Haptoglobin Acts as a TLR4 to Suppress Osteoclastogenesis via the TLR4− IFN-β Axis

This information is current as Jun-Oh Kwon, Won Jong Jin, Bongjun Kim, Hyunil Ha, of September 29, 2021. Hong-Hee Kim and Zang Hee Lee J Immunol published online 10 May 2019 http://www.jimmunol.org/content/early/2019/05/09/jimmun ol.1800661 Downloaded from

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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 © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 10, 2019, doi:10.4049/jimmunol.1800661 The Journal of Immunology

Haptoglobin Acts as a TLR4 Ligand to Suppress Osteoclastogenesis via the TLR4–IFN-b Axis

Jun-Oh Kwon,*,1 Won Jong Jin,†,1 Bongjun Kim,* Hyunil Ha,‡ Hong-Hee Kim,* and Zang Hee Lee*

Haptoglobin (Hp), a type of acute-phase , is known to have a systemic anti-inflammatory function and to modulate inflam- mation by directly affecting immune cells, such as T cells, dendritic cells, and macrophages. However, the effects of Hp on osteoclast differentiation are not well studied, even though osteoclast precursor cells belong to a macrophage-monocyte lineage. In this study, we found that the bone volume was reduced, and the number of osteoclasts was increased in Hp-deficient mice compared with wild- type mice. Moreover, our in vitro studies showed that Hp inhibits osteoclastogenesis by reducing the protein level of c-Fos at the early phase of osteoclast differentiation. We revealed that Hp-induced suppression of c-Fos was mediated by increased IFN-b levels.

Furthermore, Hp stimulated IFN-b via a TLR4-dependent mechanism. These results demonstrate that Hp plays a protective role Downloaded from against excessive osteoclastogenesis via the Hp–TLR4–IFN-b axis. The Journal of Immunology, 2019, 202: 000–000.

aptoglobin (Hp) belongs to a class of acute-phase pro- scavenging free Hb (2, 3). In addition to a systemic anti- teins that are rapidly upregulated upon stimulation inflammatory function, recent studies have shown that Hp is in- H by cytokines such as TNF-a, IL-6, and IL-1 (1). Hp is volved in the proliferation, function, and cytokine secretion of expressed in most tissues and is mainly produced by stromal cells immune cells. The binding of Hp to CD11b/CD18, which is http://www.jimmunol.org/ and hepatocytes. The primary function of Hp is to combine with expressed in various immune cells, including dendritic cells (DCs), the free (Hb) that is released during monocytes, and macrophages, suppresses the effector functions of or healthy RBC turnover, which leads to the elimination of the macrophages and DCs, such as Ag presentation and phagocytosis Hb–Hp complex by macrophages. Free Hb reacts with H2O2 to and, at the same time, reduces the secretion of proinflammatory form reactive oxygen species, such as reactive hydroxyl radicals, cytokines, such as IL-6 (4–6). In the case of T cells, Hp directly which cause damage to tissues. Hp prevents oxidative stress by binds to activated or resting CD4+ and CD8+ T cells, thereby inhibiting proliferation (7). These results imply that Hp does not only suppress the inflammatory response but physiologically plays

*Department of Cell and Developmental Biology, Dental Research Institute, School by guest on September 29, 2021 of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea; an important role in the biological processes of immune cells. †Department of Human Oncology, University of Wisconsin School of Medicine An osteoclast is a multinuclear giant cell that is formed by the ‡ and Public Health, Madison, WI 53705; and Clinical Research Division, Korea fusion of mononuclear precursor cells of a macrophage-monocyte Institute of Oriental Medicine, 483 Expo-Ro, Yuseong-Gu, Daejeon 305-811, Repub- lic of Korea lineage. Osteoclasts have a unique ability to resorb the bone di- 1J.-O.K. and W.J.J. contributed equally to this work. rectly. Two factors are essential for the differentiation from pro- genitor cells to osteoclasts: M-CSF and the receptor activator ORCIDs: 0000-0001-5454-3391 (J.-O.K.); 0000-0003-4147-9735 (W.J.J.); 0000- 0001-7198-0411 (B.K.); 0000-0001-5947-9982 (H.H.). of NF-kB ligand (RANKL) (8, 9). These factors induce the ex- Received for publication May 9, 2018. Accepted for publication April 12, 2019. pression and activation of c-Fos, a key regulator of osteoclast This work was supported by the Basic Science Research Program through the differentiation. c-Fos raises the level of RANK, the receptor of National Research Foundation of Korea funded by the Ministry of Science, RANKL, and increases the expression of NF of activated T cells 1 ICT and Future Planning (NRF-2017R1A2B2002312) and by grants from the (NFATc1), which is another key transcription factor for osteo- National Research Foundation of Korea (NRF-2017R1A2A1A17069648 and NRF-2018R1A5A2024418 to H.-H.K.). clastogenesis (10–12). Hematopoietic progenitor cells derived J.-O.K. and W.J.J. designed the research and performed experiments. J.-O.K. drafted from c-Fos–deficient mice did not have a significant problem the manuscript. B.K. participated in the research. H.H. performed statistical analyses. differentiating into macrophages but could not differentiate into H.-H.K. provided insightful guidance to design experiments. Z.H.L. provided insight- osteoclasts (13). These results indicate that c-Fos plays an es- ful guidance and critical appraisal of the manuscript. All authors read and approved the final manuscripts. sential role in the triggering of osteoclast differentiation. Address correspondence and reprint requests to Prof. Zang Hee Lee and Prof. Hong- Diseases such as lupus, rheumatoid arthritis (RA), cancer, and Hee Kim, Department of Cell and Developmental Biology, Dental Research Institute, osteoarthritis have disease-specific features but commonly have a School of Dentistry, Seoul National University, 28 Yeongon-Dong, Jongno-Gu, Seoul chronic inflammatory response (1, 14, 15). Persistent and exces- 110-749, Republic of Korea. E-mail addresses: [email protected] (Z.H.L.) and [email protected] (H.-H.K.) sive inflammatory responses increase proinflammatory cytokines, The online version of this article contains supplemental material. such as TNF-a, IL-1, and IL-6, to more than necessary levels, accelerating the recruitment of osteoclast precursor cells and Abbreviations used in this article: BMM, bone marrow–derived macrophage; mCT, microcomputed tomography; Ct.Ar, cortical bone area; DC, dendritic cell; Hb, he- osteoclastogenesis (16–19). Moreover, inflammatory responses moglobin; Hp, haptoglobin; NFATc1, NF of activated T cell 1; N.Oc./BS, number of increase RANKL expression in fibroblast-like cells and immune osteoclasts per bone surface; OCN, osteocalcin; Oc.S/BS, osteoclast surface per bone surface; P1NP, procollagen type 1 N-terminal propeptide; RA, rheumatoid cells like T cells and DCs. Thus, in diseases that are accompanied arthritis; RANK, receptor activator of NF-kB; RANKL, receptor activator of NF-kB by an inflammatory response, bone loss due to abnormal osteo- ligand; TRAP, tartrate-resistant acid phosphatase; Tt.Ar, total cross-sectional area; clast differentiation occurs frequently (20–22). Therefore, various WT, wild-type. studies have been conducted on inflammation-related molecules Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 to prevent bone loss. Nevertheless, no therapeutic agent has been

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800661 2 HAPTOGLOBIN INHIBITS OSTEOCLASTOGENESIS developed to inhibit bone loss entirely, and there is a continuing containing 10 mM b-glycerophosphate (Sigma-Aldrich), 50 mg/ml demand for therapeutic targets. Previous studies suggest that Hp ascorbate-2-phosphate (Sigma-Aldrich), and 100 ng/ml of BMP-2. The is involved in various physiological functions of immune cells, medium was replaced every 3 d. After culturing, the cells were fixed with 10% of formalin for 20 min. Osteoblast differentiation was measured by indicating that Hp may affect osteoclast differentiation. Results alkaline phosphatase or Alizarin Red S staining following manufacturer’s showing that Hp is significantly higher in patients with lupus instructions (Sigma-Aldrich). and RA than in healthy individuals emphasize the importance of ELISA studying the effect of Hp on osteoclast differentiation (1, 3). In the current study, we investigated the effect of Hp deficiency on os- To analyze the secreted Hp, osteocalcin (OCN), procollagen type 1 teoclast differentiation that affects bone phenotype and examined N-terminal propeptide (P1NP) and IFN-b levels, mouse serum, or culture supernatant medium were collected and assessed using IFN-b ELISA kit the direct effect of Hp on osteoclastogenesis. (PBL Assay Science), OCN ELISA kit (LSBio, Seattle), P1NP ELISA kit (IDS, Boldon, U.K.), or a Hp ELISA kit (Abnova, Taipei City, Taiwan) Materials and Methods according to the manufacturer’s instructions. Animal experiments FITC labeling and immunostaining All animal experiments were performed in accordance with the Animal FITC labeling of Hp was performed by using a FluoroTag FITC Conjugation Care Committee of the Institute of Laboratory Animal Resources of Seoul 2/2 Kit (Sigma-Aldrich) according to the manufacturer’s instruction. BMMs National University (Seoul, Korea). C57BL/6.Hp-deficient (Hp ) mice were cultured in 12-well plates in the presence of M-CSF (30 ng/ml). After were a kind gift from Dr. Lee Ann Garrett-Sinha (23). Eight-week-old 24 h, Hp-FITC was added to the cells and incubated at 37˚C for the in- male wild-type (WT) or Hp-deficient mice (n = 8 per group) were eu- dicated time. The cells were washed two times with PBS and fixed with thanized, and the femurs were fixed in 4% (w/v) paraformaldehyde for 10% formalin for 30 min. After blocking the cells with 1% (w/v) BSA in Downloaded from microcomputed tomography (mCT) assessment. The femurs were analyzed PBS for 30 min, the Ab against TLR4 (2 mg) in 1% BSA in PBS was by high-resolution mCT (SMX-90CT system; Shimadzu, Kyoto, Japan). added. After 1 h incubation, the cells were washed with PBS for three Scanning images from mCT were reconstructed by the VGStudio MAX times on a shaker. Then, anti-rabbit IgG Ab conjugated to Cy3 (Invitrogen) 1.2.1 program (Volume Graphics, Heidelberg, Germany). Each three- in 1% BSA in PBS was added to cells and incubated for 30 min in the dark dimensional image was analyzed to measure trabecular bone volume, room. The immunostained cells were observed under a confocal laser cortical bone volume, trabecular number, and trabecular separation by microscope (FV-300; Olympus, Tokyo, Japan). using TRI/3D-VIE (RATOC System Engineering, Kyoto, Japan). http://www.jimmunol.org/ Histology BMMs (2 3 106 cells per dish) were cultured with M-CSF (30 ng/ml) in a The samples from animal experiments were decalcified with 12% (w/v) 100-mm diameter cell-culture dish. After 24 h incubation, the cells were EDTA for 3 wk and embedded in paraffin. After histological sagittal treated with PBS, Hp (20 mg/ml), or LPS (1 mg/ml) and incubated for the sections (5-mm thickness), calvariae were stained with hematoxylin solu- indicated times. After incubation, the cells were detached by enzyme-free tion (Sigma-Aldrich, St. Louis, MO) or stained for tartrate-resistant acid cell dissociation buffer (MilliporeSigma, Billerica, MA). The cells were phosphatase (TRAP) using a Leukocyte Acid Phosphatase Assay Kit stained with anti-TLR4 Abs, followed by FITC-conjugated anti-rat IgG, (Sigma-Aldrich) according to manufacturer’s instructions. Femurs were and analyzed by flow cytometry using a FACSCalibur (BD Biosciences, stained with 0.5% methyl green solution (Sigma-Aldrich) and TRAP stain San Diego, CA). Cells stained with isotype control Abs were used as a kit. The TRAP-stained cells were analyzed by using the OsteoMeasure XP negative control. program (version 1.01; OsteoMetrics, Decatur, GA). by guest on September 29, 2021 Cell viability assay Reagents The cells (1 3 104) were cultured on 96-well culture plates. After a 12 h Recombinant human M-CSF and RANKL were obtained from PeproTech incubation, the medium was exchanged with vehicle or the indicated doses (Rocky Hill, NJ). Plasma-derived human Hp was purchased from Sigma- of Hp. After the indicated time of incubation, 10 ml of EZ-Cytox solution Aldrich. To detect target protein expression, Abs against NFATc1, c-Fos, (Daeil Lab Services, Seoul, Korea) was added to each well of the plate. and TLR4 were purchased from Santa Cruz Biotechnology (Santa Cruz, After incubation for 2 h, the absorbance was measured with Multiskan CA). Abs against TLR4, FLAG, and b-actin were purchased from (Thermo LabSystems, Philadelphia, PA) at 450 nm. Sigma-Aldrich. Secondary Abs conjugated with HRP were obtained from Sigma-Aldrich. All other Abs were from Cell Signaling Tech- Western blotting nology(Beverly,MA).ThespecificneutralizingAbagainstTLR4was purchased from eBioscience (San Diego, CA). The specific neutralizing To detect specific protein expression, the cells were lysed on ice with a Ab against IFN-b was purchased from PBL Biomedical Laboratories buffer containing 20 mM Tris-HCl, 150 mM NaCl, 1% (w/v) Triton X-100, (Piscataway, NJ). Normal mouse IgG was purchased from Santa Cruz a protease inhibitor, and phosphatase inhibitor (Sigma-Aldrich). After Biotechnology. 30 min lysis on ice, the cellular debris was removed by centrifugation at 14,000 rpm 3 g for 15 min. The supernatant was collected in the tube, and Cell culture the protein concentration was determined by the use of a DC Protein Assay 2 2 2 2 Kit (Bio-Rad Laboratories, Hercules, CA). The protein samples (20–50 mg) C57BL/6.TLR2– (TLR2 / ), C57BL/6.TLR4– (TLR4 / ), and C57BL/ 2 2 were separated by SDS-PAGE and transferred onto a nitrocellulose 6.TLR7 (TLR7 / )–deficient mice were a kind gift from Prof. Sung Joong membrane (GE Healthcare, Chalfont St. Giles, U.K.). The membrane was Lee (24). Bone marrow cells were obtained by flushing the bone marrow of blocked with 5% (w/v) skim milk, and target were incubated with the femur and tibia of 6-wk-old male mice. The nonadherent bone marrow the indicated primary Abs overnight. Blots were then incubated with cells were further cultured with M-CSF (30 ng/ml) and a-MEM for 3 d to secondary Abs conjugated with HRP and visualized by chemilumines- generate bone marrow–derived macrophages (BMMs) as described pre- cence reaction using ECL reagents (GE Healthcare). viously (25). To generate osteoclastogenesis, BMMs (4 3 104 cells/well) were cultured in 48-well plates with complete medium and a-MEM transduction and retroviral vector containing 10% (v/v) heat-inactivated FBS and 50 U/ml of penicillin in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 4 d. The Gene transduction was performed by using Platinum-E retroviral packaging medium was changed every 3 d. Multinucleated cells were observed on cells (Cell Biolabs, San Diego, CA) according to the manufacturer’s in- day 4. The cells were fixed in 10% formalin solution for 20 min and struction. Retroviral vectors pMX-c-Fos-FLAG were kindly provided permeabilized with 0.1% Triton X-100 for 1 min. After washing twice with by Prof. Nacksung Kim (27). The retroviral vectors were transiently PBS, the cells were stained by using a Leukocyte Acid Phosphatase Assay transfected into Platinum-E cells by using Genjet transfection reagent Kit from Sigma-Aldrich following the manufacturer’s instructions. Cal- (SignaGen Laboratories, Gaithersburg, MD). After incubation for 48 h, varial osteoblasts were isolated from the calvariae of newborn mice as supernatants containing retrovirus were collected. BMMs were then trans- described previously (26). To generate osteogenic differentiation, osteo- duced with the retroviral supernatants in the presence of polybrene (6 mg/ml; blasts were cultured in 48-well tissue culture plates precoated with col- Sigma-Aldrich) and M-CSF (30 ng/ml) for 12 h. The cells were further lagen at a density of 5 3 104 cells/well. After culturing for 24 h, the cells cultured with puromycin (2 mg/ml) and M-CSF (30 ng/ml) for 3 d to were further cultured in osteogenic medium: a-MEM complete medium remove uninfected cells. The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. The bone phenotype of Hp-deficient mice. The femurs of 8-wk-old WT or Hp-deficient (Hp2/2) male mice were analyzed by mCT and histological sectioning (n = 8). (A) The images represent femurs of WT or Hp2/2 mice. (B) Reconstructed three-dimensional mCT images were analyzed to measure bone parameters, such as bone volume per tissue volume (BV/TV), bone surface per bone volume (BS/BV), trabecular bone pattern factor (Tb.Pf), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). (C) Reconstructed three-dimensional mCT images were analyzed to measure cortical bone parameters, such as cortical thickness (Ct.Th), Tt.Ar, Ct.Ar, and cortical area fraction (Ct.Ar/Tt.Ar). (D) Decalcified femur bones were subjected to microdissection and stained for TRAP. Nuclei were counterstained with methyl green. Oc.S/BS, N.Oc./BS, osteoblast surface per bone surface (Ob.S/BS), and number of osteoblasts per bone surface (N.Ob./BS) were examined by using the OsteoMeasure program (black arrow, osteoclast; arrow head, osteoblast). (E) Serum levels of OCN and P1NP of 8-wk-old wild-type (WT) or Hp-deficient (Hp2/2) male mice were analyzed by ELISA. *p , 0.05, **p , 0.01, ***p , 0.001 (paired t test, two-sided).

Mouse calvarial RANKL-induced osteoclast formation model synthesis using the SuperScript II Preamplification System (Invitrogen). To assess mRNA expression, 20–30 cycles of PCR amplification were Collagen sponges that were soaked in the vehicle or Hp (5 mg/kg), in performed for each gene. PCR products were separated on a 1–1.2% combination with the vehicle or RANKL (1 mg/kg), were implanted (v/v) agarose gel, stained with ethidium bromide, and detected by UV onto the calvariae of 8-wk-old mice s.c. Each mixture was injected into light. The relative mRNA expressions were evaluated by ABI Prism the implanted collagen sponge daily for 5 d. The mice were sacrificed 7500 sequence detection system with SYBR Green PCR Master Mix 6 d after implantation. The calvariae were collected, fixed in 4% (w/v) (Applied Biosystems, Foster City, CA). Target mRNA expressions were paraformaldehyde, and histological analysis was performed as de- determined according to the 2-DD cycle threshold method using HPRT as scribed above. a reference gene. Conventional and real-time PCR Hp–TLR4 binding assay To measure relative mRNA expression level, total RNA was isolated using To identify a direct interaction between Hp and TLR4, an Hp–TLR4 TRIzol reagent (Invitrogen). Total RNA (2 mg) was used for first-strand cDNA binding assay was performed as described previously (28). Briefly, 4 HAPTOGLOBIN INHIBITS OSTEOCLASTOGENESIS Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. Hp acts in the early phase of osteoclast differentiation, inhibiting osteoclastogenesis. (A) Collagen sheets soaked with vehicle or Hp, in combination with vehicle or RANKL, were implanted onto the calvariae of 8-wk-old mice. The mice were sacrificed on day 6. Mice calvariae were stained for TRAP. Oc.S/BS and N.Oc./BS were examined by using the OsteoMeasure program. Original magnification 3200. (B) BMMs were cultured with vehicle or the indicated dose of Hp in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml). After culturing for 4 d, osteoclasts were stained for TRAP, and TRAP-positive osteoclasts containing three or more nuclei were counted. Original magnification 3100. (C and D) Hp (20 mg/ml) was treated at indicated time periods during RANKL-induced osteoclastogenesis. After 4 d of culture, the cells were fixed and stained for TRAP, and TRAP-positive osteoclasts containing three or more nuclei were counted. Original magnification 3100. *p , 0.05, **p , 0.01, ***p , 0.001. high-binding immunoassay plates (Sigma-Aldrich) were coated with mean 6 SD. The p values ,0.05 were considered significant compared BSA, Hp, or LPS for 24 h at 4˚C and then blocked in 2% BSA in PBS with their respective controls. for 1 h. After three washes with PBS containing 0.05% (v/v) Tween 20, TLR4-Fc fusion protein (2 mg) was added and incubated for 2 h at room Results temperature. The captured TLR4-Fc was detected by using an HRP- conjugated goat anti-human IgG Ab (Sigma-Aldrich). o-Phenylenediamine The bone phenotype of Hp-deficient mice dihydrochloride (Sigma-Aldrich) was added for spectrophotometric To investigate the effect of Hp on bone phenotype, we analyzed measurement at 450 nm by using Multiskan (Thermo LabSystems). the femurs of WT mice and Hp-deficient mice. Computerized Statistical analysis tomography analysis of the femurs revealed an osteoporotic phe- All quantitative experiments were performed at least in triplicate. Statistical notype in Hp-deficient mice (Fig. 1A). The bone volume per tissue significance was analyzed by Student t test or one-way ANOVA followed volume in trabecular bone, trabecular number, and trabecular by Tukey–Kramer multiple comparisons test. Values are presented as the thickness decreased significantly in Hp-deficient mice compared The Journal of Immunology 5 with WT, and the bone surface per bone volume and trabecular was carried out using osteoblast differentiation medium treated with separation increased. Furthermore, the trabecular bone pattern Hp. Experimental results showed no significant difference between factor (an index of intertrabecular connectivity) was significantly the experimental group and the control group in both the alkaline increased in Hp-deficient mice. All these indices indicate a loss phosphatase stain and the Alizarin red stain (Supplemental Fig. 1A). of trabecular bone in Hp-deficient mice (Fig. 1B). In contrast, The mRNA level of BSP and Runx2, which are markers of os- there were no significant differences of cortical bone indices, such teoblast differentiation, did not decrease compared with controls as cortical thickness, cortical bone area (Ct.Ar), and cortical area treated with the vehicle. Unexpectedly, the other osteoblastic fraction (Ct.Ar/total cross-sectional area (Tt.Ar)) (Fig. 1C). differentiation markers, Alp and Col1a1, rather marginally in- Next, we performed a TRAP stain to investigate whether the creased by Hp treatment compared with vehicle treatment osteoporotic phenotype of Hp-deficient mice was associated with (Supplemental Fig. 1B). Next, to examine whether Hp directly osteoclastogenesis. In comparison with control mice, the number of inhibits osteoclastogenesis, we investigated the effect of Hp on osteoclasts per bone surface (N.Oc./BS) and osteoclast surface per osteoclast differentiation by treating BMMs with different con- bone surface (Oc.S/BS) were significantly increased. In contrast, centrations of Hp. Osteoclast differentiation was inhibited by there were no significant differences in the number of osteoblasts 10 mg/ml of Hp, and osteoclastogenesis was almost blocked by per bone surface and osteoblast surface per bone surface (Fig. 1D). 20 mg/ml of Hp (Fig. 2B). An MTT assay was performed to Consistently, serum levels of OCN and P1NP, which are bone exclude the effect of Hp on the proliferation of BMMs. Treat- anabolism markers, showed no difference between WT mice and ment with 50 mg/ml, which is higher than the concentration used Hp-deficient mice (Fig. 1E). in the experiment, did not affect the proliferation of BMMs

(Supplemental Fig. 1C). These results suggest that the reduction Downloaded from Hp acts in the early phase of osteoclast differentiation, of bone mass in Hp-deficient mice is closely related to osteo- inhibiting osteoclastogenesis clastogenesis rather than to osteoblast differentiation. To further clarify the inhibitory effect of Hp on osteoclasto- To further investigate the inhibitory effect of Hp on osteoclas- genesis, we examined whether Hp could impede osteoclasto- togenesis, we examined the effects of Hp on the differentiation genesis in the calvarial RANKL-induced osteoclast formation phases of osteoclasts. First, we classified the process of osteoclast

model. The number of osteoclasts increased by RANKL was differentiation as early, middle, and late phases, which correspond http://www.jimmunol.org/ decreased by more than 60% in the group treated with Hp to 0–1, 1–2, and 2–3 d after RANKL treatment. In both cases, compared with the control group (Fig. 2A). To investigate the either treating the osteoclasts with Hp at each phage and then possibility that osteoblasts affected by Hp are involved in the os- maintaining treatment until mature osteoclasts were generated or teoporotic phenotype of Hp-deficient mice, we examined the effect treating the osteoclasts with Hp only at each differentiation phage, of Hp on osteoblast differentiation. The osteoblast differentiation the inhibitory effect of Hp on osteoclastogenesis was observed by guest on September 29, 2021

FIGURE 3. Hp reduces the protein level of c-Fos. Bone marrow–derived macrophages (BMMs) were cultured for the indicated times in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml) with stimulation of vehicle or Hp (20 mg/ml). (A) The mRNA expression of c-Fos and NFATc1 were analyzed by real-time PCR with b-actin mRNA as an endogenous control. (B) Total cell lysates were harvested and subjected to Western blotting with the indicated Abs. (C) BMMs transduced with pMX-FLAG-empty (Empty) or pMX-c-Fos-FLAG were cultured with or without RANKL (100 ng/ml) and Hp (20 mg/ml) in the presence of M-CSF (30 ng/ml). After culturing for 24 h, the cells were lysed, and the total cell lysates were subjected to Western blotting with the indicated Abs. (D) The trans- duced BMMs were further cultured 4 d with ve- hicle or Hp (20 mg/ml) in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml) to generate osteoclasts. The osteoclasts were then stained for TRAP, and TRAP-positive osteoclasts were counted. Original magnification 3100. ***p , 0.001 (paired t test, two-sided). 6 HAPTOGLOBIN INHIBITS OSTEOCLASTOGENESIS

FIGURE 4. IFN-b upregulated by Hp inhibits osteoclastogenesis. (A) BMMs were cultured with vehicle or Hp (20 mg/ml) in the presence of M-CSF (30 ng/ml). The mRNA expression of IFN-b was analyzed by real-time PCR with b-actin mRNA as an endogenous control. ***p , 0.001 (paired t test, two-sided). (B) BMMs were cultured with the indicated dose of Hp in the presence of M-CSF (30 ng/ml). The secreted IFN-b production from the supernatant of BMMs was measured. **p , 0.01, ***p , 0.001 versus vehicle-treated BMMs. (C) BMMs were cultured with vehicle or Hp (20 mg/ml) and normal mouse IgG (10 mg/ml) or specific

IFN-b–neutralizing Ab (10 mg/ml) in the presence Downloaded from of M-CSF (30 ng/ml) and RANKL (100 ng/ml). After culturing for 4 d, the cells were fixed and stained for TRAP. TRAP-positive osteoclasts were counted. NS and ***p , 0.001 versus IgG+Vehicle or between indicated groups. Original magnification 3100. (D) BMMs were cultured with indicated RANKL (100 ng/ml), Hp (20 mg/ml), and specific http://www.jimmunol.org/ IFN-b–neutralizing Ab (10 mg/ml) in the presence of M-CSF (30 ng/ml). After culturing for 24 h, the cells were lysed, and the total cell lysates were subjected to Western blotting with indicated Abs. (E and F) WT or IFNAR12/2 BMMs were cultured with vehicle or Hp (20 mg/ml) in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml). After culturing for 4 d, the cells were fixed and stained for

TRAP. TRAP-positive cells containing three or more by guest on September 29, 2021 nuclei were counted. Original magnification 3100. ***p , 0.001 (paired t test, two-sided).

only when osteoclasts were treated with Hp in the early differ- we hypothesized that Hp reduced the protein level of c-Fos, entiation phage (Fig. 2C, 2D). These results suggest that Hp may thereby suppressing the expression of NFATc1. To confirm this, affect the initial triggering mechanism induced by RANKL. we overexpressed the c-Fos in BMMs and differentiated osteo- clasts with or without Hp. The level of NFATc1 reduced by Hp Hp reduces the protein level of c-Fos was restored in BMMs transduced with the c-Fos retroviruses, and Based on the finding that Hp inhibits osteoclastogenesis by af- the inhibitory effect of Hp on osteoclastogenesis also disappeared fecting the early phase of osteoclast differentiation, we investigated (Fig. 3C, 3D). whether Hp changes mRNA and protein levels of c-Fos and To further investigate mechanisms, we examined whether Hp NFATc1, which are known to be key regulatory factors in the early affects MAPK signaling. Experimental results showed that the phase of osteoclast differentiation. Treatment with Hp did not alter ERK pathway and JNK pathway were not affected by Hp, but c-Fos mRNA levels but decreased c-Fos protein levels and reduced the NF-kB pathway was affected. The pretreated Hp increased the both the mRNA and protein levels of NFATc1 (Fig. 3A, 3B). basal level of p-p65 but suppressed NF-kB activation induced by Because both c-Fos and NFATc1 are essential transcription factors RANKL stimulation (Supplemental Fig. 2C). for osteoclast differentiation, the decrease of c-Fos and NFATc1 b may inhibit osteoclastogenesis by decreasing the expression of IFN- upregulated by Hp inhibits osteoclastogenesis RANK, the RANKL receptor. Thus, we examined whether Hp The inhibition of RANKL-induced c-Fos protein upregulated by changed RANK mRNA levels. The mRNA level of RANK was IFN-b, which is independent of the mRNA expression level of not significantly altered by treatment with Hp when compared c-Fos, was reported by Takanayagi group (30). Therefore, we with the vehicle (Supplemental Fig. 2A). hypothesized that Hp increased the expression of IFN-b,andthe It is well known that c-Fos binds to the promoter region of increased IFN-b acted autocrine, inhibiting osteoclastogenesis. NFATc1, regulating the transcription of NFATc1 (29). Therefore, To investigate this possibility, we examined the expression of The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 5. Hp upregulates IFN-b expression via TLR4. (A) BMMs obtained from either WT, TLR22/2, TLR42/2, or TLR72/2 mice were cultured with vehicle or Hp (20 mg/ml) in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 4 d. After culturing, the cells were fixed and stained for TRAP. (A) TRAP-positive cells containing three or more nuclei were counted. NS versus vehicle-treated WT. Original magnification 3100. (B) BMM cells were by guest on September 29, 2021 cultured with either vehicle or Hp (20 mg/ml) in the presence of M-CSF (30 ng/ml) for 24 h. The expression of IFN-b mRNA was analyzed by real-time PCR with b-actin mRNA as an endogenous control. (C) WT or TLR42/2 BMMs were cultured with vehicle or Hp (20 mg/ml) with or without RANKL (100 ng/ml) in the presence of M-CSF (30 ng/ml). After culturing for 24 h, the cells were lysed, and the total cell lysates were subjected to Western blotting with indicated Abs. (D) WT or TLR42/2 BMMs were cultured with vehicle or Hp (20 mg/ml) in the presence of M-CSF (30 ng/ml). After culturing for 12 h, the cells were lysed, and total cell lysates were subjected to Western blotting with indicated Abs. ***p , 0.001 (paired t test, two-sided).

IFN-b in BMMs after treatment with Hp. Treatment with Hp by Hp acts autocrine, inhibiting osteoclast differentiation by reduc- increased the mRNA level of IFN-b, and the amount of secreted ing the c-Fos protein level. IFN-b from BMMs was increased in proportion to the con- Hp upregulates IFN-b expression via TLR4 centration of Hp (Fig. 4A, 4B). In addition, Hp promoted the phosphorylation of STAT 1 and 2, which are known to be In previous studies, TLR signaling was shown to regulate IFN-b downstream regulators of IFN-b signaling (Supplemental Fig. expression (31). Among TLRs expressed in BMMs, we have fo- 3A). These results indicate that the autocrine loop of IFN-b was cused on TLR2, 4, and 7, which are important in regulating type formed by Hp. 1 IFN signaling and have a high rate of expression in BMMs. To investigate whether the inhibitory effect of Hp was asso- First, we investigated whether they act as a receptor for Hp. When ciated with IFN-b, we examined the effect of Hp on osteo- BMMs derived from TLR2- and TLR7-deficient mice were treated clastogenesis when IFN-b was neutralized with IFN-b Ab. The with a combination of RANKL and Hp, the inhibitory effect of Hp on IFN-b Ab significantly restored osteoclastogenesis suppressed osteoclastogenesis was maintained. However, the osteoclastogenesis by Hp (Fig. 4C). In addition, the blockade of IFN-b restored inhibitory effect of Hp was not observed in BMMs derived from the protein level of c-Fos, which was reduced by Hp (Fig. 4D). TLR4-deficient mice (Fig. 5A, Supplemental Fig. 4A). In line with However, despite the complete restoration of the c-Fos protein the result of Fig. 5A, Hp treatment to BMMs derived from TLR2- level, the inhibition of osteoclastogenesis had not entirely disappeared. and TLR7-deficient mice increased IFN-b mRNA levels without a To exclude the possibility that Hp inhibited osteoclastogenesis through significant difference when compared with WT mice. In addition, other mechanisms, the inhibitory effect of Hp on osteoclasto- osteoclast differentiation was restored by blocking IFN-b (Fig. genesis was examined using BMMs from IFNAR1-deficient 5B, Supplemental Fig. 4B). However, in the BMMs derived from mice (Fig. 4E, Supplemental Fig. 3C). The inhibitory effect of TLR4-deficient mice, the expression of IFN-b was not promoted Hp was rescued entirely in IFNAR1-deficient BMMs, and no with Hp treatment (Fig. 5B). Consistently, the deletion of TLR4 effect of Hp on osteoclastogenesis was observed (Fig. 4F). Also, restored the protein levels of c-Fos and NFATc1, which were re- the phosphorylation of STAT1 and 2 was wholly suppressed duced by Hp (Fig. 5C). Moreover, it was observed that the inhib- (Supplemental Fig. 3B). These results suggest that IFN-b increased itory effect of Hp on osteoclastogenesis entirely disappeared by 8 HAPTOGLOBIN INHIBITS OSTEOCLASTOGENESIS Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. Hp acts as a ligand for TLR4. (A) High-binding immunoassay plates were coated with the indicated dose of BSA, Hp, and LPS. Immobilized proteins were then incubated with 2 mg of the TLR4-Fc fusion protein. The ability to bind with TLR4 was measured as described in Materials and Methods. ***p , 0.001 versus vehicle. (B) BMMs were cultured in the presence of M-CSF (30 ng/ml). After culturing for 24 h, the cells were incubated with Hp-FITC (green) for the indicated times. The cells were then fixed and stained for TLR4 Ab (red) and DAPI to stain nuclei (blue). (C)BMMswere cultured with M-CSF (30 ng/ml). After culturing for 24 h, the cells were incubated with vehicle, Hp (20 mg/ml), or LPS (1 mg/ml). After incubation for the indicated time, the cells were detached and the cell surface TLR4 was analyzed by flow cytometry. *p , 0.05, **p , 0.01. ***p , 0.001 versus vehicle. blocking the TLR4 receptor with TLR4 Ab (Supplemental Fig. 4C). was maintained in BMMs derived from MyD88-deficient mice These results suggest that Hp promoted IFN-b expression through (Supplemental Fig. 4D). TLR4. Hp acts as a ligand for TLR4 It is known that IFN regulatory factor (IRF) 3 and 7 are in- volved in the regulation of IFN-b (32). Therefore, we investi- We showed that TLR4 is involved in the IFN-b expression induced gated whether Hp affects the phosphorylation of IRF3 and 7. The by Hp, but there is no report that Hp works as a ligand for TLR4. phospho-IRF3 was not detected by stimulation (data not shown). To investigate whether Hp binds directly to TLR4, we performed a In contrast, the phosphorylated form of IRF7 was significantly binding assay. LPS increased the absorbance, which indicates the increased within 12 h after Hp stimulation. However, when attachment of the TLR4 recombinant protein, depending on the BMMs derived from TLR4-deficient mice were stimulated by Hp, plate-coating dose. A similar pattern occurred for Hp (Fig. 6A). the phosphorylated form of IRF7 was not significantly increased According to previous reports, TLR4 binds with ligands to induce (Fig. 5D). In previous studies, the MyD88-dependent pathway is endocytosis, resulting in a TLR4-ligand complex entering the cell. known to be a common pathway activated by TLR4 signaling. To examine the endocytosis induced by Hp, the amount of the Therefore, there is a possibility thatTLR4signalingactivatedbyHp Hp–TLR4 complex introduced to cells was detected by confocal regulates the osteoclast differentiation through the MyD88-dependent microscopy (33). It was observed that the colocalized regions of pathway. However, the inhibitory effect of Hp on osteoclastogenesis FITC-labeled Hp and Cy3-labeled TLR4 gradually increased in The Journal of Immunology 9

FIGURE 7. The inhibitory effect of Hp on osteoclastogenesis is reduced in TLR4- deficient mice. Collagen sheets soaked with vehicle, RANKL, or RANKL+Hp were implan- Downloaded from ted onto the calvariae of 8-wk-old WT mice or TLR42/2 mice. The mice were sacrificed on day 6. (A) Mice calvariae were stained for TRAP. Original magnification 3200. (B) Oc.S/BS and N.Oc./BS were examined by using the OsteoMeasure program. (C) Graphical pre- sentation of the Hp–TLR4–IFN-b axis that in- http://www.jimmunol.org/ hibits osteoclast differentiation. ***p , 0.001. by guest on September 29, 2021

the cells depending on the length of the Hp treatment time (Fig. 6B). WT mice (Fig. 7A). In addition, osteoclast formation parameters To investigate whether colocalized regions were increased by en- such as Oc.S/BS and N.Oc./BS indicated that the inhibitory effect docytosis of the TLR4–Hp complex, we counted the cells expressing of Hp on osteoclastogenesis was decreased in TLR4-deficient TLR4 on the cell surface using FACS. It was observed that mice (Fig. 7B). TLR4-positive cells were decreased in a time-dependent man- ner after treatment with Hp and were finally decreased by 80% Discussion (Fig. 6C). These results indicated that Hp binds directly to TLR4 Previous studies have shown that the binding of Hp to CD11b/ and acts as a ligand. CD18 in macrophages, which are one of the osteoclast precur- sors, activates the NF-kB pathway (4, 34). Therefore, our initial The inhibitory effect of Hp on osteoclastogenesis is reduced in expectation was that Hp promotes osteoclastogenesis. However, TLR4-deficient mice unlike this expectation, the osteoporotic phenotype was observed To investigate whether Hp inhibits osteoclastogenesis through in the femur of the Hp-deficient mice and Hp showed a significant TLR4 in vivo, we examined osteoclast differentiation by applying inhibitory effect on osteoclast differentiation in proportion to calvarial RANKL-induced osteoclast formation model to WT and the treatment concentration (Figs. 1A, 2B). The activation of the TLR4-deficient mice. The osteoclast formation by RANKL was NF-kB pathway induced by RANKL was suppressed by Hp, and observed in the calvariae in both WT and TLR4-deficient mice the protein levels of c-Fos and NFATc1 were also decreased by TRAP staining. However, in the case of treatment with both (Fig. 3B, Supplemental Fig. 2C). In addition, we observed that the RANKL and Hp, the osteoclast formation was suppressed to the osteoclast inhibitory effect of Hp was maintained in CD11b knock vehicle-treated level in the WT mice. In the TLR4-deficient mice, the down BMMs (data not shown). These results indicate that Hp osteoclast formation was reduced by Hp in RANKL-treated mice, inhibited osteoclast differentiation in a way independent on its but the extent of reduction was much lower in TLR42/2 mice than in binding to CD11b/CD18. Our study rather showed that TLR4 is 10 HAPTOGLOBIN INHIBITS OSTEOCLASTOGENESIS the important Hp receptor in mediating the antiosteoclastogenic increases the potential to cause unwanted side effects, there is a effect of Hp. The lack of inhibitory effects of Hp on TLR42/2 continuing demand for effective and safe therapeutic agents that cells in vitro (Fig. 5C) and TLR42/2 calvariae in vivo (Fig. 7A) cure both bone loss and inflammation for inflammatory bone clearly demonstrate that Hp inhibits osteoclasts via TLR4. To our diseases. In addition to anti-inflammatory effects of Hp previ- knowledge, we are the first to report that TLR4 acts as a receptor ously reported, our results that Hp-induced IFN-b expression, for Hp, and Hp inhibits osteoclast differentiation through the inhibiting osteoclastogenesis, suggest that Hp is a good candi- TLR4–IFN-b axis (Fig. 7C). date. In particular, IFN-b has an anti-tumor effect and has been In this study, when BMMs were stimulated by Hp, phos- studied for the application to cancer therapy (41–43). It has also phorylation of IRF7 was significantly increased, whereas the been reported that IFN-b injection significantly alleviated paw phosphorylation of IRF3 was undetected. The lack of IRF3 swelling and severity of disease in animal models of RA (44, 45). phosphorylation, which has been shown to be necessary for the In further studies, we will investigate the value of Hp as a thera- expression of IFN-b, was unexpected. However, these previous peutic molecule for diseases such as cancer and RA, which cause results were obtained by using LPS as a TLR4 signaling stimulator chronic inflammation and bone loss. (35). Therefore, in the current study, using Hp as a TLR4 sig- naling stimulator, there is a possibility that IRF7 regulates IFN-b Acknowledgments expression independently of IRF3. Besides, unlike the previous We thank LeeAnn Sinha (Department of Biochemistry, State University of studies, which reported that the expression level of IRF7 was very New York, Buffalo) for kindly providing Hp-deficient mice. low in most cells and induced by type 1 IFN–mediated signaling, Downloaded from the basal protein level of IRF7 in BMMs was fairly high and did Disclosures not alter even after starvation for 6 h in our study (data not shown). The authors have no financial conflicts of interest. These results suggest that TLR4-mediated signaling induced by Hp in BMMs may be different from previous studies. Although the regulatory mechanism involved in IFN-b induction by Hp has References not been clearly elucidated, our results suggest that IRF7 activa- 1. Eckersall, P. D., and R. Bell. 2010. Acute phase proteins: biomarkers of infection and inflammation in veterinary medicine. Vet. J. 185: 23–27. tion is crucial for the Hp-stimulated IFN-b induction. http://www.jimmunol.org/ 2. 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