Wnt16 Signaling Is Required for IL-1β-Induced Matrix Metalloproteinase-13-Regulated Proliferation of Human Stem Cell-Derived Osteoblastic Cells

We established a differentiation method for homogeneous α7 integrin-positive human skeletal muscle stem cell (α7⁺hSMSC)-derived osteoblast-like (α7⁺hSMSC-OB) cells, and found that interleukin (IL)-1β induces matrix metalloproteinase (MMP)-13-regulated proliferation of these cells. These data suggest that MMP-13 plays a potentially unique physiological role in the regeneration of osteoblast-like cells. Here, we examined whether up-regulation of MMP-13 activity by IL-1β was mediated by Wingless/int1 (Wnt) signaling and increased the proliferation of osteoblast-like cells. IL-1β increased the mRNA and protein levels of Wnt16 and the Wnt receptor Lrp5/Fzd2. Exogenous Wnt16 was found to increase MMP-13 mRNA, protein and activity, and interestingly, the proliferation rate of these cells. Treatment with small interfering RNAs against Wnt16 and Lrp5 suppressed the IL-1β-induced increase in cell proliferation. We revealed that a unique signaling cascade IL-1β→Wnt16→Lrp5→MMP-13, was intimately involved in the proliferation of osteoblast-like cells, and suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation are regulated by Wnt16.     

Mycophenolate Antagonizes IFN-γ-Induced Catagen-Like Changes via β-Catenin Activation in Human Dermal Papilla Cells and Hair Follicles

Recently, various immunosuppressant drugs have been shown to induce hair growth in normal hair as well as in alopecia areata and androgenic alopecia; however, the responsible mechanism has not yet been fully elucidated. In this study, we investigate the influence of mycophenolate (MPA), an immunosuppressant, on the proliferation of human dermal papilla cells (hDPCs) and on the growth of human hair follicles following catagen induction with interferon (IFN)-γ. IFN-γ was found to reduce β-catenin, an activator of hair follicle growth, and activate glycogen synthase kinase (GSK)-3β, and enhance expression of the Wnt inhibitor DKK-1 and catagen inducer transforming growth factor (TGF)-β2. IFN-γ inhibited expression of ALP and other dermal papillar cells (DPCs) markers such as Axin2, IGF-1, and FGF 7 and 10. MPA increased β-catenin in IFN-γ-treated hDPCs leading to its nuclear accumulation via inhibition of GSK3β and reduction of DKK-1. Furthermore, MPA significantly increased expression of ALP and other DPC marker genes but inhibited expression of TGF-β2. Therefore, we demonstrate for the first time that IFN-γ induces catagen-like changes in hDPCs and in hair follicles via inhibition of Wnt/β-catenin signaling, and that MPA stabilizes β-catenin by inhibiting GSK3β leading to increased β-catenin target gene and DP signature gene expression, which may, in part, counteract IFN-γ-induced catagen in hDPCs.     

Biphasic α2β1 Integrin Expression in Breast Cancer Metastasis to Bone

Integrins participate in the pathogenesis and progression of tumors at many stages during the metastatic cascade. However, current evidence for the role of integrins in breast cancer progression is contradictory and seems to be dependent on tumor stage, differentiation status, and microenvironmental influences. While some studies suggest that loss of α2β1 enhances cancer metastasis, other studies suggest that this integrin is pro-tumorigenic. However, few studies have looked at α2β1 in the context of bone metastasis. In this study, we aimed to understand the role of α2β1 integrin in breast cancer metastasis to bone. To address this, we utilized in vivo models of breast cancer metastasis to bone using MDA-MB-231 cells transfected with an α2 expression plasmid (MDA-OEα2). MDA cells overexpressing the α2 integrin subunit had increased primary tumor growth and dissemination to bone but had no change in tumor establishment and bone destruction. Further in vitro analysis revealed that tumors in the bone have decreased α2β1 expression and increased osteolytic signaling compared to primary tumors. Taken together, these data suggest an inverse correlation between α2β1 expression and bone-metastatic potential. Inhibiting α2β1 expression may be beneficial to limit the expansion of primary tumors but could be harmful once tumors have established in bone.     

Cortisol Metabolism in Carp Macrophages: A Role for Macrophage-Derived Cortisol in M1/M2 Polarization

Macrophages are crucial not only for initiation of inflammation and pathogen eradication (classically polarized M1 macrophages), but also for inflammation inhibition and tissue regeneration (alternatively polarized M2 macrophages). Their polarization toward the M1 population occurs under the influence of interferon-γ + lipopolysaccharide (IFN-γ + LPS), while alternatively polarized M2 macrophages evolve upon, e.g., interlukin 4 (IL-4) or cortisol stimulation. This in vitro study focused on a possible role for macrophage-derived cortisol in M1/M2 polarization in common carp. We studied the expression of molecules involved in cortisol synthesis/conversion from and to cortisone like 11β-hydroxysteroid dehydrogenase type 2 and 3. (11β-HSD2 and 3) and 11β-hydroxylase (CYP11b), as well as the expression of glucocorticoid receptors (GRs) and proliferator-activated receptor gamma (PPARγ) in M1 and M2 macrophages. Lastly, we analyzed how inhibition of these molecules affect macrophage polarization. In M1 cells, upregulation of gene expression of GRs and 11β-HSD3 was found, while, in M2 macrophages, expression of 11β-hsd2 was upregulated. Moreover, blocking of cortisol synthesis/conversion and GRs or PPARγ induced changes in expression of anti-inflammatory interleukin 10 (IL-10). Consequently, our data show that carp monocytes/macrophages can convert cortisol. The results strongly suggest that cortisol, via intracrine interaction with GRs, is important for IL-10-dependent control of the activity of macrophages and for the regulation of M1/M2 polarization to finally determine the outcome of an infection.     

β2-Adrenergic Receptors Increase Cardiac Fibroblast Proliferation Through the Gαs/ERK1/2-Dependent Secretion of Interleukin-6

Fibroblasts are an important resident cell population in the heart involved in maintaining homeostasis and structure during normal conditions. They are also crucial in disease states for sensing signals and initiating the appropriate repair responses to maintain the structural integrity of the heart. This sentinel role of cardiac fibroblasts occurs, in part, through their ability to secrete cytokines. β-adrenergic receptors (βAR) are also critical regulators of cardiac function in the normal and diseased state and a major therapeutic target clinically. βAR are known to influence cytokine secretion in various cell types and they have been shown to be involved in cytokine production in the heart, but their role in regulating cytokine production in cardiac fibroblasts is not well understood. Thus, we hypothesized that βAR activation on cardiac fibroblasts modulates cytokine production to influence fibroblast function. Using primary fibroblast cultures from neonatal rats and adult mice, increased interleukin (IL)-6 expression and secretion occurred following β2AR activation. The use of pharmacological inhibitors and genetic manipulations showed that IL-6 elevations occurred through the Gαs-mediated activation of ERK1/2 and resulted in increased fibroblast proliferation. In vivo, a lack of β2AR resulted in increased infarct size following myocardial infarction and impaired wound closure in a murine dermal wound healing assay. These findings identify an important role for β2AR in regulating fibroblast proliferation through Gαs/ERK1/2-dependent alterations in IL-6 and may lead to the development of improved heart failure therapies through targeting fibrotic function of β2AR.     

Rheumatoid Arthritis-Associated MicroRNA-155 Targets SOCS1 and Upregulates TNF-α and IL-1β in PBMCs

miR-155 plays a crucial role in proinflammatory activation. This study was carried out to assess the association of abnormal expression of miR-155 in peripheral blood of patients with Rheumatoid arthritis with the expression of TNF-α and IL-1β. Release of TNF-α and IL-1β, and expression of miR-155 were determined in RA peripheral blood or peripheral blood macrophages, followed by correlation analysis of the cytokines release and miR-155 expression. Furthermore, in vitro studies indicate that miR-155 inhibited the expression of SOCS1. Our results suggest that there is a correlation between the high-level expression of miR-155 and the enhanced expression of TNF-α and IL-1β. miR-155 targets and suppresses the expression of SOCS1, and the decrease of SOCS1 may lead to the upregulation of TNF-α and IL-1β.     

Cytokine Networks in the Pathogenesis of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic systemic inflammation causing progressive joint damage that can lead to lifelong disability. The pathogenesis of RA involves a complex network of various cytokines and cells that trigger synovial cell proliferation and cause damage to both cartilage and bone. Involvement of the cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 is central to the pathogenesis of RA, but recent research has revealed that other cytokines such as IL-7, IL-17, IL-21, IL-23, granulocyte macrophage colony-stimulating factor (GM-CSF), IL-1β, IL-18, IL-33, and IL-2 also play a role. Clarification of RA pathology has led to the development of therapeutic agents such as biological disease-modifying anti-rheumatic drugs (DMARDs) and Janus kinase (JAK) inhibitors, and further details of the immunological background to RA are emerging. This review covers existing knowledge regarding the roles of cytokines, related immune cells and the immune system in RA, manipulation of which may offer the potential for even safer and more effective treatments in the future.     

Chondrocytes from Osteoarthritis Patients Adopt Distinct Phenotypes in Response to Central TH1/TH2/TH17 Cytokines

Chronic low-grade inflammation plays a central role in the pathogenesis of osteoarthritis (OA), and several pro- and anti-inflammatory cytokines have been implicated to mediate and regulate this process. Out of these cytokines, particularly IFNγ, IL-1β, IL-4 and IL-17 are associated with different phenotypes of T helper (T_H) cells and macrophages, both examples of cells known for great phenotypic and functional heterogeneity. Chondrocytes also display various phenotypic changes during the course of arthritis. We set out to study the hypothesis of whether chondrocytes might adopt polarized phenotypes analogous to T_H cells and macrophages. We studied the effects of IFNγ, IL-1β, IL-4 and IL-17 on gene expression in OA chondrocytes with RNA-Seq. Chondrocytes were harvested from the cartilage of OA patients undergoing knee replacement surgery and then cultured with or without the cytokines for 24 h. Total RNA was isolated and sequenced, and GO (Gene Ontology) functional analysis was performed. We also separately investigated genes linked to OA in recent genome wide expression analysis (GWEA) studies. The expression of more than 2800 genes was significantly altered in chondrocytes treated with IL-1β [in the C(IL-1β) phenotype] with a fold change (FC) > 2.5 in either direction. These included a large number of genes associated with inflammation, cartilage degradation and attenuation of metabolic signaling. The profile of genes differentially affected by IFNγ (the C(IFNγ) phenotype) was relatively distinct from that of the C(IL-1β) phenotype and included several genes associated with antigen processing and presentation. The IL-17-induced C(IL-17) phenotype was characterized by the induction of a more limited set of proinflammatory factors compared to C(IL-1β) cells. The C(IL-4) phenotype induced by IL-4 displayed a differential expression of a rather small set of genes compared with control, primarily those associated with TGFβ signaling and the regulation of inflammation. In conclusion, our results show that OA chondrocytes can adopt diverse phenotypes partly analogously to T_H cells and macrophages. This phenotypic plasticity may play a role in the pathogenesis of arthritis and open new therapeutic avenues for the development of disease-modifying treatments for (osteo)arthritis.     

Diagnostic Potential of Differentially Expressed Homer1, IL-1β, and TNF-α in Coronary Artery Disease

Increasing evidences suggest that inflammation plays an important role in the pathogenesis of coronary artery disease (CAD). Numerous inflammatory cytokines and related genes mediate adverse cardiovascular events in patients with CAD, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and Homer in the present study. The study was carried out on 163 CAD patients at different stages and 68 controls. The gene expression of Homer1, Homer2, Homer3, IL-1β, and TNF-α in the peripheral blood leukocytes were measured by real-time polymerase chain reaction. The mRNA levels of Homer1, IL-1β, and TNF-α in CAD patients were significantly higher than those in the control group, but not Homer2 and Homer3. However, there was no considerable difference in the mRNA levels of Homer1, IL-1β, and TNF-α among AMI, UAP, and SAP three subgroups of CAD. The receiver operating characteristic (ROC) curves showed that Homer1 had a better diagnostic value for UAP patients compared with IL-1β and TNF-α. Like IL-1β and TNF-α, Homer1 may also be an important participant of atherosclerotic plaque development and eventually rupture. The results of the present study may provide an important basis for diagnosing CAD patients, and provide new therapeutic targets for CAD.     

Inhibition of Inducible Nitric Oxide Synthase Prevents IL-1β-Induced Mitochondrial Dysfunction in Human Chondrocytes

Interleukin (IL)-1β is an important pro-inflammatory cytokine in the progression of osteoarthritis (OA), which impairs mitochondrial function and induces the production of nitric oxide (NO) in chondrocytes. The aim was to investigate if blockade of NO production prevents IL-1β-induced mitochondrial dysfunction in chondrocytes and whether cAMP and AMP-activated protein kinase (AMPK) affects NO production and mitochondrial function. Isolated human OA chondrocytes were stimulated with IL-1β in combination with/without forskolin, L-NIL, AMPK activator or inhibitor. The release of NO, IL-6, PGE₂, MMP3, and the expression of iNOS were measured by ELISA or Western blot. Parameters of mitochondrial respiration were measured using a seahorse analyzer. IL-1β significantly induced NO release and mitochondrial dysfunction. Inhibition of iNOS by L-NIL prevented IL-1β-induced NO release and mitochondrial dysfunction but not IL-1β-induced release of IL-6, PGE₂, and MMP3. Enhancement of cAMP by forskolin reduced IL-1β-induced NO release and prevented IL-1β-induced mitochondrial impairment. Activation of AMPK increased IL-1β-induced NO production and the negative impact of IL-1β on mitochondrial respiration, whereas inhibition of AMPK had the opposite effects. NO is critically involved in the IL-1β-induced impairment of mitochondrial respiration in human OA chondrocytes. Increased intracellular cAMP or inhibition of AMPK prevented both IL-1β-induced NO release and mitochondrial dysfunction.     

The Role of Cytokines Produced via the NLRP3 Inflammasome in Mouse Macrophages Stimulated with Dental Calculus in Osteoclastogenesis

Dental calculus (DC) is a common deposit in periodontitis patients. We have previously shown that DC contains both microbial components and calcium phosphate crystals that induce an osteoclastogenic cytokine IL-1β via the NLRP3 inflammasome in macrophages. In this study, we examined the effects of cytokines produced by mouse macrophages stimulated with DC on osteoclastogenesis. The culture supernatants from wild-type (WT) mouse macrophages stimulated with DC accelerated osteoclastogenesis in RANKL-primed mouse bone marrow macrophages (BMMs), but inhibited osteoclastogenesis in RANKL-primed RAW-D cells. WT, but not NLRP3-deficient, mouse macrophages stimulated with DC produced IL-1β and IL-18 in a dose-dependent manner, indicating the NLRP3 inflammasome-dependent production of IL-1β and IL-18. Both WT and NLRP3-deficient mouse macrophages stimulated with DC produced IL-10, indicating the NLRP3 inflammasome-independent production of IL-10. Recombinant IL-1β accelerated osteoclastogenesis in both RANKL-primed BMMs and RAW-D cells, whereas recombinant IL-18 and IL-10 inhibited osteoclastogenesis. These results indicate that DC induces osteoclastogenic IL-1β in an NLRP3 inflammasome-dependent manner and anti-osteogenic IL-18 and IL-10 dependently and independently of the NLRP3 inflammasome, respectively. DC may promote alveolar bone resorption via IL-1β induction in periodontitis patients, but suppress resorption via IL-18 and IL-10 induction in some circumstances.     

Interleukin-1β Modulation of the Mechanobiology of Primary Human Pulmonary Fibroblasts: Potential Implications in Lung Repair

Pro-inflammatory cytokines like interleukin-1β (IL-1β) are upregulated during early responses to tissue damage and are expected to transiently compromise the mechanical microenvironment. Fibroblasts are key regulators of tissue mechanics in the lungs and other organs. However, the effects of IL-1β on fibroblast mechanics and functions remain unclear. Here we treated human pulmonary fibroblasts from control donors with IL-1β and used Atomic Force Microscopy to unveil that IL-1β significantly reduces the stiffness of fibroblasts concomitantly with a downregulation of filamentous actin (F-actin) and alpha-smooth muscle (α-SMA). Likewise, COL1A1 mRNA was reduced, whereas that of collagenases MMP1 and MMP2 were upregulated, favoring a reduction of type-I collagen. These mechanobiology changes were functionally associated with reduced proliferation and enhanced migration upon IL-1β stimulation, which could facilitate lung repair by drawing fibroblasts to sites of tissue damage. Our observations reveal that IL-1β may reduce local tissue rigidity by acting both intracellularly and extracellularly through the downregulation of fibroblast contractility and type I collagen deposition, respectively. These IL-1β-dependent mechanical effects may enhance lung repair further by locally increasing pulmonary tissue compliance to preserve normal lung distension and function. Moreover, our results support that IL-1β provides innate anti-fibrotic protection that may be relevant during the early stages of lung repair.     

β-Defensin 2 Ameliorates Lung Injury Caused by Pseudomonas Infection and Regulates Proinflammatory and Anti-Inflammatory Cytokines in Rat

An important member of the defensin family, β-defensin 2, is believed to play an important role in defense against foreign pathogens. In the present study, we constructed lentiviral vectors to express and knockdown β-defensin 2 in rat lungs. The results showed that the infection of β-defensin 2 overexpression lentivirus and β-defensin 2 shRNA effectively increased and suppressed the expression of β-defensin 2 in rat lung, respectively. The overexpression of β-defensin 2 mediated by the lentiviral vector protected lung from infection of Pseudomonas aeruginosa, but shRNA targeting β-defensin 2 aggregated the damage of lung. In addition, we also found that β-defensin 2 overexpression increased basal expression of anti-inflammatory cytokine such as IL-4, IL-10 and IL-13 and decreased levels of proinflammatory cytokines which include IL-1α, IL-1β, IL-5, IL-6, IL-8, IL-18, and TNF-α. Moreover, in the process of cytokine regulation, NF-κB pathway may be involved. Taken together, these data suggest that β-defensin 2 has protective effects against infection of Pseudomonas aeruginosa in rat and plays a role in inflammatory regulation by adjusting cytokine levels.     

Synthesis of Oxidized 3β,3′β-Disteryl Ethers and Search after High-Temperature Treatment of Sterol-Rich Samples

It was proven that sterols subjected to high-temperature treatment can be concatenated, which results in polymeric structures, e.g., 3β,3′β-disteryl ethers. However, it was also proven that due to increased temperature in oxygen-containing conditions, sterols can undergo various oxidation reactions. This study aimed to prove the existence and perform quantitative analysis of oxidized 3β,3′β-disteryl ethers, which could form during high-temperature treatment of sterol-rich samples. Samples were heated at 180, 200 and 220 °C for 0.5 to 4 h. Quantitative analyses of the oxidized 3β,3′β-disteryl ethers were performed with liquid extraction, solid-phase extraction and liquid chromatography coupled with mass spectrometry. Additionally, to perform this analysis, the appropriate standards of all oxidized 3β,3′β-disteryl ethers were prepared. Eighteen various oxidized 3β,3′β-disteryl ethers (derivatives of 3β,3′β-dicholesteryl ether, 3β,3′β-disitosteryl ether and 3β,3′β-distigmasteryl ether) were prepared. Additionally, the influence of metal compounds on the mechanism of ether formation at high temperatures was investigated.     

NLRP2 Is Overexpressed in Spinal Astrocytes at the Peak of Mechanical Pain Sensitivity during Complete Freund Adjuvant-Induced Persistent Pain

Our earlier findings revealed that interleukin-1 receptor type-1 (IL-1R1) was overexpressed in spinal neurons, and IL-1R1-deficient mice showed significant attenuation of thermal and mechanical allodynia during the course of the Complete Freund adjuvant (CFA)-induced persistent pain model. In the present study, we found that a ligand of IL-1R1, termed interleukin-1β (IL-1β), is also significantly overexpressed at the peak of mechanical pain sensitivity in the CFA-evoked pain model. Analysis of cellular distribution and modeling using IMARIS software showed that in the lumbar spinal dorsal horn, IL-1β is significantly elevated by astrocytic expression. Maturation of IL-1β to its active form is facilitated by the formation of the multiprotein complex called inflammasome; thus, we tested the expression of NOD-like receptor proteins (NLRPs) in astrocytes. At the peak of mechanical allodynia, we found expression of the NLRP2 inflammasome sensor and its significantly elevated co-localization with the GFAP astrocytic marker, while NLRP3 was moderately present and NLRP1 showed total segregation from the astrocytic profiles. Our results indicate that peripheral CFA injection induces NLRP2 inflammasome and IL-1β expression in spinal astrocytes. The release of mature IL-1β can contribute to the maintenance of persistent pain by acting on its neuronally expressed receptor, which can lead to altered neuronal excitability.     

The Effect of Interferon-γ and Zoledronate Treatment on Alpha-Tricalcium Phosphate/Collagen Sponge-Mediated Bone-Tissue Engineering

Inflammatory responses are frequently associated with the expression of inflammatory cytokines and severe osteoclastogenesis, which significantly affect the efficacy of biomaterials. Recent findings have suggested that interferon (IFN)-γ and zoledronate (Zol) are effective inhibitors of osteoclastogenesis. However, little is known regarding the utility of IFN-γ and Zol in bone tissue engineering. In this study, we generated rat models by generating critically sized defects in calvarias implanted with an alpha-tricalcium phosphate/collagen sponge (α-TCP/CS). At four weeks post-implantation, the rats were divided into IFN-γ, Zol, and control (no treatment) groups. Compared with the control group, the IFN-γ and Zol groups showed remarkable attenuation of severe osteoclastogenesis, leading to a significant enhancement in bone mass. Histomorphometric data and mRNA expression patterns in IFN-γ and Zol-injected rats reflected high bone-turnover with increased bone formation, a reduction in osteoclast numbers, and tumor necrosis factor-α expression. Our results demonstrated that the administration of IFN-γ and Zol enhanced bone regeneration of α-TCP/CS implants by enhancing bone formation, while hampering excess bone resorption.     

Influence of Human Jaw Periosteal Cells Seeded β-Tricalcium Phosphate Scaffolds on Blood Coagulation

Tissue engineering offers auspicious opportunities in oral and maxillofacial surgery to heal bone defects. For this purpose, the combination of cells with stability-providing scaffolds is required. Jaw periosteal cells (JPCs) are well suited for regenerative therapies, as they are easily accessible and show strong osteogenic potential. In this study, we analyzed the influence of uncoated and polylactic-co-glycolic acid (PLGA)-coated β-tricalcium phosphate (β-TCP) scaffolds on JPC colonization and subsequent osteogenic differentiation. Furthermore, interaction with the human blood was investigated. This study demonstrated that PLGA-coated and uncoated β-TCP scaffolds can be colonized with JPCs and further differentiated into osteogenic cells. On day 15, after cell seeding, JPCs with and without osteogenic differentiation were incubated with fresh human whole blood under dynamic conditions. The activation of coagulation, complement system, inflammation, and blood cells were analyzed using ELISA and scanning electron microscopy (SEM). JPC-seeded scaffolds showed a dense cell layer and osteogenic differentiation capacity on both PLGA-coated and uncoated β-TCP scaffolds. SEM analyses showed no relevant blood cell attachment and ELISA results revealed no significant increase in most of the analyzed cell activation markers (β-thromboglobulin, Sc5B-9, polymorphonuclear (PMN)-elastase). However, a notable increase in thrombin-antithrombin III (TAT) complex levels, as well as fibrin fiber accumulation on JPC-seeded β-TCP scaffolds, was detected compared to the scaffolds without JPCs. Thus, this study demonstrated that besides the scaffold material the cells colonizing the scaffolds can also influence hemostasis, which can influence the regeneration of bone tissue.