Ruth A. Byrne

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.

Fluorosomes: a convenient new reagent to detect and block multivalent and complex receptor-ligand interactions

We describe for the first time fluorescent virus‐like particles decorated with biologically active mono‐ and multisubunit immune receptors of choice and the basic application of such fluorosomes (FSs) to visualize and target immune receptor‐ligand interactions. For that purpose, human embryonic kidney (HEK)‐293 cells were stably transfected with Moloney murine leukemia virus (MoMLV) matrix protein (MA) GFP fusion constructs. To produce FSs, interleukins (ILs), IL‐receptors (ILRs), and costimulatory molecules were fused to the glycosyl phosphatidyl inositol anchor acceptor sequence of CD16b and coexpressed along with MoMLV group‐specific antigen‐polymerase (gag‐pol) in MA:GFP+ HEK‐293 cells. We show that IL‐2 decorated but not control‐decorated FSs specifically identify normal and malignant IL‐2 receptor‐positive (IL‐2R+) lymphocytes by flow cytometry. In addition to cytokines and costimulatory molecules, FSs were also successfully decorated with the heterotrimeric IL‐2Rs, allowing identification of IL‐2+target cells. Specificity of binding was proven by complete inhibition with nonlabeled, soluble ligands. Moreover, IL‐2R FSs efficiently neutralized soluble IL‐2 and thus induced unresponsiveness of T cells receiving full activation stimuli via T‐cell antigen receptor and CD28. FSs are technically simple, multivalent tools for assessing and blocking mono‐ and multisubunit immune receptor‐ligand interactions with natural constituents in a plasma membrane context.—Kueng, H. J., Manta, C., Haiderer, D., Leb, V. M., Schmetterer, K. G., Neunkirchner, A., Byrne, R. A., Scheinecker, C., Steinberger, P., Seed, B., Pickl, W. F. Fluorosomes: a convenient new reagent to detect and block multivalent and complex receptor‐ligand interactions. FASEB J. 24, 1572–1582 (2010). www.fasebj.org

Squid dances: an ethogram of postures and actions of Sepioteuthis sepioidea squid with a muscular hydrostatic system

A taxonomy of the movement possibilities for any species, within the constraints of its neural and skeletal systems, should be one of the foundations of the study of its behaviour. Caribbean reef squid, Sepioteuthis sepioidea, appear to have many degrees of freedom in their movement as they live in a three-dimensional habitat and have no fixed skeleton but rather a muscular hydrostatic one. Within this apparent lack of constraints, there are regularities and patterns of common occurrences that allow this article to describe an ethogram of the movements, postures and positions of squid. Squid have a combination of bent, spread and twisted maintained postures of the eight arms and two tentacles that enhance camouflage. Their body–arm posture combinations are actively maintained in the water but also influenced by gravity. Positions related to conspecifics are stereotyped and important for communication. For locomotion, squid use a well-coordinated dual fin–jet locomotion system. This motor system uses tonic postures for camouflage and maintains body position in courtship, though squid seldom touch. The interdependence of movement control by different units is seen in fins and jet propulsion, and bilateral symmetry is maintained even for the eight arms. It is argued that the repertoire is well adapted for a soft-bodied animal in its three-dimensional, open but near-shore demersal habitat.

Correlation Between Multimodal Microscopy, Tissue Morphology, and Enzymatic Resistance in Riboflavin-UVA Cross-Linked Human Corneas.

PURPOSE To explore the utility of multimodal microscopy as a noninvasive tool to assess corneal collagen cross-linking (CXL) efficacy, we investigated the correlation between riboflavin (RF) axial profile, second harmonic generation (SHG) imaging, and histological/biochemical changes of human corneas after RF-ultraviolet A (UVA)-catalyzed CXL. METHODS De-epithelialized human corneoscleral tissues were imaged by confocal and multiphoton microscopy to study RF tissue diffusion profile and SHG-based roughness index (Rq) after CXL. We installed 0.1% RF for 5, 10, and 20 minutes, respectively, followed by UVA irradiation, while dextran drug vehicle-treated corneas served as controls. Massons trichrome staining and collagenase digestion assay were employed to assess ultrastructural modifications of collagen lamellae and bioenzymatic strength following RF-UVA CXL. RESULTS Stromal absorption of RF was significantly higher in 20 minutes compared with 5- and 10-minute drug instillations. The roughness index of SHG images was reduced after RF-UVA CXL at all RF instillation time points compared with dextran controls. Interestingly, correlation between axial profiles of RF dosage and Rq index was only observed in 10- and 20-minute RF instillations (R(2) = 0.13 and 0.28, respectively, all P < 0.05), but not in the 5-minute group. Massons trichrome staining revealed collagen fibril compaction in cross-linked corneas in an RF dose-dependent manner. Collagenase digestion assay showed significantly increased biochemical strength by higher RF doses in cross-linked corneas. CONCLUSIONS Intrastromal RF distribution profiles correlated with histological and functional property changes in RF-UVA cross-linked corneas. A riboflavin-defined threshold further determined the sensitivity of SHG imaging as a noninvasive imaging modality to assess the efficacy of RF-UVA CXL.

A Dynamic Real Time In Vivo and Static Ex Vivo Analysis of Granulomonocytic Cell Migration in the Collagen-Induced Arthritis Model

Neutrophilic granulocytes and monocytes (granulomonocytic cells; GMC) drive the inflammatory process at the earliest stages of rheumatoid arthritis (RA). The migratory behavior and functional properties of GMC within the synovial tissue are, however, only incompletely characterized. Here we have analyzed GMC in the murine collagen-induced arthritis (CIA) model of RA using multi-photon real time in vivo microscopy together with ex vivo analysis of GMC in tissue sections. GMC were abundant as soon as clinical arthritis was apparent. GMC were motile and migrated randomly through the synovial tissue. In addition, we observed the frequent formation of cell clusters consisting of both neutrophilic granulocytes and monocytes that actively contributed to the inflammatory process of arthritis. Treatment of animals with a single dose of prednisolone reduced the mean velocity of cell migration and diminished the overall immigration of GMC. In summary, our study shows that the combined application of real time in vivo microscopy together with elaborate static post-mortem analysis of GMC enables the description of dynamic migratory characteristics of GMC together with their precise location in a complex anatomical environment. Moreover, this approach is sensitive enough to detect subtle therapeutic effects within a very short period of time.

IFN-gamma promotes fibroblast-like synoviocytes motility

Rheumatoid arthritis (RA) is the most common chronic inflammatory joint disease. In RA, a systemic autoimmune response translates into an inflammatory attack on the synovium that yields the formation of an aggressive cell mass called pannus which attaches to, encroaches over and destroys the articular cartilage. Cartilage destruction is mediated by fibroblast-like synoviocytes (FLS) which are the prevailing cell type of the …

Targeted inhibition of Janus kinases abates interfon gamma-induced invasive behaviour of fibroblast-like synoviocytes

Objectives The aim was to explore the function of the T-cell cytokine IFNγ for mesenchymal tissue remodelling in RA and to determine whether IFNγ signalling controls the invasive potential of fibroblast-like synoviocytes (FLS). Methods To assess architectural responses, FLS were cultured in three-dimensional micromasses. FLS motility was analysed in migration and invasion assays. Signalling events relevant to cellular motility were defined by western blots. Baricitinib and small interfering RNA pools were used to suppress Janus kinase (JAK) functions. Results Histological analyses of micromasses revealed unique effects of IFNγ on FLS shape and tissue organization. This was consistent with accelerated migration upon IFNγ stimulation. Given that cell shape and cell motility are under the control of the focal adhesion kinase (FAK), we next analysed its activity. Indeed, IFNγ stimulation induced the phosphorylation of FAK-Y925, a phosphosite implicated in FAK-mediated cell migration. Small interfering RNA knockdown of JAK2, but not JAK1, substantially abrogated FAK activation by IFNγ. Correspondingly, IFNγ-induced FAK activation and invasion of FLS was abrogated by the JAK inhibitor, baricitinib. Conclusion Our study contributes insight into the synovial response to IFNγ and reveals JAK2 as a potential therapeutic target for FLS-mediated joint destruction in arthritis, especially in RA.

04.19 3D synovial organoid culture reveals cellular mechanisms of tissue formation and inflammatory remodelling

Background The synovial membrane is a distinctly organised tissue structure. During the course of rheumatoid arthritis (RA), the synovium becomes hyperplastic and demonstrates thickening of the lining layer and cellular condensation at the sublining layer. Using a three-dimensional synovial organ culture system, we explore cellular mechanisms of synovial tissue formation and inflammatory remodelling. Materials and methods Fibroblast-like synoviocytes (FLS) derived from patients with RA were cultured in 3D micromasses. To mimic synovial inflammation, micromasses were challenged with TNF. For histological analyses, micromasses were embedded in paraffin, sections were stained with haematoxylin and eosin; Ki67 labelling was performed to identify proliferating cells. 3D confocal micrographs were analysed using Imaris Bitplane software. mRNA levels for various genes expressed in FLS were determined by qPCR. Results Synovial micromasses demonstrated thickening of the lining layer over time. When stimulated with TNF, hyperplasia of the lining layer and cellular aggregation at the sublining layer was observed. In order to identify the origin of cells contributing to the thickening of the lining layer, proliferation studies were conducted. Intriguingly, in the early phase of the culture period, the percentage of proliferating cells in the lining layer was higher when compared to the sublining layer. This proliferative activity, however, was no longer present in the late phase, after the lining layer was established. In the presence of TNF, an increased number of proliferating cells at the lining layer was maintained for an extended period of time, consistent with higher rates of cellular proliferation at the synovial lining in sections of RA synovial tissues when compared to OA. qPCR data indicate that MMP1, MMP3, and IL-6 are differentially expressed during the early phase and the mature phase of the culture period. By contrast, lubricin, cadherin-11, CCL20, and STAT1 were not differentially expressed. Conclusions The three-dimensional FLS micromass culture reveals spontaneous cellular organisation that strikingly resembles the lining/sublining architecture of the synovium. This process involves FLS proliferation as well as expression of genes that allow for tissue remodelling. In inflammatory conditions similar cellular programs are re-activated resulting in synovial lining hyperplasia and a pannus-like condensed mass of cells.

04.01 Doings and dealings in synovitis: fibroblast-like synoviocyte cell-to-cell organelle transfer is directed by the inflammatory microenvironment

Background Fibroblast-like synoviocytes (FLS) form a complex tissue network via long-distance intercellular connexions with wide intercellular matrix spaces. The adaptive synovial tissue response to inflammation likely depends upon the concerted activity of FLS. Using an in-vitro synovial organ culture system, we explore mechanisms of FLS directed cellular cooperation. Methods Human FLS were prepared from synovial tissues obtained as discarded specimens following joint arthroplasty. Cells were labelled with cell tracker dyes or specific organelle dyes and cultured in spherical matrigel micromasses. For selected experiments, micromasses were challenged with TNF or IFNγ. Data was acquired by confocal live cell imaging. Analysis of the resulting 4D movies was done using Imaris software. Results To examine whether FLS transfer cytoplasmic cargo, we labelled 50% of FLS with green cell tracker dye and the other 50% with Mitotracker. Over time, red labelled organelles accumulated in green labelled cells with a transfer rate of 10% of newly affected cells/day. Confocal live cell imaging revealed that FLS indeed use their long-distance intercellular connexions for transfer of organelles. When micromasses were stimulated with TNF (10 ng/ml) the transfer rate increased by 2-fold when compared to control. By contrast, INFγ-stimulation resulted in decreased organelle transfer. The combined treatment of micromasses with TNF and IFNg, however, increased the transfer rate to a level beyond stimulation with TNF alone. Conclusions Our experiments suggest transfer of cytoplasmic cargo, including organelles such as mitochondria between FLS. As transfer is distinctly regulated by the cytokine milieu, organelle transfer seems to be part of the adaptive synovial response to inflammation. These studies may provide insight into how synoviocytes orchestrate their activity. Further studies will demonstrate the significance of directed cargo exchange for the function of the normal as well as the diseased synovium.

FRI0018 Targeted inhibition of janus kinases abates IFN-GAMMA-INDUCED invasive behavior of fibroblast-like synoviocytes

Background Emerging evidence suggests that fibroblast-like synoviocytes (FLS) are key effector cells in rheumatoid arthritis (RA) and research into the mechanisms defining FLS activity in RA indicate that cytokines secreted by leukocytes play a crucial role. Nevertheless, the contribution of IFNγ, which is increased in rheumatoid synovitis, to the inflammatory synovial tissue reaction is not known. Objectives To explore the function of the T-cell cytokine IFNγ for mesenchymal tissue remodeling in RA, and to determine whether IFNγ-signaling controls the invasive potential of FLS. Methods To assess architectural responses, FLS were cultured in three-dimensional micromasses. FLS motility was analyzed in migration-, spreading- and invasion assays. Signaling events relevant to cellular motility were defined by western blots. Baricitinb and siRNA pools were used to suppress Janus Kinase (JAK) functions. Results Histological analyses of micromasses revealed unique effects of IFNγ on FLS shape and tissue organization. This was consistent with accelerated migration, pronounced actin and focal adhesion (FA) re-organization upon IFNγ stimulation. Since actin and FA dynamics and, thus, cell motility are integrated by the focal adhesion kinase (FAK), we next analyzed its activity. Indeed, IFNγ stimulation induced the phosphorylation of FAK-Y925, a phosphosite implicated in FAK-mediated cell migration. siRNA knockdown of JAK2, but not JAK1, abrogated FAK activation by IFNγ. Correspondingly, IFNγ-inudced FAK activation and invasion of FLS was abrogated by the JAK-inhibitor baricitinib. Conclusions Our study contributes insight into the synovial response to IFNγ and reveals JAK2 as a potential therapeutic target for FLS-mediated joint destruction in arthritis, especially in RA. Disclosure of Interest None declared