SARS-CoV-2 NSP5 antagonizes MHC II expression by subverting histone deacetylase 2.

SARS-CoV-2 interferes with antigen presentation by downregulating major histocompatibility complex (MHC) II on antigen-presenting cells, but the mechanism mediating this process is unelucidated. Herein, analysis of protein and gene expression in human antigen-presenting cells reveals that MHC II is downregulated by the SARS-CoV-2 main protease, NSP5. This suppression of MHC II expression occurs via decreased expression of the MHC II regulatory protein CIITA. CIITA downregulation is independent of the proteolytic activity of NSP5, and rather, NSP5 delivers HDAC2 to the transcription factor IRF3 at an IRF-binding site within the CIITA promoter. 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 mechanism by which SARS-CoV-2 limits MHC II expression, thereby delaying or weakening the subsequent adaptive immune response.

Efferocytic Defects in Early Atherosclerosis Are Driven by GATA2 Overexpression in Macrophages.

The loss of efferocytosis-the phagocytic clearance of apoptotic cells-is an initiating event in atherosclerotic plaque formation. While the loss of macrophage efferocytosis is a prerequisite for advanced plaque formation, the transcriptional and cellular events in the pre-lesion site that drive these defects are poorly defined. Transcriptomic analysis of macrophages recovered from early-stage human atherosclerotic lesions identified a 50-fold increase in the expression of GATA2, a transcription factor whose expression is normally restricted to the hematopoietic compartment. GATA2 overexpression in vitro recapitulated many of the functional defects reported in patient macrophages, including deficits at multiple stages in the efferocytic process. These findings included defects in the uptake of apoptotic cells, efferosome maturation, and in phagolysosome function. These efferocytic defects were a product of GATA2-driven alterations in the expression of key regulatory proteins, including Src-family kinases, Rab7 and components of both the vacuolar ATPase and NADPH oxidase complexes. In summary, these data identify a mechanism by which efferocytic capacity is lost in the early stages of plaque formation, thus setting the stage for the accumulation of uncleared apoptotic cells that comprise the bulk of atherosclerotic plaques.

Customizable live-cell imaging chambers for multimodal and multiplex fluorescence microscopy.

Using multiple imaging modalities while performing independent experiments in parallel can greatly enhance the throughput of microscopy-based research, but requires the provision of appropriate experimental conditions in a format that meets the optical requirements of the microscope. 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 that supports the growth and imaging of cells over multiple days. This approach can also be used to produce moulds for casting microfluidic devices made of polydimethylsiloxane. 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 for producing imaging chambers that are compatible with modern microscopy techniques.

Soluble CD93 is an apoptotic cell opsonin recognized by αx ß2.

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 αx ß2 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 αx ß2 by its EGF-like repeats. The bridging of apoptotic cells to αx ß2 markedly enhanced efferocytosis by macrophages and was abrogated by αx ß2 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.