A role for Toll-like receptor 3 in lung vascular remodeling associated with SARS-CoV-2 infection.

Cardiovascular sequelae of severe acute respiratory syndrome (SARS) coronavirus-2 (CoV-2) disease 2019 (COVID-19) contribute to the complications of the disease. One potential complication is lung vascular remodeling, but the exact cause is still unknown. We hypothesized that endothelial TLR3 insufficiency contributes to lung vascular remodeling induced by SARS-CoV-2. In the lungs of COVID-19 patients and SARS-CoV-2 infected Syrian hamsters, we discovered thickening of the pulmonary artery media and microvascular rarefaction, which were associated with decreased TLR3 expression in lung tissue and pulmonary artery endothelial cells (ECs). In vitro , SARS-CoV-2 infection reduced endothelial TLR3 expression. Following infection with mouse-adapted (MA) SARS-CoV-2, TLR3 knockout mice displayed heightened pulmonary artery remodeling and endothelial apoptosis. Treatment with the TLR3 agonist polyinosinic:polycytidylic acid reduced lung tissue damage, lung vascular remodeling, and endothelial apoptosis associated with MA SARS-CoV-2 infection. In conclusion, repression of endothelial TLR3 is a potential mechanism of SARS-CoV-2 infection associated lung vascular remodeling and enhancing TLR3 signaling is a potential strategy for treatment.

Inhibiting the Deubiquitinase UCHL1 Reduces SARS-CoV-2 Viral Uptake by ACE2.

Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a significant public health burden with limited treatment options. Many ß-coronaviruses, including SARS-CoV-2, gain entry to host cells through the interaction of SARS-CoV-2 spike protein with membrane-bound ACE2 (angiotensin-converting enzyme 2). Given its necessity for SARS-CoV-2 infection, ACE2 represents a potential therapeutic target in COVID-19. However, early attempts focusing on ACE2 in COVID-19 have not validated it as a druggable target nor identified other ACE2-related novel proteins for therapeutic intervention. Here, we identify a mechanism for ACE2 protein modulation by the deubiquitinase (DUB) enzyme UCHL1 (ubiquitin carboxyl-terminal hydrolase isozyme L1). ACE2 is constitutively ubiquitinated and degraded by the proteasome in lung epithelia. SARS-CoV-2 spike protein cellular internalization increased ACE2 protein abundance by decreasing its degradation. Using an siRNA library targeting 96 human DUBs, we identified UCHL1 as a putative regulator of ACE2 function as a viral receptor. Overexpressed UCHL1 preserved ACE2 protein abundance, whereas silencing of the DUB in cells destabilized ACE2 through increased polyubiquitination. A commercially available small molecule inhibitor of UCHL1 DUB activity decreased ACE2 protein concentrations coupled with inhibition of SARS-CoV-2 infection in epithelial cells. These findings describe a unique pathway of ACE2 regulation uncovering UCHL1 as a potential therapeutic target to modulate COVID-19 viral entry as a platform for future small molecule design and testing.

The E3 ligase subunit FBXO45 binds the interferon-λ receptor and promotes its degradation during influenza virus infection.

Influenza remains a major public health challenge, as the viral infection activates multiple biological networks linked to altered host innate immunity. Following infection, IFN-λ, a ligand crucial for the resolution of viral infections, is known to bind to its cognate receptor, IFNLR1, in lung epithelia. However, little is known regarding the molecular expression and regulation of IFNLR1. Here, we show that IFNLR1 is a labile protein in human airway epithelia that is rapidly degraded after influenza infection. Using an unbiased proximal ligation biotin screen, we first identified that the Skp-Cullin-F box E3 ligase subunit, FBXO45, binds to IFNLR1. We demonstrate that FBXO45, induced in response to influenza infection, mediates IFNLR1 protein polyubiquitination and degradation through the ubiquitin-proteasome system by docking with its intracellular receptor domain. Furthermore, we found ectopically expressed FBXO45 and its silencing in cells differentially regulated both IFNLR1 protein stability and interferon-stimulated gene expression. Mutagenesis studies also indicated that expression of a K319R/K320R IFNLR1 variant in cells exhibited reduced polyubiquitination, yet greater stability and proteolytic resistance to FBXO45 and influenza-mediated receptor degradation. These results indicate that the IFN-λ-IFNLR1 receptor axis is tightly regulated by the Skp-Cullin-F box ubiquitin machinery, a pathway that may be exploited by influenza infection as a means to limit antiviral responses.

MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics.

Identifying protein-protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an "abortive" biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.

Syrian hamsters as a model of lung injury with SARS-CoV-2 infection: Pathologic, physiologic, and detailed molecular profiling.

The acute respiratory distress syndrome (ARDS) is a common complication of severe COVID-19 (coronavirus disease 2019) caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection. Knowledge of molecular mechanisms driving host responses to SARS-CoV-2 is limited by the lack of reliable preclinical models of COVID-19 that recapitulate human illness. Further, existing COVID-19 animal models are not characterized as models of experimental acute lung injury (ALI) or ARDS. Acknowledging differences in experimental lung injury in animal models and human ARDS, here we systematically evaluate a model of experimental acute lung injury as a result of SARS-CoV-2 infection in Syrian golden hamsters. Following intranasal inoculation, hamsters demonstrate acute SARS-CoV-2 infection, viral pneumonia, and systemic illness but survive infection with clearance of virus. Hamsters exposed to SARS-CoV-2 exhibited key features of experimental ALI, including histologic evidence of lung injury, increased pulmonary permeability, acute inflammation, and hypoxemia. RNA sequencing of lungs indicated upregulation of inflammatory mediators that persisted after infection clearance. Lipidomic analysis demonstrated significant differences in hamster phospholipidome with SARS-CoV-2 infection. Lungs infected with SARS-CoV-2 showed increased apoptosis and ferroptosis. Thus, SARS-CoV-2 infected hamsters exhibit key features of experimental lung injury supporting their use as a preclinical model of COVID-19 ARDS.

Interferon Lambda Signaling in Macrophages Is Necessary for the Antiviral Response to Influenza.

Interferon lambda (IFNλ) signaling is a promising therapeutic target against viral infection in murine models, yet little is known about its molecular regulation and its cognate receptor, interferon lambda receptor 1 (IFNLR1) in human lung. We hypothesized that the IFNλ signaling axis was active in human lung macrophages. In human alveolar macrophages (HAMs), we observed increased IFNLR1 expression and robust increase in interferon-stimulated gene (ISG) expression in response to IFNλ ligand. While human monocytes express minimal IFNLR1, differentiation of monocytes into macrophages with macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) increased IFNLR1 mRNA, IFNLR1 protein expression, and cellular response to IFNλ ligation. Conversely, in mice, M-CSF or GM-CSF stimulated macrophages failed to produce ISGs in response to related ligands, IFNL2 or IFNL3, suggesting that IFNLR1 signaling in macrophages is species-specific. We next hypothesized that IFNλ signaling was critical in influenza antiviral responses. In primary human airway epithelial cells and precision-cut human lung slices, influenza infection substantially increased IFNλ levels. Pretreatment of both HAMs and differentiated human monocytes with IFNL1 significantly inhibited influenza infection. IFNLR1 knockout in the myeloid cell line, THP-1, exhibited reduced interferon responses to either direct or indirect exposure to influenza infection suggesting the indispensability of IFNLR1 for antiviral responses. These data demonstrate the presence of IFNλ - IFNLR1 signaling axis in human lung macrophages and a critical role of IFNλ signaling in combating influenza infection.

A mechanistic basis for the malignant progression of salivary gland tumors.

Salivary gland tumors are diverse neoplasms, likely reflecting differences in the tissue- and cell-of-origin. 80%-90% of tumors arising in the sublingual gland (SLG) are malignant, whereas the other major glands often form benign tumors. Owing to the lack of experimental models to explore the etiology of salivary gland tumors, the cellular and molecular bases of malignancy remain unknown. Here, we generated a murine model of HRASG12V-driven salivary gland tumors amenable to examine tumor onset and malignant progression. We found that HMGA2 marks the tumor onset, and transformed-SOX2+ stem/progenitor cells expand exclusively in SLG tumors. Lineage tracing experiments showed that SLG tumor cells undergo an extensive epithelial-mesenchymal transition (EMT) and TGF-ß-responding tumor cells are a source of mesenchymal tumor cells invading the surrounding stroma. This study advances our understanding of the mechanistic basis of salivary gland malignancy and may help combat this highly heterogeneous cancer.

Response to Comment on "Tumor-initiating cells establish an IL-33-TGF-ß niche signaling loop to promote cancer progression".

Kamphuis et al argue that macrophages accumulated in the proximity of tumor-initiating cells do not express the high-affinity immunoglobulin E receptor FcεRIα. Although we cannot exclude the possibility of nonspecific binding of anti-FcεRIα antibody (clone MAR-1), we provide evidence that macrophages in squamous cell carcinomas express FcεRIα and that IL-33 induces FcεRIα expression in bone marrow cell-derived macrophages.

Tumor-initiating cells establish an IL-33-TGF-ß niche signaling loop to promote cancer progression.

Targeting the cross-talk between tumor-initiating cells (TICs) and the niche microenvironment is an attractive avenue for cancer therapy. We show here, using a mouse model of squamous cell carcinoma, that TICs play a crucial role in creating a niche microenvironment that is required for tumor progression and drug resistance. Antioxidant activity in TICs, mediated by the transcription factor NRF2, facilitates the release of a nuclear cytokine, interleukin-33 (IL-33). This cytokine promotes differentiation of macrophages that express the high-affinity immunoglobulin E receptor FcεRIα and are in close proximity to TICs. In turn, these IL-33-responding FcεRIα+ macrophages send paracrine transforming growth factor ß (TGF-ß) signals to TICs, inducing invasive and drug-resistant properties and further upregulating IL-33 expression. This TIC-driven, IL-33-TGF-ß feedforward loop could potentially be exploited for cancer treatment.

ADAP1 promotes invasive squamous cell carcinoma progression and predicts patient survival.

Invasive squamous cell carcinoma (SCC) is aggressive cancer with a high risk of recurrence and metastasis, but the critical determinants of its progression remain elusive. Here, we identify ADAP1, a GTPase-activating protein (GAP) for ARF6 up-regulated in TGF-ß-responding invasive tumor cells, as a strong predictor of poor survival in early-stage SCC patients. Using a mouse model of SCC, we show that ADAP1 overexpression promotes invasive tumor progression by facilitating cell migration and breakdown of the basement membrane. We found that ADAP1-rich, TGF-ß-responding tumor cells exhibit cytoplasmic laminin localization, which correlated with the absence of laminin and type IV collagen from the pericellular basement membrane. Interestingly, although tumors overexpressing a GAP activity-deficient mutant of ADAP1 resulted in morphologically complex tumors, those tumor cells failed to breach the basement membrane. Moreover, Adap1 deletion in tumor cells ameliorated the basement membrane breakdown and had less invading cells in the stroma. Our study demonstrates that ADAP1 is a critical mediator of TGF-ß-induced cancer invasion and might be exploited for the treatment of high-risk SCC.