Mieke F. Roelofs

Stimulation of TLR2 and TLR4 differentially skews the balance of T cells in a mouse model of arthritis

TLRs may contribute to the progression of rheumatoid arthritis through recognition of microbial or host-derived ligands found in arthritic joints. Here, we show that TLR2 and TLR4, but not TLR9, are involved in the pathogenesis of autoimmune arthritis and play distinct roles in the regulation of T cells and cytokines. We investigated the involvement of TLR2, TLR4, and TLR9 in the progression of arthritis using IL-1 receptor antagonist-knockout (IL1rn-/-) mice, which spontaneously develop an autoimmune T cell-mediated arthritis. Spontaneous onset of arthritis was dependent on TLR activation by microbial flora, as germ-free mice did not develop arthritis. Clinical and histopathological evaluation of IL1rn-/-Tlr2-/- mice revealed more severe arthritis, characterized by reduced suppressive function of Tregs and substantially increased IFN-gamma production by T cells. IL1rn-/-Tlr4-/- mice were, in contrast, protected against severe arthritis and had markedly lower numbers of Th17 cells and a reduced capacity to produce IL-17. A lack of Tlr9 did not affect the progression of arthritis. While any therapeutic intervention targeting TLR2 still seems complicated, the strict position of TLR4 upstream of a number of pathogenic cytokines including IL-17 provides an interesting potential therapeutic target for rheumatoid arthritis.

Identification of Small Heat Shock Protein B8 (HSP22) as a Novel TLR4 Ligand and Potential Involvement in the Pathogenesis of Rheumatoid Arthritis

Dendritic cells (DCs) are specialized APCs that can be activated upon pathogen recognition as well as recognition of endogenous ligands, which are released during inflammation and cell stress. The recognition of exogenous and endogenous ligands depends on TLRs, which are abundantly expressed in synovial tissue from rheumatoid arthritis (RA) patients. Furthermore TLR ligands are found to be present in RA serum and synovial fluid and are significantly increased, compared with serum and synovial fluid from healthy volunteers and patients with systemic sclerosis and systemic lupus erythematosus. Identification of novel endogenous TLR ligands might contribute to the elucidation of the role of TLRs in RA and other autoimmune diseases. In this study, we investigated whether five members of the small heat shock protein (HSP) family were involved in TLR4-mediated DC activation and whether these small HSPs were present in RA synovial tissue. In vitro, monocyte-derived DCs were stimulated with recombinant αA crystallin, αB crystallin, HSP20, HSPB8, and HSP27. Using flow cytometry and multiplex cytokine assays, we showed that both αA crystallin and HSPB8 were able to activate DCs and that this activation was TLR4 dependent. Furthermore, Western blot and immunohistochemistry showed that HSPB8 was abundantly expressed in synovial tissue from patients with RA. With these experiments, we identified sHSP αA crystallin and HSPB8 as two new endogenous TLR4 ligands from which HSPB8 is abundantly expressed in RA synovial tissue. These findings suggest a role for HSPB8 during the inflammatory process in autoimmune diseases such as RA.

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.

The orchestra of Toll-like receptors and their potential role in frequently occurring rheumatic conditions.

In the early 1990s, Janeway discussed the theory that an immune response could not occur unless antigen-presenting cells (APCs) were first activated, which he called “the immunologist’s dirty little secret.” Then, in the coup that caused the immunology community to finally take notice, and following the seminal study by Lemaitre et al (1), Janeway discovered the crucial role of pattern-recognition receptors (PRRs), which recognized evolutionary conserved molecules on infectious nonself organisms (2). Toll-like receptors (TLRs) belong to the PRR family, which was first shown to recognize microbial components, known as pathogenassociated patterns. TLRs are constitutively expressed by numerous immune cells and are designed to detect and eliminate invading pathogens by activating both innate and adaptive immune responses. Accumulating evidence indicates a role of TLRs in the recognition of “host-derived” agonists (so-called endogenous ligands or alarmins), which might be involved in various autoimmune and/or autoinflammatory syndromes. Examples of such endogenous TLR ligands are Hsp60, Hsp70, gp96, small HspB8, hyaluronic acid, and fibronectin, which are all released upon cell stress and are found in various tissues during inflammation (discussed below). TLRs are of interest to immunologists because of their important role in the initiation of immune responses. Because evidence seems to implicate innate immunity in a wide variety of rheumatic conditions that are seen in the clinical practices of rheumatologists, the question is whether TLRs deserve more of the rheumatologist’s attention as well. The role of TLR agonists in animal models of arthritis has been explored and is consistent with Janeway’s “dirty little secret.” In this light, the use of Freund’s complete adjuvant to boost arthritis has lost its magic with the discovery of TLR binding to constituents of this “miracle potion.” In this review, we discuss how recent advances in TLRassociated research have enhanced our understanding of the role of this receptor family in frequently occurring rheumatic conditions and how continued exploration of their role is likely to change the types of therapy available to battle these chronic diseases.

Novel insights in the regulation of CCL18 secretion by monocytes and dendritic cells via cytokines, Toll-like receptors and rheumatoid synovial fluid

BackgroundThe T cell attracting chemokine CCL18 is produced by antigen presenting cells and a role for CCL18 has been suggested in the pathogenesis of a variety of diseases. Rheumatoid arthritis (RA) is one of these conditions, in which abundant CCL18 production is present. Although Th2 cytokines and IL-10 are known to have an effect on CCL18 production, there are several gaps in our knowledge regarding the exact regulation of CCL18 secretion, both in general and in RA. In this study we provide new insights in the regulation of CCL18 secretion by monocytes and dendritic cells.ResultsIn contrast to a large panel of pro-inflammatory stimuli (IL-1β, TNF-α, IL-10, IL-13, IL-15, IL-17, IL-18, IFN-γ), T cell mimicking molecules (RANKL, CD40L) or TLR driven maturation, the anti-inflammatory IL-10 strongly stimulated DC to secrete CCL18. On freshly isolated monocytes, CCL18 secretion was induced by IL-4 and IL-13, in strong synergy with IL-10. This synergistic effect could already be observed after only 24 hours, indicating that not only macrophages and dendritic cells, but also monocytes secrete CCL18 under these stimulatory conditions. A high CCL18 expression was detected in RA synovial tissue and incubation of monocytes with synovial fluid from RA patients clearly enhanced the effects of IL-4, IL-13 and IL-10. Surprisingly, the effect of synovial fluid was not driven by IL-10 of IL-13, suggesting the presence of another CCL18 inducing factor in synovial fluid.ConclusionIn summary, IL-10 synergistically induces CCL18 secretion in combination with IL-4 of IL-13 on monocytes and monocyte derived cells. The effects of IL-14, IL-13 and IL-10 are strongly enhanced by synovial fluid. This synergy may contribute to the high CCL18 expression in RA.

Dendritic cells from patients with rheumatoid arthritis lack the interleukin 13 mediated increase of Fc gamma RII expression, which has clear functional consequences.

Background: Dendritic cell (DC) function is largely tailored by Fc gamma receptors (FcγR) and is critical for every immune response. Objective: To compare interleukin (IL) 13 mediated regulation of FcγRII and its related DC function between healthy controls and patients with rheumatoid arthritis (RA). Methods: DC were derived from peripheral blood mononuclear cells according to standardised protocols. FcγRI, II, and III expression and DC phenotype were assessed by FACS analysis. The level of cytokine production and chemokine expression was measured by Luminex and real time quantitative polymerase chain reaction techniques. Antigen uptake capacity was studied by DC fluorescent heat aggregated immunoglobulins and FACS analysis. Results: Replacement of IL4 by IL13 clearly increased the expression of FcγRII on DC from healthy controls (CDC), but had no effect on DC from patients with RA (RADC). The lower production of inflammatory mediators by IL13 CDC upon FcγR mediated triggering suggests that IL13 induces up regulation of specifically FcγRII. RADC co-cultured with IL4 already displayed an inhibitory DC phenotype, but this inhibitory phenotype was not augmented by the addition of IL13. The defective FcγRII regulation was further substantiated by the finding that IL13 CDC increased antigen uptake capacity, whereas IL13 RADC did not. Conclusion: IL13 regulates the expression of inhibitory FcγRII in normal subjects but not in RA, potentially resulting in a chronic proinflammatory immune reaction in RA. Unravelling the underlying mechanisms of FcγRII regulation might lead to new therapeutic targets in RA.