Inhibitors of the Janus kinases (JAKs) have been developed as anti-inflammatory and immunosuppressive agents and are currently undergoing testing in clinical trials. The JAK inhibitors CP-690,550 (tofacitinib) and INCB018424 (ruxolitinib) have demonstrated clinical efficacy in rheumatoid arthritis (RA). However, the mechanisms that mediate the beneficial actions of these compounds are not known. In this study, we examined the effects of both JAK inhibitors on inflammatory and tumor necrosis factor (TNF) responses in human macrophages (MΦs).
In vitro studies were performed with peripheral blood MΦs from healthy donors treated with TNF and synovial fluid MΦs from patients with RA. Levels of activated signal transducer and activator of transcription (STAT) proteins and other transcription factors were detected by Western blot, and gene expression was measured by real-time polymerase chain reaction. In vivo effects of JAK inhibitors were evaluated in the K/BxN serum-transfer model of arthritis.
JAK inhibitors suppressed activation and expression of STAT1 and downstream inflammatory target genes in TNF-stimulated and RA synovial macrophages. In addition, JAK inhibitors decreased nuclear localization of NF-κB subunits in TNF-stimulated and RA synovial macrophages. CP-690,550 significantly decreased IL6 expression in synovial MΦs. JAK inhibitors augmented nuclear levels of NFATc1 and cJun, followed by increased formation of osteoclast-like cells. CP-690,550 strongly suppressed K/BxN arthritis that is dependent on macrophages but not on lymphocytes.
Our findings demonstrate that JAK inhibitors suppress macrophage activation and attenuate TNF responses, and suggest that suppression of cytokine/chemokine production and innate immunity contributes to the therapeutic efficacy of JAK inhibitors.
macrophages; TNF; STAT1; rheumatoid arthritis; JAK inhibitors
Immunoreceptor tyrosine based activation motif (ITAM)-coupled receptors play an essential role in regulating macrophage activation and function by cross-regulating signaling from heterologous receptors. We investigated mechanisms by which ITAM-associated receptors inhibit type I interferon (IFN-α/β) signaling in primary human macrophages and tested the effects of simultaneous ligation of ITAM-associated receptors and TLR4 on TLR4-induced Jak-STAT signaling that is mediated by autocrine IFN-β. Preligation of ITAM-coupled β2 integrins and FcγRs inhibited proximal signaling by the type I IFN receptor IFNAR. Cross-inhibition of IFNAR signaling by β2 integrins resulted in decreased Jak1 activation and was mediated by partial downregulation of the IFNAR1 subunit and MAPK-dependent induction of USP18, which blocks the association of Jak1 with IFNAR2. Simultaneous engagement of ITAM-coupled β2 integrins or Dectin-1 with TLR4 did not affect TLR4-induced direct activation of inflammatory target genes such as TNF or IL6, but abrogated subsequent induction of IFN response genes that is mediated by autocrine IFN-β signaling. Type I IFNs promote macrophage death after infection by Listeria monocytogenes. Consequently, attenuation of IFN responses by β2 integrins protected primary human macrophages from Listeria monocytogenes induced apoptosis. These results provide a mechanism for cross-inhibition of type I IFN signaling by ITAM-coupledβ2 integrins and demonstrate that ITAM signaling qualitatively modulates macrophage responses to PAMPs and pathogens by selectively suppressing IFN responses.
monocytes/macrophages; cytokines; inflammation; TLRs; signal transduction
Acute inflammatory activation of macrophages by Toll-like and related receptors is characterized by transient activation of MAPK-, NF-κB- and IRF-mediated signaling pathways and expression of pro-inflammatory genes. This activation state is inherently unstable and often transitions into a state of `tolerance' characterized by diminished signaling, repressive chromatin modifications, and an alternative gene expression program. This Viewpoint describes signaling and epigenetic mechanisms associated with transition to tolerant states, which are proposed to correspond to alternative activation states programmed by the original inflammatory stimuli.
Alternative activation; Epige netics; Macrophages; Signal transduction; Tolerance
Endotoxin tolerance, a key mechanism for suppressing excessive inflammatory cytokine production and attendant toxicity, is induced by prior exposure of macrophages to TLR ligands. Induction of tolerance by endogenous cytokines has not been investigated. We show that prior exposure to TNF induces a tolerant state in macrophages, with diminished cytokine production on LPS challenge and protection from LPS-induced lethality. TNF-induced tolerization was mediated by coordinate action of two inhibitory mechanisms, suppression of LPS-induced signaling and chromatin remodeling. Mechanistically, TNF-induced tolerance was distinct from TLR-induced tolerance as it was dependent on GSK3, which suppressed chromatin accessibility and promoted rapid termination of NF-κB signaling by augmenting negative feedback by A20 and I-κBα. These results reveal an unexpected homeostatic function of TNF and provide a GSK3-mediated mechanism for preventing prolonged and excessive inflammation.
IL-27 is a pleiotropic cytokine with both activating and inhibitory functions on innate and acquired immunity. IL-27 is expressed at sites of inflammation in cytokine-driven autoimmune/inflammatory diseases, such as rheumatoid arthritis, psoriasis, inflammatory bowel disease, and sarcoidosis. However, its role in modulating disease pathogenesis is still unknown. In this study, we found that IL-27 production is induced by TNF-α in human macrophages (Mϕ) and investigated the effects of IL-27 on the responses of primary human Mϕ to the endogenous inflammatory cytokines TNF-a and IL-1. In striking contrast to IL-27–mediated augmentation of TLR-induced cytokine production, we found that IL-27 suppressed Mϕ responses to TNF-α and IL-1β, thus identifying an anti-inflammatory function of IL-27. IL-27 blocked the proximal steps of TNF-α signaling by downregulating cell-surface expression of the signaling receptors p55 and p75. The mechanism of inhibition of IL-1 signaling was downregulation of the ligand-binding IL-1RI concomitant with increased expression of the receptor antagonist IL-1Ra and the decoy receptor IL-1RII. These findings provide a mechanism for suppressive effects of IL-27 on innate immune cells and suggest that IL-27 regulates inflammation by limiting activation of Mf by inflammatory cytokines while preserving initial steps in host defense by augmenting responses to microbial products.
Type I IFNs have emerged as potential activators of the IFN signature and elevated STAT1 expression in RA synovium, but mechanisms that induce synovial IFN expression are unknown. Recently, TNFα was shown to induce a delayed IFN response in macrophages. Thus, we tested whether TNFα, classically thought to activate inflammatory NF-κB target genes in RA, also contributes to the ‘IFN signature’ in RA synovial macrophages.
Synovial fluid macrophages purified from patients with rheumatoid arthritis (n=24) and spondyloarthopathies (SpA) (n=18) were lysed immediately after isolation or cultured ex vivo in the absence or presence of blockade of endogenous type I IFN or TNFα. Expression of IFN-inducible target genes was measured by qPCR and ELISA.
Expression of an IFN signature and STAT1 in RA synovial macrophages was suppressed when type I IFNs or TNFα were blocked, whereas TNFα blockade did not affect expression of IFN response genes or STAT1 in SpA synovial macrophages. RA synovial fluid suppressed the IFN signature in RA synovial macrophages, and in TNFα-, IFNα- and IFNβ-stimulated control macrophages. Type I IFNs suppressed expression of IL-8 and MMP9 in RA synovial macrophages and in TNFα-stimulated control macrophages.
Our findings identify a new function for TNFα in RA synovitis by implicating TNFα as a major inducer of the RA synovial IFN response. The results suggest that the expression of IFN response genes in RA synovium is regulated by interplay between TNFα and opposing homeostatic factors expressed in the synovial microenvironment.
rheumatoid arthritis; TNFα; type I interferon; STAT1
Outside of the TLR paradigm, there is little understanding of how pathogen recognition at the cell surface is linked to functional responses in cells of the innate immune system. Recent work in this area demonstrates that the yeast particle zymosan, by binding to the β-glucan receptor Dectin-1, activates an ITAM-Syk–dependent pathway in dendritic cells, which is required for optimal cytokine production and generation of an oxidative burst. It remains unclear how activation of Syk is coupled to effector mechanisms. In human macrophages, zymosan rapidly activated a calcium-dependent pathway downstream of Dectin-1 and Syk that led to activation of calmodulin-dependent kinase II and Pyk2. Calmodulin-dependent kinase and Pyk2 transduced calcium signals into activation of the ERK–MAPK pathway, CREB, and generation of an oxidative burst, leading to downstream production of IL-10. These observations identify a new calcium-mediated signaling pathway activated by zymosan and link this pathway to both inflammatory and anti-inflammatory responses in macrophages.
TNF and type I interferons (IFNs) are induced by microbial stimuli and mediate innate immune responses. They are also involved in pathogenesis of chronic inflammatory diseases, such as rheumatoid arthritis and systemic lupus erythematosus. Activated macrophages are an important driving force of inflammatory reactions and one of the major producers of TNF in innate immunity and chronic inflammation. Despite the fact that cells at sites of damage are continuously exposed to both cytokines, little is known about mechanisms regulating TNF and type I IFN interactions during inflammation. In this review, we will discuss the role of an IFN-β-mediated autocrine loop in regulation of gene expression program induced by TNF in myeloid cells.
Activated macrophages and their inflammatory products play a key role in innate immunity and in pathogenesis of autoimmune/inflammatory diseases. Macrophage activation needs to be tightly regulated to rapidly mount responses to infectious challenges but to avoid toxicity associated with excessive activation. Rapid and potent macrophage activation is driven by cytokine-mediated feedforward loops, while excessive activation is prevented by feedback inhibition. Here we discuss feedforward mechanisms that augment macrophage responses to Toll-like receptor (TLR) ligands and cytokines that are mediated by signal transducer and activator of transcription 1 (STAT1) and induced by interferon-γ (IFN-γ). IFN-γ also drives full macrophage activation by inactivating feedback inhibitory mechanisms, such as those mediated by IL-10 and STAT3. Priming of macrophages with IFN-γ reprograms cellular responses to other cytokines, such as type I IFNs and IL-10, with a shift toward pro-inflammatory STAT1-dominated responses. Similar but partially distinct priming effects are induced by other cytokines that activate STAT1, including type I IFNs and interleukin-27. We propose a model whereby opposing feedforward and feedback inhibition loops crossregulate each other to fine tune macrophage activation. In addition, we discuss how dysregulation of the balance between feedforward and feedback inhibitory mechanisms can contribute to the pathogenesis of autoimmune and inflammatory diseases, such as rheumatoid arthritis and systemic lupus erythematosus.
monocytes/macrophages; cytokine receptors; Toll-like receptors/pattern-recognition receptors; signal transduction; inflammation
Matrix metalloproteinases (MMPs) are induced during inflammatory responses and are important for immune regulation, angiogenesis, wound healing and tissue remodeling. Expression of MMPs needs to be tightly controlled to avoid excessive tissue damage. In this study we investigated the regulation of MMP expression by inflammatory factors in primary human monocytes and macrophages. IFNγ, which augments inflammatory cytokine production in response to macrophage-activating factors such as Toll-like receptor (TLR) ligands, instead broadly suppressed TLR-induced MMP expression. Inhibition of MMP expression was dependent on STAT1 and required de novo protein synthesis. IFNγ strongly enhanced TLR-induced expression of the transcriptional repressor ATF-3 in a STAT1-dependent manner, which correlated with recruitment of ATF-3 to the endogenous MMP-1 promoter as detected by chromatin immunoprecipitation assays. RNA interference experiments further supported a role for ATF-3 in suppression of MMP-1 expression. In addition, IFNγ suppressed DNA binding by AP-1 transcription factors that are known to promote MMP expression and a combination of supershift, RNA interference and overexpression experiments implicated AP-1 family member Fra-1 in the regulation of MMP-1 expression. These results define an IFNγ-mediated homeostatic loop that limits the potential for tissue damage associated with inflammation, and identify transcriptional factors that regulate MMP expression in myeloid cells in inflammatory settings.
LPS; IFNγ; STAT1; ATF-3; Macrophages
Positive regulation of cell migration by chemotactic factors and downstream signaling pathways has been extensively investigated. In contrast, little is known about factors and mechanisms that induce migration arrest, a process important for retention of cells at inflammatory sites and homeostatic regulation of cell trafficking. In this study we found that IFN-γ directly inhibited monocyte migration by suppressing remodeling of the actin cytoskeleton and cell polarization in response to the chemokine CCL2. Inhibition was dependent on STAT1 and downstream genes, whereas STAT3 promoted migration. IFN-γ did not affect proximal CCL2 signaling, but altered monocyte responses to CCL2 by modulating the activity of the GTPases Rac and Cdc42 and the downstream PAK kinase that regulate the cytoskeleton and cell polarization. These results identify a new role for IFN-γ in arresting monocyte chemotaxis by a mechanism that involves modulation of cytoskeleton remodeling. Crosstalk between Jak-STAT and Rac/Cdc42 GTPase-mediated signaling pathways provides a molecular mechanism by which cytokines can regulate cell migration.
IFN-γ; Monocyte; Migration; STAT1; Signal transduction
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.
Emerging concepts suggest that macrophage functional phenotype is regulated by transcription factors that define alternative activation states. We found that RBP-J, the major nuclear transducer of Notch signaling, augmented TLR4-induced expression of key mediators of classically activated M1 macrophages and thus innate immune responses to L. monocytogenes. Notch-RBP-J signaling controlled expression of the transcription factor IRF8 that induced downstream M1-specific genes. RBP-J promoted IRF8 protein synthesis by selectively augmenting IRAK2-dependent TLR4 signaling to the MNK1 kinase and downstream translation initiation control through eIF4E. These results define a signaling network in which Notch-RBP-J and TLR signaling are integrated at the level of IRF8 protein synthesis and identify a mechanism by which heterologous signaling pathways can regulate TLR-induced inflammatory macrophage polarization.
Inflammation plays a key role in excessive bone loss in conditions such as rheumatoid arthritis and periodontitis. An important paradigm in immunology is that inflammatory factors activate feedback inhibition mechanisms to restrain inflammation and limit associated tissue damage. We hypothesized that inflammatory factors would activate similar feedback mechanisms to restrain bone loss in inflammatory settings. We have identified three mechanisms that inhibit osteoclastogenesis and are induced by inflammatory factors, such as toll-like receptor ligands and cytokines: downregulation of expression of costimulatory molecules such as TREM-2; induction of shedding and thereby inactivation of the M-CSF receptor c-Fms, leading to decreased RANK transcription; and induction of transcriptional repressors such as interferon regulatory factor 8. It is likely that these mechanisms work in a complementary and cooperative manner to fine tune the extent of osteoclastogenesis in inflammatory settings, and their augmentation may represent an alternative therapeutic approach to suppress bone resorption.
inflammation; osteoclasts; toll-like receptors; M-CSF; c-Fms; IRF8
The Notch-driven transcription factor RBP-J inhibits osteoclast formation in response to TNF.
Tumor necrosis factor (TNF) plays a key role in the pathogenesis of inflammatory bone resorption and associated morbidity in diseases such as rheumatoid arthritis and periodontitis. Mechanisms that regulate the direct osteoclastogenic properties of TNF to limit pathological bone resorption in inflammatory settings are mostly unknown. Here, we show that the transcription factor recombinant recognition sequence binding protein at the Jκ site (RBP-J) strongly suppresses TNF-induced osteoclastogenesis and inflammatory bone resorption, but has minimal effects on physiological bone remodeling. Myeloid-specific deletion of RBP-J converted TNF into a potent osteoclastogenic factor that could function independently of receptor activator of NF-κB (RANK) signaling. In the absence of RBP-J, TNF effectively induced osteoclastogenesis and bone resorption in RANK-deficient mice. Activation of RBP-J selectively in osteoclast precursors suppressed inflammatory osteoclastogenesis and arthritic bone resorption. Mechanistically, RBP-J suppressed induction of the master regulator of osteoclastogenesis (nuclear factor of activated T cells, cytoplasmic 1) by attenuating c-Fos activation and suppressing induction of B lymphocyte–induced maturation protein-1, thereby preventing the down-regulation of transcriptional repressors such as IRF-8 that block osteoclast differentiation. Thus, RBP-J regulates the balance between activating and repressive signals that regulate osteoclastogenesis. These findings identify RBP-J as a key upstream negative regulator of osteoclastogenesis that restrains excessive bone resorption in inflammatory settings.
Immunoreceptor tyrosine-based activation motifs (ITAMs) are used by multiple receptors to activate immune cells. However, ITAM-associated receptors can have paradoxically inhibitory effects, which have been implicated in regulation of inflammatory responses, but mechanisms of inhibitory signaling are poorly understood. New evidence shows that low avidity ligation of the ITAM-associated immunoglobulin A receptor FcαRI (transient engagement of small numbers of FcαRIs) results in translocation of FcαRI and the associated inhibitory Src homology 2 (SH2) domain–containing phosphatase–1 (SHP-1) to membrane lipid rafts. Subsequent ligation of activating receptors results in their colocalization with FcαRI and SHP-1 and trafficking to an inhibitory intracellular compartment termed the inhibisome. Thus, ITAM suppressive signals subvert the activating function of rafts to promote incorporation of receptors into supramolecular domains where signaling molecules are deactivated by SHP-1.
Bone remodeling in physiological and pathological conditions represents a balance between bone resorption mediated by osteoclasts and bone formation by osteoblasts. Bone resorption is tightly and dynamically regulated by multiple mediators, including cytokines that act directly on osteoclasts and their precursors, or indirectly by modulating osteoblast lineage cells that in turn regulate osteoclast differentiation. The critical role of cytokines in inducing and promoting osteoclast differentiation, function and survival is covered by the accompanying review by Zwerina and colleagues. Recently, it has become clear that negative regulation of osteoclastogenesis and bone resorption by inflammatory factors and cytokines, downstream signaling pathways, and a newly described network of transcriptional repressors plays a key role in bone homeostasis by fine tuning bone remodeling and restraining excessive bone resorption in inflammatory settings. In this review we discuss negative regulators of osteoclastogenesis and mechanisms by which these factors suppress bone resorption.
Systemic lupus erythematosus (SLE) is a systemic inflammatory disease characterized by autoantibody production and immune complex deposition. Interleukin-10 (IL-10), predominantly an anti-inflammatory cytokine, is paradoxically elevated in SLE patients. We hypothesize that the anti-inflammatory function of IL-10 is impaired in monocytes from SLE patients who are chronically exposed to immune complexes.
CD14+ monocytes were isolated from healthy donors and SLE patients with all experiments done in pairs. Cultured CD14+ cells were treated with heat-aggregated human IgG (HIg 325 μg/ml) in the presence or absence of IL-10 (20 ng/ml). To study gene expression, RNA was extracted 3 hours after treatment. To study cytokine production, supernatants were harvested after 8 hours. To study IL-10 signaling, cell lysates were obtained from CD14+ cells treated with HIg (325 μg/ml) for 1 hour followed by IL-10 (20 ng/ml) treatment for 10 minutes. Western blot was used to assess STAT3 phosphorylation.
SLE monocytes produced more TNFα and IL-6 than control cells when stimulated with HIg. IL-10 had less suppressive effect on HIg-induced TNFα and IL-6 production in SLE monocytes, although IL-10 receptor expression was similar in SLE and control monocytes. HIg suppressed IL-10R expression and altered IL-10 signaling in control monocytes. Like SLE monocytes, IFNα-primed control monocytes stimulated with HIg were also less responsive to IL-10.
HIg and IFNα modulate IL-10 function. In SLE monocytes, which are considered IFNα-primed and chronically exposed to immune complexes, responses to IL-10 are abnormal, limiting the anti-inflammatory effect of this cytokine.
The E26 transformation-specific (Ets) proteins are a family of transcription factors with important roles in a variety of cellular processes ranging from proliferation and differentiation to transformation and metastasis. Tissue-specific expression of Ets proteins and their ability to interact with other families of transcription factors contribute to their versatility. In this study, we investigated the regulation of Ets factors in primary human monocytes and macrophages, and their role in matrix metalloprotease (MMP) and cytokine production. The macrophage-activating Toll-like receptor ligand, lipopolysaccharide (LPS), induced the expression of Ets family members epithelium-specific Ets factor 3 (ESE-3) and TEL-2 but rapidly suppressed Friend leukemia virus integration 1 (FLI-1) expression. Modulation of FLI-1 expression using either RNA interference or forced expression identified a positive role for FLI-1 in contributing to LPS-induced expression of MMP-1, MMP-3, MMP-10, and interleukin-10 (IL-10). Thus, the rapid downregulation of FLI-1 expression after LPS stimulation attenuates the induction of various MMPs and IL-10 under inflammatory conditions. In contrast, the expression of IL-6 and TNFα and the effects of interferon (IFN)γ on LPS responses were not dependent on FLI-1. Our results define a novel FLI-1-mediated self-regulatory feedback loop that limits MMP expression and thus may attenuate extent of tissue destruction associated with inflammatory responses.
IL-1β is a key mediator of bone resorption in inflammatory settings, such as rheumatoid arthritis (RA). IL-1β promotes osteoclastogenesis by inducing RANKL expression on stromal cells and synergizing with RANKL to promote later stages of osteoclast differentiation. Because IL-1Rs share a cytosolic Toll–IL-1R domain and common intracellular signaling molecules with TLRs that can directly inhibit early steps of human osteoclast differentiation, we tested whether IL-1β also has suppressive properties on osteoclastogenesis in primary human peripheral blood monocytes and RA synovial macrophages. Early addition of IL-1β, prior to or together with RANKL, strongly inhibited human osteoclastogenesis as assessed by generation of TRAP+ multinucleated cells. IL-1β acted directly on human osteoclast precursors (OCPs) to strongly suppress expression of RANK, of the costimulatory triggering receptor expressed on myeloid cells 2 receptor, and of the B cell linker adaptor important for transmitting RANK-induced signals. Thus, IL-1β rendered early-stage human OCPs refractory to RANK stimulation. Similar inhibitory effects of IL-1β were observed using RA synovial macrophages. One mechanism of RANK inhibition was IL-1β–induced proteolytic shedding of the M-CSF receptor c-Fms that is required for RANK expression. These results identify a homeostatic function of IL-1β in suppressing early OCPs that contrasts with its well-established role in promoting later stages of osteoclast differentiation. Thus, the rate of IL-1–driven bone destruction in inflammatory diseases, such as RA, can be restrained by its direct inhibitory effects on early OCPs to limit the extent of inflammatory osteolysis.
Several signaling pathways including the Notch pathway can modulate Toll-like receptor (TLR) activation to achieve responses most appropriate for the environment. One mechanism of TLR-Notch crosstalk is TLR-induced expression of Notch ligands Jagged and Delta that feed back to engage Notch receptors on TLR-activated cells. In this study, we investigated mechanisms by which TLRs induce Notch ligand expression in primary macrophages. TLRs induced Jagged1 expression rapidly and independently of new protein synthesis. Jagged1 induction was augmented by interferon (IFN)-γ, was partially dependent on canonical TLR-activated NF-κB and MAPK signaling pathways, and elevated Jagged1 expression augmented TLR-induced IL-6 production. Strikingly, TLR-induced Jagged1 expression was strongly dependent on the Notch master transcriptional regulator RBP-J, and also on upstream components of the Notch pathway γ-secretase and Notch1 and Notch2 receptors. Thus, Jagged1 is an RBP-J target gene that is activated in a binary manner by TLR and Notch pathways. Early and direct cooperation between TLR and Notch pathways leads to Jagged1-RBP-J-mediated autoamplification of Notch signaling that can modulate later phases of the TLR response.