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Originally published online as doi:10.1189/jlb.1006625 on April 5, 2007

Published online before print April 5, 2007
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(Journal of Leukocyte Biology. 2007;82:124-132.)
© 2007 by Society for Leukocyte Biology

Recombinant ST2 boosts hepatic Th2 response in vivo

Aldo Amatucci1, Tatiana Novobrantseva, Kevin Gilbride, Margot Brickelmaier, Paula Hochman and Alexander Ibraghimov

Biogen Idec, Cambridge, Massachusetts, USA

1 Correspondence: Biogen Idec, 14 Cambridge Center, Cambridge, MA 02142, USA. E-mail: aldo.amatucci{at}biogenidec.com


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ABSTRACT
 
Excessive scarring or fibrosis is a common feature of a wide spectrum of diseases characterized by an exaggerated Th2 response. The TLR/IL-1 receptor (IL-1R)-related protein ST2 is expressed in a membrane-bound form selectively by Th2 cells and was shown to be indispensable for some in vivo Th2 responses. ST2 was also found to block TLR signaling. We addressed the impact of the ST2 pathway on fibrogenesis using a mouse model of hepatic injury and fibrosis induced by carbon tetrachloride (CCl4). We showed that cytokine production by intrahepatic lymphocytes from CCl4-injured liver is abrogated in the absence of TLR-4. Interfering with the ST2 pathway using an ST2-Fc fusion protein accelerated and enhanced hepatic fibrosis, paralleled by the increasing ex vivo secretion of Th2 cytokines IL-4, -5, -10, and -13 by intrahepatic lymphocytes of ST2-Fc-treated, CCl4-gavaged mice. Absence of IL-4/13 signaling in IL-4R{alpha}-deficient mice obliterated this ST2-Fc effect on fibrogenesis. Moreover, depletion of CD4+ T cells abrogated ST2-Fc-enhanced Th2 cytokines and accelerated fibrosis. Thus, ST2-Fc caused overproduction of Th2 cytokines by intrahepatic CD4+ T cells, possibly by modifying TLR-4 signaling in injured liver. This ST2-Fc-driven Th2 response exacerbated CCl4-induced hepatic fibrosis.

Key Words: liver • fibrosis • TLR/IL-1 receptor related protein • CCl4


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INTRODUCTION
 
The IL-1 receptor (IL-1R)-related gene ST2 encodes a secreted (sST2) and a membrane-bound (ST2L) protein [1 ]. sST2 was discovered originally in fibroblasts and was later found to be secreted by human and mouse macrophages and by human Th2 cells [1 2 3 4 ]. It is most important that ST2L appears to be a stable and selective marker expressed by mouse and human Th2 cells (characterized by a discrete cytokine pattern including IL-4, -5, -9, -10, -13, -25, and -31), but not by Th1 cells [4 5 6 ]. ST2L is also expressed by mouse IL-10-secreting Type 1 regulatory T cells, IL-4-producing mastocytes, and human Type 2 cytotoxic and NK2 cell lines [7 8 9 10 ]. In vitro, ST2L expression by Th2 cells is up-regulated significantly by proinflammatory cytokines IL-6, IL-1, and TNF-{alpha} and suppressed by a Th1 cytokine IFN-{gamma} [11 , 12 ].

ST2 appears to participate in two different pathways, a direct, positive regulation of Th2 function, and suppression of Th1 inflammatory response. In the former pathway, cross-linking of ST2L on Th2 cells stimulates production of Type 2 cytokines [11 ]. The underlying mechanism remained unclear until the recent discovery of IL-33, a ST2L ligand-activating NF-{kappa}B and MAPK, and driving IL-4, -5, and -13 production by Th2 cells [13 ].

In murine respiratory mucosa, positive signaling through ST2L is indispensable for triggering and/or maintenance of Th2 response, and soluble ST2-Fc works as a decoy receptor [14 15 16 17 18 ]. Positive regulation of pulmonary Th2 response by ST2L holds true for "Th2-prone" BALB/c and for "Th1-prone" C57Bl/6 mice [5 , 14 , 16 , 18 19 20 ].

In addition to stimulating the Th2 response directly, sST2 was shown to suppress expression of TLR-1, -2, -4, and -9 by macrophages. sST2 is postulated to signal through an unknown receptor, which is up-regulated upon TLR-4 engagement [21 , 22 ]. As a result, proinflammatory cytokines IL-6, IL-12, IL-1, and TNF-{alpha} are down-regulated, and mice are protected from endotoxic shock [21 , 22 ]. Moreover, the intracellular Toll-IL-1R domain of ST2L was shown to inhibit signaling through IL-1R and TLR-4 by sequestering their adaptor protein MyD88 and MyD88 adaptor-like protein [22 ]. Thus, sST2 and ST2L are able to negatively regulate a TLR-supported Th1 response. In accordance with this model, ST2-Fc injected in vivo suppressed Th1-driven, collagen-induced arthritis and down-regulated IL-6, IL-12, IFN-{gamma}, and TNF-{alpha} [23 ].

However, the role of the ST2 pathway in regulation of Th1/Th2 balance is not always clear. Effector Th2 response to Nippostrongylus brasiliensis in the intestinal tract of ST2 knockout (KO) or sST2 transgenic (TG) mice is intact [24 ]. Similarly, treatment with ST2-Fc or with anti-ST2 mAb fails to abrogate the Th2 response in footpads of Leishmania major-infected BALB/c mice [25 ]. Together, these data suggest that the role of the ST2 pathway in immune/inflammatory reactions varies in different tissues. To evaluate this postulate, we studied ST2 regulation of repeated injury, inflammation, and resultant fibrosis in the liver, which unlike lung, has no strong predisposition to Th2 responses.

Tissue fibrosis is a pathologic process characterized by the abnormal accumulation of extracellular matrix in the interstitium, ultimately resulting in loss of function. Fibrotic tissue remodeling has been shown to depend on a Type 2-immune response in a variety of tissues, including lung [26 27 28 ], skin [29 ], and liver [30 , 31 ]. To consider the role of the ST2 pathway in hepatic Type 2 response and tissue remodeling, we studied the effects of administration of ST2-Fc on hepatic inflammation and fibrosis induced by carbon tetrachloride (CCl4), which is a known hepatotoxin, inducing acute centrilobular liver necrosis, followed by tissue repair and fibrogenesis. Our study demonstrates that in the context of CCl4-induced liver injury, exogenous ST2-Fc greatly enhances expression of Type 2 cytokines by CD4+ intrahepatic lymphocytes (IHL) and leads to a significant acceleration and increased level of hepatic fibrosis.


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MATERIALS AND METHODS
 
Mice
Male BALB/c wild-type (WT), TLR-4 mutant, CD1 KO, IL-4 KO, and IL-4R{alpha} KO mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). JH KO mice from Taconic (Germantown, NY, USA) were kept in a specific, pathogen-free facility at Biogen Idec (Cambridge, MA, USA). The Biogen Idec Institutional Animal Care and Use Committee approved all animal experimentation. Mice were 6–8 weeks old at the initiation of experiments.

Antibodies and reagents
Anti-CD4(RM4-5)-FITC, -PE, -CyChrome, and -allophycoyanin (APC); anti-CD8{alpha} (53-6.7)-FITC, -PE, and -APC; anti-CD8ß(53-5.8)-FITC, -PE, and -CyChrome; anti-CD11a(2D7)-FITC and -PE; anti-CD11b (M1/70)-FITC, -PE, and -APC; anti-CD16/32(2.4G2)-FITC; anti-CD18(C71/16)-PE; anti-CD19(1D3)-FITC and -PE; anti-CD25(PC61)-PE and -APC; anti-CD44(IM7)-FITC, -PE, and -CyChrome; anti-CD45RB(16A)-FITC and -PE; anti-CD49a (Ha31/8)-FITC; anti-CD49b (Ha1/29)-FITC; anti-CD49b(DX5)-FITC and -PE; anti-CD49d (R1-2)-PE; anti-62L(MEL-14)-FITC, -PE, and -APC; anti-CD54(3E2)-PE; anti-CD69 (H1.2F3)-FITC and -PE; anti-CD90.2(53-2.1)-PE; anti-CD103(M290)-PE; anti-CD122 (5H4)-FITC; anti-CD223(C9B7W)-PE; anti-Ly-6G(RB6-8C5)-PE and -APC; anti-TCRCß (H57-597)-CyChrome and -Allophycocyanin; and anti-{gamma}{delta}TCR(GL3)-FITC and -PE mAb were purchased from BD Biosciences (San Diego, CA, USA). Rat anti-CD4 mAb GK1.5 for in vivo cell depletion and anti-T cell Ig mucin (TIM)-3(8B.2C12)-PE were purchased from eBioscience (San Diego, CA, USA). Mineral oil, CCl4, LPS, and collagenase IV were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Passive lysis buffer and luciferase substrate were purchased from Promega (Madison, WI, USA). Percoll was purchased from Amersham (Uppsala, Sweden). ELISA kits for detection of mouse IL-2, -4, -5, -6, -10, -12, -13, -17, and -21, TNF-{alpha}, IFN-{gamma}, and RANTES were purchased from R&D Systems (Minneapolis, MN, USA). ELISA antibodies and purified standards for IL-9 and IL-18 were purchased from BD PharMingen (San Diego, CA, USA).

Generation of recombinant mouse ST2-Fc
The DNA sequence containing the extracellular region of murine ST2L was generated by RT-PCR from the total RNA of BALB-3T3 cells serum-starved for 36 h, followed by stimulation with 10 µg/ml cyclohexamide and 10% serum for 20 h. The RT-PCR reaction was performed using primers 5'-TGCCATTGCCATAGAGAGAC-3' (forward) and 5'-TAGTAGATGCTTCGGTGATC-3' (reverse) to amplify the region encoding amino acids 1–336 (GenBank Accession Number D13695). The obtained ST2L fragment was linked to a murine (m)IgG2a-Fc fragment and cloned into the CH269 transient expression vector. ST2-Fc protein was purified using protein A affinity purification.

Induction of hepatic fibrosis and mouse treatment
To induce chronic liver injury, mice were administered CCl4 in mineral oil by gavage (1.75 ml/kg) in a total volume of 200 µl weekly for 5 or 6 weeks. Control mice were treated with mineral oil only. In acute experiments, mice were killed on Days 1, 3, or 5 after a single dose of CCl4 and in some experiments, 1 day after the second weekly dose of CCl4 (Day 8). Some mice were injected i.p. with 200 µg ST2-Fc or control mouse IgG2a at Day –3, relative to the first dose of CCl4, and then twice per week for the duration of CCl4 treatment. In acute experiments, mice were injected with ST2-Fc on Days –3 and 0. Hepatotoxicity was assessed by alanyltransferase (ALT) analysis in sera 24 h after the first dose of CCl4.

Immunohistochemistry (IHC)
Three different liver lobes were taken from each mouse and incubated in 4% paraformaldehyde in PBS for 2 days prior to embedding for immunohistochemical analysis. General tissue damage was illustrated by H&E staining and apoptotic areas at early time-points by TUNEL staining.

TUNEL staining was performed using an ApopTag in situ apoptosis detection kit (Chemicon International, Temecula, CA, USA) according to the manufacturer’s instructions. Labeled apoptotic cells were detected using 3',3'-diaminobenzidine (DAB)/nickel chloride as the substrate. Slides were counterstained for 5 min with methyl green (Vector Laboratories, Burlingame, CA, USA). Hepatic collagen deposition was visualized using Sirius red staining of paraffin-embedded liver sections [32 ].

Kupffer cells and infiltrating macrophages were detected by staining for F4/80, and F4/80-specific antibody (Clone CI:A3-1, Serotec Inc., Raleigh, NC, USA) was used at 20 µg/ml. Tissue sections were pretreated with proteinase K (DakoCytomation, Denmark) for 5 min at room temperature. Binding of primary antibody was detected using a Vector Elite ABC kit (Vector Laboratories), using DAB substrate. Slides were counterstained with Mayer’s hematoxylin for 1 min.

Transformed stellate cells were detected by staining for {alpha}-smooth muscle actin ({alpha}SMA). Antibody specific for {alpha}SMA (Clone 1A4, DakoCytomation) was used at a 1:50 dilution with 30-min incubation. Heat-induced epitope retrieval pretreatment of tissue sections was performed in 10 mM citrate buffer, pH 6.0, for 30 s at 125°C, kept at 90°C for 10 s, and cooled to room temperature for an additional 20 min prior to immunostaining. Binding of primary antibodies to tissue elements was detected using a Mouse on Mouse biotinylation kit (Biocare Medical, Concord, CA, USA) with DAB substrate. Slides were counterstained with Mayer’s hematoxylin for 1 min.

Interstitial collagen quantification
A total of three sections from the liver (each from a different lobe) was stained for each mouse. Black and white pictures of Sirius red staining were made in polarized light at x250 magnification. Tissue occupied the entire area captured by the camera so that total image area was identical in each picture (four to 10 pictures per liver). Vasculature normally containing collagen was removed electronically from each image, and the amount of interstistial collagen (white staining) was quantified using MetaMorph image analysis software (Universal Imaging Corp., Downingtown, PA, USA). Quantification is displayed in arbitrary units (one correlates to 1000 pixels).

Isolation and in vitro culture of intrahepatic lymphocytes
Livers were perfused through portal vein with PBS and excised, gall bladders removed, and the rest of the tissue homogenized in RPMI/5% FBS, centrifuged, resuspended in 0.02% collagenase IV, and incubated for 1 h at 37°C with constant shaking. Samples were diluted with cold RPMI, and hepatocytes were sedimented at 500 rpm for 2.5 min and discarded. Lymphocytes were purified using discontinuous Percoll gradient 45%/70%, washed in sterile media, and used for four-color flow cytometric analysis or for in vitro culture with or without anti-CD3{epsilon} mAb for 48 h. Supernatants were analyzed for cytokines by ELISA.

In vitro inhibition of TLR-4 signaling by ST2-Fc
RAW264.7/NF-{kappa}B.luc Clone 31 was generated at Biogen Idec. Briefly, RAW 264.7 cells (ATCC TIB-71, American Type Culture Collection, Manassas, VA, USA) were cotransfected using Lipofectamine 2000 with the reporter NF-{kappa}B luciferase (Clontech, Palo Alto, CA, USA) and a plasmid containing theneomycin-resistance gene (10:1), subjected to selection in 250 mg/ml Geneticin (G418), and cloned. For the assay, cells plated in a 96-well plate at 8 x 104 cells/well overnight in triplicates were supplemented with media containing 10 ng/ml LPS alone, in combination with the ascending amounts of ST2-Fc or control IgG or with media alone. After incubation for 4 h at 37°C and removal of the media, cells were lysed, luciferase substrate was added, and the reading was performed on a luminometer Wallac 1450 MicroBeta Jet.


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RESULTS
 
Recombinant ST2-Fc favors a Type 2 response by intrahepatic lymphocytes in CCl4-treated mice
To explore the effect of ST2-Fc on an inflammatory liver response, mice gavaged with CCl4 at Days 0 and 7 were injected i.p. with 200 µg soluble ST2-Fc or control mouse IgG Days –3, 0, 3, and 7. Livers harvested from mice killed on study at Days 1, 3, 5, and 8 were used for isolation of IHL and for histology studies. IHL were analyzed by flow cytometry and stimulated in vitro with anti-CD3{epsilon} mAb for 48 h. Supernatants from ex vivo-stimulated IHL were screened for the presence of Th1 cytokines IL-2, -12, and -17 and IFN-{gamma}, Th2 cytokines IL-4, -5, -9, -10, -13, and -21, and proinflammatory cytokines IL-6, TNF-{alpha}, and RANTES. As shown in Figure 1A , CCl4-induced liver injury without ST2-Fc treatment results in a nonpolarized response, as judged by a modest production of Th1 cytokines (IL-2 and –17 and IFN-{gamma}), Th2 cytokines (IL-4, -10, and -13), as well as IL-6 and RANTES. ST2-Fc but not control IgG treatment selectively enhanced secretion of Type 2 cytokines IL-4, -5, -10, and -13, but not IL-9 and IL-21, by ex vivo-stimulated IHL from CCl4-injured livers (Fig. 1A , and not shown). These data suggest that exogenous ST2-Fc promotes a Th2 response in IHL activated by an ongoing hepatic injury. The requirement for activation in vivo is supported by the following observation. Following CCl4-inflicted injury at Day 0, the Th2-enhancing effects of ST2-Fc begin to fade from Days 1–5 and only rebound following the second CCl4 gavage at Day 7, in spite of the ST2-Fc boost at Day 4.


Figure 1
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  		Figure 1. ST2-Fc promotes a Th2 response in IHL from CCl4-injured liver. IHL isolated from individual livers 24 h after 1.75 ml/kg CCl4 gavage were cultured in duplicates for 48 h in the presence of soluble anti-CD3{epsilon} antibody. Supernatants from individual samples were analyzed for cytokines by ELISA. Each treatment group contained four animals. Samples from control (oil or oil+ST2-Fc-treated) mice cultured with anti-CD3{epsilon} as well as all samples cultured without anti-CD3{epsilon} did not produce cytokines, except for background levels of IL-6 and IL-10 and are not shown. Bars show a mean of quadruplicate values. Error bars show standard deviation. Solid bars, (A and B) CCl4 + ST2-Fc (200 µg per dose i.p.)-treated group; (C) CCl4 + control IgG-treated group. (A) Cytokines were measured in IHL samples from mice killed 24 h following gavage with CCl4 on Day 0 ± ST2-Fc injected on Days –3 and 0. Measurement for IL-9, IL-12, IL-18, IL-21, and TNF-{alpha} never yielded any activity above background. A representative experiment out of 10 is shown. (B) IL-4 and IL-13 were measured for IHL samples from mice treated with CCl4 on Days 0 and 7 ± ST2-Fc on Days –3, 0, 3, and 7 and killed on Days 1, 3, 5, and 8. A representative experiment out of four is shown. (C) IL-4 was measured for IHL isolated on Day 1 from groups of mice with CCl4 treatment on Day 0 combined with ST2-Fc on Days –1, –2, –3, or –3 and 0. Solid and open bars represent individual mice (two per group) and show a mean from duplicate culture samples. Data from a representative experiment out of two are shown.

A kinetic study showed that enhancement of the Th2 response by IHL isolated 1 day after CCl4 gavage is observed following a single dose of ST2-Fc on Day –3 (relative to gavage; Fig. 1C ). However, delaying ST2-Fc treatment to Days –2 or –1 or to the day of CCl4 gavage resulted in little if any effect. These data suggest that it takes several days for ST2-Fc to preset conditions needed for Th2 polarization of IHL, but once the milieu is created, polarization occurs within 24 h postinjury.

Recombinant ST2-Fc does not affect hepatic injury but accelerates the onset of fibrosis in CCl4-treated mice
Analysis of ALT activity in sera 24 h post-CCl4-induced injury shows no shift in hepatotoxicity of ST2-Fc-treated mice (Fig. 2A ). This observation is in good agreement with TUNEL and H&E staining, both of which fail to reveal a difference in the amount of apoptotic or otherwise damaged cells in liver at Day 1 (Fig. 2B , and data not shown). Immunohistochemical staining for F4/80 at Days 1–8 postinjury shows no significant difference in numbers or distribution of Kupffer cells (a potential source of TGF-ß) in injured livers of CCl4/ST2-Fc-treated mice as compared with CCl4/control IgG-treated mice (Fig. 2C) . Nevertheless, staining for {alpha}SMA shows more activated myofibroblasts (collagen-producing cells) in ST2-Fc-exposed, injured livers by Day 5 (Fig. 2D) . In accordance with that, Sirius red staining reveals a significant increase in the level of collagen deposition of ST2-Fc-treated livers 8 days after CCl4 injury (Fig. 3 ). Different types of tissue staining shown at specific time-points reveal the expected succession of events from initial injury to macrophage/Kupffer cell activation, which in turn induces transformation of slellate cells into myofibroblasts and result in collagen deposition (Supplemental Fig. 1 ). Together, with the in vitro data, this suggests that ST2-Fc treatment affects neither early CCl4-induced hepatic injury nor the magnitude of macrophage activation but rather enhances Th2 response to accelerate hepatic fibrosis.


Figure 2
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  		Figure 2. Effect of ST2-Fc on hepatic injury and repair. Mice were treated with oil (n=2), oil + ST2-Fc (n=2), CCl4 + ST2-Fc (n=7), or with CCl4 + control IgG (n=7). Oil or CCl4 was given by gavage at Day 0 and 200 µg ST2-Fc or IgG at Days –3, 0, and 3 relative to oil or CCl4 gavage. The experiment was performed four times with similar results. (A) CCl4 + IgG and CCl4 + ST2-Fc groups were bled 24 h after CCl4 gavage, and ALT levels in sera were measured. Magnification for representative sections for apoptotic cell-specific TUNEL was performed at Day 1 (B), macrophage/Kupffer cell-specific F4/80 performed at Day 5 (C), and myofibroblast-specific {alpha}SMA performed at Day 5 (D); original, x50. All time-points were relative to oil or CCl4 administration.


Figure 3
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  		Figure 3. ST2-Fc enhances early fibrogenesis in injured liver. Mice were killed, and liver sections were prepared on Day 8 from oil, CCl4 + control IgG, or CCl4 + ST2-Fc-treated mice. Oil or CCl4 was given by gavage at Days 0 and 7 and 200 µg ST2-Fc or IgG at Days –3, 0, 3, and 7. Each group contained three mice. The experiment was performed twice with similar results. Collagen-specific Sirius red staining was performed to visualize interstitial collagen deposition. A representative section for each treatment group containing three mice is shown at original magnification, x50. Quantification of collagen was performed using MetaMorph image analysis software. CCl4 + control IgG and CCl4 + ST2-Fc treatment groups, respectively, revealed a threefold and 4.5-fold increase in the amount of interstitial collagen deposition relative to the oil treatment group.

Recombinant ST2-Fc maintains Th2 commitment and enhances collagen deposition in chronically injured liver
We next assessed whether the ST2-Fc-enhanced Th2 response can be maintained in chronically injured liver to accelerate fibrosis as measured by increased levels of collagen deposition. Mice gavaged weekly with CCl4 for 5 weeks were dosed with ST2-Fc or control IgG twice a week for the duration of the experiment. At the termination of experiment, ex vivo-stimulated IHL from CCl4/ST2-Fc-treated but not from CCl4/IgG-treated mice demonstrated a selectively exaggerated Th2 response, including IL-4, IL-13, and to a lesser extent, IL-10. Th1 or inflammatory cytokines such as IFN-{gamma}, IL-6, and RANTES were not affected by ST2-Fc (Fig. 4A ). All the changes were similar to that seen during the first week of treatment. Sirius red staining revealed a profound increase in the amount of fibrillar collagen depositions in the livers of the ST2-Fc-treated mice as compared with livers of the control group of mice (Fig. 4B and 4C) .


Figure 4
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  		Figure 4. ST2-Fc enhances fibrosis in chronically injured liver. Mice were treated for 5 weeks with weekly doses of oil (0.2 ml) or CCl4 (1.75 ml/kg) in combination with biweekly doses of control IgG or ST2-Fc (200 µg). The first dose of antibody was delivered 3 days before the first oil/CCl4 dose. Twenty-four hours after the fifth weekly gavage with CCl4 or oil, three mice from each group of eight were killed, IHL were isolated, and cytokine production by IHL was analyzed. Twenty-four hours after the fifth weekly gavage with CCl4 or oil, five mice of each group were killed, and collagen-specific Sirius red staining of liver sections was performed to quantify interstitial collagen deposition. (A) Supernatants from ex vivo-stimulated IHL were analyzed for secretion of IFN-{gamma}, IL-6, RANTES, IL-10, IL-4, and IL-13. Bars show a mean of triplicate values; error bars show SD. (B) A representative section for CCl4 + IgG and for CCl4 + ST2-Fc groups is shown at original magnification, x50. (C) Quantification of stained collagen was performed using MetaMorph image analysis software. A column of symbols represents a series of sections from one mouse. Mean values are depicted as bars.

CD4+ cells are required for ST2-Fc-induced Type 2 response and fibrosis in CCl4-injured liver
Extensive four-color flow cytometry analyses of the IHL isolated from CCl4-injured livers were performed. Major T cell subsets ({alpha}ßTCR+CD4+, {alpha}ßTCR+CD8{alpha}+ß+, {alpha}ßTCR+CD8{alpha}+ß–, {alpha}ßTCR+CD4–CD8{alpha}–, {gamma}{delta}TCR+CD8{alpha}+, and {gamma}{delta}TCR+CD8{alpha}–) were analyzed for the expression of major T cell coreceptors (CD2, CD5, CD28, and CD90), activation markers (CD25, CD44, CD54, CD62L, CD69, and CD223), and integrins/differentiation markers (CD11a, CD18, CD29, CD49a, CD49b, CD49d, ß7, CD103, CD16/32, CD122, and TIM-3). Additional analysis included IH B cells, macrophages/Kupffer cells, and granulocytes. Significant differences in IHL composition resulting from ST2-Fc treatment were not observed at early time-points or after 5 weeks of treatment.

To address the cellular source of Type 2 cytokines, we used a depleting anti-CD4 mAb to render WT BALB/c mice CD4+ T cell-deficient. Administration of the anti-CD4 antibody at Days –5, –3, and 0, relative to CCl4 gavage, eliminates 93–97% of CD4+ cells in spleen and liver (data not shown) and results in complete abrogation of IL-4, -5, -10, and -13 secretion by IHL from the CCl4/ST2-Fc-treated group (Fig. 5A ). Furthermore, staining hepatic tissue for collagen at Day 8 post-CCl4 gavage shows that CD4+ T cell depletion prevents the acceleration of fibrosis caused by ST2-Fc treatment (Fig. 5B) .


Figure 5
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  		Figure 5. ST2-enhanced Th2 response and fibrosis are mediated by CD4+ IH T cells. Mice were injected i.p. with 100 µg depleting anti-CD4 antibody or with a control rat IgG on Days –5, –3, and 0, gavaged with 1.75 ml/kg CCl4 on Days 0 and 7, and injected with 200 µg ST2-Fc or control mIgG on Days –3, 0, 3, and 7. Data from a representative experiment of three are shown. (A) Supernatants from ex vivo-stimulated IHL harvested at Day 1 were analyzed for secretion of IL-4, IL-5, IL-10, and IL-13. Bars show a mean of triplicate values; error bars show SD. (B) Collagen-specific Sirius red staining of liver sections was performed to quantify interstitial collagen deposition. Quantification of stained collagen was performed using MetaMorph image analysis software. A column of symbols represents a series of sections from one mouse. Mean values are depicted as bars. *, P < 0.01.

We wanted to discriminate between CD4+ Th lymphocytes and CD4+ NKT cells, which constitute up to 50% of the total IHL CD4+ population. To address a possible role of invariant (i)NKT cells, we used CD1 KO mice, where iNKT cells do not develop. As shown in Figure 6 , the absence of iNKT cells does not result in a significant shift in Th2 cytokine production stimulated by ST2-Fc.


Figure 6
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  		Figure 6. IH iNKT cells are not the major source of ST2-enhanced Th2 response. WT and CD1 KO Balb/c mice were gavaged with oil or with 1.75 ml/kg CCl4 on Day 0, injected i.p. with 200 µg control IgG or ST2-Fc on Days –3 and 0, and killed on Day 1. A representative experiment out of three is shown. Supernatants from ex vivo-stimulated IHL were analyzed for secretion of IL-4, IL-5, IL-6, IL-10, and IL-13. Bars show a mean of quadruplicate values; error bars show SD. When averaging all the experiments performed, we came up with the 2.5, 0.7, 0.9, 0.6, and 1.2 mean fold reduction in the cytokine levels for IL-4, IL-5, IL-6, IL-10, and IL-13, respectively, in CD1–/–, when compared with WT. None of the differences seen is statistically significant.

These results indicate that the CD4+ T lymphocyte compartment of IHL is the major factor of accelerated fibrogenesis and an important source of Type 2 cytokines induced by ST2-Fc in injured liver and suggest that it is through their Th2 cytokine response that CD4+ IH T lymphocytes enhance hepatic fibrosis.

Profibrotic activity of ST2-Fc is driven by Th2 cytokines
To evaluate whether enhanced Th2 cytokine production was indeed a trigger of fibrogenesis or rather a side-effect of ST2-Fc treatment, we extended our studies of CCl4 or CCl4/ST2-Fc treatment to IL-4R{alpha} KO mice. The IL-4R{alpha} subunit is shared by the IL-4R and IL-13R, and as a result, IL-4R{alpha} KO mice lack IL-4 and IL-13 signaling. An accelerated hepatic collagen deposition caused by ST2-Fc by Day 8 postinjury is attenuated significantly in IL-4R{alpha} KO livers (Fig. 7 ). Thus, the ability of ST2-Fc to trigger hepatic fibrosis in injured liver is in fact mediated by Th2 cytokines.


Figure 7
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  		Figure 7. ST2-Fc uses IL-4/IL-13 signaling to enhance hepatic fibrosis. IL-4R{alpha} KO and WT Balb/c mice were gavaged with oil or with 1.75 ml/kg CCl4 on Days 0 and 7 and injected i.p. with 200 µg control IgG or ST2-Fc on Days –3, 0, 3, and 7 and were killed on Day 8. The experiment was performed three times with similar results. A representative section for each treatment group stained for interstitial collagen with Sirius red is shown at original magnification, x50.

Cytokine response in CCl4-injured liver is TLR-4-mediated
As ST2 activity was attributed to its ability to modulate TLR-2, -4, and -9 signaling [22 ], and TLR-4 engagement was shown to tolerize against subsequent CCl4-induced liver injury [33 ], we wanted to see whether TLR-4 is an important factor in CCl4-triggered inflammation. To address this question, we assessed the effects of ST2-Fc administration to TLR-4-deficient, CCl4-gavaged BALB/c mice. In contrast to WT mice, in the absence of TLR-4, cytokine secretion by IHL from CCl4-treated and CCl4/ST2-Fc-treated groups was abrogated completely or reduced drastically (Fig. 8 ). The fact that dramatic reduction occurs to all Th1, Th2, and inflammatory factors points to TLR-4 as an indispensable mediator of acute inflammation in CCl4-injured liver.


Figure 8
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  		Figure 8. Cytokine response of IHL from CCl4-injured liver is TLR-4-dependent. WT and TLR-4-deficient Balb/c mice were gavaged with oil or with 1.75 ml/kg CCl4 on Day 0. IgG or ST2-Fc (200 µg) was injected i.p. on Days –3 and 0, and IHL were harvested on Day 1. A representative experiment out of three is shown. Supernatants from ex vivo-stimulated IHL were analyzed for secretion of IL-2, IL-4, IL-6, IL-10, IL-13, IL-17, and IFN-{gamma}. Bars show a mean of triplicate values; error bars show SD.

Although the mechanism, whereby ST2L can regulate TLRs and IL-1R, was ascribed to its ability to sequester adaptor protein MyD88 [22 ], similar activity of soluble ST2 remains less characterized. To demonstrate that ST2-Fc can interfere directly with TLR-4 signaling and to address a mechanism of its action, we used a TLR-4+ monocyte cell line RAW264.7 with a luciferase gene introduced under the control of the NF-{kappa}B promoter. NF-{kappa}B activation, induced in RAW cells by LPS, was assessed by luciferase activity. In our experiments, ST2-Fc demonstrated significant inhibition of TLR-4-mediated NF-{kappa}B activation in a dose-dependent manner (Supplemental Fig. 2 ), suggesting that soluble ST2 is able to disrupt the proinflammatory cascade downstream from TLR-4.


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DISCUSSION
 
In the present study, we investigated the effect of exogenous IL-1R-related protein ST2 on an intrahepatic Th2 response. The results presented in this paper show that ST2 selectively stimulated production of Type 2 cytokines IL-4, -5, -10, and -13 by intrahepatic CD4+ T cells of chemically injured liver. Although ST2-Fc was injected i.p., it had no effect on splenic or mesenteric lymph nodes’ lymphocytes and elicited Th2 response in the liver only as long as this tissue sustained massive injury 24–72 h post-ST2-Fc injection. Th2 cytokines peaked 24 h after injury and then faded in spite of repeated injections of ST2-Fc. These data suggested that ST2-Fc induced mediator(s), which in the milieu created by massive hepatocyte death, drove rapid Th2 differentiation.

Continuous ST2-Fc treatment, accompanied by repeated CCl4-induced hepatic injury, leads to accelerated and enhanced liver fibrosis as compared with mice receiving CCl4 and control IgG. Acceleration of hepatic fibrosis by ST2-Fc was mediated by CD4+ T cells and by IL-4/IL-13 signaling through IL-4R{alpha}.

The fact that an exaggerated Th2 response resulted in enhanced hepatic fibrosis is not particularly surprising. Fibrosis affects different tissues with a domination of Type 2 cytokine production, including IL-4, -5, -10, and -13 [30 , 34 35 36 37 38 ]. There are several pathways used by Type 2 cytokines to support fibrogenesis. IL-13 can induce tissue fibrosis via direct stimulation of collagen production by activated fibroblasts [35 , 36 ], as well as through activation of the major fibrogenic cytokine TGF-ß [38 ]. Moreover, IL-4 and IL-13 were shown to trigger an arginase I-dependent synthetic pathway in alternatively activated macrophages and dendritic cells (DC) [39 , 40 ], leading to production of proline, an essential amino acid for collagen synthesis. IL-10 has also demonstrated a fibrogenic activity in lung, reportedly mediated by IL-4 and IL-13 [37 ].

The ability of ST2-Fc to enhance a Th2 response reported in this paper is intriguing. ST2 interference with Th1/Th2 balance has been under investigation, as ST2 was found to be a selective marker of Th2 cells [5 , 6 ]. Multiple in vivo studies addressing this issue produced seemingly contradictory data. Overproduction of TG sST2 or injection of recombinant ST2-Fc efficiently blocked allergen-induced Th2 response in pulmonary mucosa [5 , 14 , 18 ] or suppressed Th1 inflammation in arthritic joints [23 ] but failed to affect Th2 responses against L. major in footpads [25 ] or against N. brasiliensis in intestinal mucosa [24 ] and greatly enhanced the Th2 response in injured liver (this study). To reconcile these data, we suggest that there are two distinct modes of action of the ST2 pathway as related to regulation of inflammation and underlying Th1/Th2 balance.

In one mode, interaction between IL-33 or an unknown ligand expressed by DC and ST2L on Th2 cells delivers positive signaling into the latter, thus eliciting a Th2 response [13 , 14 ]. Overproduced TG sST2 or exogenous ST2-Fc as well as anti-ST2 mAb or ST2 deficiency in KO mice disrupt this circuit and block the Type 2 response in pulmonary mucosa [5 , 14 , 16 , 18 19 20 ], suggesting that the ST2 pathway is indispensable for triggering/maintenance of Th2 reactions in this tissue. This is probably why other important Th2-supporting pathways such as B7RP-1/ICOS appear redundant in the lung [41 ]. Conversely, the lack of effect of ST2 on intestinal Th2 response induced by N. brasiliensis could be ascribed to this organism’s ability to secrete its own factors driving Th2 differentiation [42 ], similar to other helminthic parasites Schistosoma mansoni [43 ] and Acanthocheilonema viteae [44 ]. It is tempting to speculate that L. major also produces a factor triggering the Th2 response in BALB/c mice, thus making the ST2 pathway redundant in parasite-infested tissue.

In a different mode, sST2 or ST2L disrupts TLR-mediated signaling and thus blocks the Th1 response supported by the TLR pathway. In accordance with this paradigm, endogenous sST2 was demonstrated to be an important mediator in a negative-feedback loop controlling a TLR-4-driven, inflammatory response through an unknown, ST2-binding receptor expressed by macrophages [21 ]. Similarly, up-regulation of ST2 occurred upon TLR-2/TLR-1 or TLR-2/TLR-6 engagement and was accompanied by a decrease in Th1 cytokines IL-12 and IL-18 [45 ]. Regulatory activity of ST2 was expanded to TLR-2 and -9 and IL-1R and was ascribed to the ability of ST2L to sequester adaptor protein MyD88 shared by TLR-2, -4, and -9 and IL-1R [22 ]. Consistent with the known propensity of endogenous sST2 to modulate acute tissue inflammation [3 ], exogenous ST2-Fc, similar to sST2, was shown to suppress Th1-driven murine collagen-induced arthritis [23 ], endotoxic shock [21 , 22 ], and polymicrobial peritonitis [45 ].

We find our data consistent with the TLR-blocking effect of ST2-Fc, which could result in subdued Th1 and enhanced Th2 responses. CCl4-induced hepatic injury appears to be at least in part mediated by TLR signaling [33 ], and in our experiments, CCl4-induced cytokine production by IHL was abrogated completely in TLR-4-deficient BALB/c mice. Moreover, a prior engagement of TLR-4 or IL-1R, known to induce sST2 as a negative regulatory factor [21 , 22 ], attenuates hepatic injury by CCl4 [33 ]. It is important that in mice deficient in TLR signaling, Th1-immune response is compromised [46 ] and in some cases, defaults to a Th2 response [47 ]. In the latest support of this concept, S. mansoni infection of livers of MyD88 KO mice (with disrupted TLR signaling) leads to increased Th2 response as manifested by IL-13 production and results in enhanced hepatic fibrosis [48 ]. It is thus tempting to speculate that in our study, recombinant ST2-Fc also modifies CCl4-induced TLR signaling and lets a fibrogenic Th2 response prevail. The exact mechanism of involvement of the ST2 pathway in hepatic injury will require further studies for its clarification. In summary, we have demonstrated for the first time that exogenous ST2 elicits a strong Th2 response in chemically injured liver.


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ACKNOWLEDGEMENTS
 
We thank Lynn Jackson and the Biogen Idec animal facility staff for excellent mouse colony maintenance and Humphrey Gardner, Thomas Crowell, and the Research Pathology Laboratory for the immunohistological staining and blood serum analysis. We also thank Juanita Campos and Jose Sancho for maintaining the FACS facility as well as Richard Tizard and the sequencing lab for sequencing DNA samples and oligonucleotide synthesis. Finally, we are grateful to Leonid Gorelik for helpful discussion and analysis. The authors also declare that they have no conflicting financial interests.

Received October 10, 2006; revised March 8, 2007; accepted March 18, 2007.


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