Necrotic Bone Stimulates Proinflammatory Responses in Macrophages through the Activation of Toll-Like Receptor 4

Necrotic Bone Stimulates Proinflammatory Responses in Macrophages through the Activation of Toll-Like Receptor 4

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The American Journal of Pathology, Vol. -, No. -, - 2016

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Necrotic Bone Stimulates Proinflammatory Responses in Macrophages through the Activation of Toll-Like Receptor 4 Q40

Naga Suresh Adapala,*y Ryosuke Yamaguchi,*y Matthew Phipps,* Olumide Aruwajoye,* and Harry K.W. Kim*y From the Center for Excellence in Hip Disorders,* Texas Scottish Rite Hospital for Children, Dallas; and the Department of Orthopedic Surgery,y University of Texas Southwestern Medical Center, Dallas, Texas Accepted for publication June 28, 2016.

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Address correspondence to Harry K.W. Kim, Texas Scottish Rite Hospital for Children, 2222 Welborn St., Dallas, TX 75219. E-mail: harry.kim@ tsrh.org.

In children with Legg-Calvé-Perthes disease, loss of blood supply to the femoral head results in ischemic osteonecrosis. Generally, macrophages play important roles in the inflammatory response to tissue necrosis, but their role in ischemic osteonecrosis of the femoral head (ONFH) is not known. The purpose of this study was to determine the macrophage-inflammatory responses after ONFH and the receptor mechanisms involved in sensing the necrotic bone, using a piglet model of Legg-Calvé-Perthes disease. Induction of ONFH resulted in increased numbers of CD14þ macrophages (4.2-fold, P Z 0.007) in the fibrovascular repair tissue compared with normal, as determined by immunohistochemistry. Quantitative real-time PCR analysis of macrophages isolated by laser capture microdissection showed significantly increased expression of proinflammatory cytokines (IL-1b: 3.9-fold, P < 0.0001; tumor necrosis factor-a: 2.3-fold, P Z 0.0006; IL-6: 3.6-fold, P Z 0.04) in ONFH compared with normal. Because Tolllike receptors (TLRs 2, 4, 9) mediate macrophage-inflammatory responses in other inflammatory conditions, we determined their gene expression in macrophages and found significantly increased levels of TLR4 (5.3-fold, P Z 0.003) but not TLR2 (P Z 0.46) and TLR9 (P Z 0.9) in ONFH. Mechanistically, in vitro, bone marrow-derived macrophages treated with necrotic bone showed increased extracellular signal-regulated kinases 1/2 and Ik kinase-a phosphorylation, increased proliferation and migration, and inflammatory cytokine expression, which were blocked by a TLR4 inhibitor, TAK242, and by TLR4 ablation in macrophages using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease method. In conclusion, necrotic bone stimulates macrophage-inflammatory responses through TLR4 activation. (Am J Pathol 2016, -: 1e13; http:// dx.doi.org/10.1016/j.ajpath.2016.06.024)

Legg-Calvé-Perthes disease (LCPD) is a pediatric hip disorder in which a loss of blood supply to the femoral head results in ischemic osteonecrosis, followed by a development of femoral head deformity and early osteoarthritis.1,2 During the repair of the necrotic bone, several features suggestive of chronic inflammatory repair process have been observed both in LCPD patients and in a piglet model of ischemic osteonecrosis of femoral head (ONFH), including excessive resorption of the necrotic bone, delayed new bone formation, and replacement of the necrotic bone by a fibrovascular tissue, which persists over time.2 Patients with LCPD have also been shown to have chronic hip synovitis and significantly elevated levels of the proinflammatory

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cytokine, IL-6, in the synovial fluid.3 However, ONFH has not been previously considered to have a chronic inflammatory repair process, and no study has investigated the inflammatory component of the repair process. Macrophages are known to play a central role in the chronic inflammatory repair processes.4,5 After tissue necrosis in conditions such as myocardial infarction, stroke, and chemical injuries to lung and liver, macrophages are known to sense tissue damage using pattern Supported by the Intramural Research Program of Texas Scottish Rite Hospital for Children (H.K.W.K.). Q2 Disclosures: None declared. Q3

Copyright ª 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2016.06.024

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recognition receptors, most commonly Toll-like receptors (TLRs).6e8 Among the TLRs, the role of TLR4 is most well characterized. The stimulation of TLR4 results in the activation of myeloid differentiation primary response 88 (Myd88)-extracellular regulated kinase1/2 (ERK1/2), Ik kinase (IKK)aeNF-kB pathway and, TIR domaincontaining adapter-inducing interferon (IFN)-b (TRIF) and IFN regulatory factor 3 signaling pathways.9 Importantly, the activation of NF-kB and ERK1/2 signaling pathways result in the gene expression of molecules involved in migration, proliferation, and inflammatory cytokine responses in macrophages10 and TRIFdependent signaling results in IFN production and subsequent activation of STAT1-mediated transcription. The involvement of macrophages in the repair process after ONFH and the receptor mechanisms involved in sensing the ischemic bone damage are unknown. An assessment of the role of macrophages after ONFH would reveal potential mechanisms responsible for chronic inflammation and novel therapeutic approaches to target the inflammatory responses. Macrophages can perform proinflammatory (causing further tissue damage) or anti-inflammatory (resulting in tissue repair) roles depending on the environment. The predominance of a proinflammatory M1 phenotype [express inducible nitric oxide synthase, IL-12b, CD68 and proinflammatory cytokines IL-1b, tumor necrosis factor (TNF)-a, IL-6, and IFN-g] or an anti-inflammatory M2 phenotype (express Arg1, Ym1, CD163 and antiinflammatory cytokines IL-4, IL-10) has been suggested to correlate with further tissue damage or resolution of inflammation, respectively.11,12 After ONFH, it is not known whether the macrophages in the repair environment show a proinflammatory M1 or an anti-inflammatory M2 phenotype. The purpose of this study was to determine the inflammatory response of macrophages to the necrotic bone and the pattern recognition receptors involved in the sensing of necrotic bone using a well-established piglet model of ONFH.13e15 We hypothesized that after ONFH, necrotic bone stimulates the proliferation, migration, and inflammatory cytokine expression in macrophages and that macrophages in necrotic repair environment have a proinflammatory phenotype.

Materials and Methods Animals The animal study was approved by Institutional Animal Care and Use Committee at University of Texas Southwestern Medical Center. Yorkshire piglets (6 to 8 weeks old) were obtained from a local breeder and were housed at the University of Texas Southwestern animal care facility. A total of 20 piglets were used in this study as described below.

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Induction of ONFH ONFH was induced in the right femoral head (n Z 12 piglets) by placing a suture ligature tightly around the femoral neck and by transection of the ligamentum teres, as described previously.13e15 The left femoral head was unoperated and served as the control group. The piglets were sacrificed, and samples were collected at 8 weeks after the surgery, a time point at which there is a persistence of fibrovascular repair tissue in the femoral head and a severe collapse of the femoral head similar to that observed in patients with LCPD.13e15 The number of macrophages and levels of gene expression of inflammatory cytokines were used as primary outcome measures. For micro computed tomography (CT) imaging, histology, and immunohistochemistry (n Z 6 piglets), for flow cytometry, frozen sectioning, and laser capture microdissection (LCM) and the preparation of necrotic bone material in the osteonecrosis group (n Z 6 piglets), the indicated number of animals were used. For the sham operation group (n Z 3 piglets), the femoral neck was exposed but no ligation or transection was performed. Bone marrow was collected from the tibia of unoperated piglets (n Z 5 piglets) for the generation of macrophages in vitro.

Micro CT Imaging The femoral heads were bisected coronally and gross images were taken. The bisected femoral heads were fixed in 10% neutral buffered formalin. A micro CT scanning was performed at 100 kV and 100 mA (SkyScan 1172; SkyScan, Kontich, Belgium) at a set resolution of 26.6 mm per pixel, and rotation steps were 0.80 degrees over a 180-degree range. The three-dimensional reconstruction was performed using NRecon Reconstruction software version 1.6.8 and Q9 visualized in CT Vox 2.6.0 r908 BrukerMicroCT software Q10 (Kartuizersweg, Kontich, Belgium).

Histology and Immunohistochemistry Femoral heads were decalcified using 14% EDTA, and embedded in paraffin. Three micron-thick sections were cut using a microtome (Shandon Finesse; Thermo Scientific, Waltham, MA). Hematoxylin and eosin staining was performed using a standard method. For immunohistochemistry, paraffin sections were processed with trypsin digestion, inhibition of endogenous peroxidase activity, and blocking with goat serum (Dako, Carpentaria, CA). Frozen sections for immunohistochemistry or LCM were prepared at 10 mm thickness using a cryostat (MICROM HM560; Thermo Scientific). Mouse monoclonal antibodies at 1:100 or 1:200 dilutions were used against CD14 (MIL-2) (AbD Serotec, Raleigh, NC). Rabbit polyclonal antibodies were used against IL-1b (ab9722), TNF-a (ab6671), IL-6 (ab6672), and TLR4 (HTA-125) (Abcam, Cambridge, MA). As secondary antibodies, a goat anti-mouse IgG

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biotinylated antibody and a goat anti-rabbit IgG biotinylated antibody (Millipore, Billerica, MA) diluted in phosphatebuffered saline (dilution 1:500) were used, and the staining was visualized with, 3,30 -diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MO) for 10 minutes before brief counterstaining with hematoxylin (Invitrogen, Carlsbad, CA). Mouse and rabbit IgG were used as isotype controls (Abcam). The numbers of macrophages were quantitated using ImageJ software (NIH, Bethesda, MD), using 20 images per section, captured at 20 magnification with a Nikon Eclipse E800M microscope (Nikon, Tokyo, Japan).

Flow Cytometry

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Femoral head samples were digested with collagenase (Thermo Scientific; 2 mg/mL) and hyaluronidase (SigmaAldrich; 2 mg/mL). The red blood cells were lysed using ACK (0.15 mol/L ammonium chloride, 1 mol/L potassium bicarbonate, 0.1 mol/L EDTA) lysis buffer, washed, and single-cell suspensions were labeled using a porcine CD14-fluorescein isothiocyanate (MIL-2) antibody (GeneTex, Irvine, CA) after FcR blockade (Miltenyi Biotec, San Diego, CA), in the fluorescence-activated cell sorting staining medium containing 1  Hanks’ balanced salt solution, 0.01 mol/L HEPES (pH 7.4) and 2% fetal calf serum (Gibco, Waltham, MA). 7-Aminoactinomycin Depositive dead cells were identified and excluded before analysis. Flow cytometry was performed using BD-FACS LSRII (BD Biosciences, San Jose, CA), and the data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR).

LCM LCM was performed on frozen sections. To facilitate the visualization of macrophages, nonspecific esterase (NSE) staining was performed.16 NSE-positive cells were then harvested by the LCM method17 under strict RNase-free conditions using an ArcturusXT LCM System (Life Technologies, Carlsbad, CA). Low-power laser conditions were used (70 mW, 15 milliseconds, spot size of 25 mm). Approximately 500 NSE-positive cells were obtained from the frozen sections for each animal (n Z 6 piglets) and immediately preserved at 80 C in lysis buffer (ArcturusPicoPure Frozen RNA isolation kit; Life Technologies) until further use.

LCM RNA Preparation, Linear Amplification, and Quantitative Real-Time PCR RNA isolation was performed according to the manufacturer’s protocol for the ArcturusPicoPure Frozen RNA isolation kit. The quantity of the RNA was assessed by Nanodrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE), with a typical RNA yield of 0.9 to 1.7 ng/mL from each pig sample, with 260:280 ratios in the range of

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1.5 to 2.0. The RNA integrity number was assessed using an Agilent Bioanalyzer (Santa Clara, CA) (Supplemental Figure S1 and Supplemental Table S1). An analysis of the mRNA expression in LCM samples was performed in the following three steps: i) reverse transcription used 10 ng of RNA, RT reaction was performed using Superscript First Strand Synthesis System for RT-PCR (Invitrogen) in a 30-mL reaction volume; ii) linear amplification used a LCM primer pool for genes of interest, a linear amplification was performed using 12 mL of cDNA from RT reaction, using a Platinum Taq polymerase (Life Technologies); and iii) quantitative RT-PCR used a 1:10 dilution of linear ampli- Q14 fication product from step 2 for quantitative RT-PCR reaction, using forward and reverse LCM primers for each gene of interest using a Bio-Rad iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA) and Applied Biosystems 7500 Real Time PCR System (Thermo Scientific). Data analysis was performed using the deltadelta CT method. The sequence information for the LCMspecific primers used in this study is shown in Table 1. ½T1

Immunofluorescence Staining and Fluorescence Microscopy Frozen sections or macrophages cultured on glass coverslips were treated with appropriate primary antibodies as mentioned above, followed by incubation in a mix of a goat anti-mouse IgG 20 antibody conjugated with Alexa Fluor 488 (dilution 1:200) and a goat anti-rabbit IgG 20 antibody conjugated with Alexa Fluor 594 (dilution 1:500; Thermo Scientific). This step was followed by a nuclear staining using DAPI (Vector Laboratories, Burlingame, CA). Stained sections were examined using an Axioskop2 MOT microscope (Carl Zeiss, Oberkochen, Germany).

Preparation of Normal Bone and Necrotic Bone for in Vitro Treatment Studies Normal and necrotic bone were curetted from proximal femoral epiphysis under aseptic, strictly sterile conditions using endotoxin-free equipment and sterile saline containing protease inhibitors (Sigma-Aldrich). The bone samples were sectioned into pieces to release cells and tissue fluid into the saline suspension. The samples were centrifuged at 2000  g for 5 minutes at 4 C. The supernatant fraction (necrotic bone fluid) that may contain soluble proteins such as inflammatory cytokines was separated from the bone sediment fraction (necrotic bone). The necrotic bone was pulverized into a powder using liquid nitrogen and re-suspended in saline containing protease inhibitors. Both the supernatant and sediment fractions from the normal and necrotic bone samples were tested for endotoxin contamination, which can activate TLRs. The endotoxin levels were undetectable by a limulus amebocyte lysate test (below 0.1 Q15 endotoxin units/mL; Pierce, Waltham, MA).

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Adapala et al Table 1 RT-PCR Gene NOS2 IL-12b CD68 Arg1 Ym1 CD163 IL-1b TNF-a IL-6 IFN-g IL-4 IL-10 TLR2 TLR4 TLR9 GAPDH

Sequence Information for the Sus scrofa Laser Capture Microdissection Primers Used in the Linear Amplification and Quantitative Forward primer 0

Reverse primer 0

5 -TGAATCTGGGTGAAGAGCCC-3 50 -GCATTGGGGTACCAGTCCAA-30 50 -AATCTCCCTTCTTGCACCCC-30 50 -AGCCCAGCAAGTTCATACCT-30 50 -GGGTCCTCAGCCACACATAC-30 50 -CACGAGTCCCACCTTTCACT-30 50 -CAGCCAGTCTTCATTGTTCAGG-30 50 -GGCCCAAGGACTCAGATCAT-30 50 -CCCACCAGGAACGAAAGAGA-30 50 -TCAGCTTTGCGTGACTTTGTG-30 50 -ACACAAGTGCGACATCACCT-30 50 -CGGCCCAGTGAAGAGTTTCT-30 50 -CGGTGTGCTGCAAGGTCAA-30 50 -CGTGCAGGTGGTTCCTAACA-30 50 -TGTCATGGGCCCCCGC-30 50 -GACCAGGTTGTGTCCTGTGA-30

50 -TCATCCTGTGTTACTGGAACAAT-30 50 -GGAGCTTATAGCCTGACCCC-30 50 -TGTAGCAGAGGGGAGGACAA-30 50 -ACCAGCCAGCTTTGTCAGAT-30 50 -CTCTGTGGCCTCTCCTCAAC-30 50 -GTGTATCTTGAGCAGACTACGCC-30 50 -GGTCATTATTGTTGTCACCGTAGT-30 50 -CTGTCCCTCGGCTTTGACAT-30 50 -TGAAGGCGCTTGTGGAGAG-30 50 -TGAGGTACTTGCATTAAAATAGTCC-30 50 -TGCACGAGTTCTTTCTCGCT-30 50 -GGCAACCCAGGTAACCCTTA-30 50 -CAATCCCCAAGACCCATGCT-30 50 -GTGGGGATGTTGTCAGGGATT-30 50 -CTGGGCCAGAGTCGCAG-30 50 -AGCTTGACGAAGTGGTCGTT-30

Porcine specific exon-exon spanning laser capture microdissection primers were designed using National Center for Biotechnology Information primer design resources.

In Vitro Studies of Bone MarroweDerived Macrophages To obtain pig macrophages in culture, total bone marrow cells were cultured in a petri dish in the presence of 20 ng/ mL recombinant human macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN) in a-minimum essential medium containing 10% fetal bovine serum (Sigma-Aldrich), 1% penicillin-streptomycin 10,000 U/mL (Gibco, Waltham, MA). Macrophages were exposed to the necrotic bone fluid or the necrotic bone or normal bone. For the in vitro mRNA expression analysis, total RNA was isolated at 6 or 24 hours. To determine the effect of TLR4 inhibition, macrophages were treated with 100 mg/mL necrotic bone in the presence or absence of TLR4 inhibitor TAK242 (Calbiochem, Temecula, CA). The EC50 of the inhibitor TAK242 was determined in a dose-response study using the Transwell migration assay (Supplemental Figure S2). For migration assay, macrophages were allowed to migrate for 4 hours in a 5-m Transwell plate (Corning, Corning, NY) according to the manufacturer’s instructions. The percentages of seeded cells that have migrated to the lower surface were determined, and the data are expressed as fold difference compared with basal migration in a-minimum essential medium only (ie, no stimulation). For proliferation assay, macrophages were plated in quadruplicates in a 96-well tissue culture plate at a density of 2  104 cells per well and allowed to proliferate for 48 hours. The proliferation assay was performed using CellTiter 96 AQueous One Solution (Promega, Madison, WI) according to the manufacturer’s instructions. For Western blot analysis, macrophages were serumstarved for 60 minutes, followed by stimulation with necrotic bone (100 mg/mL). For inhibitor studies, cells were

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treated with TLR4 inhibitor TAK242 for 30 minutes, which were replenished during the treatment with necrotic bone for an additional 30 minutes. For clustered regularly inter- Q16 spaced short palindromic repeats (CRISPR)eCRISPRassociated protein-9 endonuclease (Cas9) TLR4 study, total bone marrow was cultured on petri dish in the presence of MSCF (20 ng/mL) for 48 hours. The adherent macrophage precursor cells were transfected with either the control or TLR4 CRISPR-Cas9 plasmids as per the manufacturer’s instructions (Santa Cruz Biotechnology, Dallas, TX). The transfection efficiency was determined by green fluorescent protein signal. After 48 hours, macrophages were serumstarved and were stimulated as described above for Western blot experiments using TAK242. The deletion of TLR4 by CRISPR-Cas9 TLR4 but not by control plasmid was confirmed by Western blot analysis. Total cell lysates were probed for phosphorylated and total forms of ERK1/2 (Cell Signaling Technology, Danvers, MA), IKKa (Thermo Scientific), or STAT1 (Santa Cruz Biotechnology) using specific antibodies. For the determination of TLR4 protein expression, macrophages were cultured in the presence of normal or necrotic bone for 24 hours, followed by Western blot analysis using TLR antibody (Abcam) and b-actin (Cell Signaling Technology) antibody. The scanning and quantitation of blots was performed using a LICOR imaging system (Lincoln, NE).

Statistical Analysis For the determination of the piglet sample size, an a of 0.05 and at least 80% power to detect a difference of 1.8 SDs by a standard t-test were used. Statistical analyses were performed using GraphPad Prism software version 6.0 (GraphPad Software Inc., La Jolla, CA). For Q17

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comparison of two groups, a Student’s t-test and for more than two groups, analysis of variance and Tukey’s post hoc test were used. P < 0.05 was considered statistically significant.

Results Repair Tissue in the Necrotic Femoral Head Contains Significantly Increased Number of Proinflammatory M1 Macrophages A severe flattening deformity of the necrotic femoral head was observed at 8 weeks after the induction of ONFH ½F1 (n Z 6 piglets) (Figure 1A). Bisection of the femoral heads

revealed a fibrovascular tissue in the necrotic femoral heads (Figure 1A), and extensive bone loss was observed by micro CT imaging (Figure 1A). Histologic analysis using hematoxylin and eosin staining showed a fibrovascular repair tissue invading the necrotic marrow space and remnants of the necrotic trabecular bone (Figure 1B). For the detection of macrophages, an immunohistochemistry was performed using the porcine CD14 antibody18 (Figure 1C). In the fibrovascular tissue, macrophages were mainly present around small blood vessels. The quantitation of the number of macrophages showed a 4.2-fold increase in the number of CD14þ macrophages (P Z 0.007) in the repair tissue compared with normal bone (Figure 1D). The increase in the number of macrophages

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Figure 1 Development of a flattening deformity of the femoral head after ischemic ON and the presence of macrophages in the fibrovascular repair tissue. A: Appearance of normal and ON femoral heads at 8 weeks after the induction of ischemic (top row). Gross morphology of the bisected femoral heads (middle row) showing presence of fibrovascular tissue and a three-dimensional micro CT image (bottom row) showing resorption of the bone in some areas of the necrotic femoral head. B: Hematoxylin and eosin staining of the femoral head sections. A fibrovascular repair tissue that contained fragments of necrotic bone was present in the necrotic femoral head. Areas in dashed boxes are shown at higher magnification below. C: CD14 immunohistochemistry followed by fast green and hematoxylin staining showing aggregates of macrophages adjacent to small vessels. Arrows indicates the color of positive staining. Areas in dashed boxes are shown at higher magnification below. D: Quantitation of CD14þ cells in the perivascular region. Representative images are shown. n Z 6 piglets. Statistical analysis was performed using a Student’s t-test: **P < 0.01. Scale bars: 2 mm (B, top row); 100 mm (B, bottom row, and C, top row); 20 mm (C, bottom row). Original magnification: 0.5 (B, top row); 10 (B, bottom row); 4 (C, top row); 40 (C, bottom row). CT, computed tomography; H&E, hematoxylin and eosin; IHC, immunohistochemistry; ON, osteonecrosis.

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621 was confirmed by flow cytometry (P Z 0.001; n Z 6 expression levels of anti-inflammatory cytokines IL-4 622 piglets) (Supplemental Figure S3). (P Z 0.0003) and IL-10 (P < 0.0001) were significantly 623 To assess the gene expression of M1, M2 markers and decreased. These results indicate that the repair tissue in the 624 inflammatory cytokines, macrophages were captured using necrotic femoral head contains increased numbers of 625 proinflammatory macrophages. ½F2 NSE-stained frozen sections by LCM (Figure 2, A and B) 626 Sham-operated piglets did not show ischemic osteonecrosis (n Z 6 piglets). A quantitative RT-PCR analysis revealed 627 or the presence of fibrovascular tissue (Supplemental Figure S5). significantly increased mRNA levels (Figure 2C) of proin628 flammatory M1 macrophage markers (NOS2, IL-12b, Ciita, 629 CD68) in macrophages in the repair tissue, whereas M2 Necrotic Bone Environment Stimulates the Gene 630 631 macrophage markers Arg1, Ym1, and CD16312,17 remained Expression of Proinflammatory Cytokines in 632 unaltered. Furthermore, a significantly increased expression Macrophages 633 of proinflammatory cytokines (Figure 2D and Supplemental 634 Figure S4) IL-1b (P < 0.0001), TNF-a (P Z 0.0006), and To investigate whether necrotic bone environment 635 IL-6 (P Z 0.04) but not IFN-g was observed. The activates proinflammatory responses in macrophages, bone 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 Figure 2 Gene expression levels of M1, M2 markers and cytokines in macrophages after ischemic ON of the femoral head. A: A NSE staining of frozen 674 sections showing the macrophages in the normal bone or necrotic bone repair tissue. Arrows show the typical NSE-positive cells. Areas in dashed boxes are 675 shown at higher magnification below. B: NSE-positive cells were isolated by LCM method. The captured macrophages were collected on the LCM cap, and RNA 676 isolation was performed. Areas in dashed boxes are shown at higher magnification below. C: mRNA levels of markers for M1 and M2 macrophages were 677 assessed by quantitative real-time RT-PCR. D: Levels of gene expression of proinflammatory and anti-inflammatory cytokines in normal and ON samples are 678 shown. Abundance of mRNA levels for the individual genes over GAPDH is shown. n Z 6 piglets for each group. Representative images are shown. Paired t-test: 679 *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bars: 100 mm (A, top row); 20 mm (A, bottom row, and B, top right panel and bottom row); 50 mm (B, top 680 left panel). Original magnification: 4 (A, top row); 40 (A, bottom row, and B, top right panel and bottom row); 10 (B, top left panel). GAPDH, 681 glyceraldehyde-3-phosphate dehydrogenase; NSE; nonspecific esterase; LCM, laser capture microdissection; ON, osteonecrosis. 682

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Necrotic Bone Activates TLR4 on Inflammatory Macrophages 807 808 809 810 811 812 Necrotic Bone Stimulates the Expression of TLR4 in 813 Macrophages and Activates TLR4 Signaling Pathways 814 815 To determine the molecular mechanisms responsible for the 816 817 activation of macrophages in the necrotic bone environment, 818 we determined the expression and activation of TLRs on 819 macrophages. The expression levels of TLR4 (P Z 0.03; 820 n Z 6 piglets) were significantly increased in macrophages 821 isolated from the necrotic environment compared with 822 normal bone (Figure 4A) by the LCM method. The increase ½F4 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 Figure 3 Stimulation of inflammatory cytokine expression in macrophages exposed to the osteonecrosis repair environment. AeD: Macrophages were Q34 862 exposed to the soluble factors in the necrotic bone fluid or the necrotic bone at the indicated concentrations and mRNA levels of proinflammatory cytokines IL863 1b (A and C) and TNF-a (B and D) were assessed. The mRNA levels of IL-1b and TNF-a were normalized to GAPDH, and the levels in macrophages exposed to 864 the necrotic environment are shown as fold difference compared with the untreated control. E: The mRNA levels of IL-1b, TNF-a, and IL-6 were determined in 865 the macrophages exposed to 100 mg/mL concentration of the normal bone or the necrotic bone. Data normalized to GAPDH mRNA levels are shown as fold 866 difference in treated samples compared with untreated samples. Statistical analysis was performed by analysis of variance and Tukey’s post hoc test, 867 *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. A representative of three independent experiments is shown. TNF, tumor necrosis factor. 868

marrow-derived macrophages were exposed to the necrotic bone fluid (soluble fraction) or the necrotic bone (sediment fraction) in vitro. The gene expression of proinflammatory cytokine IL-1b was significantly induced by both the ½F3 necrotic bone fluid and the necrotic bone (Figure 3, A and C), whereas the gene expression of TNF-a was stimulated only by necrotic bone (Figure 3, B and D). These results indicate that necrotic bone stimulated the inflammatory cytokine expression from macrophages consistent with the in vivo findings using the LCM method. To verify whether the increased inflammatory gene expression is specific to the exposure to the necrotic bone, but not to the normal bone, macrophages were exposed to either the normal or the necrotic bone. The gene expression of proinflammatory cytokines IL-1b, TNF-a, and IL-6 was

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significantly increased by the necrotic bone but not by normal bone (Figure 3E), suggesting that necrotic but not normal bone stimulates the expression of proinflammatory cytokines in macrophages.

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869 in the TLR4 protein expression was evidenced by immu870 nohistochemistry (Figure 4B). The mRNA levels of TLR2 871 were increased but were not statistically significant 872 (P Z 0.46). TLR9 expression levels were similar in 873 macrophages in the normal and repair tissue. 874 In vitro, treatment of macrophages with the necrotic bone 875 but not normal bone resulted in a significant increase in the 876 mRNA and protein levels of TLR4 (Figure 4, C and D). The 877 increased expression of TLR4 on macrophages was 878 879 confirmed by the double-immunofluorescence method using 880 CD14 antibody and TLR4 antibody (Supplemental 881 Figure S6). These results indicate that necrotic bone stim882 ulates the expression of TLR4 in macrophages. 883 884 Necrotic Bone Stimulates Macrophages through the 885 Activation of TLR4 886 887 TLR4 activation stimulates Myd88-dependent ERK1/2 and 888 889 IKK-a pathways which leads to NF-kBemediated transcrip890 tion, whereas TRIF-dependent pathway involves IFN-g 891 production and subsequent phosphorylation of STAT1.8 892 Treatment of macrophages with necrotic bone stimulated the 893 phosphorylation of ERK1/2 and IKK-a but not STAT1 894 compared with the normal bone, suggesting a predominant 895 activation of Myd88-dependent TLR4-signaling pathways 896 (Figure 5A). To confirm the role of TLR4, a CRISPR-Cas9 ½F5 897 genetic approach was used to ablate TLR4 expression. The 898 activation of ERK1/2, IKK-a but not STAT1, in response to the 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930

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treatment with necrotic bone was abrogated in macrophages transfected with CRISPR-Cas9 TLR4 plasmid, indicating that TLR4 activation is a major receptor mechanism responsible for necrotic bone-mediated stimulation of macrophages. Corresponding to TLR4 signaling pathways, the increased gene expression of IL-1b, TNF-a, and IL-6 was significantly suppressed in macrophages transfected with CRISPR-Cas9 TLR4 plasmid but not with control plasmid (Figure 5B).

Necrotic Bone Stimulates the Proliferation, Migration, and Proinflammatory Cytokine Responses in Macrophages through the Activation of TLR4 To determine whether activation of TLR4 by necrotic bone is responsible for the stimulation of macrophage inflammatory responses, we treated macrophages with necrotic bone in the presence or absence of the TLR4 inhibitor TAK242. Treatment of macrophages with necrotic bone stimulated the phosphorylation of ERK1/2 and IKK-a, suggesting the activation of TLR4-signaling pathways (Figure 6A and Supplemental Figure S7). The activation of ½F6 ERK1/2 and IKK-a but not STAT1 in response to the treatment with necrotic bone was abrogated in the presence of specific TLR4 inhibitor TAK242,19 indicating that TLR4 activation is a major receptor mechanism responsible for necrotic bone-mediated activation of macrophages and that necrotic bone activates TLR4 signaling pathways in Figure 4 Gene and protein expression levels of TLR4 in macrophages exposed to the necrotic bone. A: Quantitative real-time RT-PCR analysis of mRNA levels of TLRs in the nonspecific esterasepositive cells isolated by the LCM method. The mRNA levels for individual genes were normalized to GAPDH, and fold differences in osteonecrosis were compared with normal. B: Detection of TLR4 protein expression in the cells in normal femoral head or repair tissue of necrotic femoral head by immunohistochemistry. Paraffin sections were stained with IgG control antibody or TLR4 antibody followed by counterstaining with light green and hematoxylin. Arrow indicates the color of positive staining. C: TLR4 gene expression in the macrophages treated with normal bone or necrotic bone was detected by quantitative RT-PCR analysis. The mRNA levels of TLR4 were normalized to GAPDH levels, and the data are shown as a fold-difference compared with untreated control. D: Western blot analysis of TLR4 protein expression in macrophages exposed to 100 ng/mL normal or necrotic bone at 24 hours. The blots were probed using anti-TLR4 antibody and b-actin for loading control. Data are expressed as means  SD. n Z 6 piglets for each group (B); n Z 3 blots from independent experiments (C and D). *P < 0.05 compared with untreated control, analysis of variance and Tukey’s post hoc test (t-test), **P < 0.01. Scale bars Z 20 mm. Original magnification, 40. LCM, laser capture microdissection; TLR, Toll-like receptor.

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Necrotic Bone Activates TLR4 on Inflammatory Macrophages

Figure 5 Necrotic bone stimulates macrophages through the activation of TLR4 signaling pathways. A: Western blot analysis of TLR4 activation in macrophages exposed to the necrotic bone. TLR4 gene deletion was performed using the CC9 method. Macrophages were transfected with control plasmid or CC9-TLR4 plasmid. The cells were exposed to normal bone or necrotic bone (100 mg/mL) for 30 minutes. LPS (100 ng/mL) was used as positive control for TLR4 activation. The blots were probed for TLR4 and b-actin as loading control, phosphorylated forms and total proteins ERK1/2, IKK-a, and STAT1. B: The images were captured by LICOR-Odyssey system. mRNA levels of proinflammatory cytokines IL-1b, TNF-a, and IL-6 were normalized to GAPDH mRNA levels. Data are shown as the fold difference in treated samples compared with untreated samples. n Z 3 independent representative experiments. *P < 0.05, ***P < 0.001, and ****P < 0.0001, analysis of variance and Tukey’s post hoc test. CC9, clustered regularly interspaced short palindromic repeats (CRISPR)eCRISPRassociated protein-9 endonuclease (Cas9); Ctrl, control; ERK, extracellular regulated kinase; IKK, Ik kinase; LPS, lipopolysaccharide; MEM, minimum essential medium; Pl, plasmid; TLR, Toll-like receptor; TNF, tumor necrosis factor.

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macrophages. Corresponding to TLR4 signaling, TAK242 inhibited IL-1b, TNF-a, and IL-6 gene expression in macrophages mediated by necrotic bone (Figure 6B). We also determined the proliferation and migration responses of macrophages on exposure to necrotic bone. The necrotic bone significantly increased macrophage proliferation (fold-increase: 1.0  0.2; P < 0.001) compared with the untreated control and the macrophage-colony stimulating factor control (Figure 6C). The macrophage proliferation stimulated by the necrotic bone was significantly inhibited in the presence of TAK242 (fold-increase: 0.3  0.08; P < 0.01). Similarly, in the Transwell migration assay (Figure 6D and Supplemental Figure S8), the addition of necrotic bone significantly increased the migration of macrophages (fold-increase: 6.3  0.3; P < 0.0001) compared with the negative control and the macrophage colony-stimulating factor control, which was significantly inhibited (2.2-fold decrease; P < 0.0001) in the presence of TAK242 compared with the addition of necrotic bone. These results indicate that necrotic bone stimulates the proliferation, migration, and proinflammatory cytokine expression of macrophages through TLR4.

Discussion The major conclusions of the present study are that ONFH results in the formation of a fibrovascular repair tissue

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containing increased numbers of proinflammatory macrophages. Necrotic bone stimulates proinflammatory responses from macrophages through the activation of a specific pattern recognition receptor TLR4. We demonstrated that the numbers of proinflammatory M1 macrophages were increased in the repair tissue, which showed increased expression of proinflammatory cytokines IL-1b, TNF-a, and IL-6 and the pattern recognition receptor TLR4. Necrotic bone activated the TLR4-signaling pathways in macrophages, and the blockade of TLR4 abrogated macrophage proliferation, migration, and proinflammatory cytokine expression. The results from this study are consistent with our recent demonstration of the persistence of inflammatory macrophages in the mouse model of ischemic osteonecrosis.20 Few studies have previously addressed the role of macrophages in the repair process after ONFH. In patients with ONFH21 and in canine and murine models22 of ischemic osteonecrosis,23 only the presence of macrophages in the repair tissue has been reported, but the phenotypic characterization of these cells and receptor mechanisms have not been investigated. In bisphosphonate-related osteonecrosis of the jaw and steroid-induced osteonecrosis, which are distinct disorders from ONFH, both the presence of macrophages and the possible association with M1 macrophage activation have been proposed.24,25 Our study shows that necrotic bone stimulates inflammatory responses in macrophages through the activation of

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Figure 6 TLR4 inhibition prevents the proinflammatory responses in macrophages induced by necrotic bone. A: Western blot analysis of macrophage activation mediated by the necrotic bone. Macrophages were treated with necrotic bone (100 mg/mL) with or without TLR4 inhibitor TAK242 (1 mmol/L). LPS (100 ng/mL) was used as positive control for TLR4 activation. The blots were probed for phosphorylated forms and total proteins ERK1/2, IKK-a, and the images were captured by LICOR-Odyssey system. B: Macrophages were exposed to the necrotic bone (100 mg/mL) with or without TLR4 inhibitor TAK242 (1 mmol/L), and mRNA levels of proinflammatory cytokines IL-1b, TNF-a, and IL-6 were normalized to GAPDH mRNA levels. C: Macrophage proliferation in response to treatment with normal or necrotic bone for 48 hours in the presence or absence of TLR4 inhibitor. D: Migration assay, MCSF was used as positive control. Data are expressed as the fold difference in treated samples compared with untreated samples (B). Percentage of migrated cells among total cells is shown as the fold difference compared with the basal migration with no stimulus (a-MEM) (D). n Z 3 independent experiments is shown. *P < 0.05, **P < 0.01, and ****P < 0.0001, analysis of variance and Tukey’s post hoc test. ERK, extracellular regulated kinase; IKK, Ik kinase; LPS, lipopolysaccharide; MCSF, macrophage-colony stimulating factor; MEM, minimum essential medium; TLR, Toll-like receptor; TNF, tumor necrosis factor.

TLR4, suggesting that necrotic bone components are recognized by TLR4. In previous studies in mice, the injection of normal bone particles resulted in a systemic inflammatory response with an increase in the serum levels of inflammatory cytokines. This elevation in inflammatory cytokine levels was attenuated in TLR4-knockout mice, suggesting a role for TLR4 in recognizing the bone particles.26 In our study, the necrotic bone significantly induced macrophage inflammatory responses through TLR4 activation. After ischemic injury, the damaged cell-endogenous and matrix molecules serve as alarmins (or damageassociated molecular patterns, DAMPs) which can recruit and activate macrophages.6e8

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The activation of macrophages by the necrotic bone suggests that the ischemic injury to bone results in the generation of necrotic bone alarmins, which are recognized by TLR4. Interestingly, in TLR4 signaling after necrotic bone stimulation, Myd88-dependent NF-kB pathway, which includes activation of phospho-ERK, and phospho-IKK-a, was stimulated by necrotic bone (Figure 5A) but not TRIF-dependent pathway, which leads to IFN-g production and STAT1 phosphorylation.7 The reason for this observation could be that several DAMPs including Tenascin-C27 and biglycans28 which are likely to be present in necrotic bone, preferentially activate Myd88-dependent NF-kB (pERK, pIKK-a) pathway but not TRIF pathway (Figures 5A and 6A) which leads to

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IFN-g production and phosphorylation of STAT1.7,8 In these studies by Midwood et al27 and Schaefer et al,28 it was demonstrated that the loss of Myd88 in the cells derived from Myd88/ mice abrogated TLR4-dependent responses to Tenascin-c and biglycan. Another important implication of the absence of STAT1 phosphorylation response to necrotic bone is that the stimulation of TLRs by pathogen-associated molecular patterns from infectious agents and DAMPs (cell-endogenous and matrix components) could be different. Several previous studies, which were reviewed by Piccinini and Midwood8 and Schaefer,29 suggest that pathogen-associated molecular patterns and DAMPs might elicit different responses while interacting with the same TLRs.30 These differences could emanate from ligand recognition and/or signaling mechanisms. In the study by Frey et al,31 several proteoglycans (biglycan, decorin, lumican, versican, hyaluronan), which might be present in necrotic bone as DAMPs, were described to mediate inflammatory activation through TLR2/4. Thus, the mechanisms of production of IFN-g downstream of TLRs such as TLR7, TLR8, and TLR9 might be distinct between infections and sterile inflammation, such as after ischemic osteonecrosis.6e10,27e31 The possible involvement of alarmins and chronic inflammation was also suggested in osteonecrosis of jaw in a diabetic mouse model, which showed NLRP3/caspase-1-dependent IL-1b production, but the role of TLR4 was not investigated in this study.32 In LCPD patients, a recent study evaluated the possible genetic link between TLR4 polymorphisms and the incidence of LCPD,33 but no statistically significant association was found. Because of a small sample size and the possibility of type II statistical error, these findings do not exclude the role of TLR4 activation in the pathologic repair process resulting in chronic inflammation. Interestingly, injection of lipopolysaccharide alone, whose inflammatory effects require TLR4 activation,34 or in combination with corticosteroids35 resulted in ONFH in a rabbit model. These studies further support the pathogenic role of TLR4 activation in osteonecrosis. The involvement of TLR4 in the stimulation of macrophages by necrotic bone may be therapeutically significant, because TLR4 inhibition is a potential therapeutic strategy to limit inflammatory tissue damage.36 In addition, TLR4 activation can result in bone loss due to the effects on the formation of osteoblasts and osteoclasts. The activation of TLR4 signaling pathway inhibits osteoblast differentiation,37 whereas TLR4 inhibition decreases osteoclast formation.38 Furthermore, TLR4-deficient mice show enhanced bone healing in a calvarial defect model.39 Further studies are required to determine the effects of the necrotic bone-mediated macrophage activation on the formation and function of osteoclasts and osteoblasts and whether the blockade of TLR4 (using TLR4 inhibitor, TAK242) might rescue the bone loss and femoral head collapse after ONFH.

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Our previous studies showed that the pathologic bone remodeling after ONFH is dominated by the inhibition of new bone formation and excessive bone resorption,13,15 which are also characteristic of inflammatory bone loss.40 These observations were confirmed by studies in which the new bone formation was promoted by the inhibition of bone resorption and increasing the new bone formation.41e43 The formation of a fibrovascular tissue that does not provide mechanical support similar to bone tissue, leads to a mechanical weakening of the femoral head and the development of a flattening deformity.13,15 Our results in this study suggest that the pathologic repair process after ONFH may be modulated by macrophages that promote chronic inflammatory responses marked by tissue fibrosis instead of tissue regeneration, that is, new bone formation (Supplemental Figure S9). This study has some limitations. We examined the involvement of TLRs in ONFH because these receptor mechanisms are known to be crucial in the stimulation of inflammatory responses in macrophages. Among several pattern recognition receptors, only the involvement of TLR2, TLR4, and TLR9 in ONFH were studied. The possible roles of the other pattern recognition receptors (eg, response receptor for advanced glycosylation end products) and the cellular mechanisms (eg, NLRP3 inflammasome) in ONFH requires further investigation.44,45 The molecular mechanism by which necrotic bone specifically increased TLR4 expression but not TLR2 or TLR9 needs further evaluation. The in vitro evaluation of the macrophage activation mechanism in response to necrotic bone was studied using chemical inhibitor of TLR4, TAK242, and by genetic ablation of TLR4 in macrophages using CRISPR-Cas9 method. As such, these in vitro assessments limit the ability to conclude a role for this receptor in vivo. For this purpose, future studies will assess the in vivo role of TLR4 activation using the mouse model of ischemic osteonecrosis.20 Another limitation of the study is that only an 8-week time point after the induction of ONFH was studied, because this stage of repair corresponds to fibrovascular tissue formation that suggests chronic inflammation. The assessment of macrophage responses at earlier time points is a subject of further studies.

Conclusions In conclusion, ONFH results in the formation of a fibrovascular repair tissue that contains increased numbers of proinflammatory macrophages. Necrotic bone stimulates inflammatory responses in the macrophages through TLR4 activation. Future studies are needed to determine the therapeutic potential of TLR4 blockade to reduce inflammatory damage after ONFH.

Acknowledgments We thank Amanda McLerran for animal care and surgical assistance, Reuel Cornelia and Richard Banlaygas for

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Adapala et al assistance with histology, Ila Oxendine and Yang Li for assistance in the in vitro studies. N.S.A. and H.K.W.K. designed experiments; R.Y. performed the immunofluorescence studies; M.P. did the in vitro quantitative RT-PCR analysis; O.A. performed the microcomputed tomography; N.S.A. and H.K.W.K. reviewed the data; N.S.A., H.K.W.K., and R.Y. wrote the manuscript.

Supplemental Data Supplemental material for this article can be found at http://dx.doi:10.1016/j.ajpath.2016.06.024.

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Necrotic Bone Activates TLR4 on Inflammatory Macrophages

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to steroid-induced femoral head osteonecrosis. BMC Musculoskelet Disord 2014, 15:18 Hennessy EJ, Parker AE, O’Neill LA: Targeting Toll-like receptors: emerging therapeutics? Nat Rev Drug Discov 2010, 9: 293e307 Bandow K, Maeda A, Kakimoto K, Kusuyama J, Shamoto M, Ohnishi T, Matsuguchi T: Molecular mechanisms of the inhibitory effect of lipopolysaccharide (LPS) on osteoblast differentiation. Biochem Biophys Res Commun 2010, 402:755e761 Nakamura H, Fukusaki Y, Yoshimura A, Shiraishi C, Kishimoto M, Kaneko T, Hara Y: Lack of Toll-like receptor 4 decreases lipopolysaccharide-induced bone resorption in C3H/HeJ mice in vivo. Oral Microbiol Immunol 2008, 23:190e195 Wang D, Gilbert JR, Cray JJ Jr, Kubala AA, Shaw MA, Billiar TR, Cooper GM: Accelerated calvarial healing in mice lacking Toll-like receptor 4. PLoS One 2012, 7:e46945 Redlich K, Smolen JS: Inflammatory bone loss: pathogenesis and therapeutic intervention. Nat Rev Drug Discov 2012, 11: 234e250

The American Journal of Pathology

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41. Kim HK, Morgan-Bagley S, Kostenuik P: RANKL inhibition: a novel strategy to decrease femoral head deformity after ischemic osteonecrosis. J Bone Miner Res 2006, 21:1946e1954 42. Kim HK, Randall TS, Bian H, Jenkins J, Garces A, Bauss F: Ibandronate for prevention of femoral head deformity after ischemic necrosis of the capital femoral epiphysis in immature pigs. J Bone Joint Surg Am 2005, 87:550e557 43. Kim HK, Aruwajoye O, Du J, Kamiya N: Local administration of bone morphogenetic protein-2 and bisphosphonate during non-weightbearing treatment of ischemic osteonecrosis of the femoral head: an experimental investigation in immature pigs. J Bone Joint Surg Am 2014, 96:1515e1524 44. Zhou Z, Xiong WC: RAGE and its ligands in bone metabolism. Front Biosci (Schol Ed) 2011, 3:768e776 45. Qu C, Bonar SL, Hickman-Brecks CL, Abu-Amer S, McGeough MD, Pena CA, Broderick L, Yang C, Grimston SK, Kading J, Abu-Amer Y, Novack DV, Hoffman HM, Civitelli R, Mbalaviele G: NLRP3 mediates osteolysis through inflammation-dependent and -independent mechanisms. FASEB J 2015, 29:1269e1279

ajp.amjpathol.org

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1613 Supplemental Figure S1 RNA assessment after LCM of macrophages. A: Agilent Bioanalyzer gel images. Positive control: lane 1, macrophage RNA 1614 (in vitro). Synovium control: bone tissue (lanes 2 and 3). LCM tissue sections: lane 4. B: cDNA prepared using LCM macrophage RNA. Positive control: cDNA 1615 (in vitro cell culture study). C: Quantitative RT-PCR amplification curve (efficiency). D: Melting curve (PCR product characteristics). A sample 96-well plate 1616 reaction: IL-1b, IL-6, TNF-a, IL-12b, iNOS, GAPDH. FU, arbitrary fluorescent unit; iNOS, inducible nitric oxide synthase; L, RNA ladder; LCM, laser capture 1617 microdissection; RIN, RNA integrity number; TNF, tumor necrosis factor. 1618 1619 Supplemental Figure S2 Titration of TLR4 blocker (TAK242) to determine the dose-response effect on macrophage migration. Macrophages were 1620 exposed to necrotic bone (100 mg/mL) with or without TAK242 (4 hours, 5-m Transwell migration chamber). Numbers of macrophages that migrated to the 1621 lower surface are expressed as the average numbers of cells/. n Z 3 independent experiments. *P < 0.05 versus 0 mmol/L inhibitor, analysis of variance1622 Q21 Tukey’s post hoc test. Original magnification, 10. HPF, high-power field; TLR, Toll-like receptor. 1623 1624 Supplemental Figure S3 Quantitation of the number of macrophages by flow cytometry. A: Cells isolated from femoral heads at 8 weeks after ischemic 1625 osteonecrosis were stained with porcine CD14-FITC antibody and analyzed by flow cytometry. Gated cells for quantitation of CD14þ macrophages are shown 1626 with a dashed box. B: Flow cytometric quantitation of the percentage of CD14þ cells among the total number of live cells. n Z 6 piglets. ***P < 0.001 1627 (t-test). FITC, fluorescein isothiocyanate; ON, osteonecrosis; SSC, side scatter. 1628 1629 Supplemental Figure S4 Double immunofluorescence staining of inflammatory cytokine protein levels after ischemic osteonecrosis of the femoral head. 1630 Q22 Frozen sections of osteonecrosis femoral heads were stained with CD14 followed by IL-1b, TNF-a, or IL-6. The top row (green) shows CD14þ cells, the middle 1631 row shows individual cytokines (red), and the bottom row shows a merge of green and red. The blue color represents nuclei with DAPI staining. n Z 6 piglets. 1632 Scale bars Z 10 mm. Original magnification, 100. TNF, tumor necrosis factor. 1633 1634 Supplemental Figure S5 Sham surgery does not induce ischemic osteonecrosis of the femoral head. A: Hematoxylin and eosin staining showing normal 1635 Q23 Q24 bone tissue and bone marrow in nonoperated (left column) or sham-operated (right column) femoral heads. Areas in dashed boxes are shown at higher 1636 magnification below. B: CD163 immunohistochemistry demonstrating the absence of any increase in macrophage numbers after sham surgery. Isotype control 1637 antibody staining is shown as IgG control. n Z 3 piglets. Scale bar: 100 mm (A, top row); 20 mm (A, bottom row, and B). Original magnification: 10 (A, top 1638 row) 40 (A, bottom row, and B). H&E, hematoxylin and eosin. 1639 1640 Supplemental Figure S6 Double immunofluorescence staining to determine TLR4 expression on CD14þ macrophages exposed to necrotic bone. Mac1641 rophages were exposed to necrotic bone (100 mg/mL), and TLR4 protein expression was detected by double immunofluorescence using CD14 and TLR4 an1642 tibodies. IgG control (left column). Green: CD14 (top row); red: TLR4 (middle row) merged image; blue: DAPI-stained nuclei. Yellow: CD14 and TLR4þ cells. 1643 Q25 Scale bars Z 10 mm. Original magnification, 100. TLR, Toll-like receptor. 1644 1645 Supplemental Figure S7 Effects of necrotic bone stimulation and TAK242-mediated TLR4 blockade on STAT1 phosphorylation. Macrophages were 1646 exposed to necrotic bone (100 mg/mL) for 30 minutes in the presence or absence of TLR4 blocker TAK242. LPS (100 ng/mL) was used as a positive control for 1647 TLR4 activation. The blots were probed for phosphorylated forms of STAT1. The images were captured by the LICOR-Odyssey system. LPS, lipopolysaccharide; 1648 MEM, minimum essential medium; TLR, Toll-like receptor. 1649 1650 Supplemental Figure S8 Macrophage migration assay. Hematoxylin staining of migrated cells in the Transwell chamber is shown. MCSF was used as a 1651 Q26 positive control. Scale bars Z 50 mm. Original magnification, 20. MCSF, macrophage colony-stimulating factor; MEM, minimum essential medium; TLR, 1652 Toll-like receptor. 1653 1654 Supplemental Figure S9 A proposed model depicting a possible mechanism of chronic inflammation during the repair process after ischemic osteo1655 necrosis of the femoral head and its role in the development of a flattened deformity of the femoral head. Necrosis of the bone tissue in the femoral head due 1656 to the loss of blood supply results in the release of alarmins, which are recognized by TLR4 on macrophages. The activation of TLR4 induces the inflammatory 1657 responses in macrophages by increasing proliferation and migration, and inflammatory cytokine production. The resulting chronic inflammation promotes 1658 fibrovascular repair tissue formation, inhibits new bone formation, and increases bone resorption. Consequently, because of the loss of mechanical strength, 1659 the femoral head develops a flattened deformity. Blockade of TLR4 might be a potential therapeutic target to decrease chronic inflammation and promote new 1660 bone formation. TLR4, Toll-like receptor 4.

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