Impaired local intrinsic immunity to SARS-CoV-2 infection in severe COVID-19

Infection with SARS-CoV-2, the virus that causes COVID-19, can lead to severe lower respiratory illness including pneumonia and acute respiratory distress syndrome, which can result in profound morbidity and mortality. However, many infected individuals are either asymptomatic or have isolated upper respiratory symptoms, which suggests that the upper airways represent the initial site of viral infection, and that some individuals are able to largely constrain viral pathology to the nasal and oropharyngeal tissues. Which cell types in the human nasopharynx are the primary targets of SARS-CoV-2 infection, and how infection influences the cellular organization of the respiratory epithelium remains incompletely understood. Here, we present nasopharyngeal samples from a cohort of 35 individuals with COVID-19, representing a wide spectrum of disease states from ambulatory to critically ill, as well as 23 healthy and intubated patients without COVID-19. Using standard nasopharyngeal swabs, we collected viable cells and performed single-cell RNA-sequencing (scRNA-seq), simultaneously profiling both host and viral RNA. We find that following infection with SARS-CoV-2, the upper respiratory epithelium undergoes massive reorganization: secretory cells diversify and expand, and mature epithelial cells are preferentially lost. Further, we observe evidence for deuterosomal cell and immature ciliated cell expansion, potentially representing active repopulation of lost ciliated cells through coupled secretory cell differentiation. Epithelial cells from participants with mild/moderate COVID-19 show extensive induction of genes associated with anti-viral and type I interferon responses. In contrast, cells from participants with severe lower respiratory symptoms appear globally muted in their anti-viral capacity, despite substantially higher local inflammatory myeloid populations and equivalent nasal viral loads. This suggests an essential role for intrinsic, local epithelial immunity in curbing and constraining viral-induced pathology. Using a custom computational pipeline, we characterized cell-associated SARS-CoV-2 RNA and identified rare cells with RNA intermediates strongly suggestive of active replication. Both within and across individuals, we find remarkable diversity and heterogeneity among SARS-CoV-2 RNA+ host cells, including developing/immature and interferon-responsive ciliated cells, KRT13+ "hillock"-like cells, and unique subsets of secretory, goblet, and squamous cells. Finally, SARS-CoV-2 RNA+ cells, as compared to uninfected bystanders, are enriched for genes involved in susceptibility (e.g., CTSL, TMPRSS2) or response (e.g., MX1, IFITM3, EIF2AK2) to infection. Together, this work defines both protective and detrimental host responses to SARS-CoV-2, determines the direct viral targets of infection, and suggests that failed anti-viral epithelial immunity in the nasal mucosa may underlie the progression to severe COVID-19.

Functional regulation of the cardiac ryanodine receptor by suramin and calmodulin involves multiple binding sites.

Suramin and structurally related compounds increase not only the open probability (P(o)) of ryanodine receptor (RyR) channels but also the single-channel conductance in a unique characteristic manner. In this report, we examine the mechanisms underlying the complex changes to cardiac RyR channel function caused by suramin and the evidence that these changes result from an interaction with calmodulin (CaM) binding sites. In the presence of 100 microM cytosolic Ca(2+), we demonstrate that suramin exerts a triphasic effect on P(o), indicating the presence of high-, intermediate-, and low-affinity suramin binding sites. The effects of suramin binding to high-affinity sites are Ca(2+)-dependent; P(o) is decreased and seems to result from a reduction in the sensitivity of the channel to cytosolic Ca(2+). We suggest that this site is the CaM inhibition site. Suramin also binds to intermediate-affinity sites that mediate an increase in P(o) and an increase in conductance. Cytosolic Ca(2+) is not an absolute requirement for the effects mediated via intermediate-affinity suramin sites. The suramin-induced increase in P(o) and conductance are both concentration-dependent. The correlation between the increase in P(o) and increase in conductance indicates that the binding events which produce an increase in P(o) also lead to an increase in conductance and, because the effect is concentration-dependent, multiple suramin molecules must bind to produce the maximum effect. The low-affinity suramin binding sites are inhibition sites and mediate a reduction in P(o) caused by changes to both open and closed lifetimes.

DIDS modifies the conductance, gating, and inactivation mechanisms of the cardiac ryanodine receptor.

The effects of the covalent modifier of amino groups, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on the single-channel properties of purified sheep cardiac ryanodine receptors (RyR) incorporated into planar phospholipid bilayers were investigated. DIDS increased single-channel conductance and open probability (P(o)) and induced unique modifications to the voltage-dependence of gating. The effects of DIDS on conduction and gating were irreversible within the time scale of the experiments, and both effects were dependent on the permeant ion. DIDS induced a greater increase in conductance with Ca(2+) (20%) compared with K(+) (8%) as the permeant ion. After modification by DIDS, all channels could be rapidly inactivated in a voltage-dependent manner. The open probability of the DIDS-modified channel decreased with increasing positive or negative transmembrane potentials; however, inactivation was only observed at negative potentials. Our results demonstrate that inactivation of RyR channels is dependent on the ligand activating the channel, and this will have consequences for the control and termination of sarcoplasmic reticulum Ca(2+) release in cardiac cells.