Rheumatoid arthritis (RA) is an autoimmune disease characterized by persistent synovial inflammation. The major drivers of synovial inflammation are cytokines and chemokines. Among these molecules, TNF activates fibroblast-like synoviocytes (FLSs), which leads to the production of inflammatory mediators. Here, we show that TNF regulates the expression of the transcription factor interferon regulatory factor 1 (IRF1) in human FLSs as well as in a TNF transgenic arthritis mouse model. Transcriptomic analyses of IRF1-deficient, TNF-stimulated FLSs define the interferon (IFN) pathway as a major target of IRF1. IRF1 expression is associated with the expression of IFNβ, which leads to the activation of the JAK-STAT pathway. Blocking the JAK-STAT pathway with the Janus kinase inhibitor (JAKinib) baricitinib or tofacitinib reduces the expression of IFN-regulated genes (IRGs) in TNF-activated FLSs. Therefore, we conclude that TNF induces a distinct inflammatory cascade, in which IRGs are key elements, in FLSs. The IFN-signature might be a promising biomarker for the efficient and personalized use of new treatment strategies for RA, such as JAKinibs.

We demonstrate that the integration of complex human synovial organ cultures in a lab-on-a-chip provides reproducible and reliable information on how systemic stress factors affect synovial tissue architectures using light scatter biosensing.

Protein kinase C (PKC) is known to regulate epithelial barrier function. However, the effect of specific PKC isozymes, and their mechanism of action, are largely unknown. We determined that the nonphorbol ester PKC agonist bryostatin-1 increased transepithelial electrical resistance (TER), a marker of barrier function, in confluent T84 epithelia. Bryostatin-1, which has been shown to selectively activate PKC-alpha, -epsilon, and -delta (34), was associated with a shift in the subcellular distribution of the tight junction proteins claudin-1 and ZO-2 from a detergent-soluble fraction into a detergent-insoluble fraction. Bryostatin-1 also led to the appearance of a higher-molecular-weight form of occludin previously shown to correspond to protein phosphorylation. These changes were attenuated by the conventional and novel PKC inhibitor Gö-6850 but not the conventional PKC inhibitor Gö-6976 or the PKC-delta inhibitor röttlerin, implicating a novel isozyme, likely PKC-epsilon. The results suggest that enhanced epithelial barrier function induced by bryostatin-1 involves a PKC-epsilon-dependent signaling pathway leading to recruitment of claudin-1 and ZO-2, and phosphorylation of occludin, into the tight junctional complex.

Rheumatoid arthritis is characterised by a progressive, intermittent inflammation at the synovial membrane, which ultimately leads to the destruction of the synovial joint. The synovial membrane, which is the joint...

Tumor necrosis factor (TNF) increases epithelial permeability in many model systems. Protein kinase C (PKC) isozymes regulate epithelial barrier function and alter ligand-receptor interactions. We sought to define the impact of PKC on TNF-induced barrier dysfunction in T84 intestinal epithelia. TNF induced a dose- and time-dependent fall in transepithelial electrical resistance (TER) and an increase in [(3)H]mannitol flux. The TNF-induced fall in TER was not PKC mediated but was prevented by pretreatment with bryostatin-1, a PKC agonist. As demonstrated by a pattern of sensitivity to pharmacological inhibitors of PKC, this epithelial barrier preservation was mediated by novel PKC isozymes. Bryostatin-1 reduced TNF receptor (TNF-R1) surface availability, as demonstrated by radiolabeled TNF binding and cell surface biotinylation assays, and increased TNF-R1 receptor shedding. The pattern of sensitivity to isozyme-selective PKC inhibitors suggested that these effects were mediated by activation of PKC-epsilon. In addition, after bryostatin-1 treatment, PKC-delta and TNF-R1 became associated, as determined by mutual coimmunoprecipitation assay, which has been shown to lead to receptor desensitization in neutrophils. TNF-induced barrier dysfunction occurs independently of PKC, but selective modulation of novel PKC isozymes may regulate TNF-R1 signaling.

In the present work, we combine experimental and computational methods to define the critical shear stress as an alternative parameter for nanotoxicological and nanomedical evaluations using an in vitro microfluidic vascular model. We demonstrate that our complementary in vitro and in silico approach is well suited to assess the fluid flow velocity above which clathrin-mediated (active) nanoparticle uptake per cell decreases drastically although higher numbers of nanoparticles per cell are introduced. Results of our study revealed a critical shear stress of 1.8 dyn/cm, where maximum active cellular nanoparticle uptake took place, followed by a 70% decrease in uptake of 249 nm nanoparticles at 10 dyn/cm, respectively. The observed nonlinear relationship between flow velocity and nanoparticle uptake strongly suggests that fluid mechanical forces also need to be considered in order to predict potential in vivo distribution, bioaccumulation, and clearance of nanomaterials and novel nanodrugs.

Besides being regulated by G-protein–coupled receptors, the activity of heterotrimeric G proteins is modulated by many cytoplasmic proteins. GIV/Girdin and DAPLE (Dvl-associating protein with a high frequency of leucine) are the best-characterized members of a group of cytoplasmic regulators that contain a Gα-binding and -activating (GBA) motif and whose dysregulation underlies human diseases, including cancer and birth defects. GBA motif–containing proteins were originally reported to modulate G proteins by binding Gα subunits of the Gi/o family (Gαi) over other families (such as Gs, Gq/11, or G12/13), and promoting nucleotide exchange in vitro. However, some evidence suggests that this is not always the case, as phosphorylation of the GBA motif of GIV promotes its binding to Gαs and inhibits nucleotide exchange. The G-protein specificity of DAPLE and how it might affect nucleotide exchange on G proteins besides Gαi remain to be investigated. Here, we show that DAPLE's GBA motif, in addition to Gαi, binds efficiently to members of the Gs and Gq/11 families (Gαs and Gαq, respectively), but not of the G12/13 family (Gα12) in the absence of post-translational phosphorylation. We pinpointed Met-1669 as the residue in the GBA motif of DAPLE that diverges from that in GIV and enables better binding to Gαs and Gαq. Unlike the nucleotide-exchange acceleration observed for Gαi, DAPLE inhibited nucleotide exchange on Gαs and Gαq. These findings indicate that GBA motifs have versatility in their G-protein–modulating effect, i.e. they can bind to Gα subunits of different classes and either stimulate or inhibit nucleotide exchange depending on the G-protein subtype.

Rheumatoid arthritis is characterised by a progressive, intermittent inflammation at the synovial membrane, which ultimately leads to the destruction of the synovial joint. The synovial membrane, which is the joint capsule's inner layer, is lined with fibroblast-like synoviocytes that are the key player supporting persistent arthritis leading to bone erosion and cartilage destruction. While microfluidic models that model molecular aspects of bone erosion between bone-derived cells and synoviocytes have been established, the synovial-chondral axis in rheumatoid arthritis has yet not been realised using a microfluidic 3D model based on human patient in vitro cultures. Consequently, we established a chip-based three-dimensional tissue co-culture model that simulates the reciprocal cross-talk between individual synovial and chondral organoids. We now demonstrate that chondral organoids, when co-cultivated with synovial organoids, induce a higher degree of physiological cartilage architecture and show differential cytokine response compared to their respective monocultures highlighting the importance of reciprocal tissue-level cross-talk in the modelling of arthritic diseases.