Stephan Blüml

Essential role of microRNA-155 in the pathogenesis of autoimmune arthritis in mice

OBJECTIVE MicroRNAs (miRNA) are a new class of regulatory elements. Altered expression of miRNA has been demonstrated in the inflamed joints of patients with rheumatoid arthritis (RA). The aim of this study was to examine the role of miRNA in the pathogenesis of autoimmune arthritis, using 2 murine models. METHODS Collagen-induced arthritis (CIA) and K/BxN serum-transfer arthritis were induced in wild-type (WT) and miR-155-deficient (miR-155(-/-) ) mice. The severity of arthritis was determined clinically and histologically. Anticollagen antibodies and cytokines were measured by enzyme-linked immunosorbent assay. The cellular composition of the draining lymph nodes after induction of CIA was measured by flow cytometry. RESULTS The miR-155(-/-) mice did not develop CIA. Deficiency in miR-155 prevented the generation of pathogenic autoreactive B and T cells, since anticollagen antibodies and the expression levels of antigen-specific T cells were strongly reduced in miR-155(-/-) mice. Moreover, Th17 polarization of miR-155(-/-) mouse T cells was impaired, as shown by a significant decrease in the levels of interleukin-17 (IL-17) and IL-22. In the K/BxN serum-transfer arthritis model, which only depends on innate effector mechanisms, miR-155(-/-) mice showed significantly reduced local bone destruction, attributed to reduced generation of osteoclasts, although the severity of joint inflammation was similar to that in WT mice. CONCLUSION These results demonstrate that miR-155 is essentially involved in the adaptive and innate immune reactions leading to autoimmune arthritis, and therefore miR-155 might provide a novel target for the treatment of patients with RA.

CD14 is a coreceptor of Toll-like receptors 7 and 9

CD14 interacts with and is essential for the functions of endosomal TLR7 and TLR9 in mice.

B-cell targeted therapeutics in clinical development

B lymphocytes are the source of humoral immunity and are thus a critical component of the adaptive immune system. However, B cells can also be pathogenic and the origin of disease. Deregulated B-cell function has been implicated in several autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis. B cells contribute to pathological immune responses through the secretion of cytokines, costimulation of T cells, antigen presentation, and the production of autoantibodies. DNA-and RNA-containing immune complexes can also induce the production of type I interferons, which further promotes the inflammatory response. B-cell depletion with the CD20 antibody rituximab has provided clinical proof of concept that targeting B cells and the humoral response can result in significant benefit to patients. Consequently, the interest in B-cell targeted therapies has greatly increased in recent years and a number of new biologics exploiting various mechanisms are now in clinical development. This review provides an overview on current developments in the area of B-cell targeted therapies by describing molecules and subpopulations that currently offer themselves as therapeutic targets, the different strategies to target B cells currently under investigation as well as an update on the status of novel therapeutics in clinical development. Emerging data from clinical trials are providing critical insight regarding the role of B cells and autoantibodies in various autoimmune conditions and will guide the development of more efficacious therapeutics and better patient selection.

Multi-Hit Inhibition of Circulating and Cell-Associated Components of the Toll-Like Receptor 4 Pathway by Oxidized Phospholipids

Objective—Oxidized phospholipids (OxPLs) that are abundant in atherosclerotic lesions are increasingly recognized as context-dependent lipid mediators demonstrating both pro- and antiinflammatory activities. Molecular mechanisms of their effects are largely unknown. Here we present novel information on the mechanisms whereby OxPLs modulate activation of TLR4 by lipopolysaccharide (LPS). Methods and Results—We show, using several cell types and various inflammatory genes as readouts, that different classes and molecular species of OxPLs do not stimulate TLR4 but exert prominent inhibitory effects on LPS-induced reactions. Our data demonstrate that binding of OxPLs to the LPS-binding protein (LBP) and CD14 prevents recognition of LPS by these proteins, thus impairing activation of TLR4. In addition, OxPLs inhibited LBP- and CD14-independent activation of TLR4 by the synthetic TLR4 agonist E6020 indicating that in parallel with LBP and CD14, OxPLs target cell-associated steps in TLR4 cascade. Conclusions—Our data suggest that OxPLs inhibit action of LPS via a multi-hit mechanism. These results support the notion that OxPLs are endogenous inhibitors of TLR4 produced in response to oxidative stress.

Regulation of autoantibody activity by the IL-23-TH17 axis determines the onset of autoimmune disease

The checkpoints and mechanisms that contribute to autoantibody-driven disease are as yet incompletely understood. Here we identified the axis of interleukin 23 (IL-23) and the TH17 subset of helper T cells as a decisive factor that controlled the intrinsic inflammatory activity of autoantibodies and triggered the clinical onset of autoimmune arthritis. By instructing B cells in an IL-22- and IL-21-dependent manner, TH17 cells regulated the expression of β-galactoside α2,6-sialyltransferase 1 in newly differentiating antibody-producing cells and determined the glycosylation profile and activity of immunoglobulin G (IgG) produced by the plasma cells that subsequently emerged. Asymptomatic humans with rheumatoid arthritis (RA)-specific autoantibodies showed identical changes in the activity and glycosylation of autoreactive IgG antibodies before shifting to the inflammatory phase of RA; thus, our results identify an IL-23–TH17 cell–dependent pathway that controls autoantibody activity and unmasks a preexisting breach in immunotolerance.

Targeting TNF receptors in rheumatoid arthritis

Tumour necrosis factor (TNF) is a pro-inflammatory cytokine that signals through two distinct receptors, TNFR1 and TNFR2. TNF is essentially involved in the pathogenesis of various inflammatory and autoimmune diseases. Blocking TNF, in turn, has been proven to be highly effective in treating a variety of diseases. However, the role of its two receptors in these conditions is not very well understood. It is established that TNFR1 is mainly responsible for the detrimental effects of TNF. However, accumulating evidence suggests differential or even opposing effects of TNFR2 in the pathogenesis of a number of inflammatory and autoimmune conditions. In this review, we summarize the available data concerning biological and functional properties of the two TNF receptors and potential therapeutic consequences of these insights.

Macrophage PTEN Regulates Expression and Secretion of Arginase I Modulating Innate and Adaptive Immune Responses

The activation of innate immune cells triggers numerous intracellular signaling pathways, which require tight control to mount an adequate immune response. The PI3K signaling pathway is intricately involved in innate immunity, and its activation dampens the expression and release of proinflammatory cytokines in myeloid cells. These signaling processes are strictly regulated by the PI3K antagonist, the lipid phosphatase, PTEN, a known tumor suppressor. Importantly, PTEN is responsible for the elevated production of cytokines such as IL-6 in response to TLR agonists, and deletion of PTEN results in diminished inflammatory responses. However, the mechanisms by which PI3K negatively regulates TLR signaling are only partially resolved. We observed that Arginase I expression and secretion were markedly induced by PTEN deletion, suggesting PTEN−/− macrophages were alternatively activated. This was mediated by increased expression and activation of the transcription factors C/EBPβ and STAT3. Genetic and pharmacologic experimental approaches in vitro, as well as in vivo autoimmunity models, provide convincing evidence that PI3K/PTEN-regulated extracellular Arginase I acts as a paracrine regulator of inflammation and immunity.

Antiinflammatory effects of tumor necrosis factor on hematopoietic cells in a murine model of erosive arthritis

OBJECTIVE To investigate the mechanisms leading to the influx of inflammatory hematopoietic cells into the synovial membrane and the role of tumor necrosis factor receptor I (TNFRI) and TNFRII in this process in an animal model of rheumatoid arthritis (RA). METHODS We performed bone marrow transplantations in human TNF-transgenic mice using hematopoietic cells from wild-type, TNFRI(-/-), TNFRII(-/-), and TNFRI/II(-/-) mice as donors and assessed the severity of arthritis histologically. Generation of osteoclasts from the different genotypes was analyzed in vitro and in vivo. Apoptosis was analyzed using annexin V staining as well as TUNEL assays. RESULTS Despite lacking responsiveness to TNF in their hematopoietic compartment, mice not only developed full-blown erosive arthritis but even showed increased joint destruction when compared with mice with a TNF-responsive hematopoietic compartment. We demonstrated different roles of the 2 different TNFRs in the regulation of these processes. The absence of TNFRI on hematopoietic cells did not affect joint inflammation but markedly attenuated erosive bone destruction via reduced synovial accumulation of osteoclast precursors. In contrast, the absence of TNFRII on hematopoietic cells increased joint inflammation as well as erosive bone destruction via the regulation of osteoclast precursor apoptosis. CONCLUSION Our findings indicate that selective blockade of TNFRI, leaving the antiinflammatory effects of TNFRII unaltered instead of unselectively blocking TNF, might be advantageous in patients with RA.

Tumor necrosis factor-inhibiting therapy preferentially targets bone destruction but not synovial inflammation in a tumor necrosis factor-driven model of rheumatoid arthritis.

OBJECTIVE To investigate how tumor necrosis factor (TNF)-inhibiting therapy affects bone destruction and inflammation in a TNF-driven mouse model of rheumatoid arthritis. METHODS In order to evaluate the influence of TNF on osteoclastogenesis in vitro, different concentrations of TNF were added to spleen cell-derived monocytes in the absence or presence of different concentrations of RANKL. In addition, the effects of TNF inhibition on osteoclast precursors as well as local bone destruction in vivo were assessed by treating TNF-transgenic mice with different doses of adalimumab. RESULTS TNF stimulated osteoclastogenesis mainly by increasing the number of osteoclast precursor cells in vitro. This TNF effect was independent of the presence of RANKL. In the hTNF-transgenic mouse model of destructive arthritis, low-dose TNF-inhibiting therapy with adalimumab had no effect on synovial inflammation but significantly inhibited local bone destruction and the generation of osteoclasts. This inhibition was accompanied by a reduction in the number of c-Fms-positive osteoclast precursor cells in the bone marrow and a reduction of the osteoclast precursor pools in the blood and inflamed synovial membrane of hTNF-transgenic mice. CONCLUSION Low-dose TNF-inhibiting therapy significantly reduces bone erosions by reducing the number of circulating and joint-invading osteoclast precursors. This effect is uncoupled from its antiinflammatory action.

Abatacept (CTLA‐4IG) treatment reduces the migratory capacity of monocytes in patients with rheumatoid arthritis

OBJECTIVE The binding of abatacept (CTLA-4Ig) to the B7 ligands CD80 and CD86 prevents the engagement of CD28 on T cells and thereby prevents effector T cell activation. In addition, a direct effect of CTLA-4Ig on antigen-presenting cells (APCs) could contribute to the therapeutic effect. To further elucidate the mechanism of CTLA-4Ig, we performed phenotype and functional analyses of APCs in patients with rheumatoid arthritis (RA) before and after the initiation of CTLA-4Ig therapy. METHODS Peripheral blood mononuclear cells were analyzed before and at 2 and 4 weeks after the initiation of CTLA-4Ig therapy. Proportions of APCs were determined by flow cytometry. CD14+ monocytes were further analyzed for the expression of costimulatory and adhesion molecules and for their transendothelial migratory capacity in vitro. In addition, CD14+ monocytes from healthy controls were analyzed for their migratory and spreading capacity. RESULTS Proportions and absolute numbers of monocytes were significantly increased in RA patients treated with CTLA-4Ig. The expression of several adhesion molecules was significantly diminished. In addition, monocytes displayed a significant reduction in their endothelial adhesion and transendothelial migratory capacity upon treatment with CTLA-4Ig. Likewise, isolated monocytes from healthy controls revealed a significant reduction in their migratory and spreading activity after preincubation with CTLA-4Ig or anti-CD80 and anti-CD86 antibodies. CONCLUSION We describe direct effects of CTLA-4Ig therapy on phenotype and functional characteristics of monocytes in RA patients that might interfere with the migration of monocytes to the synovial tissue. This additional mechanism of CTLA-4Ig might contribute to the beneficial effects of CTLA-4Ig treatment in RA patients.