The Effect of Antibody Size and Mechanical Loading on Solute Diffusion Through the Articular Surface of Cartilage.

Because of the heterogeneous nature of articular cartilage tissue, penetration of potential therapeutic molecules for osteoarthritis (OA) through the articular surface (AS) is complex, with many factors that affect transport of these solutes within the tissue. Therefore, the goal of this study is to investigate how the size of antibody (Ab) variants, as well as application of cyclic mechanical loading, affects solute transport within healthy cartilage tissue. Penetration of fluorescently tagged solutes was quantified using confocal microscopy. For all the solutes tested, fluorescence curves were obtained through the articular surface. On average, diffusivities for the solutes of sizes 200 kDa, 150 kDa, 50 kDa, and 25 kDa were 3.3, 3.4, 5.1, and 6.0 µm2/s from 0 to 100 µm from the articular surface. Diffusivities went up to a maximum of 16.5, 18.5, 20.5, and 23.4 µm2/s for the 200 kDa, 150 kDa, 50 kDa, and 25 kDa molecules, respectively, from 225 to 325 µm from the surface. Overall, the effect of loading was very significant, with maximal transport enhancement for each solute ranging from 2.2 to 3.4-fold near 275 µm. Ultimately, solutes of this size do not diffuse uniformly nor are convected uniformly, through the depth of the cartilage tissue. This research potentially holds great clinical significance to discover ways of further optimizing transport into cartilage and leads to effective antibody-based treatments for OA.

BET bromodomain inhibition suppresses transcriptional responses to cytokine-Jak-STAT signaling in a gene-specific manner in human monocytes.

Disruption of the interaction of bromo and extraterminal (BET) proteins with acetylated histones using small molecule inhibitors suppresses Myc-driven cancers and TLR-induced inflammation in mouse models. The predominant mechanism of BET inhibitor action is to suppress BET-mediated recruitment of positive transcription elongation factor b and, thus, transcription elongation. We investigated the effects of BET inhibitor I-BET151 on transcriptional responses to TLR4 and TNF in primary human monocytes and also on responses to cytokines IFN-ß, IFN-γ, IL-4, and IL-10, which activate the JAK-STAT signaling pathway and are important for monocyte polarization and inflammatory diseases. I-BET151 suppressed TLR4- and TNF-induced IFN responses by diminishing both autocrine IFN-ß expression and transcriptional responses to IFN-ß. I-BET151 inhibited cytokine-induced transcription of STAT targets in a gene-specific manner without affecting STAT activation or recruitment. This inhibition was independent of Myc or other upstream activators. IFN-stimulated gene transcription is regulated primarily at the level of transcription initiation. Accordingly, we found that I-BET151 suppressed the recruitment of transcriptional machinery to the CXCL10 promoter and an upstream enhancer. Our findings suggest that BET inhibition reduces inflammation partially through suppressing cytokine activity and expands the understanding of the inhibitory and potentially selective immunosuppressive effects of inhibiting BET proteins.

Inhibition of osteoclastogenesis and inflammatory bone resorption by targeting BET proteins and epigenetic regulation.

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.

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.

Regulation of inflammatory responses in tumor necrosis factor-activated and rheumatoid arthritis synovial macrophages by JAK inhibitors.

OBJECTIVE: JAK inhibitors have been developed as antiinflammatory 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. The purpose of this study was to examine the effects of both JAK inhibitors on inflammatory and tumor necrosis factor (TNF) responses in human macrophages. METHODS: In vitro studies were performed using peripheral blood macrophages derived from healthy donors and treated with TNF and using synovial fluid macrophages derived from patients with RA. Levels of activated STAT proteins and other transcription factors were detected by Western blotting, and gene expression was measured by real-time polymerase chain reaction analysis. The in vivo effects of JAK inhibitors were evaluated in the K/BxN serum-transfer model of arthritis. RESULTS: JAK inhibitors suppressed the activation and expression of STAT-1 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 the expression of interleukin-6 in synovial macrophages. JAK inhibitors augmented nuclear levels of NF-ATc1 and cJun, followed by increased formation of osteoclast-like cells. CP-690,550 strongly suppressed K/BxN serum-transfer arthritis, which is dependent on macrophages, but not lymphocytes. CONCLUSION: Our findings demonstrate that JAK inhibitors suppress macrophage activation and attenuate TNF responses and further suggest that suppression of cytokine/chemokine production and innate immunity contribute to the therapeutic efficacy of JAK inhibitors.

TNF activates calcium-nuclear factor of activated T cells (NFAT)c1 signaling pathways in human macrophages.

Acute activation of cells by tumor necrosis factor (TNF) has been well characterized, but little is known about later phases of TNF responses that are relevant for cells exposed to TNF for several days during inflammation. We found that prolonged exposure of human macrophages to TNF resulted in a wave of delayed but sustained activation of c-Jun and nuclear factor κB (NF-κB) proteins and of calcium oscillations that became apparent 1-3 d after TNF stimulation. These signaling events culminated in the induction and activation of the calcium-dependent transcription factor, nuclear factor of activated T cells (NFAT)c1, which mediated a gene expression program leading to cell fusion and osteoclast differentiation. TNF-induced NFATc1 activity primed macrophages for enhanced osteoclastogenesis in response to RANKL. High NFATc1 expression was apparent in synovial macrophages in a subset of patients with TNF-driven inflammatory arthritis. Thus, long-term exposure to TNF activates calcium-dependent signaling and an NFATc1-mediated gene activation program important for cell fusion and osteoclastogenesis. These findings identify a signaling pathway activated by TNF that is important for myeloid cell differentiation and suggest a role for TNF-induced calcium and NFAT signaling in chronic inflammation and associated bone resorption.

Type I interferon: a new player in TNF signaling.

TNF and type I interferons (IFNs) are induced by microbial stimuli and mediate innate immune responses. They are also involved in the 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 discuss the role of an IFN-beta-mediated autocrine loop in the regulation of gene expression program induced by TNF in myeloid cells.

Regulation of STAT pathways and IRF1 during human dendritic cell maturation by TNF-alpha and PGE2.

Maturation of dendritic cells (DCs) by TLR ligands induces expression of IFN-beta and autocrine activation of IFN-inducible Stat1-dependent genes important for DC function. In this study, we analyzed the regulation of STAT signaling during maturation of human DCs by TNF-alpha and PGE2, which induced maturation of human DCs comparably with LPS but did not induce detectable IFN-beta production or Stat1 tyrosine phosphorylation. Consistent with these results, TNF-alpha and PGE2 did not induce Stat1 DNA binding to a standard Stat1-binding oligonucleotide. Instead, TNF-alpha and PGE2 increased Stat1 serine phosphorylation and Stat4 tyrosine phosphorylation and activated expression of the NF-kappaB and Stat1 target gene IFN regulatory factor 1 (IRF1), which contributes to IFN responses. TNF-alpha and PGE2 induced a complex that bound an oligonucleotide derived from the IRF1 promoter that contains a STAT-binding sequence embedded in a larger palindromic sequence, and this complex was recognized by Stat1 antibodies. These results suggest that TNF-alpha and PGE2 activate STAT-mediated components of human DC maturation by alternative pathways to the IFN-beta-mediated autocrine loop used by TLRs.

TNF activates an IRF1-dependent autocrine loop leading to sustained expression of chemokines and STAT1-dependent type I interferon-response genes.

Rapid induction of inflammatory genes by tumor necrosis factor (TNF) has been well studied, but little is known about delayed and chronic TNF responses. Here we investigated the kinetics of primary macrophage responses to TNF and discovered that TNF initiates an interferon-beta-mediated autocrine loop that sustains expression of inflammatory genes and induces delayed expression of interferon-response genes such as those encoding the transcription factors STAT1 and IRF7, which enhance macrophage responses to stimulation of cytokines and Toll-like receptors. TNF-induced interferon-beta production depended on interferon-response factor 1, and downstream gene expression was mediated by synergy between small amounts of interferon-beta and canonical TNF-induced signals. Thus, TNF activates a 'feed-forward' loop that sustains inflammation but avoids the potential toxicity associated with the high interferon production induced by stimulation of Toll-like receptors.

Suppression of the effector phase of inflammatory arthritis by double-stranded RNA is mediated by type I IFNs.

Innate immune receptors that recognize nucleic acids, such as TLRs and RNA helicases, are potent activators of innate immunity that have been implicated in the induction and exacerbation of autoimmunity and inflammatory arthritis. Polyriboinosine-polyribocytidylic acid sodium salt (poly(IC)) is a mimic of dsRNA and viral infection that activates TLR3 and the RNA helicases retinoic acid-induced gene-1 and melanoma differentiation-associated gene-5, and strongly induces type I IFN production. We analyzed the effects of systemic delivery of poly(IC) on the inflammatory effector phase of arthritis using the collagen Ab-induced and KRN TCR-transgenic mouse serum-induced models of immune complex-mediated experimental arthritis. Surprisingly, poly(IC) suppressed arthritis, and suppression was dependent on type I IFNs that inhibited synovial cell proliferation and inflammatory cytokine production. Administration of exogenous type I IFNs was sufficient to suppress arthritis. These results suggest a regulatory role for innate immune receptors for dsRNA in modulating inflammatory arthritis and provide additional support for an anti-inflammatory function of type I IFNs in arthritis that directly contrasts with a pathogenic role in promoting autoimmunity in systemic lupus.

IFN-gamma suppresses IL-10 production and synergizes with TLR2 by regulating GSK3 and CREB/AP-1 proteins.

The control of IL-10 production and mechanisms that mediate synergy between IFN-gamma and TLR ligands are not well understood. We report that IFN-gamma augments induction of TNFalpha by TLR ligands, immune complexes, and zymosan by suppressing IL-10 production and thereby interrupting Stat3-mediated feedback inhibition. IFN-gamma altered TLR2-induced signal transduction by increasing GSK3 activity and suppressing MAPK activation, leading to diminished IL-10 production. Inhibition of GSK3 or ablation of the GSK3beta gene ameliorated TLR2-induced peritonitis and arthritis. IFN-gamma suppressed the activity of CREB and AP-1, transcription factors that induce IL-10 expression and are regulated in part by MAPKs and GSK3. These results yield insight into mechanisms by which IFN-gamma regulates IL-10 production and TLR2-mediated inflammatory responses and identify inhibition of CREB and AP-1 as part of the macrophage response to IFN-gamma. GSK3 and CREB/AP-1 are key players in integrating IFN-gamma and TLR2 responses in innate immunity and inflammation.

High expression of ILT3 and ILT4 is a general feature of tolerogenic dendritic cells.

The direct interaction between antigen specific CD8(+) CD28(-) T suppressor cells (T(S)) with dendritic cells (DC) results in the tolerization of DC by inducing the upregulation of immunologlobulin like transcript 3 (ILT3) and ILT4. We show here that such tolerogenic DC anergize alloreactive CD4(+) CD45RO(+) CD25(+) T cells converting them into regulatory T cells (T(R)), which in turn, continue the cascade of suppression by tolerizing other DC. Interleukin 10 (IL-10) and interferon-alpha (IFN-alpha) also induce ILT3 and ILT4 upregulation in DC, rendering them tolerogenic. This implies a common mechanism of DC-mediated suppression. This finding and the observation that in organ allograft recipients quiescence is associated with the presence in the circulation of donor-specific T(S) and T(R) emphasize the importance of the cross talk between tolerogenic DC and T cells in suppression of the immune response.