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Risk factors associated with disease severity and length of hospital stay in COVID-19 patients Dear Editor,We read with interest the article in this journal which revealed the critical role of timely supply of medical resources for COVID-19 patients. 1 The pandemic of COVID-19 has placed an enormous burden on health authorities across the world. The virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; previously known as 2019-nCoV), causes acute respiratory disease with common signs of infection being respiratory symptoms, fever, cough and breathing difficulties. In more severe cases, infection causes pneumonia, lung failure, septic shock, organ failure and risk of death. The WHO reports that 80% of those infected will develop mild symptoms, 14% severe symptoms and 6% will become critically ill. Given the wide clinical spectrum of COVID-19, a key challenge faced by frontline clinical staff is prioritisation of stretched resources. Thus, there is a critical need for robust risk assessment for clinical management.

Idiopathic pulmonary fibrosis (IPF), the prototypic progressive fibrotic interstitial lung disease, is thought to be a consequence of repetitive micro-injuries to an ageing, susceptible alveolar epithelium. Ageing is a risk factor for IPF and incidence has been demonstrated to increase with age. Decreased (macro)autophagy with age has been reported extensively in a variety of systems and diseases, including IPF. However, it is undetermined whether the role of faulty autophagy is causal or coincidental in the context of IPF. Here, we report that in alveolar epithelial cells inhibition of autophagy promotes epithelial-mesenchymal transition (EMT), a process implicated in embryonic development, wound healing, cancer metastasis and fibrosis. We further demonstrate that this is attained, at least in part, by increased p62/SQSTM1 expression that promotes p65/RELA mediated-transactivation of an EMT transcription factor, Snail2 (SNAI2), which not only controls EMT but also regulates the production of locally acting profibrogenic mediators. Our data suggest that reduced autophagy induces EMT of alveolar epithelial cells and can contribute to fibrosis via aberrant epithelial-fibroblast crosstalk.

ganglion (DRG) sensory neurons, including >90% of C-nociceptors (pain-sensing neurons) and C-low-threshold mechanoreceptors, as well as a lower percentage of Ad-nociceptors and Ab afferents. At age 10 weeks (n ¼ 5) and at age 26 weeks (n ¼ 5), mice were perfused transcardially with paraformaldehyde, and the right knees were collected, post fixed and decalcified. Twenty-mm thick frozen sections were collected at mid-joint level. Consecutive sections were stained with hematoxylin & eosin. Age-matched heterozygous C57BL/6 Pirt-GCaMP3 mice were used to confirm innervation patterns. These mice express the green fluorescent calcium indicator, GCaMP3, in~90% of all sensory DRG neurons (including the Na v 1.8 population), and not in other peripheral or central tissues, through the Pirt promoter. Results: Examination of the knees of 10-week old Na V 1.8-TdTomato mice revealed areas of dense innervation by Na V 1.8-expressing sensory fibers, most notably the bone marrow, the lateral synovium, and the connective tissue layer (epiligament) surrounding the cruciate ligaments, including the areas of attachment. Other structures, such as the medial synovium and the collagenous substance of the cruciate ligaments, were less densely innervated. Na V 1.8 nociceptors were also present in the outer third of the lateral meniscus. The articular cartilage, the inner two thirds of the lateral meniscus, and the medial meniscus did not show innervation. Figure 1 shows an example of these features in one mouse-but these findings were remarkably reproducible in n ¼ 5 mice. Assessment of Na V 1.8 signal in knees of 26-week old mice revealed marked changes in innervation density (not shown). Compared to 10-week old knees, 26-week old knees showed a dramatic decline in Na V 1.8-expressing nociceptors in the lateral synovium, as well as in the epiligament and attachment areas of the cruciate ligaments. Similar age-related changes in the innervation were also detected in the knees of 26-week old Pirt-GCaMP3 mice compared to 10-week old knees, providing independent evidence that the chosen markers are specific for nerve fibers. Conclusions: This study reproducibly shows, for the first time, that the nociceptive innervation of specific murine knee tissues dramatically declines with age. Remarkably, this occurs quite early on in the life of the mouse, where we find dense innervation at 10 weeks and a marked decline by 26 weeks. Ongoing studies are aimed at monitoring innervation with more advanced age. The biological significance of these findings needs to be explored, as well as the relationship with pathogenesis of osteoarthritis.

Osteoarthritis (OA) is the most common joint disease, characterized by progressive destruction of the articular cartilage. The surface of joint cartilage is the first defensive and affected site of OA, but our knowledge of genesis and homeostasis of this superficial zone is scarce. EGFR signaling is important for tissue homeostasis. Immunostaining revealed that its activity is mostly dominant in the superficial layer of healthy cartilage but greatly diminished when OA initiates. To evaluate the role of EGFR signaling in the articular cartilage, we studied a cartilage-specific Egfr-deficient (CKO) mouse model (Col2-Cre EgfrWa5/flox). These mice developed early cartilage degeneration at 6 mo of age. By 2 mo of age, although their gross cartilage morphology appears normal, CKO mice had a drastically reduced number of superficial chondrocytes and decreased lubricant secretion at the surface. Using superficial chondrocyte and cartilage explant cultures, we demonstrated that EGFR signaling is critical for maintaining the number and properties of superficial chondrocytes, promoting chondrogenic proteoglycan 4 (Prg4) expression, and stimulating the lubrication function of the cartilage surface. In addition, EGFR deficiency greatly disorganized collagen fibrils in articular cartilage and strikingly reduced cartilage surface modulus. After surgical induction of OA at 3 mo of age, CKO mice quickly developed the most severe OA phenotype, including a complete loss of cartilage, extremely high surface modulus, subchondral bone plate thickening, and elevated joint pain. Taken together, our studies establish EGFR signaling as an important regulator of the superficial layer during articular cartilage development and OA initiation.EGFR | articular cartilage | chondrocyte | lubrication | osteoarthritis

Mechanical loading on articular cartilage can induce many physical and chemical stimuli on chondrocytes residing in the extracellular matrix (ECM). Intracellular calcium ([Ca ] ) signaling is among the earliest responses of chondrocytes to physical stimuli, but the [Ca ] signaling of in situ chondrocytes in loaded cartilage is not fully understood due to the technical challenges in [Ca ] imaging of chondrocytes in a deforming ECM. This study developed a novel bi-directional microscopy loading device that enables the record of transient [Ca ] responses of in situ chondrocytes in loaded cartilage. It was found that compressive loading significantly promoted [Ca ] signaling in chondrocytes with faster [Ca ] oscillations in comparison to the non-loaded cartilage. Seven [Ca ] signaling pathways were further investigated by treating the cartilage with antagonists prior to and/or during the loading. Removal of extracellular Ca ions completely abolished the [Ca ] responses of in situ chondrocytes, suggesting the indispensable role of extracellular Ca sources in initiating the [Ca ] signaling in chondrocytes. Depletion of intracellular Ca stores, inhibition of PLC-IP pathway, and block of purinergic receptors on plasma membrane led to significant reduction in the responsive rate of cells. Three types of ion channels that are regulated by different physical signals, TRPV4 (osmotic and mechanical stress), T-type VGCCs (electrical potential), and mechanical sensitive ion channels (mechanical loading) all demonstrated critical roles in controlling the [Ca ] responses of in situ chondrocyte in the loaded cartilage. This study provided new knowledge about the [Ca ] signaling and mechanobiology of chondrocytes in its natural residing environment. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:730-738, 2018.

Traumatic joint injuries often result in elevated proinflammatory cytokine (such as IL-1β) levels in the joint cavity, which can increase the catabolic activities of chondrocytes and damage cartilage. This study investigated the early genetic responses of healthy in situ chondrocytes under IL-1β attack with a focus on cell cycle and calcium signaling pathways. RNA sequencing analysis identified 2,232 significantly changed genes by IL-1β, with 1,259 upregulated and 973 downregulated genes. Catabolic genes related to ECM degeneration were promoted by IL-1β, consistent with our observations of matrix protein loss and mechanical property decrease during 24-day in vitro culture of cartilage explants. IL-1β altered the cell cycle (108 genes) and Rho GTPases signaling (72 genes) in chondrocytes, while chondrocyte phenotypic shift was observed with histology, cell volume measurement, and MTT assay. IL-1β inhibited the spontaneous calcium signaling in chondrocytes, a fundamental signaling event in chondrocyte metabolic activities. The expression of 24 genes from 6 calcium-signaling related pathways were changed by IL-1β exposure. This study provided a comprehensive list of differentially expressed genes of healthy in situ chondrocytes in response to IL-1β attack, which represents a useful reference to verify and guide future cartilage studies related to the acute inflammation after joint trauma.

Chemically defined serum-free medium has been shown to maintain the mechanical integrity of articular cartilage explants better than serum-supplemented medium during long-term in vitro culture, but little is known about its effect on cellular mechanisms. We hypothesized that the chemically defined culture medium can regulate the spontaneous calcium signaling of in situ chondrocytes, which may modulate the cellular metabolic activities. Bovine cartilage explants were cultured in chemically defined serum-free or serum-supplemented medium for four weeks. The spontaneous intracellular calcium ([Ca2+]i) signaling of in situ chondrocytes was longitudinally measured together along with the biomechanical properties of the explants. The spontaneous [Ca2+]i oscillations in chondrocytes were enhanced at the initial exposure of serum-supplemented medium, but were significantly dampened afterwards. In contrast, cartilage explants in chemically defined medium preserved the level of calcium signaling, and showed more responsive cells with higher and more frequent [Ca2+]i peaks after one to four week culture in comparison to those in serum medium. Regardless of the culture medium that the explants were exposed, a positive correlation was detected between the [Ca2+]i responsive rate and the stiffness of cartilage (Spearman's rank correlation coefficient = 0.762). A stable pattern of [Ca2+]i peaks was revealed for each chondrocyte, i.e., the spatiotemporal features of [Ca2+]i peaks from a cell were highly consistent during the observation period (15 minutes). This study showed that the beneficial effect of chemically defined culture of cartilage explants is associated with the spontaneous [Ca2+]i signaling of chondrocytes in cartilage.