Cell-autonomous requirement for ACE2 across organs in lethal mouse SARS-CoV-2 infection.

Angiotensin-converting enzyme 2 (ACE2) is the cell-surface receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). While its central role in Coronavirus Disease 2019 (COVID-19) pathogenesis is indisputable, there remains significant debate regarding the role of this transmembrane carboxypeptidase in the disease course. These include the role of soluble versus membrane-bound ACE2, as well as ACE2-independent mechanisms that may contribute to viral spread. Testing these roles requires in vivo models. Here, we report humanized ACE2-floxed mice in which hACE2 is expressed from the mouse Ace2 locus in a manner that confers lethal disease and permits cell-specific, Cre-mediated loss of function, and LSL-hACE2 mice in which hACE2 is expressed from the Rosa26 locus enabling cell-specific, Cre-mediated gain of function. Following exposure to SARS-CoV-2, hACE2-floxed mice experienced lethal cachexia, pulmonary infiltrates, intravascular thrombosis and hypoxemia-hallmarks of severe COVID-19. Cre-mediated loss and gain of hACE2 demonstrate that neuronal infection confers lethal cachexia, hypoxemia, and respiratory failure in the absence of lung epithelial infection. In this series of genetic experiments, we demonstrate that ACE2 is absolutely and cell-autonomously required for SARS-CoV-2 infection in the olfactory epithelium, brain, and lung across diverse cell types. Therapies inhibiting or blocking ACE2 at these different sites are likely to be an effective strategy towards preventing severe COVID-19.

Cerebral cavernous malformations are driven by ADAMTS5 proteolysis of versican.

Cerebral cavernous malformations (CCMs) form following loss of the CCM protein complex in brain endothelial cells due to increased endothelial MEKK3 signaling and KLF2/4 transcription factor expression, but the downstream events that drive lesion formation remain undefined. Recent studies have revealed that CCM lesions expand by incorporating neighboring wild-type endothelial cells, indicative of a cell nonautonomous mechanism. Here we find that endothelial loss of ADAMTS5 reduced CCM formation in the neonatal mouse model. Conversely, endothelial gain of ADAMTS5 conferred early lesion genesis in the absence of increased KLF2/4 expression and synergized with KRIT1 loss of function to create large malformations. Lowering versican expression reduced CCM burden, indicating that versican is the relevant ADAMTS5 substrate and that lesion formation requires proteolysis but not loss of this extracellular matrix protein. These findings identify endothelial secretion of ADAMTS5 and cleavage of versican as downstream mechanisms of CCM pathogenesis and provide a basis for the participation of wild-type endothelial cells in lesion formation.

The histone variant macroH2A confers functional robustness to the intestinal stem cell compartment.

A stem cell's epigenome directs cell fate during development, homeostasis, and regeneration. Epigenetic dysregulation can lead to inappropriate cell fate decisions, aberrant cell function, and even cancer. The histone variant macroH2A has been shown to influence gene expression, guide cell fate, and safeguard against genotoxic stress. Interestingly, mice lacking functional macroH2A histones (hereafter referred to as macroH2A DKO) are viable and fertile; yet suffer from increased perinatal death and reduced weight and size compared to wildtype (WT). Here, we ask whether the ostensible reduced vigor of macroH2A DKO mice extends to intestinal stem cell (ISC) function during homeostasis, regeneration, and oncogenesis. Lgr5-eGFP-IRES-CreERT2 or Hopx-CreERT2::Rosa26-LSL-tdTomato ISC reporter mice or the C57BL/6J-Apcmin/J murine intestinal adenoma model were bred into a macroH2A DKO or strain-matched WT background and assessed for ISC functionality, regeneration and tumorigenesis. High-dose (12Gy) whole-body γ-irradiation was used as an injury model. We show that macroH2A is dispensable for intestinal homeostasis and macroH2A DKO mice have similar numbers of active crypt-base columnar ISCs (CBCs). MacroH2A DKO intestine exhibits impaired regeneration following injury, despite having significantly more putative reserve ISCs. DKO reserve ISCs disproportionately undergo apoptosis compared to WT after DNA damage infliction. Interestingly, a macroH2A DKO background does not significantly increase tumorigenesis in the Apcmin model of intestinal adenoma. We conclude that macroH2A influences reserve ISC number and function during homeostasis and regeneration. These data suggest macroH2A enhances reserve ISC survival after DNA damage and thus confers functional robustness to the intestinal epithelium.

Protein arginylation targets alpha synuclein, facilitates normal brain health, and prevents neurodegeneration.

Alpha synuclein (α-syn) is a central player in neurodegeneration, but the mechanisms triggering its pathology are not fully understood. Here we found that α-syn is a highly efficient substrate for arginyltransferase ATE1 and is arginylated in vivo by a novel mid-chain mechanism that targets the acidic side chains of E46 and E83. Lack of arginylation leads to increased α-syn aggregation and causes the formation of larger pathological aggregates in neurons, accompanied by impairments in its ability to be cleared via normal degradation pathways. In the mouse brain, lack of arginylation leads to an increase in α-syn's insoluble fraction, accompanied by behavioral changes characteristic for neurodegenerative pathology. Our data show that lack of arginylation in the brain leads to neurodegeneration, and suggests that α-syn arginylation can be a previously unknown factor that facilitates normal α-syn folding and function in vivo.

Extranodal NK/T-Cell Lymphoma, Nasal Type, Presenting as Refractory Pseudomonas aeruginosa Facial Cellulitis.

Extranodal natural killer T-cell lymphoma, nasal type (ENKL), formerly called lethal midline granuloma or angiocentric T-cell lymphoma, is a predominantly extranodal non-Hodgkin lymphoma characterized by vascular damage, necrosis, and an association with Epstein-Barr virus. In the United States, it is more frequently seen in Asian, Asian Pacific Islander, and Hispanic descent populations and is more prevalent in males in their fifth decade. Clinical presentation of NK nasal lymphoma most commonly involves epistaxis; obstruction; discharge; destructive mass in sinuses, palate, and nose; and skin ulceration. These symptoms can mimic invasive fungal infections and other sinonasal disorders. Furthermore, ENKL has a broad cytologic spectrum and induces a mixture of inflammatory cells, causing difficulty in establishing the diagnosis, especially in initial biopsies. We present a case of refractory Pseudomonas aeruginosa facial cellulitis in a young woman whose treatment course was complicated by septic shock and resistance to multiple antibiotics, resulting in a delayed diagnosis of ENKL nasal type.

Quantitative role of the human papillomavirus type 16 E5 gene during the productive stage of the viral life cycle.

Human papillomaviruses (HPVs) are small circular DNA viruses that cause warts. Infection with high-risk anogenital HPVs, such as HPV type 16 (HPV16), is associated with human cancers, specifically cervical cancer. The life cycle of HPVs is intimately tied to the differentiation status of the host epithelium and has two distinct stages: the nonproductive stage and the productive stage. In the nonproductive stage, which arises in the poorly differentiated basal epithelial compartment of a wart, the virus maintains itself as a low-copy-number nuclear plasmid. In the productive stage, which arises as the host cell undergoes terminal differentiation, viral DNA is amplified; the capsid genes, L1 and L2, are expressed; and progeny virions are produced. This stage of the viral life cycle relies on the ability of the virus to reprogram the differentiated cells to support DNA synthesis. Papillomaviruses encode multiple oncoproteins, E5, E6, and E7. In the present study, we analyze the role of one of these viral oncogenes, E5, in the viral life cycle. To assess the role of E5 in the HPV16 life cycle, we introduced wild-type (WT) or E5 mutant HPV16 genomes into NIKS, a keratinocyte cell line that supports the papillomavirus life cycle. By culturing these cells under conditions that allow them to remain undifferentiated, a state similar to that of basal epithelial cells, we determined that E5 does not play an essential role in the nonproductive stage of the HPV16 life cycle. To determine if E5 plays a role in the productive stage of the viral life cycle, we cultured keratinocyte populations in organotypic raft cultures, which promote the differentiation and stratification of epithelial cells. We found that cells harboring E5 mutant genomes displayed a quantitative reduction in the percentage of suprabasal cells undergoing DNA synthesis, compared to cells containing WT HPV16 DNA. This reduction in DNA synthesis, however, did not prevent amplification of viral DNA in the differentiated cellular compartment. Likewise, late viral gene expression and the perturbation of normal keratinocyte differentiation were retained in cells harboring E5 mutant genomes. These data demonstrate that E5 plays a subtle role during the productive stage of the HPV16 life cycle.