Modeling Rheumatoid Arthritis In Vitro: From Experimental Feasibility to Physiological Proximity

Rheumatoid arthritis (RA) is a chronic, inflammatory, and systemic autoimmune disease that affects the connective tissue and primarily the joints. If not treated, RA ultimately leads to progressive cartilage and bone degeneration. The etiology of the pathogenesis of RA is unknown, demonstrating heterogeneity in its clinical presentation, and is associated with autoantibodies directed against modified self-epitopes. Although many models already exist for RA for preclinical research, many current model systems of arthritis have limited predictive value because they are either based on animals of phylogenetically distant origin or suffer from overly simplified in vitro culture conditions. These limitations pose considerable challenges for preclinical research and therefore clinical translation. Thus, a sophisticated experimental human-based in vitro approach mimicking RA is essential to (i) investigate key mechanisms in the pathogenesis of human RA, (ii) identify targets for new therapeutic approaches, (iii) test these approaches, (iv) facilitate the clinical transferability of results, and (v) reduce the use of laboratory animals. Here, we summarize the most commonly used in vitro models of RA and discuss their experimental feasibility and physiological proximity to the pathophysiology of human RA to highlight new human-based avenues in RA research to increase our knowledge on human pathophysiology and develop effective targeted therapies

Optimization of a Tricalcium Phosphate-Based Bone Model Using Cell-Sheet Technology to Simulate Bone Disorders

Bone diseases such as osteoporosis, delayed or impaired bone healing, and osteoarthritis still represent a social, financial, and personal burden for affected patients and society. Fully humanized in vitro 3D models of cancellous bone tissue are needed to develop new treatment strategies and meet patient-specific needs. Here, we demonstrate a successful cell-sheet-based process for optimized mesenchymal stromal cell (MSC) seeding on a beta-tricalcium phosphate (TCP) scaffold to generate 3D models of cancellous bone tissue. Therefore, we seeded MSCs onto the beta-TCP scaffold, induced osteogenic differentiation, and wrapped a single osteogenically induced MSC sheet around the pre-seeded scaffold. Comparing the wrapped with an unwrapped scaffold, we did not detect any differences in cell viability and structural integrity but a higher cell seeding rate with osteoid-like granular structures, an indicator of enhanced calcification. Finally, gene expression analysis showed a reduction in chondrogenic and adipogenic markers, but an increase in osteogenic markers in MSCs seeded on wrapped scaffolds. We conclude from these data that additional wrapping of pre-seeded scaffolds will provide a local niche that enhances osteogenic differentiation while repressing chondrogenic and adipogenic differentiation. This approach will eventually lead to optimized preclinical in vitro 3D models of cancellous bone tissue to develop new treatment strategies

JAK/STAT Activation: A General Mechanism for Bone Development, Homeostasis, and Regeneration

The Janus kinase (JAK) signal transducer and activator of transcription (STAT) signaling pathway serves as an important downstream mediator for a variety of cytokines, hormones, and growth factors. Emerging evidence suggests JAK/STAT signaling pathway plays an important role in bone development, metabolism, and healing. In this light, pro-inflammatory cytokines are now clearly implicated in these processes as they can perturb normal bone remodeling through their action on osteoclasts and osteoblasts at both intra- and extra-articular skeletal sites. Here, we summarize the role of JAK/STAT pathway on development, homeostasis, and regeneration based on skeletal phenotype of individual JAK and STAT gene knockout models and selective inhibition of components of the JAK/STAT signaling including influences of JAK inhibition in osteoclasts, osteoblasts, and osteocytes

Linear optical properties of organic microcavity polaritons with non-Markovian Quantum State Diffusion

Hybridisation of the cavity modes and the excitons to polariton states together with the coupling to the vibrational modes determine the linear optical properties of organic semiconductors in microcavities. In this article we compute the refractive index for such system using the Holstein-Tavis-Cummings model and determine then the linear optical properties using the transfer matrix method. We first extract the parameters for the exciton in our model from fitting to experimentally measured absorption of a 2,7-bis [9,9-di(4-methylphenyl)-fluoren-2-yl]-9,9-di(4-methylphenyl) fluorene (TDAF) molecular thin film. Then we compute the reflectivity of such a thin film in a metal clad microcavity system by including the dispersive microcavity mode to the model. We compute susceptibility of the model systems evolving just a single state vector by using the non-Markovian Quantum State Diffusion. The computed location and height of the lower and upper polaritons agree with the experiment within the estimated errorbars for small angles ($\leq 30^\circ$). For larger angles the location of the polariton resonances are within the estimated error.Comment: 10 pages, 4 figure

The In Vitro Human Fracture Hematoma Model - A Tool for Preclinical Drug Testing

The aim of the study was to establish an in vitro fracture hematoma (FH) model, which mimics the in vivo situation of the human fracture gap in order to assess drug efficacy and effectiveness for the treatment of fracture healing disorders. Therefore, human peripheral blood and mesenchymal stromal cells (MSCs) were coagulated to produce in vitro FH models, incubated in osteogenic medium under normoxia/hypoxia, and analyzed for cell composition, gene expression and cytokine/chemokine secretion. To evaluate the model, we studied the impact of dexamethasone (impairing fracture healing) and deferoxamine (promoting fracture healing). Under hypoxic conditions, MSCs represented the predominant cell population, while the frequencies of leukocytes decreased. Marker gene expression of osteogenesis, angiogenesis, inflammation, migration and hypoxic adaptation increased significantly over time and compared to normoxia while cytokine/chemokine secretion remained unchanged. Finally, dexamethasone favored the frequency of immune cells compared to MSCs, suppressed osteogenic and pro-angiogenic gene expression and enhanced the secretion of inflammatory cytokines. Conversely, deferoxamine favored the frequency of MSCs over that of immune cells and enhanced the expression of the osteogenic marker RUNX2 and markers of the hypoxic adaptation. In summary, we demonstrate that hypoxia is an important factor for in vitro modeling the initial phase of fracture healing, that both fracture-healing disrupting and promoting substances can influence the in vitro model comparable to the in vivo situation. Therefore, we conclude that our model is able to mimic in part the human FH and to reduce the number of animal experiments in early preclinical studies

Impact of Janus Kinase Inhibition with Tofacitinib on Fundamental Processes of Bone Healing

Both inflammatory diseases like rheumatoid arthritis (RA) and anti-inflammatory treatment of RA with glucocorticoids (GCs) or non-steroidal anti-inflammatory drugs (NSAIDs) negatively influence bone metabolism and fracture healing. Janus kinase (JAK) inhibition with tofacitinib has been demonstrated to act as a potent anti-inflammatory therapeutic agent in the treatment of RA, but its impact on the fundamental processes of bone regeneration is currently controversially discussed and at least in part elusive. Therefore, in this study, we aimed to examine the effects of tofacitinib on processes of bone healing focusing on recruitment of human mesenchymal stromal cells (hMSCs) into the inflammatory microenvironment of the fracture gap, chondrogenesis, osteogenesis and osteoclastogenesis. We performed our analyses under conditions of reduced oxygen availability in order to mimic the in vivo situation of the fracture gap most optimal. We demonstrate that tofacitinib dose-dependently promotes the recruitment of hMSCs under hypoxia but inhibits recruitment of hMSCs under normoxia. With regard to the chondrogenic differentiation of hMSCs, we demonstrate that tofacitinib does not inhibit survival at therapeutically relevant doses of 10-100 nM. Moreover, tofacitinib dose-dependently enhances osteogenic differentiation of hMSCs and reduces osteoclast differentiation and activity. We conclude from our data that tofacitinib may influence bone healing by promotion of hMSC recruitment into the hypoxic microenvironment of the fracture gap but does not interfere with the cartilaginous phase of the soft callus phase of fracture healing process. We assume that tofacitinib may promote bone formation and reduce bone resorption, which could in part explain the positive impact of tofacitinib on bone erosions in RA. Thus, we hypothesize that it will be unnecessary to stop this medication in case of fracture and suggest that positive effects on osteoporosis are likely

Production of IL-6 and Phagocytosis Are the Most Resilient Immune Functions in Metabolically Compromised Human Monocytes

At sites of inflammation, monocytes carry out specific immune functions while facing challenging metabolic restrictions. Here, we investigated the potential of human monocytes to adapt to conditions of gradually inhibited oxidative phosphorylation (OXPHOS) under glucose free conditions. We used myxothiazol, an inhibitor of mitochondrial respiration, to adjust two different levels of decreased mitochondrial ATP production. At these levels, and compared to uninhibited OXPHOS, we assessed phagocytosis, production of reactive oxygen species (ROS) through NADPH oxidase (NOX), expression of surface activation markers CD16, CD80, CD11b, HLA-DR, and production of the inflammatory cytokines IL-1 beta, IL-6 and TNF-alpha in human monocytes. We found phagocytosis and the production of IL-6 to be least sensitive to metabolic restrictions while surface expression of CD11b, HLA-DR, production of TNF-alpha, IL-1 beta and production of ROS through NOX were most compromised by inhibition of OXPHOS in the absence of glucose. Our data demonstrate a short-term hierarchy of immune functions in human monocytes, which represents novel knowledge potentially leading to the development of new therapeutics in monocyte-mediated inflammatory diseases

Serum proteomics in giant cell arteritis in response to a three-day pulse of glucocorticoid followed by tocilizumab monotherapy (the GUSTO trial).

OBJECTIVE Proteome analyses in patients with newly diagnosed, untreated giant cell arteritis (GCA) have not been reported previously, nor are changes of protein expression upon treatment with glucocorticoids (GC) and/or tocilizumab (TCZ) known. The GUSTO trial allows to address these questions, provides the opportunity to learn about the differential effects of GC and TCZ on proteomics and may help to identify serum proteins to monitor disease activity. METHODS Serum samples obtained from 16 patients with new-onset GCA at different time points (day 0, 3, 10, and week 4, 24, 52) during the GUSTO trial (NCT03745586) were examined for 1436 differentially expressed proteins (DEPs) based on proximity extension assay technology. The patients received 500 mg methylprednisolone intravenously for 3 consecutive days followed by TCZ monotherapy. RESULTS When comparing day 0 (before the first GC infusion) with week 52 (lasting remission), 434 DEPs (213↑, 221↓) were identified. In response to treatment, the majority of changes occurred within 10 days. GC inversely regulated 25 proteins compared to remission. No difference was observed between weeks 24 and 52 during established remission and ongoing TCZ treatment. Expression of CCL7, MMP12, and CXCL9 was not regulated by IL6. CONCLUSION Disease-regulated serum proteins improved within 10 days and were normalized within 24 weeks, showing a kinetic corresponding to the gradual achievement of clinical remission. The proteins inversely regulated by GC and TCZ shed light on the differential effects of the two drugs. CCL7, CXCL9, and MMP12 are biomarkers that reflect disease activity despite normalized C-reactive protein levels

Plant-derived pectin nanocoatings to prevent inflammatory cellular response of osteoblasts following Porphyromonas gingivalis infection

Background: Bioengineered plant-derived Rhamnogalacturonan-Is (RG-Is) from pectins are potential candidates for surface nanocoating of medical devices. It has recently been reported that RG-I nanocoatings may prevent bacterial infection and improve the biocompatibility of implants. The aim of the study was to evaluate in vitro impact of bioengineered RG-I nanocoatings on osteogenic capacity and proinflammatory cytokine response of murine osteoblasts following Porphyromonas gingivalis infection. Methods: Murine MC3T3-E1 osteoblasts and isolated primary calvarial osteoblasts from C57BL/6J (B6J osteoblasts) mice were infected with P. gingivalis and incubated on tissue culture polystyrene plates with or without nanocoatings of unmodified RG-Is isolated from potato pulps (PU) or dearabinanated RG-Is (PA). To investigate a behavior of infected osteoblasts cultured on RG-Is cell morphology, proliferation, metabolic activity, mineralization and osteogenic and pro-inflammatory gene expression were examined. Results: Following P. gingivalis infection, PA, but not PU, significantly promoted MC3T3-E1 and BJ6 osteoblasts proliferation, metabolic activity, and calcium deposition. Moreover, Il-1b, Il-6, TNF-α, and Rankl gene expressions were downregulated in cells cultured on PU and to a higher extent on PA as compared to the corresponding control, whereas Runx, Alpl, Col1a1, and Bglap gene expressions were upregulated vice versa. Conclusion: Our data clearly showed that pectin RG-Is nanocoating with high content of galactan (PA) reduces the osteoblastic response to P. gingivalis infection in vitro and may, therefore, reduce a risk of inflammation especially in immunocompromised patients with rheumatoid or periodontal disorders