Greenhill et al. Arthritis Research & Therapy 2014, 16:419 http://arthritis-research.com/content/16/4/419
RESEARCH
Open Access
Interleukin-10 regulates the inflammasome-driven augmentation of inflammatory arthritis and joint destruction Claire J Greenhill1, Gareth W Jones1, Mari A Nowell1, Zarabeth Newton1, Ann K Harvey1, Abdul N Moideen1, Fraser L Collins1, Anja C Bloom1, Rebecca C Coll2, Avril AB Robertson3, Matthew A Cooper3, Marcela Rosas1, Philip R Taylor1, Luke A O’Neill2, Ian R Humphreys1, Anwen S Williams1 and Simon A Jones1*
Abstract Introduction: Activation of the inflammasome has been implicated in the pathology of various autoinflammatory and autoimmune diseases. While the NLRP3 inflammasome has been linked to arthritis progression, little is known about its synovial regulation or contribution to joint histopathology. Regulators of inflammation activation, such as interleukin (IL)-10, may have the potential to limit the inflammasome-driven arthritic disease course and associated structural damage. Hence, we used IL-10-deficient (IL-10KO) mice to assess NLRP3 inflammasome-driven arthritic pathology. Methods: Antigen-induced arthritis (AIA) was established in IL-10KO mice and wild-type controls. Using histological and radiographic approaches together with quantitative real-time PCR of synovial mRNA studies, we explored the regulation of inflammasome components. These were combined with selective blocking agents and ex vivo investigative studies in osteoclast differentiation assays. Results: In AIA, IL-10KO mice display severe disease with increased histological and radiographic joint scores. Here, focal bone erosions were associated with increased tartrate-resistant acid phosphatase (TRAP)-positive cells and a localized expression of IL-1β. When compared to controls, IL-10KO synovium showed increased expression of Il1b, Il33 and NLRP3 inflammasome components. Synovial Nlrp3 and Casp1 expression further correlated with Acp5 (encoding TRAP), while neutralization of IL-10 receptor signaling in control mice caused increased expression of Nlrp3 and Casp1. In ex vivo osteoclast differentiation assays, addition of exogenous IL-10 or selective blockade of the NLRP3 inflammasome inhibited osteoclastogenesis. Conclusions: These data provide a link between IL-10, synovial regulation of the NLRP3 inflammasome and the degree of bone erosions observed in inflammatory arthritis.
Introduction As new biologics enter the clinical arena and advances in synovial histopathology identify divergent mechanisms of arthritis progression, it is essential to understand how the cytokine network governs the pattern of synovial inflammation [1,2]. Innate sensing mechanisms involving pattern recognition receptors are increasingly implicated in autoimmunity and promote cytokine responses associated with * Correspondence: [email protected] 1 Cardiff Institute of Infection and Immunity, The School of Medicine, Cardiff University, Heath Park Campus, Cardiff CF14 4XN, Wales, UK Full list of author information is available at the end of the article
rheumatoid arthritis. Although these pathways represent promising therapeutic targets, further investigation is required to understand the expression and functional contribution of pattern recognition receptors in autoimmunity. Pattern recognition receptors were initially characterized as sensors of microbial products of bacterial, fungal or viral infection. These include the Toll-like receptors (TLR); nucleotide-binding domain and leucine-rich repeat containing receptors (NLR), Rig-I-like receptors (RLR) and C-type lectins [3,4]. Activation of these receptors promotes the inflammatory regulation of various interleukins, tumour necrosis family members and type-1 interferons
© 2014 Greenhill et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Greenhill et al. Arthritis Research & Therapy 2014, 16:419 http://arthritis-research.com/content/16/4/419
[3]. They therefore represent innate sensing mechanisms, which shape the adaptive immune response to chronic disease, allergy, cancer and infection. As a consequence, various processes have evolved to protect against the prolonged activation of these receptors. For example, interleukin (IL)-10 limits the duration and intensity of their signaling in myeloid cells [5,6]. Here, IL-10 inhibits pattern recognition receptor signaling through mechanisms, which include downregulation of MyD88 expression [7], and the ubiquitination and subsequent degradation of MyD88-dependent signaling molecules such as TRAF6 [8]. In experimental models of inflammatory arthritis, IL10 is protective and mice deficient in IL-10 show exacerbated joint inflammation [9,10]. These data are consistent with the characterization of IL-10 as a cytokine synthesis inhibitory factor, which acts as an immunomodulatory cytokine affecting both innate and cellular immunity [11,12]. For example, IL-10 inhibits nuclear factor kappa B (NF-κB) signaling in response to TLR agonists to block expression of certain proinflammatory mediators associated with arthritis progression. Interleukin-10 is abundantly expressed in synovial fluids of RA patients and has been linked with the control of bone resorption through inhibition of osteoclastogenesis in vitro [13-15]. While IL-10 is renowned for its ability to inhibit TLR signaling, its impact on innate sensing receptors, including the NLR family, is less documented. Here, caspase 1 activity is essential for the processing of cytokine precursors (for example, pro-IL-1β, pro-IL-18 and potentially pro-IL33) into functionally active mature forms [16]. Activation of caspase 1 by the NLR family member NLRP3, acting in association with its adaptor protein ASC, leads to the secretion of IL-1β and IL-18 [16]. Each of these cytokines affect arthritis progression [17,18], which suggests that innate sensing complexes (termed the inflammasome) must be regulated during the course of disease. Various particulate and crystalline agonists activate the NLRP3 inflammasome. For example, monosodium urate crystals and calcium pyrophosphate dihydrate crystals trigger arthritis symptoms in inflammatory forms of gout and pseudogout [19-21], while basic calcium phosphate (hydroxyapatite) crystals are seen in 70% of osteoarthritis (OA) cases [22,23]. A role for the NLRP3 inflammasome in arthritic disease is illustrated by several in vitro studies, which show that crystals from the joints of OA patients and basic calcium phosphate crystals induce IL-1β production by macrophages [24,25]. Also, a recent report shows that there is modulation of the NLRP3 inflammasome in peripheral blood mononuclear cells in RA patients and that single nucleotide polymorphisms (SNPs) in NLRP3 are associated with disease severity [26]. However, little is known about the regulation and activation of inflammasome components in inflammatory arthritis. We now show that the exacerbated joint pathology seen in IL-
Page 2 of 10
10KO mice during antigen-induced arthritis (AIA) is associated with increased synovial expression of NLRP3 inflammasome components and a localized expression of IL-1β at sites of focal bone erosions. Our data supports a role for IL-10 as a negative regulator of the inflammasome and highlights a role for the inflammasome in osteoclastogenesis during inflammatory arthritis.
Materials and methods Mouse strains
Inbred C57BL/6 mouse strains from The Jackson Laboratory (Bar Harbor, ME, USA) were bred and maintained in-house under high barrier and pathogen-free conditions. All animal studies were performed in the United Kingdom. Experiments were performed on eightto twelve-week-old male mice in accordance with UK Home Office Project License PPL-30/2361 and 30/2928. The ethical approval of these licenses covers all aspects of the study and all experiments conducted. Induction of murine AIA
Mice were immunized (subcutaneous (s.c.)) with an emulsion containing 1 mg/ml methylated bovine serum albumin (mBSA) in phosphate-buffered saline (PBS) and Freud’s complete adjuvant (CFA) (Sigma-Aldrich, St Louis, MO, USA). Concurrently, mice were injected (intraperitoneal (i.p.)) with 200 ng of heat-inactivated Bordetella pertussis toxin adjuvant (Sigma-Aldrich, Poole, UK). The immune response was boosted one week later with a second injection (s.c.) of mBSA emulsified in CFA. Arthritis was induced two weeks later with an intra-articular (i.a.) injection of 10 μl of mBSA (10 mg/ml) into the right knee joint. Arthritis progression was monitored using a micrometer to measure changes in knee joint swelling. Radiology
A Kodak in vivo Imaging System FX was used to take radiographs of the mouse knee joints. Both arthritic (right) and non-arthritic joints (left) were compared. Radiographic scores were independently assigned by an orthopedic registrar and based on visible bone erosions (0; normal, 1; mild, 2; moderate, 3; severe). Histology
Joints were fixed in neutral-buffered formalin saline, decalcified with formic acid at 4°C and embedded in paraffin. Midsagittal sections (8 μm) were stained with haematoxylin, safranin-O and Fast Green. Two independent observers scored histology sections for subsynovial inflammation (0 = normal, to 5 = ablation of adipose tissue due to leukocyte infiltrate), synovial exudate (0 = normal, to 3 = substantial number of cells with large fibrin deposits), synovial hyperplasia (0 = normal 1 to 3 cells thick, to 3 = over three layers thick with overgrowth onto joint surfaces with evidence of
Greenhill et al. Arthritis Research & Therapy 2014, 16:419 http://arthritis-research.com/content/16/4/419
cartilage/bone erosion), cartilage/bone erosion (0 = normal, 3 = destruction of a significant part of the bone). Cartilage integrity was determined in histological sections using a Mankin scoring system. Two independent observers evaluated cartilage irregularity and cleft formation (0 = normal, to 6 = complete disorganization of glycoproteins with clefts into the cartilage), cellularity (0 = normal, to 3 = hypocellularity), proteoglycan depletion (0 = normal, to 4 = complete proteoglycan degradation with no dye apparent) and tidemark integrity (0 = intact, to 1 = tidemark crossed by blood vessels). The total sum of these scores resulted in a maximum score of 14. For detection of tartrate-resistant acid phosphatase (TRAP) activity, slides were rehydrated after decalcification, incubated with TRAP staining solution (0.2 M acetate buffer, 50 mM sodium tartrate, 0.5 mg/ml naphthol AS-MX phosphate, 1.1 mg/ml Fast Red Violet LB salt) and counterstained with haematoxylin. Immunohistochemistry
Antigen retrieval was performed on paraffin-embedded sections using either trypsin (0.1%) for 30 mins at 37°C, or 10 mM citrate buffer (pH 6) for 40 mins at 95°C. Endogenous peroxidase and biotin activity was blocked using 3% H202 and an avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA, USA) respectively. Sections were incubated in 10% (v:v) rabbit serum for 1 hour before staining with rat anti-mouse F4/80 (1:50 dilution, Santa Cruz Technology, Santa Cruz, CA, USA). Antibody binding to sections was detected with rabbit anti-rat biotin-conjugated secondary antibody and streptavidinhorseradish peroxidase (HRP) complex (Vector Laboratories). Diaminobenzidine substrate (Dako, Glostrup, Denmark) was used to develop sections and haematoxylin was used as a counterstain. Image analysis
Immunohistochemistry was viewed with a Leica DMLB light microscope (Milton Keynes, UK). Analysis across five random fields of view was performed using the Leica digital image capture program. Values are expressed as a percentage of total immunoperoxidase staining. Osteoclast cell culture and TRAP stain
Bone marrow cells from femurs of WT and IL-10KO mice were re-suspended in alpha minimum essential medium (αMEM) supplemented with 10% (v:v) foetal calf serum (FCS) and seeded at a density of 6.4 × 106 cells/ml in 24-well plates. Following adhesion culture media was supplemented with macrophage colony-stimulating factor (MCSF) and receptor activator of NF-κβ ligand (RANKL). IL-10 and inflammasome inhibitors were added as indicated in figure legends. TRAP-positive cells were detected after seven days and total RNA isolated from subsequent analysis.
Page 3 of 10
Quantitative real-time PCR (qPCR)
Synovial membranes were dissected from the underpinning cartilage of knee joints [27]. Total RNA was extracted from samples using TRI Reagent (Sigma-Aldrich) and cDNA derived from 1 μg of total RNA using a reverse transcription kit (Primer Design, Southampton, UK) [27]. Gene expression analyses were performed on triplicate samples with SYBR Green (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) using an ABI Prism 7900HT instrument (Applied Biosystems, Thermo Fisher Scientific, Carlsbad, CA, USA). Details of oligonucleotide primer sequences are presented in Additional file 1. Data analysis was performed using the Sequence Detection System Version 2.3 software (Applied Biosystems). Enzyme-linked immunosorbent assay (ELISA)
Serum dickkopf-1 (DKK1) was quantified using a commercial enzyme-linked immunosorbent assay (ELISA) (R&D Systems Inc, Minneapolis, MN, USA) as per the manufacturer’s instructions. Statistical analysis
Data was evaluated using the non-parametric MannWhitney U test and an unpaired Student t test. In all cases, P
RESEARCH
Open Access
Interleukin-10 regulates the inflammasome-driven augmentation of inflammatory arthritis and joint destruction Claire J Greenhill1, Gareth W Jones1, Mari A Nowell1, Zarabeth Newton1, Ann K Harvey1, Abdul N Moideen1, Fraser L Collins1, Anja C Bloom1, Rebecca C Coll2, Avril AB Robertson3, Matthew A Cooper3, Marcela Rosas1, Philip R Taylor1, Luke A O’Neill2, Ian R Humphreys1, Anwen S Williams1 and Simon A Jones1*
Abstract Introduction: Activation of the inflammasome has been implicated in the pathology of various autoinflammatory and autoimmune diseases. While the NLRP3 inflammasome has been linked to arthritis progression, little is known about its synovial regulation or contribution to joint histopathology. Regulators of inflammation activation, such as interleukin (IL)-10, may have the potential to limit the inflammasome-driven arthritic disease course and associated structural damage. Hence, we used IL-10-deficient (IL-10KO) mice to assess NLRP3 inflammasome-driven arthritic pathology. Methods: Antigen-induced arthritis (AIA) was established in IL-10KO mice and wild-type controls. Using histological and radiographic approaches together with quantitative real-time PCR of synovial mRNA studies, we explored the regulation of inflammasome components. These were combined with selective blocking agents and ex vivo investigative studies in osteoclast differentiation assays. Results: In AIA, IL-10KO mice display severe disease with increased histological and radiographic joint scores. Here, focal bone erosions were associated with increased tartrate-resistant acid phosphatase (TRAP)-positive cells and a localized expression of IL-1β. When compared to controls, IL-10KO synovium showed increased expression of Il1b, Il33 and NLRP3 inflammasome components. Synovial Nlrp3 and Casp1 expression further correlated with Acp5 (encoding TRAP), while neutralization of IL-10 receptor signaling in control mice caused increased expression of Nlrp3 and Casp1. In ex vivo osteoclast differentiation assays, addition of exogenous IL-10 or selective blockade of the NLRP3 inflammasome inhibited osteoclastogenesis. Conclusions: These data provide a link between IL-10, synovial regulation of the NLRP3 inflammasome and the degree of bone erosions observed in inflammatory arthritis.
Introduction As new biologics enter the clinical arena and advances in synovial histopathology identify divergent mechanisms of arthritis progression, it is essential to understand how the cytokine network governs the pattern of synovial inflammation [1,2]. Innate sensing mechanisms involving pattern recognition receptors are increasingly implicated in autoimmunity and promote cytokine responses associated with * Correspondence: [email protected] 1 Cardiff Institute of Infection and Immunity, The School of Medicine, Cardiff University, Heath Park Campus, Cardiff CF14 4XN, Wales, UK Full list of author information is available at the end of the article
rheumatoid arthritis. Although these pathways represent promising therapeutic targets, further investigation is required to understand the expression and functional contribution of pattern recognition receptors in autoimmunity. Pattern recognition receptors were initially characterized as sensors of microbial products of bacterial, fungal or viral infection. These include the Toll-like receptors (TLR); nucleotide-binding domain and leucine-rich repeat containing receptors (NLR), Rig-I-like receptors (RLR) and C-type lectins [3,4]. Activation of these receptors promotes the inflammatory regulation of various interleukins, tumour necrosis family members and type-1 interferons
© 2014 Greenhill et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Greenhill et al. Arthritis Research & Therapy 2014, 16:419 http://arthritis-research.com/content/16/4/419
[3]. They therefore represent innate sensing mechanisms, which shape the adaptive immune response to chronic disease, allergy, cancer and infection. As a consequence, various processes have evolved to protect against the prolonged activation of these receptors. For example, interleukin (IL)-10 limits the duration and intensity of their signaling in myeloid cells [5,6]. Here, IL-10 inhibits pattern recognition receptor signaling through mechanisms, which include downregulation of MyD88 expression [7], and the ubiquitination and subsequent degradation of MyD88-dependent signaling molecules such as TRAF6 [8]. In experimental models of inflammatory arthritis, IL10 is protective and mice deficient in IL-10 show exacerbated joint inflammation [9,10]. These data are consistent with the characterization of IL-10 as a cytokine synthesis inhibitory factor, which acts as an immunomodulatory cytokine affecting both innate and cellular immunity [11,12]. For example, IL-10 inhibits nuclear factor kappa B (NF-κB) signaling in response to TLR agonists to block expression of certain proinflammatory mediators associated with arthritis progression. Interleukin-10 is abundantly expressed in synovial fluids of RA patients and has been linked with the control of bone resorption through inhibition of osteoclastogenesis in vitro [13-15]. While IL-10 is renowned for its ability to inhibit TLR signaling, its impact on innate sensing receptors, including the NLR family, is less documented. Here, caspase 1 activity is essential for the processing of cytokine precursors (for example, pro-IL-1β, pro-IL-18 and potentially pro-IL33) into functionally active mature forms [16]. Activation of caspase 1 by the NLR family member NLRP3, acting in association with its adaptor protein ASC, leads to the secretion of IL-1β and IL-18 [16]. Each of these cytokines affect arthritis progression [17,18], which suggests that innate sensing complexes (termed the inflammasome) must be regulated during the course of disease. Various particulate and crystalline agonists activate the NLRP3 inflammasome. For example, monosodium urate crystals and calcium pyrophosphate dihydrate crystals trigger arthritis symptoms in inflammatory forms of gout and pseudogout [19-21], while basic calcium phosphate (hydroxyapatite) crystals are seen in 70% of osteoarthritis (OA) cases [22,23]. A role for the NLRP3 inflammasome in arthritic disease is illustrated by several in vitro studies, which show that crystals from the joints of OA patients and basic calcium phosphate crystals induce IL-1β production by macrophages [24,25]. Also, a recent report shows that there is modulation of the NLRP3 inflammasome in peripheral blood mononuclear cells in RA patients and that single nucleotide polymorphisms (SNPs) in NLRP3 are associated with disease severity [26]. However, little is known about the regulation and activation of inflammasome components in inflammatory arthritis. We now show that the exacerbated joint pathology seen in IL-
Page 2 of 10
10KO mice during antigen-induced arthritis (AIA) is associated with increased synovial expression of NLRP3 inflammasome components and a localized expression of IL-1β at sites of focal bone erosions. Our data supports a role for IL-10 as a negative regulator of the inflammasome and highlights a role for the inflammasome in osteoclastogenesis during inflammatory arthritis.
Materials and methods Mouse strains
Inbred C57BL/6 mouse strains from The Jackson Laboratory (Bar Harbor, ME, USA) were bred and maintained in-house under high barrier and pathogen-free conditions. All animal studies were performed in the United Kingdom. Experiments were performed on eightto twelve-week-old male mice in accordance with UK Home Office Project License PPL-30/2361 and 30/2928. The ethical approval of these licenses covers all aspects of the study and all experiments conducted. Induction of murine AIA
Mice were immunized (subcutaneous (s.c.)) with an emulsion containing 1 mg/ml methylated bovine serum albumin (mBSA) in phosphate-buffered saline (PBS) and Freud’s complete adjuvant (CFA) (Sigma-Aldrich, St Louis, MO, USA). Concurrently, mice were injected (intraperitoneal (i.p.)) with 200 ng of heat-inactivated Bordetella pertussis toxin adjuvant (Sigma-Aldrich, Poole, UK). The immune response was boosted one week later with a second injection (s.c.) of mBSA emulsified in CFA. Arthritis was induced two weeks later with an intra-articular (i.a.) injection of 10 μl of mBSA (10 mg/ml) into the right knee joint. Arthritis progression was monitored using a micrometer to measure changes in knee joint swelling. Radiology
A Kodak in vivo Imaging System FX was used to take radiographs of the mouse knee joints. Both arthritic (right) and non-arthritic joints (left) were compared. Radiographic scores were independently assigned by an orthopedic registrar and based on visible bone erosions (0; normal, 1; mild, 2; moderate, 3; severe). Histology
Joints were fixed in neutral-buffered formalin saline, decalcified with formic acid at 4°C and embedded in paraffin. Midsagittal sections (8 μm) were stained with haematoxylin, safranin-O and Fast Green. Two independent observers scored histology sections for subsynovial inflammation (0 = normal, to 5 = ablation of adipose tissue due to leukocyte infiltrate), synovial exudate (0 = normal, to 3 = substantial number of cells with large fibrin deposits), synovial hyperplasia (0 = normal 1 to 3 cells thick, to 3 = over three layers thick with overgrowth onto joint surfaces with evidence of
Greenhill et al. Arthritis Research & Therapy 2014, 16:419 http://arthritis-research.com/content/16/4/419
cartilage/bone erosion), cartilage/bone erosion (0 = normal, 3 = destruction of a significant part of the bone). Cartilage integrity was determined in histological sections using a Mankin scoring system. Two independent observers evaluated cartilage irregularity and cleft formation (0 = normal, to 6 = complete disorganization of glycoproteins with clefts into the cartilage), cellularity (0 = normal, to 3 = hypocellularity), proteoglycan depletion (0 = normal, to 4 = complete proteoglycan degradation with no dye apparent) and tidemark integrity (0 = intact, to 1 = tidemark crossed by blood vessels). The total sum of these scores resulted in a maximum score of 14. For detection of tartrate-resistant acid phosphatase (TRAP) activity, slides were rehydrated after decalcification, incubated with TRAP staining solution (0.2 M acetate buffer, 50 mM sodium tartrate, 0.5 mg/ml naphthol AS-MX phosphate, 1.1 mg/ml Fast Red Violet LB salt) and counterstained with haematoxylin. Immunohistochemistry
Antigen retrieval was performed on paraffin-embedded sections using either trypsin (0.1%) for 30 mins at 37°C, or 10 mM citrate buffer (pH 6) for 40 mins at 95°C. Endogenous peroxidase and biotin activity was blocked using 3% H202 and an avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA, USA) respectively. Sections were incubated in 10% (v:v) rabbit serum for 1 hour before staining with rat anti-mouse F4/80 (1:50 dilution, Santa Cruz Technology, Santa Cruz, CA, USA). Antibody binding to sections was detected with rabbit anti-rat biotin-conjugated secondary antibody and streptavidinhorseradish peroxidase (HRP) complex (Vector Laboratories). Diaminobenzidine substrate (Dako, Glostrup, Denmark) was used to develop sections and haematoxylin was used as a counterstain. Image analysis
Immunohistochemistry was viewed with a Leica DMLB light microscope (Milton Keynes, UK). Analysis across five random fields of view was performed using the Leica digital image capture program. Values are expressed as a percentage of total immunoperoxidase staining. Osteoclast cell culture and TRAP stain
Bone marrow cells from femurs of WT and IL-10KO mice were re-suspended in alpha minimum essential medium (αMEM) supplemented with 10% (v:v) foetal calf serum (FCS) and seeded at a density of 6.4 × 106 cells/ml in 24-well plates. Following adhesion culture media was supplemented with macrophage colony-stimulating factor (MCSF) and receptor activator of NF-κβ ligand (RANKL). IL-10 and inflammasome inhibitors were added as indicated in figure legends. TRAP-positive cells were detected after seven days and total RNA isolated from subsequent analysis.
Page 3 of 10
Quantitative real-time PCR (qPCR)
Synovial membranes were dissected from the underpinning cartilage of knee joints [27]. Total RNA was extracted from samples using TRI Reagent (Sigma-Aldrich) and cDNA derived from 1 μg of total RNA using a reverse transcription kit (Primer Design, Southampton, UK) [27]. Gene expression analyses were performed on triplicate samples with SYBR Green (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) using an ABI Prism 7900HT instrument (Applied Biosystems, Thermo Fisher Scientific, Carlsbad, CA, USA). Details of oligonucleotide primer sequences are presented in Additional file 1. Data analysis was performed using the Sequence Detection System Version 2.3 software (Applied Biosystems). Enzyme-linked immunosorbent assay (ELISA)
Serum dickkopf-1 (DKK1) was quantified using a commercial enzyme-linked immunosorbent assay (ELISA) (R&D Systems Inc, Minneapolis, MN, USA) as per the manufacturer’s instructions. Statistical analysis
Data was evaluated using the non-parametric MannWhitney U test and an unpaired Student t test. In all cases, P
