Some patients infected with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) develop severe pneumonia and the acute respiratory distress syndrome (ARDS) 1 . Distinct clinical features in these patients have led to speculation that the immune response to virus in the SARS-CoV-2-infected alveolus differs from other types of pneumonia 2 . We collected bronchoalveolar lavage fluid samples from 88 patients with SARS-CoV-2-induced respiratory failure and 211 patients with known or suspected pneumonia from other pathogens and subjected them to flow cytometry and bulk transcriptomic profiling. We performed single-cell RNA-seq on 10 bronchoalveolar lavage fluid samples collected from patients with severe COVID-19 within 48 hours of intubation. In the majority of patients with SARS-CoV-2 infection, the alveolar space was persistently enriched in T cells and monocytes. Bulk and single-cell transcriptomic profiling suggested that SARS-CoV-2 infects alveolar macrophages, which in turn respond by producing T cell chemoattractants. These T cells produce interferon-gamma to induce inflammatory cytokine release from alveolar macrophages and further promote T cell activation. Collectively, our results suggest that SARS-CoV-2 causes a slowly-unfolding, spatially limited alveolitis in which alveolar macrophages harboring SARS-CoV-2 and T cells form a positive feedback loop that drives persistent alveolar inflammation.

We previously described the phenomenon of retinal ischemic preconditioning (IPC) and we have shown the role of various signaling proteins in the protective pathways, including the mitogen-activated protein kinase p38. In this study we examined the role in IPC of mitogen-activated protein kinase phosphatase-1 (MKP-1), which inactivates p38. Ischemia was produced by elevation of intraocular pressure above systolic arterial blood pressure in adult Wistar rats. Preconditioning was produced by transient retinal ischemia for 5 min, 24 h prior to ischemia. Small interfering RNA (siRNA) to MKP-1 or a control non-silencing siRNA, was injected into the vitreous 6 h prior to IPC. Recovery was assessed by electroretinography (ERG) and histology. The a- and b-waves, and oscillatory potentials (OPs), measured before and 1 week after ischemia, were then normalized relative to pre-ischemic baseline, and corrected for diurnal variation in the normal non-ischemic eye. The P2, or post-photoreceptor component of the ERG (which reflects function of the rod bipolar cells in the inner retina), was derived using the Hood-Birch model. MKP-1 was localized in specific retinal cells using immunohistochemistry; levels of mitogen-activated protein kinases were measured using Western blotting. Injection of siRNA to MKP-1 significantly attenuated the protective effect of IPC as reflected by decreased recovery of the electroretinogram a- and b-waves and the P2 after ischemia. The injection of siRNA to MKP-1 reduced the number of cells in the retinal ganglion cell and outer nuclear layers after IPC and ischemia. Blockade of MKP-1 by siRNA also increased the activation of p38 at 24 h following IPC. MKP-1 siRNA did not alter the levels of phosphorylated jun N-terminal kinase (JNK) or extracellular signal-regulated kinase (ERK) after IPC. The results suggest the involvement of dual-specificity phosphatase MKP-1 in IPC and that MKP-1 is involved in IPC by regulating levels of activated MAPK p38.

Ischemic pre-conditioning (IPC) provides neuroprotection in the rat retina from the damaging effects of severe ischemia. Recently, neuroprotection by retinal ischemic post-conditioning (Post-C), i.e., transient ischemia after more lengthy, damaging ischemia, was described, but its mechanisms are not yet known. One possible explanation of the effectiveness of Post-C is that it augments intrinsic neuroprotective mechanisms initiated during ischemia. Increasing duration of the damaging ischemic insult may therefore impact the effectiveness of Post-C. IPC, in contrast, sets in motion a series of neuroprotective events prior to the onset of ischemia. Thus, IPC and Post-C may operate by differing mechanisms. Accordingly, we examined the effect of retinal ischemic duration on post-ischemic outcome in vivo in rats after adding Post-C, and the impact of combining pre- and post-conditioning. Recovery after ischemia performed 24 h after IPC, or after Post-C performed 5 min after ischemia ended, was assessed functionally (electroretinography) and histologically at 7 days after ischemia. Durations of ischemia of 45 and 55 min were studied. Since recovery with IPC or Post-C alone, with 55 min of ischemia, did not achieve the same degree of effect (i.e., not complete recovery) exhibited in our previous studies of IPC using a different ischemia model, we also combined IPC and Post-C to test the hypothesis of the possible additive effects of the IPC and Post-C. We found that the recovery after Post-C was enhanced to a greater degree when ischemia was of longer duration. Post-C led to greater post-ischemic recovery compared to IPC. Both IPC and Post-C also attenuated structural damage to the retina. Contrary to our hypothesis, IPC and Post-C did not combine to enhance recovery after ischemia. In earlier studies, IPC attenuated post-ischemic apoptosis. To begin to examine the mechanism of Post-C, we studied its impact on apoptosis following ischemia. We examined apoptosis by determining the percentage of TUNEL-positive cells at 24 h after ischemia. Post-C attenuated apoptosis, but when combined with IPC, TUNEL was similar in the combined group to that of ischemia alone. We also examined the role of the recruitment of an inflammatory response in ischemia and Post-C. We found that inflammatory markers increased by ischemia were not altered by Post-C. We conclude that Post-C effectiveness depends upon the duration of ischemia; Post-C is not additive with IPC, and Post-C functions, in part, by preventing apoptotic damage to the inner retina. Post-C has considerable promise for clinical translation to eye diseases that cause blindness by ischemia.

In previous studies, it was shown that post-conditioning, a transient period of brief ischemia following prolonged severe ischemia in the retina, could provide significant improvement in post-ischemic recovery, attenuation of cell loss, and decreased apoptosis. These studies showed that post-conditioning effectively prevented damage after retinal ischemia when it was instituted early (within one hour) in the post-ischemic period. While post-ischemic conditioning holds high promise of clinical translation, patients often present late after the onset of retinal ischemia and therefore immediate application of this anti-ischemic maneuver is generally not feasible. In this study, we examined the hypothesis that application of a post-conditioning stimulus at 24 h or greater following the end of prolonged ischemia would decrease the extent of ischemic injury. Ischemia was induced in rat retina in vivo. Recovery after ischemia followed by 5 minutes of post-conditioning brief ischemia 24 or 48 h after prolonged ischemia was assessed functionally (electroretinography) and histologically at 7 days after ischemia and post-conditioning or sham post-conditioning. We found that the brief ischemic stimulus applied 24, but not 48 h after prolonged ischemia significantly improved functional recovery and decreased histological damage induced by prolonged ischemia. We conclude that within a defined time window, delayed post-ischemic conditioning ameliorated post-ischemic injury in rats. Compared to earlier studies, the present work demonstrates for the first time the novel ability of a significantly delayed ischemic stimulus to provide robust neuroprotection in the retina following ischemia.

The complex cellular heterogeneity of the lung poses a unique challenge to researchers in the field. While the use of bulk RNA sequencing has become a ubiquitous technology in systems biology, the technique necessarily averages out individual contributions to the overall transcriptional landscape of a tissue. Single-cell RNA sequencing (scRNA-seq) provides a robust, unbiased survey of the transcriptome comparable to bulk RNA sequencing while preserving information on cellular heterogeneity. In just a few years since this technology was developed, scRNA-seq has already been adopted widely in respiratory research and has contributed to impressive advancements such as the discoveries of the pulmonary ionocyte and of a profibrotic macrophage population in pulmonary fibrosis. In this review, we discuss general technical considerations when considering the use of scRNA-seq and examine how leading investigators have applied the technology to gain novel insights into respiratory biology, from development to disease. In addition, we discuss the evolution of single-cell technologies with a focus on spatial and multi-omics approaches that promise to drive continued innovation in respiratory research.