Kyung-Hyun Park-Min

Inhibition of RANK expression and osteoclastogenesis by TLRs and IFN-γ in human osteoclast precursors

TLRs have been implicated in promoting osteoclast-mediated bone resorption associated with inflammatory conditions. TLRs also activate homeostatic mechanisms that suppress osteoclastogenesis and can limit the extent of pathologic bone erosion associated with infection and inflammation. We investigated mechanisms by which TLRs suppress osteoclastogenesis. In human cell culture models, TLR ligands suppressed osteoclastogenesis by inhibiting expression of receptor activator of NF-κB (RANK), thereby making precursor cells refractory to the effects of RANKL. Similar but less robust inhibition of RANK expression was observed in murine cells. LPS suppressed generation of osteoclast precursors in mice in vivo, and adsorption of LPS onto bone surfaces resulted in diminished bone resorption. Mechanisms that inhibited RANK expression were down-regulation of RANK transcription, and inhibition of M-CSF signaling that is required for RANK expression. TLRs inhibited M-CSF signaling by rapidly down-regulating cell surface expression of the M-CSF receptor c-Fms by a matrix metalloprotease- and MAPK-dependent mechanism. Additionally, TLRs cooperated with IFN-γ to inhibit expression of RANK and of the CSF1R gene that encodes c-Fms, and to synergistically inhibit osteoclastogenesis. Our findings identify a new mechanism of homeostatic regulation of osteoclastogenesis that targets RANK expression and limits bone resorption during infection and inflammation.

Tumor necrosis factor induces GSK3 kinase-mediated cross-tolerance to endotoxin in macrophages

Endotoxin tolerance, a key mechanism for suppressing excessive inflammatory cytokine production, is induced by prior exposure of macrophages to Toll-like receptor (TLR) ligands. Induction of cross-tolerance to endotoxin by endogenous cytokines has not been investigated. Here we show that prior exposure to tumor necrosis factor (TNF) induced a tolerant state in macrophages, with less cytokine production after challenge with lipopolysaccharide (LPS) and protection from LPS-induced death. TNF-induced cross-tolerization was mediated by suppression of LPS-induced signaling and chromatin remodeling. TNF-induced cross-tolerance was dependent on the kinase GSK3, which suppressed chromatin accessibility and promoted rapid termination of signaling via the transcription factor NF-κB by augmenting negative feedback by the signaling inhibitors A20 and IκBα. Our results demonstrate an unexpected homeostatic function for TNF and a GSK3-mediated mechanism for the prevention of prolonged and excessive inflammation.

iRHOM2 is a critical pathogenic mediator of inflammatory arthritis

iRHOM2, encoded by the gene Rhbdf2, regulates the maturation of the TNF-α convertase (TACE), which controls shedding of TNF-α and its biological activity in vivo. TACE is a potential target to treat TNF-α-dependent diseases, such as rheumatoid arthritis, but there are concerns about potential side effects, because TACE also protects the skin and intestinal barrier by activating EGFR signaling. Here we report that inactivation of Rhbdf2 allows tissue-specific regulation of TACE by selectively preventing its maturation in immune cells, without affecting its homeostatic functions in other tissues. The related iRHOM1, which is widely expressed, except in hematopoietic cells, supported TACE maturation and shedding of the EGFR ligand TGF-α in Rhbdf2-deficient cells. Remarkably, mice lacking Rhbdf2 were protected from K/BxN inflammatory arthritis to the same extent as mice lacking TACE in myeloid cells or Tnfa-deficient mice. In probing the underlying mechanism, we found that two main drivers of K/BxN arthritis, complement C5a and immune complexes, stimulated iRHOM2/TACE-dependent shedding of TNF-α in mouse and human cells. These data demonstrate that iRHOM2 and myeloid-expressed TACE play a critical role in inflammatory arthritis and indicate that iRHOM2 is a potential therapeutic target for selective inactivation of TACE in myeloid cells.

Inhibition of Interleukin 10 Signaling after Fc Receptor Ligation and during Rheumatoid Arthritis

Interleukin-10 (IL-10) is a potent deactivator of myeloid cells that limits the intensity and duration of immune and inflammatory responses. The activity of IL-10 can be suppressed during inflammation, infection, or after allogeneic tissue transplantation. We investigated whether inflammatory factors suppress IL-10 activity at the level of signal transduction. Out of many factors tested, only ligation of Fc receptors by immune complexes inhibited IL-10 activation of the Jak-Stat signaling pathway. IL-10 signaling was suppressed in rheumatoid arthritis joint macrophages that are exposed to immune complexes in vivo. Activation of macrophages with interferon-γ was required for Fc receptor–mediated suppression of IL-10 signaling, which resulted in diminished activation of IL-10–inducible genes and reversal of IL-10–dependent suppression of cytokine production. The mechanism of inhibition involved decreased cell surface IL-10 receptor expression and Jak1 activation and was dependent on protein kinase C delta. These results establish that IL-10 signaling is regulated during inflammation and identify Fc receptors and interferon-γ as important regulators of IL-10 activity. Generation of macrophages refractory to IL-10 can contribute to pathogenesis of inflammatory and infectious diseases characterized by production of interferon-γ and immune complexes.

Expression and function of semaphorin 3A and its receptors in human monocyte-derived macrophages

Semaphorins are a large family of secreted and membrane-bound proteins. Recently, several roles of semaphorins in the immune system have emerged. Several semaphorins and their receptors are expressed in a variety of lymphoid and myeloid cells and affect immune cell functions, including cell proliferation, differentiation, chemotaxis, and cytokine production. However, the roles of class 3 semaphorins in human myeloid cells are not well known. Here we examined the regulation of expression of class 3 semaphorins and their receptors by inflammatory stimuli and their function in human macrophages. We show that the expression of Sema3A receptors (neuropilin-1 (NRP-1), NRP-2, plexin A1, plexin A2, and plexin A3) significantly increased during M-CSF-mediated differentiation of monocytes into macrophages under conditions that promote an M2 alternatively activated macrophage phenotype. Consistent with increased NRP-1 expression, cell surface binding of Sema3A increased during M2 differentiation. Interferon (IFN)-gamma and lipopolysaccharide, which promote classical M1 macrophage activation affected expression of NRP-1, NRP-2 and plexin A1. IFN-gamma decreased NRP-1 expression and LPS suppressed NRP-2 and plexin A1 expression. Furthermore we show that Sema3A induced apoptosis in monocyte-derived macrophages and cooperated with anti-Fas CH11 antibody to augment apoptosis. Our results suggest that Sema3A plays a role in induction of apoptosis in monocyte-derived macrophages that are resistant to Fas-induced apoptosis, and that its function can be modulated in inflammatory conditions.

IL-10 Suppresses Calcium-Mediated Costimulation of Receptor Activator NF-κB Signaling during Human Osteoclast Differentiation by Inhibiting TREM-2 Expression

Induction of effective osteoclastogenesis by RANK (receptor activator of NF-κB) requires costimulation by ITAM-coupled receptors. In humans, the TREM-2 (triggering receptor expressed on myeloid cells 2) ITAM-coupled receptor plays a key role in bone remodeling, as patients with TREM-2 mutations exhibit defective osteoclastogenesis and bone lesions. We have identified a new rapidly induced costimulatory pathway for RANK signaling that is dependent on TREM-2 and mediated by calcium signaling. TREM-2-dependent calcium signals are required for RANK-mediated activation of calcium/calmodulin-dependent protein kinase (CaMK)II and downstream MEK and ERK MAPKs that are important for osteoclastogenesis. IL-10 inhibited RANK-induced osteoclastogenesis and selectively inhibited calcium signaling downstream of RANK by inhibiting transcription of TREM-2. Down-regulation of TREM-2 expression resulted in diminished RANKL-induced activation of the CaMK-MEK-ERK pathway and decreased expression of the master regulator of osteoclastogenesis NFATc1. These findings provide a new mechanism of inhibition of human osteoclast differentiation. The results also yield insights into crosstalk between ITAM-coupled receptors and heterologous receptors such as RANK, and they identify a mechanism by which IL-10 can suppress cellular responses to TNFR family members.

Interleukin‐27 inhibits human osteoclastogenesis by abrogating RANKL‐mediated induction of nuclear factor of activated T cells c1 and suppressing proximal RANK signaling

OBJECTIVE Interleukin-27 (IL-27) has stimulatory and regulatory immune functions and is expressed in rheumatoid arthritis (RA) synovium. This study was undertaken to investigate the effects of IL-27 on human osteoclastogenesis, to determine whether IL-27 can stimulate or attenuate the osteoclast-mediated bone resorption that is a hallmark of RA. METHODS Osteoclasts were generated from blood-derived human CD14+ cells. The effects of IL-27 on osteoclast formation were evaluated by counting the number of tartrate-resistant acid phosphatase-positive multinucleated cells and measuring the expression of osteoclast-related genes. The induction of nuclear factor of activated T cells c1 (NFATc1) and the activation of signaling pathways downstream of RANK were measured by immunoblotting. The expression of key molecules implicated in osteoclastogenesis (NFATc1, RANK, costimulatory receptors, and immunoreceptor tyrosine-based activation motif-harboring adaptor proteins) was measured by real-time reverse transcription-polymerase chain reaction. Murine osteoclast precursors obtained from mouse bone marrow and synovial fluid macrophages derived from RA patients were also tested for their responsiveness to IL-27. RESULTS IL-27 inhibited human osteoclastogenesis, suppressed the induction of NFATc1, down-regulated the expression of RANK and triggering receptor expressed on myeloid cells 2 (TREM-2), and inhibited RANKL-mediated activation of ERK, p38, and NF-kappaB in osteoclast precursors. Synovial fluid macrophages from RA patients were refractory to the effects of IL-27. In contrast to the findings in humans, IL-27 only moderately suppressed murine osteoclastogenesis, and this was likely attributable to low expression of the IL-27 receptor subunit WSX-1 on murine osteoclast precursors. CONCLUSION IL-27 inhibits human osteoclastogenesis by a direct mechanism that suppresses the responses of osteoclast precursors to RANKL. These findings suggest that, in addition to its well-known antiinflammatory effects, IL-27 plays a homeostatic role in restraining bone erosion. This homeostatic function is compromised under conditions of chronic inflammation such as in RA synovitis.

Tumor Necrosis Factor α Induces Sustained Signaling and a Prolonged and Unremitting Inflammatory Response in Rheumatoid Arthritis Synovial Fibroblasts

OBJECTIVE The nonresolving character of synovial inflammation in rheumatoid arthritis (RA) is a conundrum. To identify the contribution of fibroblast-like synoviocytes (FLS) to the perpetuation of synovitis, we investigated the molecular mechanisms that govern the tumor necrosis factor α (TNFα)-driven inflammatory program in human FLS. METHODS FLS obtained from the synovial tissues of patients with RA or osteoarthritis were stimulated with TNFα and assayed for gene expression and cytokine production by real-time quantitative reverse transcription-polymerase chain reaction analysis and enzyme-linked immunosorbent assay. NF-κB signaling was evaluated by Western blotting. Histone acetylation, chromatin accessibility, and NF-κB p65 and RNA polymerase II (Pol II) occupancy at the interleukin-6 (IL-6) promoter were measured by chromatin immunoprecipitation and restriction enzyme accessibility assays. RESULTS In FLS, TNFα induced prolonged transcription of messenger RNA (mRNA) for IL-6 and progressive accumulation of IL-6 protein over 4 days. Similarly, induction of mRNA for CXCL8/IL-8, CCL5/RANTES, matrix metalloproteinase 1 (MMP-1), and MMP-3 after TNFα stimulation was sustained for several days. This contrasted with the macrophage response to TNFα, which characteristically involved a transient increase in the expression of proinflammatory genes. In FLS, TNFα induced prolonged activation of NF-κB signaling and sustained transcriptional activity, as indicated by increased histone acetylation, chromatin accessibility, and p65 and Pol II occupancy at the IL-6 promoter. Furthermore, FLS expressed low levels of the feedback inhibitors A20-binding inhibitor of NF-κB activation 3 (ABIN-3), IL-1 receptor-associated kinase M (IRAK-M), suppressor of cytokine signaling 3 (SOCS-3), and activating transcription factor 3 (ATF-3), which terminate inflammatory responses in macrophages. CONCLUSION TNFα signaling is not effectively terminated in FLS, which leads to an uncontrolled inflammatory response. The results suggest that prolonged and sustained inflammatory responses by FLS in response to synovial TNFα contribute to the persistence of synovial inflammation in RA.

IL-10 suppresses calcium-mediated costimulation of receptor activator NF-kappa B signaling during human osteoclast differentiation by inhibiting TREM-2 expression.

Induction of effective osteoclastogenesis by RANK (receptor activator of NF-κB) requires costimulation by ITAM-coupled receptors. In humans, the TREM-2 (triggering receptor expressed on myeloid cells 2) ITAM-coupled receptor plays a key role in bone remodeling, as patients with TREM-2 mutations exhibit defective osteoclastogenesis and bone lesions. We have identified a new rapidly induced costimulatory pathway for RANK signaling that is dependent on TREM-2 and mediated by calcium signaling. TREM-2-dependent calcium signals are required for RANK-mediated activation of calcium/calmodulin-dependent protein kinase (CaMK)II and downstream MEK and ERK MAPKs that are important for osteoclastogenesis. IL-10 inhibited RANK-induced osteoclastogenesis and selectively inhibited calcium signaling downstream of RANK by inhibiting transcription of TREM-2. Down-regulation of TREM-2 expression resulted in diminished RANKL-induced activation of the CaMK-MEK-ERK pathway and decreased expression of the master regulator of osteoclastogenesis NFATc1. These findings provide a new mechanism of inhibition of human osteoclast differentiation. The results also yield insights into crosstalk between ITAM-coupled receptors and heterologous receptors such as RANK, and they identify a mechanism by which IL-10 can suppress cellular responses to TNFR family members.

IFN-γ-Primed Macrophages Exhibit Increased CCR2-Dependent Migration and Altered IFN-γ Responses Mediated by Stat1

Priming of macrophages with IFN-γ increases cellular responsiveness to inflammatory stimuli, including IFN-γ itself. We described previously that priming with subactivating concentrations of IFN-γ increased Stat1 expression and resulted in enhanced activation of Stat1 and of a subset of IFN-γ-responsive genes when primed macrophages were restimulated with low doses of IFN-γ. In this study, we determined the effects of IFN-γ priming on the macrophage transcriptome and on transcriptional responses to high saturating concentrations of IFN-γ. At baseline, primed macrophages expressed a small subset of IFN-γ-inducible genes, including CCR2, and exhibited increased migration in response to CCL2. Activation of gene expression by high concentrations of IFN-γ was altered in primed macrophages, such that activation of a subset of IFN-γ-inducible genes was attenuated. A majority of genes in this “less induced” category corresponded to genes that are induced by IFN-γ via Stat1-independent but Stat3-dependent pathways and have been implicated in inflammatory tissue destruction. One mechanism of attenuation of gene expression was down-regulation of Stat3 function by increased levels of Stat1. These results reveal that priming enhances migration to inflammatory chemokines and identify IFN-γ-inducible genes whose expression is attenuated by high levels of Stat1. The increase in Stat1 expression during priming provides a mechanism by which physiological regulation of the relative abundance of Stat1 and Stat3 impacts on gene expression. Our results also suggest that, in addition to inducing hypersensitivity to inflammatory stimuli, IFN priming delivers a homeostatic signal by attenuating IFN-γ induction of certain tissue-destructive genes.