Innate immunology in COVID-19-a living review. Part II: dysregulated inflammation drives immunopathology

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a global health crisis and will likely continue to impact public health for years. As the effectiveness of the innate immune response is crucial to patient outcome, huge efforts have been made to understand how dysregulated immune responses may contribute to disease progression. Here we have reviewed current knowledge of cellular innate immune responses to SARS-CoV-2 infection, highlighting areas for further investigation and suggesting potential strategies for intervention. We conclude that in severe COVID-19 initial innate responses, primarily type I interferon, are suppressed or sabotaged which results in an early interleukin (IL)-6, IL-10 and IL-1β-enhanced hyperinflammation. This inflammatory environment is driven by aberrant function of innate immune cells: monocytes, macrophages and natural killer cells dispersing viral pathogen-associated molecular patterns and damage-associated molecular patterns into tissues. This results in primarily neutrophil-driven pathology including fibrosis that causes acute respiratory distress syndrome. Activated leukocytes and neutrophil extracellular traps also promote immunothrombotic clots that embed into the lungs and kidneys of severe COVID-19 patients, are worsened by immobility in the intensive care unit and are perhaps responsible for the high mortality. Therefore, treatments that target inflammation and coagulation are promising strategies for reducing mortality in COVID-19

Innate immunology in COVID-19?a living review. Part I: viral entry, sensing and evasion

The coronavirus infectious disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a world health concern and can cause severe disease and high mortality in susceptible groups. While vaccines offer a chance to treat disease, prophylactic and anti-viral treatments are still of vital importance, especially in context of the mutative ability of this group of viruses. Therefore, it is essential to elucidate the molecular mechanisms of viral entry, innate sensing and immune evasion of SARS-CoV-2, which control the triggers of the subsequent excessive inflammatory response. Viral evasion strategies directly target anti-viral immunity, counteracting host restriction factors and hijacking signalling pathways to interfere with interferon production. In Part I of this review, we examine SARS-CoV-2 viral entry and the described immune evasion mechanisms to provide a perspective on how the failure in initial viral sensing by infected cells can lead to immune dysregulation causing fatal COVID-19, discussed in Part II

The role and uses of antibodies in COVID-19 infections: a living review

Coronavirus disease 2019 has generated a rapidly evolving field of research, with the global scientific community striving for solutions to the current pandemic. Characterizing humoral responses towards SARS-CoV-2, as well as closely related strains, will help determine whether antibodies are central to infection control, and aid the design of therapeutics and vaccine candidates. This review outlines the major aspects of SARS-CoV-2-specific antibody research to date, with a focus on the various prophylactic and therapeutic uses of antibodies to alleviate disease in addition to the potential of cross-reactive therapies and the implications of long-term immunity

T cell phenotypes in COVID-19 - a living review

COVID-19 is characterized by profound lymphopenia in the peripheral blood, and the remaining T cells display altered phenotypes, characterized by a spectrum of activation and exhaustion. However, antigen-specific T cell responses are emerging as a crucial mechanism for both clearance of the virus and as the most likely route to long-lasting immune memory that would protect against re-infection. Therefore, T cell responses are also of considerable interest in vaccine development. Furthermore, persistent alterations in T cell subset composition and function post-infection have important implications for patients’ long-term immune function. In this review, we examine T cell phenotypes, including those of innate T cells, in both peripheral blood and lungs, and consider how key markers of activation and exhaustion correlate with, and may be able to predict, disease severity. We focus on SARS-CoV-2-specific T cells to elucidate markers that may indicate formation of antigen-specific T cell memory. We also examine peripheral T cell phenotypes in recovery and the likelihood of long-lasting immune disruption. Finally, we discuss T cell phenotypes in the lung as important drivers of both virus clearance and tissue damage. As our knowledge of the adaptive immune response to COVID-19 rapidly evolves, it has become clear that while some areas of the T cell response have been investigated in some detail, others, such as the T cell response in children remain largely unexplored. Therefore, this review will also highlight areas where T cell phenotypes require urgent characterisation

P127 Disrupting the cartilage mechanostat: the role of the ciliary protein IFT88 in the adolescent growth plate

Background/sims: as long bone elongation draws to a close, the cartilaginous growth plate begins to ossify in preparation of growth plate fusion. Previous embryonic developmental in vivo work has identified the crucial Parathyroid Hormone-related Protein-Indian hedgehog (PTHrP-Ihh) feedback loop that is responsible for the proliferation of chondrocytes at the epiphysis, whilst also allowing for the hypertrophic differentiation of chondrocytes before ossification at the diaphysis. Indian hedgehog signalling relies upon the microtubule-based organelle the primary cilium, as disruption to either results in similar musculoskeletal phenotypes. Here, we asked for the first time whether juvenile and adolescent primary cilia disruption affected chondrocyte differentiation in the growth plate.Methods: we used a chondrocyte-specific conditional knockout (AggrecanCreERT2; Ift88fl/fl, cKO) of a key primary ciliary protein (Ift88) administering tamoxifen at (4, 6, 8 weeks-of-age) to both cKO and control (Ift88fl/fl) animals, collecting two weeks later (6, 8, 10-weeks-of-age). Immunohistochemistry was performed using type X collagen (ColX), a specific marker of hypertrophic chondrocytes.Results: deletion of IFT88 resulted in large bi-lateral cartilaginous regions filled with disorganised ColX positive hypertrophic chondrocytes, indicating failed ossification. Our results indicate that deletion of IFT88 does not impact hypertrophic differentiation, but disrupts ossification processes downstream at the chondro-osseous junction, such as matrix remodelling and angiogenesis, necessary for growth plate closure. Interestingly, this phenotype was observed only in the bi-lateral most loaded regions of the tibia whilst the middle was unaffected.Conclusion: this observation indicates that the primary cilium could be involved in transducing mechanically regulated biophysical and signalling cues in the adolescent growth plate

Developmentally inspired model of endochondral ossification

Introduction: In postnatal development, chondro-osseous transitions such as endochondral ossification (EO) are regulated by rapid matrix turnover, mineralisation and chondro-osseous transdifferentiation, all disrupted in osteoarthritis (OA). We are exploring how force governs these transitions of cartilage to bone; previous studies from our group indicate cartilage matrix and chondrocyte phenotypic plasticity in the growth plate, and stability in articular cartilage, is mechanoregulated. Here, we describe exploitation of a human ‘developmental biology-inspired platform’ alongside in vivo studies, to study cartilage mineralisation.Materials and Methods: In vivo studies reveal chondro-osseous transitions are inhibited by forces. We synthesise GelMA using click chemistry to generate hydrogels with compressive moduli of 3–5 kPa. Buoyancy-driven gradients of BMP-2 within hydrogels seeded with hMSCs were cultured for 28 days. qPCR, histology and immunohistochemistry assessed, cartilage, hypertrophic and bone markers. Uniaxial cyclic compression (0.5 Hz, 10% strain) was applied using Electroforce5500. Cells from Confetti-UBCre mice are being used for lineage tracing studies.Results: MSC-laden GelMA constructs showed differential gene expression across the gradient, indicating tri-phasic osteochondral tissue formation within 28 days. Runx2/Sox9 immunostaining confirmed osteochondral differentiation, increased expression of SPP1, SP7, Runx2, Col1 indicated osteogenesis at one end, while distinct expression of Col10 and Runx2 in the central region marked cellular hypertrophy. The effects of cyclic loading on cell signalling, hyaline cartilage formation and thickness, calcification and phenotypic plasticity/stability are being assessed and compared with in vivo findings.Discussion: These data validate the formation of a humanised osteochondral gradient recapitulating developmental processes, in vitro. It is hypothesised that the generated osteochondral tissues will reflect the results of phenotypic changes in cells and ECM regulation, under physiological and pathological loads. The model will be integrated with snRNA sequencing and lineage tracing, to study trans-differentiation. This approach provides an experimentally tractable mechanobiology model and clinically conformant osteochondral tissue development model enabling fundamental biology and disease modelling across scales

Techniques for visualization and quantification of primary cilia in chondrocytes

Primary cilia regulate and coordinate a variety of cell signaling pathways important in chondrocyte physiology and cartilage development, health, and disease. Despite this, the chondrocyte primary cilium and its associated role in cartilage biology remains poorly understood. Key to elucidating primary cilia structure and function in chondrocytes is the ability to visualize this unique structure. Here we describe materials and methods for immunofluorescence labeling, microscopy, and measurement of chondrocyte primary cilia

Cilia protein IFT88 regulates extracellular protease activity by optimizing LRP-1–mediated endocytosis

Matrix protease activity is fundamental to developmental tissue patterning and remains influential in adult homeostasis. In cartilage, the principal matrix proteoglycan is aggrecan, the protease-mediated catabolism of which defines arthritis; however, the pathophysiologic mechanisms that drive aberrant aggrecanolytic activity remain unclear. Human ciliopathies exhibit altered matrix, which has been proposed to be the result of dysregulated hedgehog signaling that is tuned within the primary cilium. Here, we report that disruption of intraflagellar transport protein 88 (IFT88), a core ciliary trafficking protein, increases chondrocyte aggrecanase activity in vitro. We find that the receptor for protease endocytosis in chondrocytes, LDL receptor–related protein 1 (LRP-1), is unevenly distributed over the cell membrane, often concentrated at the site of cilia assembly. Hypomorphic mutation of IFT88 disturbs this apparent hot spot for protease uptake, increases receptor shedding, and results in a reduced rate of protease clearance from the extracellular space. We propose that IFT88 and/or the cilium regulates the extracellular remodeling of matrix—independently of Hedgehog regulation—by enabling rapid LRP-1–mediated endocytosis of proteases, potentially by supporting the creation of a ciliary pocket. This result highlights new roles for the cilium’s machinery in matrix turnover and LRP-1 function, with potential relevance in a range of diseases.—Coveney, C. R., Collins, I., Mc Fie, M., Chanalaris, A., Yamamoto, K., Wann, A. K. T. Cilia protein IFT88 regulates extracellular protease activity by optimizing LRP-1–mediated endocytosis

Ciliary proteins specify the cell inflammatory response by tuning NFκB signaling, independently of primary cilia

Complex inflammatory signalling cascades define the response to tissue injury but also control development and homeostasis, limiting these pathways as therapeutic targets. Primary cilia are sub-cellular regulators of cellular signalling, controlling how signalling is organized, encoded and, in some instances, driving or influencing pathogenesis. Our previous research revealed that disruption of ciliary intraflagellar transport (IFT), altered the cell response to IL-1β, supporting a putative link emerging between cilia and inflammation. Here, we show that IFT88 depletion affects specific cytokine-regulated behaviors, changing cytosolic NFκB translocation dynamics, but leaving MAPK unaffected. RNAseq analysis indicates IFT88 regulates one third of the genome-wide targets, including the pro-inflammatory genes Nos2, Il6 and Tnf. By microscopy, we find altered NFκB dynamics are independent to assembly of a ciliary axoneme. Indeed, depletion of IFT88 inhibits the inflammatory responses in the non-ciliated macrophage. We propose ciliary proteins, including IFT88, KIF3A, TTBK2 and NPHP4, act outside of the ciliary axoneme, to tune cytoplasmic NFκB signalling, and specify the downstream cell response. This is thus a non-canonical function for ciliary proteins in shaping cellular inflammation