The YAP1-TEAD axis promotes autophagy against intracellular Staphylococcus aureus in vitro.

Previously considered as an exclusive extracellular bacterium, Staphylococcus aureus has been shown to be able to invade many cells in vitro and in humans. Once inside the host cell, both cytosolic and endosome-associated S. aureus strongly induce macroautophagy/autophagy. Whether autophagy fosters S. aureus intracellular survival or clearance remains unclear. The YAP1-TEAD axis regulates the expression of target genes controlling the cell fate (e.g., proliferation, migration, cell cycle …). Growing evidence indicates that YAP1-TEAD also regulates autophagy and lysosomal pathways. Recently we showed that the YAP1-TEAD axis promotes autophagy and lysosome biogenesis to restrict S. aureus intracellular replication. We also discovered that the C3 exoenzyme-like EDIN-B toxin produced by the pathogenic S. aureus ST80 strain inhibits YAP1 nuclear translocation resulting in a strong increase of intracellular S. aureus burden.

YAP promotes cell-autonomous immune responses to tackle intracellular Staphylococcus aureus in vitro.

Transcriptional cofactors YAP/TAZ have recently been found to support autophagy and inflammation, which are part of cell-autonomous immunity and are critical in antibacterial defense. Here, we studied the role of YAP against Staphylococcus aureus using CRISPR/Cas9-mutated HEK293 cells and a primary cell-based organoid model. We found that S. aureus infection increases YAP transcriptional activity, which is required to reduce intracellular S. aureus replication. A 770-gene targeted transcriptomic analysis revealed that YAP upregulates genes involved in autophagy/lysosome and inflammation pathways in both infected and uninfected conditions. The YAP-TEAD transcriptional activity promotes autophagic flux and lysosomal acidification, which are then important for defense against intracellular S. aureus. Furthermore, the staphylococcal toxin C3 exoenzyme EDIN-B was found effective in preventing YAP-mediated cell-autonomous immune response. This study provides key insights on the anti-S. aureus activity of YAP, which could be conserved for defense against other intracellular bacteria.

YAP Transcriptional Activity Dictates Cell Response to TNF In Vitro.

YAP/TAZ are transcription co-factors recently described responsive to pro-inflammatory cytokines and involved in inflammatory-related disorders. However, the role of tumor necrosis factor (TNF), a major pro-inflammatory cytokine, on YAP signaling is not well understood and controversial. Here, we observe in vitro, using wild type and YAP knockout HEK293 cells, that TNF triggers YAP nuclear translocation and transcriptional activity, thus being dependent on Rho family of GTPases. In response to TNF, YAP transcriptional activity orientates cell fate toward survival. Transcriptional analysis with Nanostring technology reveals that YAP modulates TNF-induced increase in fibro-inflammatory pathways such as NF-κB, inflammasomes, cytokines or chemokines signaling and pro-fibrotic pathways involving TGF-ß and extracellular matrix remodeling. Therefore, in response to TNF, YAP acts as a sustainer of the inflammatory response and as a molecular link between inflammation and fibrotic processes. This work identifies that YAP is critical to drive several biological effects of TNF which are involved in cancer and inflammatory disorders.

YAP/TAZ: Key Players for Rheumatoid Arthritis Severity by Driving Fibroblast Like Synoviocytes Phenotype and Fibro-Inflammatory Response.

Objective: The role of YAP/TAZ, two transcriptional co-activators involved in several cancers, was investigated in rheumatoid arthritis (RA). Methods: Fibroblast like synoviocytes (FLS) from patients with RA or osteoarthritis were cultured in 2D or into 3D synovial organoids. Arthritis rat model (n=28) and colitis mouse model (n=21) were used. YAP/TAZ transcriptional activity was inhibited by verteporfin (VP). Multiple techniques were used to assess gene and/or protein expression and/or localization, cell phenotype (invasion, proliferation, apoptosis), bone erosion, and synovial stiffness. Results: YAP/TAZ were transcriptionally active in arthritis (19-fold increase for CTGF expression, a YAP target gene, in RA vs. OA organoids; p<0.05). Stiff support of culture or pro-inflammatory cytokines further enhanced YAP/TAZ transcriptional activity in RA FLS. Inhibiting YAP/TAZ transcriptional activity with VP restored a common phenotype in RA FLS with a decrease in apoptosis resistance, proliferation, invasion, and inflammatory response. Consequently, VP blunted hyperplasic lining layer formation in RA synovial organoids. In vivo, VP treatment strongly reduced arthritis severity (mean arthritic index at 3.1 in arthritic group vs. 2.0 in VP treated group; p<0.01) by restoring synovial homeostasis and decreasing systemic inflammation. YAP/TAZ transcriptional activity also enhanced synovial membrane stiffening in vivo, thus creating a vicious loop with the maintenance of YAP/TAZ activation over time in FLS. YAP/TAZ inhibition was also effective in another inflammatory model of mouse colitis. Conclusion: Our work reveals that YAP/TAZ were critical factors during arthritis. Thus, their transcriptional inhibition could be relevant to treat inflammatory related diseases.

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds.

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment. At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.

Parathyroid Hormone Remodels Bone Transitional Vessels and the Leptin Receptor-Positive Pericyte Network in Mice.

Intermittent parathyroid hormone (iPTH) is anti-osteoporotic and affects bone vessels. Transitional capillaries close to the bone surface, which express both endomucin (Edm) and CD31, bear leptin receptor-expressing (LepR) perivascular cells that may differentiate into osteoblasts. Increased numbers of type H endothelial cells (THEC; ie, Edmhi /CD31hi cells assessed by flow cytometry, FACS) are associated with higher bone formation in young mice. We hypothesized that iPTH administration impacts transitional vessels by expanding THECs. Four-month-old C57/Bl6J female mice were injected with PTH 1-84 (100 µg/kg/d) or saline (CT) for 7 or 14 days. We quantified LepR+ , CD31+ , Edm+ cells and THECs by FACS in hindlimb bone marrow, and Edm/LepR double immunolabelings on tibia cryosections. Additionally, we analyzed bone mRNA expression of 87 angiogenesis-related genes in mice treated with either intermittent or continuous PTH (iPTH/cPTH) or saline (CT) for 7, 14, and 28 days. iPTH dramatically decreased the percentage of THECs by 78% and 90% at days 7 and 14, respectively, and of LepR+ cells at day 14 (-46%) versus CT. Immunolabeling quantification showed that the intracortical Edm+ -vessel density increased at day 14 under iPTH. In the bone marrow, perivascular LepR+ cells, connected to each other via a dendrite network, were sparser under iPTH at day 14 (-58%) versus CT. iPTH decreased LepR+ cell coverage of transitional vessels only (-51%), whereas the number of LepR+ cells not attached to vessels increased in the endocortical area only (+ 49%). Transcriptomic analyses showed that iPTH consistently upregulated PEDF, Collagen-18α1, and TIMP-1 mRNA expression compared with CT and cPTH. Finally, iPTH increased immunolabeling of endostatin, a Collagen-18 domain that can be cleaved and become antiangiogenic, in both endocortical (79%) and peritrabecular transitional microvessels at day 14. Our results show that iPTH specifically remodels transitional vessels and suggest that it promotes LepR+ cell mobilization from these vessels close to the bone surface. © 2019 American Society for Bone and Mineral Research.

Porphyromonas gingivalis experimentally induces periodontis and an anti-CCP2-associated arthritis in the rat.

OBJECTIVES: Association between periodontal disease (PD) and rheumatoid arthritis (RA) has been extensively described, but direct evidence of causal involvement of PD in RA is missing. We investigated the priming role of oral Porphyromonas gingivalis (P. gingivalis) in PD and subsequent RA and we assessed biomarkers of bone resorption and arthritis development in rats. METHODS: Lewis rats were orally exposed to either P. gingivalis, Prevotella intermedia or control gel for 1 month and then followed for 8 months. The onset and development of PD was assessed by serology, gingivitis severity and micro-CT (µCT). We investigated arthritis development using circulating proinflammatory markers, anticyclic citrullinated peptide (CCP), anticitrullinated protein antibody (ACPA), ankle histology and µCT. RESULTS: PD was only observed in the P. gingivalis treated rats, as early as 1 month postexposure. Joint and systemic inflammation were detected only in the P. gingivalis group after 4 and 8 months. At 8 months, inflammatory cell infiltrate was observed in ankle joints and paralleled cortical erosions and overall cortical bone reduction. Furthermore, anti-CCP2 correlated with local and systemic bone loss. CONCLUSIONS: In our long-term study, PD induced by oral exposure to P. gingivalis triggered seropositive arthritis, with systemic inflammation and bone erosions. This is the first in vivo demonstration of arthritis induced by oral priming with P. gingivalis.

Early sclerostin expression explains bone formation inhibition before arthritis onset in the rat adjuvant-induced arthritis model.

Periarticular bone loss in rheumatoid arthritis (RA) is considered to be mainly related to synovial inflammation. However, strong bone loss has also described at the time of arthritis onset. Recently, a paradoxical exacerbation of joint damage was described when blocking sclerostin in various arthritis models. Thus, we aimed to determine kinetics of bone loss and its mechanisms in the adjuvant induced arthritis (AIA) rat model of RA. AIA was induced (n = 35) or not (n = 35) at day 0. In addition to well-known arthritis at day 12, we showed with 3D-imaging and histomorphometry that bone microstructural alterations occurred early from day 8 post-induction, characterized by cortical porosity and trabecular bone loss. Active osteoclastic surfaces were increased from day 8 with RANKL upregulation. More surprisingly SOST and DKK1 were overexpressed from day 6 and followed by a dramatic decrease in bone formation from day 8. At the time of arthritis onset, SOST and DKK1 returned to control values, but frizzled related protein 1 (SFRP1), proinflammatory cytokines, and MMPs started to increase. Bone alterations before arthritis onset reinforce the hypothesis of an early bone involvement in arthritis. Kinetics of osteocyte markers expression should be considered to refine Wnt inhibitor treatment strategies.