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Multiple type I interferons and interferon-γ (IFN-γ) are expressed under physiological conditions and are increased by stress and infections, and in autoinflammatory and autoimmune diseases. Interferons activate the Jak–STAT signaling pathway and induce overlapping patterns of expression, called ‘interferon signatures’, of canonical interferon-stimulated genes (ISGs) encoding molecules important for antiviral responses, antigen presentation, autoimmunity and inflammation. It has now become clear that interferons also induce an ‘interferon epigenomic signature’ by activating latent enhancers and ‘bookmarking’ chromatin, thus reprogramming cell responses to environmental cues. The interferon epigenomic signature affects ISGs and other gene sets, including canonical targets of the transcription factor NF-κB that encode inflammatory molecules, and is involved in the priming of immune cells, tolerance and the training of innate immune memory. Here we review the mechanisms through which interferon signatures and interferon epigenomic signatures are generated, as well as the expression and functional consequences of these signatures in homeostasis and autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis and systemic sclerosis.

Autologous blood-derived products such as platelet-rich plasma (PRP) are widely used to treat musculoskeletal conditions, including knee osteoarthritis (OA). However, the clinical outcomes after PRP administration are often variable, and there is limited information about the specific characteristics of PRP that impact bioactivity and clinical responses. In this study, we aimed to develop an integrative workflow to evaluate responses to PRP in vitro, and to assess if the in vitro responses to PRP are associated with the PRP composition and clinical outcomes in patients with knee OA. To do this, we used a coculture system of macrophages and fibroblasts paired with transcriptomic analyses to comprehensively characterize the modulation of inflammatory responses by PRP in vitro. Relying on patient-reported outcomes and achievement of minimal clinically important differences in OA patients receiving PRP injections, we identified responders and non-responders to the treatment. Comparisons of PRP from these patient groups allowed us to identify differences in the composition and in vitro activity of PRP. We believe that our integrative workflow may enable the development of targeted approaches that rely on PRP and other orthobiologics to treat musculoskeletal pathologies.

Epigenetic and transcriptional analyses of B cell subsets from patients with systemic lupus erythematosus reveal extensive effects of the disease environment and identify previously unknown connections and regulators of DN2 B cells and ABC-like cells.

Background Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. Methods Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. Results Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. Conclusions We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers. Electronic supplementary material The online version of this article (10.1186/s13075-018-1631-y) contains supplementary material, which is available to authorized users.

Fibroblasts regulate tissue homeostasis, coordinate inflammatory responses, and mediate tissue damage. In rheumatoid arthritis (RA), synovial fibroblasts maintain chronic inflammation which leads to joint destruction. Little is known about fibroblast heterogeneity or if aberrations in fibroblast subsets relate to pathology. Here, we show functional and transcriptional differences between fibroblast subsets from human synovial tissues using bulk transcriptomics of targeted subpopulations and single-cell transcriptomics. We identify seven fibroblast subsets with distinct surface protein phenotypes, and collapse them into three subsets by integrating transcriptomic data. One fibroblast subset, characterized by the expression of proteins podoplanin, THY1 membrane glycoprotein and cadherin-11, but lacking CD34, is threefold expanded in patients with RA relative to patients with osteoarthritis. These fibroblasts localize to the perivascular zone in inflamed synovium, secrete proinflammatory cytokines, are proliferative, and have an in vitro phenotype characteristic of invasive cells. Our strategy may be used as a template to identify pathogenic stromal cellular subsets in other complex diseases., Synovial fibroblasts are thought to be central mediators of joint destruction in rheumatoid arthritis (RA). Here the authors use single-cell transcriptomics and flow cytometry to identify synovial fibroblast subsets that are expanded and display distinct tissue distribution and function in patients with RA.

Hypoxia and glycolysis have long been appreciated to promote immune cell activation. In 2019, several studies highlighted a counterbalancing homeostatic function for the glycolytic metabolite lactate. Lactate directly suppresses signalling pathways and modifies histones to play an important role in regulating macrophage polarization, tumour immunity and antiviral responses. Studies in the field of inflammation in 2019 have highlighted a counterbalancing homeostatic function for the glycolytic metabolite lactate, which is produced in hypoxic conditions, such as in tumours and chronic inflammation. Lionel Ivashkiv describes how lactate suppresses inflammatory signalling pathways and regulates macrophage polarization.

Interferon-γ (IFN-γ) primes macrophages for enhanced microbial killing and inflammatory activation by Toll-like receptors (TLRs), but little is known about the regulation of cell metabolism or mRNA translation during this priming. We found that IFN-γ regulated the metabolism and mRNA translation of human macrophages by targeting the kinases mTORC1 and MNK, both of which converge on the selective regulator of translation initiation eIF4E. Physiological downregulation of mTORC1 by IFN-γ was associated with autophagy and translational suppression of repressors of inflammation such as HES1. Genome-wide ribosome profiling in TLR2-stimulated macrophages showed that IFN-γ selectively modulated the macrophage translatome to promote inflammation, further reprogram metabolic pathways and modulate protein synthesis. These results show that IFN-γ-mediated metabolic reprogramming and translational regulation are key components of classical inflammatory macrophage activation.

Type I interferons (IFNs) activate intracellular antimicrobial programmes and influence the development of innate and adaptive immune responses. Canonical type I IFN signalling activates the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway, leading to transcription of IFN-stimulated genes (ISGs). Host, pathogen and environmental factors regulate the responses of cells to this signalling pathway and thus calibrate host defences while limiting tissue damage and preventing autoimmunity. Here, we summarize the signalling and epigenetic mechanisms that regulate type I IFN-induced STAT activation and ISG transcription and translation. These regulatory mechanisms determine the biological outcomes of type I IFN responses and whether pathogens are cleared effectively or chronic infection or autoimmune disease ensues.

Emerging concepts suggest that the functional phenotype of macrophages is regulated by transcription factors that define alternative activation states. We found that RBP-J, the main nuclear transducer of signaling via Notch receptors, augmented Toll-like receptor 4 (TLR4)-induced expression of key mediators of classically activated M1 macrophages and thus of innate immune responses to Listeria monocytogenes. Notch-RBP-J signaling controlled expression of the transcription factor IRF8 that induced downstream M1 macrophage-associated genes. RBP-J promoted the synthesis of IRF8 protein by selectively augmenting kinase IRAK2-dependent signaling via TLR4 to the kinase MNK1 and downstream translation-initiation control through eIF4E. Our results define a signaling network in which signaling via Notch-RBP-J and TLRs is integrated at the level of synthesis of IRF8 protein and identify a mechanism by which heterologous signaling pathways can regulate the TLR-induced inflammatory polarization of macrophages.

Bone metabolism results from a balance between osteoclast-driven bone resorption and osteoblast-mediated bone formation. Diseases such as periodontitis and rheumatoid arthritis are characterized by increased bone destruction due to enhanced osteoclastogenesis1,2. Here we report that interferon regulatory factor 8 (IRF8), a transcription factor expressed in immune cells, is a key regulatory molecule for osteoclastogenesis. IRF8 expression in osteoclast precursors was downregulated during the initial phase of osteoclast differentiation induced by receptor activator of nuclear factor κB ligand (RANKL, also called TRANCE, ODF, and OPGL), which is encoded by the Tnfsf11 gene. Mice deficient in IRF8 exhibited severe osteoporosis due to increased numbers of osteoclasts, and enhanced bone destruction following lipopolysaccharide (LPS) administration. Irf8–/– osteoclast precursors underwent increased osteoclastogenesis in response to RANKL and tumor necrosis factor α (TNFα). IRF8 suppressed osteoclastogenesis by inhibiting the function and expression of nuclear factor of activated T cells c1 (NFATc1). Our results show that IRF8 inhibits osteoclast formation under physiological and pathological conditions, and suggest a model where downregulation of inhibitory factors like IRF8 contributes to RANKL-mediated osteoclastogenesis.

An important function of receptors that signal through immunoreceptor tyrosine-based activation motifs (ITAMs) is to regulate signaling by heterologous receptors. This review describes mechanisms by which ITAM-associated receptors modulate signaling by Toll-like receptors (TLRs), tumor necrosis factor receptor family members and cytokine receptors that use the Jak-STAT signaling pathway, and the biological importance of this signal transduction cross-talk. ITAM-mediated cross-regulation can either augment or dampen signaling by other receptors. Conversely, TLRs and cytokines modulate ITAM-mediated signaling, by means including activation of beta2 integrins that are coupled to the ITAM-containing adaptors DAP12 and FcRgamma. Integration of ITAM signaling into signaling networks through cross-talk with other signal transduction pathways results in tight regulation and fine tuning of cellular responses to various extracellular stimuli and contributes to induction of specific activation and differentiation pathways.

Semaphorins were originally discovered in the nervous system, and have been implicated in repulsive axon guidance during the development of nervous system. Semaphorins are also implicated in tumor progression, by affecting adhesion, migration of malignant cells and angiogenesis, and are involved in normal cardiovascular development. Recently, 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. This review focuses on recent work on the functions of semaphorins in the immune system and autoimmune diseases.

Immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors modulate the amplitude and nature of macrophage responses to Toll-like receptor and cytokine receptor stimulation. However, the molecular mechanisms enabling this receptor crosstalk are not known. Here we investigated the function of the calcium-dependent kinases CaMK and Pyk2 'downstream' of ITAM-associated receptors in the regulation of cytokine-induced activation of Jak kinases and STAT transcription factors. CaMK and Pyk2 relayed signals from integrins and the ITAM-containing adaptor DAP12 to augment interleukin 10- and interferon-alpha-induced Jak activation and STAT1-dependent gene expression. CaMK inhibition suppressed STAT1-mediated interferon-alpha signaling in a mouse model of systemic lupus erythematosus. Our results associate Pyk2 and Jak kinases with the linkage of signals emanating from cytokine and heterologous ITAM-dependent receptors.

A key function of interferons is priming multiple cell types for enhanced activation by cytokines and inflammatory factors, including tumor necrosis factor, bacterial lipopolysaccharide and interferons themselves. Here we show that interferon-alpha (IFN-alpha)-induced activation of the transcriptional activator STAT1 and inflammatory STAT1 target genes was enhanced in IFN-gamma-primed macrophages. Enhanced IFN-alpha signaling and proinflammatory function were dependent on the tyrosine kinase Syk and on adaptor proteins that activate Syk through immunoreceptor tyrosine activation motifs. Increased STAT1 expression contributed to enhanced IFN-alpha-induced STAT1 activation in primed macrophages. These results identify a mechanism by which crosstalk between cytokine and immune cell-specific immunoreceptor tyrosine activation motif-dependent signaling pathways regulates macrophage responses to IFN-alpha.

Emerging evidence suggests that RANKL-induced changes in chromatin state are important for osteoclastogenesis, but these epigenetic mechanisms are not well understood and have not been therapeutically targeted. In this study, we find that the small molecule I-BET151 that targets bromo and extra-terminal (BET) proteins that 'read' chromatin states by binding to acetylated histones strongly suppresses osteoclastogenesis. I-BET151 suppresses pathologic bone loss in TNF-induced inflammatory osteolysis, inflammatory arthritis and post-ovariectomy models. Transcriptome analysis identifies a MYC-NFAT axis important for osteoclastogenesis. Mechanistically, I-BET151 inhibits expression of the master osteoclast regulator NFATC1 by suppressing expression and recruitment of its newly identified upstream regulator MYC. MYC is elevated in rheumatoid arthritis macrophages and its induction by RANKL is important for osteoclastogenesis and TNF-induced bone resorption. These findings highlight the importance of an I-BET151-inhibited MYC-NFAT axis in osteoclastogenesis, and suggest targeting epigenetic chromatin regulators holds promise for treatment of inflammatory and oestrogen deficiency-mediated pathologic bone resorption.

TNF-α is a key pathogenic factor in inflammatory arthritis. Rapid and transient signaling and functional responses of cells to TNF-α, such as activation of NF-γB and MAPKs, are well known. These signaling mechanisms are widely assumed to be functional in cells chronically exposed to TNF-α and to mediate the pathogenic effects of TNF-α in chronic inflammation. We investigated the responses of primary macrophages to TNF-α over the course of several days and compared patterns of signaling and gene expression to RA synovial macrophages. The acute inflammatory response to TNF-α subsided after several hours and was followed by an IFN response characterized by sustained expression of STAT1 and downstream target genes. TNF-α-mediated induction of an IFN response was mediated by IFN-β and was sensitive to inhibition by Jak inhibitors. Concomitantly TNF-α induced a state of macrophage resistance to the homeostatic cytokines IL-10 and IL-27. Microarray analysis demonstrated that sustained TNF-α signaling induced expression of novel genes not appreciated to be 'TNF-inducible', but are highly expressed in RA synovial macrophages. Induction of an IFN response and abrogation of homeostatic cytokine signaling was also observed in RA synovial macrophages and likely contributes to the pathogenic actions of TNF-α during arthritis. Subsequently and surprisingly, TNF-α induced a tolerant state in macrophages, with diminished cytokine production on lipopolysaccharide (LPS) challenge and protection from LPS-induced lethality. TNF-α-induced cross-tolerization was mediated by coordinate action of two inhibitory mechanisms, suppression of LPS-induced signaling and chromatin remodeling. Mechanistically, TNF-α-induced cross-tolerance was distinguished from TLR-induced tolerance by strong dependence on the nuclear kinase GSK3, which suppressed chromatin accessibility and promoted rapid termination of NF-γB signaling by augmenting negative feedback by A20 and IγBα. These results reveal an unexpected homeostatic function of TNF-α and provide a GSK3-mediated mechanism for preventing prolonged and excessive inflammation. This homeostatic mechanism may be compromised during RA synovitis, possibly by hypomorphic alleles of TNFAIP3 (encodes A20) or by cytokines that suppress A20 expression or antagonize its function. These data suggest that augmenting homeostatic functions and signals and thereby rebalancing the pro- versus anti-inflammatory profile of TNF-α may represent an efficacious alternative therapeutic approach to suppress chronic inflammation. Overall, the data reveal novel signals and functions of TNF-α and that are likely operative during chronic inflammation and RA synovitis. Targeted inhibition of these non-traditional functional components of the TNF-α response may be efficacious in alleviating chronic inflammation while preserving acute TNF-α responses and host defense against infections.

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.

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.

A variety of cytokines and growth factors use the Janus kinase (Jak)–STAT signaling pathway to transmit extracellular signals to the nucleus. STATs (signal transducers and activators of transcription) are latent cytoplasmic transcription factors. There are seven mammalian STATs and they have critical, nonredundant roles in mediating cellular transcriptional responses to cytokines. The physiological roles of STATs have been elucidated by analysis of mice rendered deficient in STAT genes. STAT activation is regulated and can be modulated in a positive or negative fashion; it can be reprogrammed to drive different cellular responses. Several auto-regulatory and signaling crosstalk mechanisms for regulating Jak–STAT signaling have been described. Understanding and manipulation of the function of STATs will help in the development of therapeutic strategies for diseases that are regulated by cytokines.