Efferocytosis is essential for homeostasis and prevention of the inflammatory and autoimmune diseases resulting from apoptotic cell lysis. CD93 is a transmembrane glycoprotein previously implicated in efferocytosis, with mutations in CD93 predisposing patients to efferocytosis‐associated diseases. CD93 is a cell surface protein, which is proteolytically shed under inflammatory conditions, but it is unknown how CD93 mediates efferocytosis or whether its efferocytic activity is mediated by the soluble or membrane‐bound form. Herein, using cell lines and human monocytes and macrophages, we demonstrate that soluble CD93 (sCD93) potently opsonizes apoptotic cells but not a broad range of microorganisms, whereas membrane‐bound CD93 has no phagocytic, efferocytic, or tethering activity. Using mass spectrometry, we identified αxβ2 as the receptor that recognizes sCD93, and via deletion mutagenesis determined that sCD93 binds to apoptotic cells via its C‐type lectin‐like domain and to αxβ2 by its EGF‐like repeats. The bridging of apoptotic cells to αxβ2 markedly enhanced efferocytosis by macrophages and was abrogated by αxβ2 knockdown. Combined, these data elucidate the mechanism by which CD93 regulates efferocytosis and identifies a previously unreported opsonin‐receptor system utilized by phagocytes for the efferocytic clearance of apoptotic cells.

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Using multiple imaging modalities while performing independent experiments in parallel can greatly enhance the throughput of microscopy-based research, but requires provision of appropriate experimental conditions in a format that meets the microscopy’s optical requirements. Although customized imaging chambers can meet these challenges, the difficulty of manufacturing custom chambers and the relatively high cost and design inflexibility of commercial chambers has limited the adoption of this approach. Herein, we demonstrate the use of 3D printing to produce inexpensive, customized live-cell imaging chambers that are compatible with a range of imaging modalities including super-resolution microscopy. In this approach, biocompatible plastics are used to print imaging chambers designed to meet the specific needs of an experiment, followed by adhesion of the printed chamber to a glass coverslip, producing a chamber that is impermeant to liquids and which supports the growth and imaging of cells over multiple days. This approach can also be used to produce moulds for casting PDMS microfluidic devices. The utility of these chambers is demonstrated using designs for multiplex microscopy, imaging under shear, chemotaxis, and general cellular imaging. Together, this approach represents an inexpensive yet highly customizable approach to produce imaging chambers that are compatible with modern microscopy techniques.

The clearance of SARS-CoV-2 requires a multi-faceted immune response that is initiated by innate immune cells, with infection ultimately resolved by adaptive immune mechanisms. Induction of adaptive immunity to SARS-CoV-2 is dependent on the presentation of viral antigens on MHC II by professional antigen presenting cells such as dendritic cells and macrophages, to induce robust activation of CD4+ T cells. SARS-CoV-2 interferes with antigen presentation by downregulating MHC II on the antigen presenting cells of COVID-19 patients, but the molecular mechanism mediating this process is unelucidated. In this study, analysis of protein and gene expression in human antigen presenting cells reveals that the expression of MHC II is inhibited by the SARS-CoV-2 main protease, NSP5. Suppression of MHC II expression occurs via downregulation of the transcription factor CIITA, which is required for MHC II expression. This downregulation of CIITA is independent of NSP5's proteolytic activity, and rather, NSP5 delivers HDAC2 to the CIITA promoter via interactions with IRF3, Here, HDAC2 deacetylates and inactivates the CIITA promoter. This loss of CIITA expression prevents further expression of MHC II, with this suppression alleviated by ectopic expression of CIITA or knockdown of HDAC2. These results identify a novel mechanism by which SARS-CoV-2 can limit antigen presentation on MHC II, thereby delaying or weakening the subsequent adaptive immune response.

While new high-resolution microscopy techniques are continually developed, adoption of these methods is often difficult due to an inability to meet the experimental conditions required for an experiment in a format which also meets the demanding optical requirements of these microscopy techniques. Although specialized imaging chambers can meet these challenges, the difficulty of manufacturing customized chambers in-house and the relatively high cost and design inflexibility of commercial chambers has limited the incorporation of imaging chambers into fluorescence and super-resolution microscopy experiments. Herein, we demonstrate the use of fused deposition modeling (3D printing) for producing inexpensive, customized imaging chambers that are compatible with long-duration live-cell imaging using fluorescence and super-resolution microscopy techniques. In this approach, biocompatible 3D printing plastics are used to generate imaging chambers designed to meet the specific needs of an experiment, followed by adhesion of the printed chamber to a glass coverslip suitable for fluorescence and super-resolution imaging. This technique produces a chamber that is impermeant to liquids that can support the growth and imaging of cells over multiple days. The utility of these chambers is then demonstrated using designs for multiplex microscopy, imaging under shear, chemotaxis, and general cellular imaging. Together, this approach represents an inexpensive yet highly customizable approach to produce imaging chambers that are compatible with many modern microscopy techniques.