George D. Kalliolias

Indirect Inhibition of Toll-like Receptor and Type I Interferon Responses by ITAM-Coupled Receptors and Integrins

An important function of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors is cross-regulation of heterologous receptor signaling, but mechanisms of cross-inhibition are poorly understood. We show that high-avidity ligation of ITAM-coupled beta2 integrins and FcgammaRs in macrophages inhibited type I interferon receptor and Toll-like receptor (TLR) signaling and induced expression of interleukin-10 (IL-10); signaling inhibitors SOCS3, ABIN-3, and A20; and repressors of cytokine gene transcription STAT3 and Hes1. Induction of inhibitors was dependent on a pathway composed of signaling molecules DAP12, Syk, and Pyk2 that coupled to downstream kinases p38 and MSKs and required integration of IL-10-dependent and -independent signals. ITAM-induced inhibitors abrogated TLR responses by cooperatively targeting distinct steps in TLR signaling. Inhibitory signaling was suppressed by IFN-gamma and attenuated in inflammatory arthritis synovial macrophages. These results provide an indirect mechanism of cross-inhibition of TLRs and delineate a signaling pathway important for deactivation of macrophages.

IL-27 Activates Human Monocytes via STAT1 and Suppresses IL-10 Production but the Inflammatory Functions of IL-27 Are Abrogated by TLRs and p38

IL-27 is a member of the IL-12 family of cytokines that activates the Jak-STAT signaling pathway in a context-dependent manner and has pleiotropic effects on acquired immunity. IL-27 has the capacity to promote early stages of Th1 generation, but recent evidence has suggested a predominant suppressive effect on Th1, Th2, and Th17 differentiation. Although modest suppressive effects of IL-27 on myeloid lineage cells have been observed, there is limited knowledge about the role of IL-27 in the regulation of innate immunity. In this study we report that although in resting murine macrophages IL-27 had minimal if any effects, in resting human monocytes IL-27 had profound proinflammatory functions. IL-27 activated a STAT1-dominant pattern of signaling in human monocytes with the consequent activation of STAT1-dependent inflammatory target genes. IL-27 primed monocytes for augmented responses to TLR stimulation in a STAT1-dependent manner, altered IL-10 signaling, and attenuated IL-10-induced gene expression. Strikingly, IL-27 strongly suppressed TLR-induced IL-10 production in human monocytes. However, the proinflammatory effects of IL-27 on human monocytes were rapidly abrogated by LPS via a p38-mediated mechanism that inhibited IL-27 signaling. Our findings identify a predominantly proinflammatory function for IL-27 in human monocytes and suggest a mechanism by which the activating effects of IL-27 on innate immunity are attenuated as an immune response proceeds and IL-27 transitions to predominantly suppressive effects on acquired immunity.

Proliferative lesions and metalloproteinase activity in murine lupus nephritis mediated by type I interferons and macrophages

Glomerulonephritis is a major cause of morbidity in patients with systemic lupus erythematosus. Although substantial progress has been made in the identification of pathogenic triggers that result in autoantibody production, little is known about the pathogenesis of aggressive proliferative processes that lead directly to irreversible glomerular damage and compromise of renal function. In this study, we describe a model of polyinosinic: polycytidylic acid-accelerated lupus nephritis in NZB/W mice that is characterized by severe glomerular proliferative lesions with de novo crescent formation, findings that are linked with decreased survival and adverse outcomes in lupus. Proliferative glomerulonephritis was associated with infiltrating kidney macrophages and renal expression of IFN-inducible genes, matrix metalloproteinases (MMPs), and growth factors. Crescent formation and renal MMP and growth factor expression were dependent on renal macrophages that expressed Il10, MMPs, osteopontin, and growth factors, including Pdgfc and Hbegf. Infiltrating macrophages and renal MMP expression were induced by type I IFN. These findings reveal a role for type I IFNs and alternatively activated macrophages in aggressive proliferative lesions of lupus nephritis.

The future of the IL-1 receptor antagonist anakinra: from rheumatoid arthritis to adult-onset Still's disease and systemic-onset juvenile idiopathic arthritis

Background: IL-1 receptor antagonist (IL-1Ra) is a naturally occurring IL-1RI-binding molecule that blocks the biologic effects of the proinflammatory cytokine IL-1. A recombinant form of human IL-1Ra, anakinra (Kineret), has been approved for use in rheumatology initially to manage rheumatoid arthritis (RA) patients that are refractory to more conventional forms of treatment. Objective: This review summarizes the experience with anakinra in the treatment of patients with rheumatic diseases emphasizing its beneficial effects in novel applications. Methods: English-language trials of anakinra were searched using MEDLINE and abstracts from rheumatology scientific meetings. Results/conclusions: In the treatment of patients with RA anakinra is effective but inferior to TNF-α blocking agents. Over the last few years it has become increasingly evident that anakinra is highly effective and safe in patients with systemic-onset juvenile idiopathic arthritis, adult-onset Stills disease, hereditary autoinflammatory syndromes, Schnitzlers syndrome and recently in gouty attacks.

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.

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.

Suppression of TNF-α and IL-1 Signaling Identifies a Mechanism of Homeostatic Regulation of Macrophages by IL-27

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-α 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 Mϕ by inflammatory cytokines while preserving initial steps in host defense by augmenting responses to microbial products.

Overview of the biology of type I interferons

Type I interferons are pleiotropic cytokines with antiviral, antitumor and immunoregulatory functions. An aspect of their complex biology is the paradox that, depending on context, type I interferons can be anti-inflammatory and tissue protective or can be proinflammatory and promote autoimmunity. Along these lines, the activation of type I interferon pathways is effective in suppressing disease activity in patients with multiple sclerosis and in animal models of arthritis and colitis, while there is an expectation that blockade of the same pathways will be beneficial in the treatment of patients with systemic lupus erythematosus.

Modulation of TNF-Induced Macrophage Polarization by Synovial Fibroblasts

Mesenchymal stromal cells have emerged as powerful modulators of the immune system. In this study, we explored how the human macrophage response to TNF is regulated by human synovial fibroblasts, the representative stromal cell type in the synovial lining of joints that become activated during inflammatory arthritis. We found that synovial fibroblasts strongly suppressed TNF-mediated induction of an IFN-β autocrine loop and downstream expression of IFN-stimulated genes (ISGs), including chemokines CXCL9 and CXCL10 that are characteristic of classical macrophage activation. TNF induced the production of soluble synovial fibroblast factors that suppressed the macrophage production of IFN-β, and cooperated with TNF to limit the responsiveness of macrophages to IFN-β by suppressing activation of Jak–STAT signaling. Genome-wide transcriptome analysis showed that cocultured synovial fibroblasts modulate the expression of approximately one third of TNF-regulated genes in macrophages, including genes in pathways important for macrophage survival and polarization toward an alternatively activated phenotype. Pathway analysis revealed that gene expression programs regulated by synovial fibroblasts in our coculture system were also regulated in rheumatoid arthritis synovial macrophages, suggesting that these fibroblast-mediated changes may contribute to rheumatoid arthritis pathogenesis. This work furthers our understanding of the interplay between innate immune and stromal cells during an inflammatory response, one that is particularly relevant to inflammatory arthritis. Our findings also identify modulation of macrophage phenotype as a new function for synovial fibroblasts that may prove to be a contributing factor in arthritis pathogenesis.