellular pathways that mediate the pathogenic function of iNKT cells remain obscure. In this study, we demonstrate that TPL2 kinase is a crucial signaling factor in iNKT cells and major mediator of hepatic inflammation.

A functional link between TPL2 and iNKT cell–mediated pathology was inferred by the observation that TPL2 is required for the accumulation of CD3⁺NK1.1⁺ NKT cells in the liver and the development of fulminant hepatitis in mice exposed to Con A (Figs. 1–3). Although NKT cells are phenotypically diverse, we have focused on CD3⁺NK1.1⁺ NKT cells given their established pathogenic role in liver injury in C57BL/6 mice (5). Increased numbers of hepatic NKT cells have been documented in patients with autoimmune liver diseases (38) and genetic ablation of NKT cells in the mouse alleviates the deleterious effects of Con A on liver injury (5). Activated iNKT cells produce large amounts of the effector cytokines IFN-γ and IL-4 that play important roles in Con A–induced liver pathology by mediating hepatocyte killing (6, 21, 22) and neutrophil infiltration (23), respectively. Data presented in this study show that TPL2 regulates hallmarks of iNKT cell activation in vivo, as evidenced by the reduction in circulating levels of IL-4 and IFN-γ and in hepatic accumulation of neutrophils in Con A–treated tpl2^−/− mice (Fig. 3, Supplemental Fig. 3A). In line with these associations, the adoptive transfer of tpl2^−/− NKT cells in NKT cell–deficient animals was largely ineffective in restoring susceptibility to the disease compared with that of TPL2-proficient NKT cells (Fig. 4). However, the observation that TPL2 ablation alleviates but does not abolish Con A–mediated liver injury (Fig. 1) indicates the operation of additional kinase pathways controlling immune-mediated hepatitis in this mouse model.

iNKT cells respond to glycolipid Ags presented by CD1d on APCs, including αGalCer (4, 25, 26). Mice immunized with αGalCer undergo mild hepatitis associated with elevated levels of liver transaminases and proinflammatory cytokines, IL-4–dependent neutrophil infiltration and induction of hepatocyte killing (27, 39). TNF-α and IFN-γ have been identified as important mediators of hepatic injury in this model, an effect mediated by increased expression of Fas ligand (CD178) in iNKT cells (27) and Fas (CD95) and STAT1-IFN regulatory factor 1 in affected hepatocytes (39). Data presented in this paper demonstrate that TPL2 ablation alleviates the aforementioned pathogenic effects of the iNKT-specific ligand αGalCer in vivo, including TNF-α synthesis (Fig. 5), and reduces IL-4 and IFN-γ production in primary splenocyte and LMNC cultures in vitro (Fig. 6B). Using a coculture system of tpl2^+/+ and tpl2^−/− DCs and NKT cells, we have excluded defects in αGalCer presentation by DCs as drivers of reduced effector cytokine synthesis by tpl2^−/− iNKT cells (Fig. 6C). Collectively, these observations directly link TPL2 to pathogenic iNKT cell function.

The profound protective outcomes of TPL2 ablation in experimental models of fulminant (Con A) and mild (αGalCer) hepatitis discussed above, coupled with the absence of an effect on iNKT cell development (Fig. 7), highlight TPL2 as putative target for the management of immune-driven liver pathology.

In line with this concept, administration of a TPL2 kinase inhibitor attenuates the pathological manifestations of experimental hepatitis in vivo (Figs. 1, 3) and the production of IFN-γ and IL-4 by iNKT cells in vitro (Fig. 8A, 8B, 8E). Thus, further studies are warranted to evaluate the potential of TPL2 kinase inhibitors (40, 41) to minimize the severity of inflammatory liver diseases and their comorbidities.

Which are the molecular pathways that link TPL2 to effector cytokine synthesis in NKT cells? The physiological role of TPL2 in signal transduction has largely been explored downstream of Toll-like and TNF receptors in macrophages, dendritic, and hepatic stellate cells (10–12). These studies have shown that TPL2 ablation ameliorates ERK activation and renders macrophages defective in the production of TNF and other proinflammatory molecules (10, 11). Data presented in this paper demonstrate that TPL2 is required for ERK signaling also downstream of the iNKT TCR. We show that signals transduced via the TPL2-ERK pathway regulate the transcription factor JunB (Fig. 9A), which controls the expression of the IFN-γ gene in iNKT cells and influences αGalCer-induced inflammatory liver disease in the mouse (34). Our findings also uncover a novel physiological role for TPL2 in Akt activation and show that this signaling axis promotes the nuclear accumulation of NFAT in αGalCer-challenged iNKT cells (Figs. 8, 9). Pertinent to these observations, the NFAT:JunB complex represents a major component of the il4 transcriptional machinery (32, 33). Therefore, TPL2 coordinates effector cytokine synthesis in NKT cells via ERK- and Akt-transduced signals (Fig. 9C).

An important aspect of TPL2 signaling is its interplay with regulators of the NF-κB pathway such as the IKKβ, which is required for TPL2 activation downstream of TLR4 and TNFR1 (8, 42, 43). Data presented in this paper show that αGalCer-challenged iNKT cells engage TPL2-dependent ERK signaling in the absence of an effect on the NF-κB pathway (Supplemental Fig. 4A). It is thus conceivable that alternative mechanisms may operate to regulate TPL2 signal transduction downstream of the iNKT TCR, a hypothesis that merits further investigations. The existence of alternative mechanism(s) of TPL2 activation is supported by a recent study describing TPL2-dependent but IKKβ-independent activation of ERK during TLR3 and TLR9 signaling in macrophages (44).

The findings reported in this paper thus expand our understanding of the mechanisms of iNKT cell activation by defining TPL2 as a crucial signaling factor and mediator of iNKT effector cytokine expression. The protective effects of TPL2 ablation or inhibition of its kinase activity on hepatic inflammation may have important ramifications for the development of therapies for liver diseases. Given the capacity of iNKT cells to orchestrate the licensing and activation of other immune cells, our results may also help to better appreciate the diverse and often contradictory effects of TPL2 on immune functions.

We thank Z. Vlata, N. Gounalaki, M. Ioannou, and E. Stagakis for technical support; Albert Bendelac, M. Verykokakis, and B. L Kee (University of Chicago, Chicago, IL) for reagents; and C. Tsatsanis, C. Mamalaki, and E. Drakos for helpful discussions.

The authors have no financial conflicts of interest.