IFITM dependency of SARS-CoV-2 variants of concern

We have recently shown that a SARS-CoV-2 strain isolated in the Netherlands in February 2020 (NL-02-2020) hijacks interferon-induced transmembrane proteins, especially IFITM2, as entry cofactors for efficient infection. Here, we examined whether SARS-CoV-2 'variants of concern' (VOCs), including the currently dominating delta variant, maintained the dependency on IFITMs for efficient replication. Depletion of IFITM2 reduced viral RNA production from 31- (B.1.1.7) to 755-fold (P.1). In comparison, silencing of IFITM1 had little effect, while knock-down of IFITM3 resulted in an intermediate phenotype. Strikingly, silencing of IFITM2 generally reduced infectious virus production in Calu-3 cells to near background levels. An antibody directed against the N-terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells. In conclusion, endogenously expressed IFITM proteins (especially IFITM2) are important cofactors for replication of genuine SARS-CoV-2 VOCs, including the Delta variant.

Oxygen and Steroids Affect the Regulatory Role of Natriuretic Peptide Receptor-C on Surfactant Secretion by Type II Cells

Atrial natriuretic peptide (ANP) and its receptors Natriuretic peptide receptor (NPR)-A and NRP-C are all highly expressed in alveolar epithelial type II cells (AEC2s) in the late gestation ovine fetal lung and are dramatically decreased postnatally. However, of all the components, NPR-C stimulation inhibits ANP-mediated surfactant secretion. Since alveolar oxygen increases dramatically after birth, and steroids are administered to mothers antenatally to enhance surfactant lung maturity, we investigated the effects of O2 concentration and steroids on NPR-C-mediated surfactant secretion in AEC2s. NPR-C expression was highest at 5% O2, while being suppressed by 21% O2, in cultured mouse lung epithelial cells (MLE-15s) and/or human primary AEC2s. Surfactant protein-B (SP-B) was significantly elevated in media from both in vitro and ex-vivo culture at 13% O2 versus 21% O2 in the presence of ANP or terbutaline (TER). Both ANP and C-ANP (an NPR-C agonist) attenuated TER-induced SP-B secretion; this effect was reversed by dexamethasone (DEX) pretreatment in AEC2s and by transfection with NPR-C siRNA in MLE-15 cells. DEX markedly reduced AEC2 NPR-C expression, and pregnant ewes treated with betamethasone showed reduced ANP in fetal sheep lung fluid. These data suggest that elevated O2 downregulates AEC2 NPR-C, and that steroid-mediated NPR-C downregulation in neonatal lungs may provide a novel mechanism for their effect on perinatal surfactant production.

Expression of SARS-CoV-2 Entry Factors in Human Alveolar Type II Cells in Aging and Emphysema

Alveolar type II (ATII) cells proliferate and restore the injured epithelium. It has been described that SARS-CoV-2 infection causes diffuse alveolar damage in the lungs. However, host factors facilitating virus infection in ATII cells are not well known. We determined the SARS-CoV-2-related genes and protein expression using RT-PCR and Western blotting, respectively, in ATII cells isolated from young and elderly non-smokers, smokers, and ex-smokers. Cells were also obtained from lung transplants of emphysema patients. ACE2 has been identified as the receptor for SARS-CoV-2, and we found significantly increased levels in young and elderly smokers and emphysema patients. The viral entry depends on TMPRSS2 protease activity, and a higher expression was detected in elderly smokers and ex-smokers and emphysema patients. Both ACE2 and TMPRSS2 mRNA levels were higher in this disease in comparison with non-smokers. CD209L serves as a receptor for SARS-CoV-2, and we found increased levels in ATII cells obtained from smokers and in emphysema patients. Also, our data suggest CD209L regulation by miR142. Endoplasmic reticulum stress was detected in ATII cells in this disease. Our results suggest that upregulation of SARS-CoV-2 entry factors in ATII cells in aging, smokers, and emphysema patients may facilitate infection.

MORPHOMETRIC STUDY OF ISOLATED DOGIEL TYPE II CELLS OF THE INTERMUSCULAR PLEXUS

Aim of the study was to calculate the morphometric parameters of isolated Dogiel type II cells from the intermuscular plexus of the small intestine. Materials and methods: 3D models of the oval-shaped Dogiel type II cells from the intermuscular plexus of a rat (n = 1) were constructed and studied. Neurocytes were identified by the impregnation method on the frontal and horizontal sections of the wall of the small intestine. Results: The results showed that the total number of the nodes in the virtual model was 36534, and the mesh elements — 156595. The resulting 3D model of the cell and nucleus was reduced 900 times to obtain a threedimensional cell and nucleus with absolute dimensions, with a ratio of 1:1 to their true size. The volume of Dogiel type II cell was 2785.11 μm³, the volume of the nucleus was 647.7 μm³ and the volume of its perikaryon was 2785.11 μm³. Conclusion: Dogiel type II cells from the intermuscular plexus of the rat small intestine has an ovoid shaped threedimensional structure. These cells are flattened in transverse direction and elongated in longitudinal direction.

Evaluation of Stable LifeAct-mRuby2- and LAMP1-NeonGreen Expressing A549 Cell Lines for Investigation of Aspergillus fumigatus Interaction with Pulmonary Cells

Inhaled Aspergillus fumigatus spores can be internalized by alveolar type II cells. Cell lines stably expressing fluorescently labeled components of endocytic pathway enable investigations of intracellular organization during conidia internalization and measurement of the process kinetics. The goal of this report was to evaluate the methodological appliance of cell lines for studying fungal conidia internalization. We have generated A549 cell lines stably expressing fluorescently labeled actin (LifeAct-mRuby2) and late endosomal protein (LAMP1-NeonGreen) following an evaluation of cell-pathogen interactions in live and fixed cells. Our data show that the LAMP1-NeonGreen cell line can be used to visualize conidia co-localization with LAMP1 in live and fixed cells. However, caution is necessary when using LifeAct-mRuby2-cell lines as it may affect the conidia internalization dynamics.