Mark H. Wenink

Proteome-wide Analysis and CXCL4 as a Biomarker in Systemic Sclerosis

BACKGROUND Plasmacytoid dendritic cells have been implicated in the pathogenesis of systemic sclerosis through mechanisms beyond the previously suggested production of type I interferon. METHODS We isolated plasmacytoid dendritic cells from healthy persons and from patients with systemic sclerosis who had distinct clinical phenotypes. We then performed proteome-wide analysis and validated these observations in five large cohorts of patients with systemic sclerosis. Next, we compared the results with those in patients with systemic lupus erythematosus, ankylosing spondylitis, and hepatic fibrosis. We correlated plasma levels of CXCL4 protein with features of systemic sclerosis and studied the direct effects of CXCL4 in vitro and in vivo. RESULTS Proteome-wide analysis and validation showed that CXCL4 is the predominant protein secreted by plasmacytoid dendritic cells in systemic sclerosis, both in circulation and in skin. The mean (±SD) level of CXCL4 in patients with systemic sclerosis was 25,624±2652 pg per milliliter, which was significantly higher than the level in controls (92.5±77.9 pg per milliliter) and than the level in patients with systemic lupus erythematosus (1346±1011 pg per milliliter), ankylosing spondylitis (1368±1162 pg per milliliter), or liver fibrosis (1668±1263 pg per milliliter). CXCL4 levels correlated with skin and lung fibrosis and with pulmonary arterial hypertension. Among chemokines, only CXCL4 predicted the risk and progression of systemic sclerosis. In vitro, CXCL4 down-regulated expression of transcription factor FLI1, induced markers of endothelial-cell activation, and potentiated responses of toll-like receptors. In vivo, CXCL4 induced the influx of inflammatory cells and skin transcriptome changes, as in systemic sclerosis. CONCLUSIONS Levels of CXCL4 were elevated in patients with systemic sclerosis and correlated with the presence and progression of complications, such as lung fibrosis and pulmonary arterial hypertension. (Funded by the Dutch Arthritis Association and others.).

Increased Frequency and Compromised Function of T Regulatory Cells in Systemic Sclerosis (SSc) Is Related to a Diminished CD69 and TGFβ Expression

Background Regulatory T cells (Tregs) are essential in the control of tolerance. Evidence implicates Tregs in human autoimmune conditions. Here we investigated their role in systemic sclerosis (SSc). Methods/Principal Findings Patients were subdivided as having limited cutaneous SSc (lcSSc, n = 20) or diffuse cutaneous SSc (dcSSc, n = 48). Further subdivision was made between early dcSSc (n = 24) and late dcSSc (n = 24) based upon the duration of disease. 26 controls were studied for comparison. CD3+ cells were isolated using FACS and subsequently studied for the expression of CD4, CD8, CD25, FoxP3, CD127, CD62L, GITR, CD69 using flow cytometry. T cell suppression assays were performed using sorted CD4CD25highCD127- and CD4CD25lowCD127high and CD3+ cells. Suppressive function was correlated with CD69 surface expression and TGFβ secretion/expression. The frequency of CD4+CD25+ and CD25highFoxP3highCD127neg T cells was highly increased in all SSc subgroups. Although the expression of CD25 and GITR was comparable between groups, expression of CD62L and CD69 was dramatically lower in SSc patients, which correlated with a diminished suppressive function. Co-incubation of Tregs from healthy donors with plasma from SSc patients fully abrogated suppressive activity. Activation of Tregs from healthy donors or SSc patients with PHA significantly up regulated CD69 expression that could be inhibited by SSc plasma. Conclusions/Significance These results indicate that soluble factors in SSc plasma inhibit Treg function specifically that is associated with altered Treg CD69 and TGFβ expression. These data suggest that a defective Treg function may underlie the immune dysfunction in systemic sclerosis.

Type I interferons might form the link between Toll-like receptor (TLR) 3/7 and TLR4-mediated synovial inflammation in rheumatoid arthritis (RA)

Background: Rheumatoid arthritis (RA) has been associated with an increased risk of infections, but the underlying pathways have not yet been identified. Toll-like receptors (TLR) probably play a role in synovial inflammation and may also contribute to the understanding of the role of infections in RA. Objectives: To investigate if the synovial expression of TLR3 and TLR7 in RA correlates with that of inflammatory cytokines, and to assess whether this has functional consequences for local cytokine production and to study potential links between the TLR3/7 axis and TLR4 in RA synovium. Methods: Immunohistochemistry was used to study the expression of TLR3, TLR7, interferon α (IFNα), tumour necrosis factor α (TNFα) and interleukins IL1β, IL12, IL17 and IL18 in RA synovium obtained by arthroscopy from 34 patients with RA. Monocytes, monocyte-derived dendritic cells (MoDCs) and RA synovial fibroblasts were stimulated via TLR3 (poly-IC) and TLR7 (loxorubin), after which IL1β, IL6 and TNFα were measured by Luminex bead array technology. Following preincubation with IFNα, IL1β and IL18, TLR3 and TLR7 mRNA expression was assessed using real-time PCR. Cytokine production after preincubation with IFNα and subsequent TLR stimulation was measured. Results: Synovial TLR3/7 expression was co-expressed with IFNα, IL1β and IL18, but not with TNFα, IL12 and IL17. Stimulation of TLR3/TLR7 on monocytes, MoDCs or synovial fibroblasts led to secretion of type I IFN but no biologically active IL1β or IL18 could be detected. Type I IFNα increased TLR3/7 mRNA expression whereas IL1β and IL18 did not. In spite of the fact that the mRNA level of TLR4 remained unchanged, IFNα enhanced the response to TLR4 agonists, a phenomenon that was clearly more marked in patients with RA. Conclusion: Type I interferons are highly co-expressed with TLR3/TLR7 in RA synovium. They enhance TLR3/TLR7-mediated cytokine production and also TLR4-mediated responses.

TLR2 promotes Th2/Th17 responses via TLR4 and TLR7/8 by abrogating the Type I IFN amplification loop

TLR2 plays an important role in the removal of Gram-positive bacteria; contrastingly, it also appears to have important protective effects against unrestrained inflammation and subsequent organ injury during infection and autoimmunity. We hypothesized that TLR2 tunes the phenotype of dendritic cells (DCs) activated through other TLRs, thereby fulfilling a crucial role in the modulation of the immune response. TLR2 potently inhibited TLR4- and TLR7/8-induced cytokine production by human DCs. The inhibitory effect of TLR2 on the release of TNF-α but not of IL-12p70 was mediated by PI3K. TLR2 inhibits the production of IL-12p70 by dampening the type 1 IFN amplification loop. When DCs were triggered with the potent synergistic combination of LPS (TLR4) and R848 (TLR7/8) in conjunction with a TLR2 ligand, a clear shift to more Th2- and Th17-prone responses in the naive and memory T cell subpopulations was observed. This shift in T cell responses was inherent to the inability of TLR2-stimulated DCs to produce IL-12p70 and was dependent on the production of IL-1 and IL-6.

The inhibitory Fc gamma IIb receptor dampens TLR4-mediated immune responses and is selectively up-regulated on dendritic cells from rheumatoid arthritis patients with quiescent disease.

Rheumatoid arthritis (RA) is a common autoimmune disease leading to profound disability and premature death. Although a role for FcγRs and TLRs is accepted, their precise involvement remains to be elucidated. FcγRIIb is an inhibitory FcR important in the maintenance of tolerance. We hypothesized that the inhibitory FcγRIIb inhibits TLR responses on monocyte-derived dendritic cells (DC) and serves as a counterregulatory mechanism to dampen inflammation, and we surmised that this mechanism might be defective in RA. The expression of the inhibitory FcγRIIb was found to be significantly higher on DCs from RA patients having low RA disease activity in the absence of treatment with antirheumatic drugs. The expression of activating FcγRs was similarly distributed among all RA patients and healthy controls. Intriguingly, only DCs with a high expression of FcγRIIb were able to inhibit TLR4-mediated secretion of proinflammatory cytokines when stimulated with immune complexes. In addition, when these DCs were coincubated with the combination of a TLR4 agonist and immune complexes, a markedly inhibited T cell proliferation was apparent, regulatory T cell development was promoted, and T cells were primed to produce high levels of IL-13 compared with stimulation of the DCs with the TLR4 agonist alone. Blocking FcγRIIb with specific Abs fully abrogated these effects demonstrating the full dependence on the inhibitory FcγRIIb in the induction of these phenomena. This TLR4-FcγRIIb interaction was shown to dependent on the PI3K and Akt pathway.

Perception of self : Distinguishing autoimmunity from autoinflammation

Rheumatic diseases can be divided in two groups, autoinflammatory and autoimmune disorders. The clinical presentation of both types of diseases overlap, but the pathological pathways underlying rheumatic autoinflammation and autoimmunity are distinct and are the subject of ongoing research. There are a number of ways in which these groups of diseases differ in terms of disease mechanisms and therapeutic responses. First, autoinflammatory diseases are driven by endogenous danger signals, metabolic mediators and cytokines, whereas autoimmunity involves the activation of T and B cells, the latter requiring V-(D)-J recombination of receptor-chain gene segments for maturation. Second, the efficacy of biologic agents directed against proinflammatory cytokines (for example IL-1β and TNF) also highlights differences between autoinflammatory and autoimmune processes. Finally, whereas autoinflammatory diseases are mostly driven by inflammasome-induced IL-1β and IL-18 production, autoimmune diseases are associated with type I interferon (IFN) signatures in blood. In this Review, we provide an overview of the monocyte intracellular pathways that drive autoinflammation and autoimmunity. We convey recent findings on how the type I IFN pathway can modulate IL-1β signalling (and vice versa), and discuss why IL-1β-mediated autoinflammatory diseases do not perpetuate into autoimmunity. The origins of intracellular autoantigens in autoimmune disorders are also discussed. Finally, we suggest how new mechanistic knowledge of autoinflammatory and autoimmune diseases might help improve treatment strategies to benefit patient care.

Brief report: Enrichment of activated group 3 innate lymphoid cells in psoriatic arthritis synovial fluid

Innate lymphoid cells (ILCs) are a recently discovered group of cells that are essential to epithelial homeostasis and are implicated in psoriasis pathogenesis, yet they have never been reported in psoriatic arthritis (PsA).

Toll-like receptors in rheumatic diseases: Are we paying a high price for our defense against bugs?

In the last decade Toll‐like receptor (TLR) research has led to new insights in the pathogenesis of many rheumatic diseases. In autoimmune diseases like systemic lupus erythematosus, rheumatoid arthritis and systemic sclerosis TLR signaling is likely to be involved in tolerance breakthrough and chronic inflammation via combined Fc gamma receptors and TLR recognition of immune complexes. Furthermore, inflammatory diseases like psoriatic arthritis and gout also show more and more evidence for TLR involvement. In this review we will discuss the involvement of TLR signaling in several rheumatic diseases and stress their similarities and differences based on recent findings.

Soluble immune complexes shift the TLR-induced cytokine production of distinct polarized human macrophage subsets towards IL-10.

Background Costimulation of murine macrophages with immune complexes (ICs) and TLR ligands leads to alternative activation. Studies on human myeloid cells, however, indicate that ICs induce an increased pro-inflammatory cytokine production. This study aimed to clarify the effect of ICs on the pro- versus anti-inflammatory profile of human polarized macrophages. Materials and Methods Monocytes isolated from peripheral blood of healthy donors were polarized for four days with IFN-γ, IL-4, IL-10, GM-CSF, M-CSF, or LPS, in the presence or absence of heat aggregated gamma-globulins (HAGGs). Phenotypic polarization markers were measured by flow cytometry. Polarized macrophages were stimulated with HAGGs or immobilized IgG alone or in combination with TLR ligands. TNF, IL-6, IL-10, IL-12, and IL-23 were measured by Luminex and/or RT-qPCR. Results HAGGs did not modulate the phenotypic polarization and the cytokine production of macrophages. However, HAGGs significantly altered the TLR-induced cytokine production of all polarized macrophage subsets, with the exception of MΦIL-4. In particular, HAGGs consistently enhanced the TLR-induced IL-10 production in both classically and alternatively polarized macrophages (M1 and M2). The effect of HAGGs on TNF and IL-6 production was less pronounced and depended on the polarization status, while IL-23p19 and IL-12p35 expression was not affected. In contrast with HAGGs, immobilized IgG induced a strong upregulation of not only IL-10, but also TNF and IL-6. Conclusion HAGGs alone do not alter the phenotype and cytokine production of in vitro polarized human macrophages. In combination with TLR-ligands, however, HAGGs but not immobilized IgG shift the cytokine production of distinct macrophage subsets toward IL-10.

Abatacept modulates proinflammatory macrophage responses upon cytokine-activated T cell and Toll-like receptor ligand stimulation

Objectives We investigated whether Abatacept might reduce proinflammatory cytokine production by macrophages upon contact with cytokine activated T cells and/or stimulation with TLR ligands. Methods Macrophages and cytokine stimulated T cells (Tck) were added together in the presence of Abatacept or a control Ig, with or without TLR ligands. The production of cytokines was determined by luminex. Results Abatacept reduced Tck-induced production of TNFa by macrophages. Tck and TLR ligands synergistically induced the production of proinflammatory cytokines by macrophages, especially IL-12p70. The production of IL-12p70 coincided with the production of IFNg, which were both reduced in the presence of Abatacept. Conclusions Tck induce the production of TNFa by macrophages and facilitate the highly increased production of proinflammatory cytokines in the presence of TLR ligands. Abatacept was shown to potently suppress these pathways suggesting that its role may extend beyond antigen specific T cell mediated effector function.