Comparative spatial transcriptomic profiling of severe acute respiratory syndrome coronavirus 2 Delta and Omicron variants infections in the lungs of cynomolgus macaques.

Recently emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants are generally less pathogenic than previous strains. However, elucidating the molecular basis for pulmonary immune response alterations is challenging owing to the virus's heterogeneous distribution within complex tissue structure. Here, we revealed the spatial transcriptomic profiles of pulmonary microstructures at the SARS-CoV-2 infection site in the nine cynomolgus macaques upon inoculation with the Delta and Omicron variants. Delta- and Omicron-infected lungs had upregulation of genes involved in inflammation, cytokine response, complement, cell damage, proliferation, and differentiation pathways. Depending on the tissue microstructures (alveoli, bronchioles, and blood vessels), there were differences in the types of significantly upregulated genes in each pathway. Notably, a limited number of genes involved in cytokine and cell damage response were differentially expressed between bronchioles of the Delta- and Omicron-infection groups. These results indicated that despite a significant antigenic shift in SARS-CoV-2, the host immune response mechanisms induced by the variants were relatively consistent, with limited transcriptional alterations observed only in large airways. This study may aid in understanding the pathogenesis of SARS-CoV-2 and developing a clinical strategy for addressing immune dysregulation by identifying potential transcriptional biomarkers within pulmonary microstructures during infection with emerging variants.

Lung-on-a-Chip Models of the Lung Parenchyma.

Since the publication of the first lung-on-a-chip in 2010, research has made tremendous progress in mimicking the cellular environment of healthy and diseased alveoli. As the first lung-on-a-chip products have recently reached the market, innovative solutions to even better mimic the alveolar barrier are paving the way for the next generation lung-on-chips. The original polymeric membranes made of PDMS are being replaced by hydrogel membranes made of proteins from the lung extracellular matrix, whose chemical and physical properties exceed those of the original membranes. Other aspects of the alveolar environment are replicated, such as the size of the alveoli, their three-dimensional structure, and their arrangement. By tuning the properties of this environment, the phenotype of alveolar cells can be tuned, and the functions of the air-blood barrier can be reproduced, allowing complex biological processes to be mimicked. Lung-on-a-chip technologies also provide the possibility of obtaining biological information that was not possible with conventional in vitro systems. Pulmonary edema leaking through a damaged alveolar barrier and barrier stiffening due to excessive accumulation of extracellular matrix proteins can now be reproduced. Provided that the challenges of this young technology are overcome, there is no doubt that many application areas will benefit greatly.

Clathrin-Mediated Albumin Clearance in Alveolar Epithelial Cells of Murine Precision-Cut Lung Slices.

A hallmark of acute respiratory distress syndrome (ARDS) is an accumulation of protein-rich alveolar edema that impairs gas exchange and leads to worse outcomes. Thus, understanding the mechanisms of alveolar albumin clearance is of high clinical relevance. Here, we investigated the mechanisms of the cellular albumin uptake in a three-dimensional culture of precision-cut lung slices (PCLS). We found that up to 60% of PCLS cells incorporated labeled albumin in a time- and concentration-dependent manner, whereas virtually no uptake of labeled dextran was observed. Of note, at a low temperature (4 °C), saturating albumin receptors with unlabeled albumin and an inhibition of clathrin-mediated endocytosis markedly decreased the endocytic uptake of the labeled protein, implicating a receptor-driven internalization process. Importantly, uptake rates of albumin were comparable in alveolar epithelial type I (ATI) and type II (ATII) cells, as assessed in PCLS from a SftpcCreERT2/+: tdTomatoflox/flox mouse strain (defined as EpCAM+CD31-CD45-tdTomatoSPC-T1α+ for ATI and EpCAM+CD31-CD45-tdTomatoSPC+T1α- for ATII cells). Once internalized, albumin was found in the early and recycling endosomes of the alveolar epithelium as well as in endothelial, mesenchymal, and hematopoietic cell populations, which might indicate transcytosis of the protein. In summary, we characterize albumin uptake in alveolar epithelial cells in the complex setting of PCLS. These findings may open new possibilities for pulmonary drug delivery that may improve the outcomes for patients with respiratory failure.

Impaired cell-cell communication and axon guidance because of pulmonary hypoperfusion during postnatal alveolar development.

BACKGROUND: Pulmonary hypoperfusion is common in children with congenital heart diseases (CHDs) or pulmonary hypertension (PH) and causes adult pulmonary dysplasia. Systematic reviews have shown that some children with CHDs or PH have mitigated clinical outcomes with COVID-19. Understanding the effects of pulmonary hypoperfusion on postnatal alveolar development may aid in the development of methods to improve the pulmonary function of children with CHDs or PH and improve their care during the COVID-19 pandemic, which is characterized by cytokine storm and persistent inflammation. METHODS AND RESULTS: We created a neonatal pulmonary hypoperfusion model through pulmonary artery banding (PAB) surgery at postnatal day 1 (P1). Alveolar dysplasia was confirmed by gross and histological examination at P21. Transcriptomic analysis of pulmonary tissues at P7(alveolar stage 2) and P14(alveolar stage 4) revealed that the postnatal alveolar development track had been changed due to pulmonary hypoperfusion. Under the condition of pulmonary hypoperfusion, the cell-cell communication and axon guidance, which both determine the final number of alveoli, were lost; instead, there was hyperactive cell cycle activity. The transcriptomic results were further confirmed by the examination of axon guidance and cell cycle markers. Because axon guidance controls inflammation and immune cell activation, the loss of axon guidance may explain the lack of severe COVID-19 cases among children with CHDs or PH accompanied by pulmonary hypoperfusion. CONCLUSIONS: This study suggested that promoting cell-cell communication or supplementation with guidance molecules may treat pulmonary hypoperfusion-induced alveolar dysplasia, and that COVID-19 is less likely to cause a cytokine storm in children with CHD or PH accompanied by pulmonary hypoperfusion.

Histopathological pulmonary findings of survivors and autopsy COVID-19 cases: A bi-center study.

The coronavirus disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), evolved into a global pandemic. As ACE2 on the surface of alveolar cells of the lung epithelium is one of the potential target receptors for SARS-CoV-2, the respiratory symptoms are the most common presentation of COVID-19. The aim of our study was to investigate the morphological findings in lung tissue after being infected by SARS-CoV-2 and compare histopathologic changes in patients with COVID-19 infection history who died to those who survived. We analyzed lung tissue samples from 9 patients who died from COVID-19 and from 35 patients with COVID-19 infection history who survived and had undergone lung surgery for different reasons. Most of histopathological changes in autopsy and survivors' cases overlapped; however, they occurred with different frequency. The predominant histologic finding both in the case of the deceased and the survivors was patchy distribution of foamy macrophages in the alveolar spaces. In comparison with autopsy cases viral cytopathic-like changes in hyperplastic pneumocytes were rarely observed in the survivors' lung tissue. Pulmonary edema, fibrin deposition within alveoli, bronchopneumonia, small vessel thrombosis and type II pneumocyte hyperplasia were also more often observed within autopsy cases. Life-threatening complications such as hyaline membrane formations and diffuse alveolar damage were present only within the deceased, whereas changes requiring enough time to progress to the fibrotic phase, such as organizing pneumonia, bronchiolization of the alveoli, and interstitial fibrosis were observed in the lung parenchyma only in survivors. Additionally, 14 cases of pulmonary pneumo-hematocele in patients with COVID-19 infection history who survived were observed. It is a newly observed entity in the form of a cystic lesion formed by large accumulation of blood and fibrin between the collapsed and rejected lung parenchyma and/or present with air-fluid levels. The thin wall of pneumo-hematocele is formed by the inter lobar interstitial fibroconnective tissue and has no epithelial lining or bronchial wall elements. As the COVID-19 pandemic continues, new complications following SARS-CoV-2 infection are identified. Newly observed entity in patients with COVID-19 infection history who survived is pulmonary pneumo-hematocele. The appearance of these lesion has become increasingly frequent.

Wnt5a/ß-catenin axis is involved in the downregulation of AT2 lineage by PAI-1.

Failure to regenerate injured alveoli functionally and promptly causes a high incidence of fatality in coronavirus disease 2019 (COVID-19). How elevated plasminogen activator inhibitor-1 (PAI-1) regulates the lineage of alveolar type 2 (AT2) cells for re-alveolarization has not been studied. This study aimed to examine the role of PAI-1-Wnt5a-ß catenin cascades in AT2 fate. Dramatic reduction in AT2 yield was observed in Serpine1Tg mice. Elevated PAI-1 level suppressed organoid number, development efficiency, and total surface area in vitro. Anti-PAI-1 neutralizing antibody restored organoid number, proliferation and differentiation of AT2 cells, and ß-catenin level in organoids. Both Wnt family member 5A (Wnt5a) and Wnt5a-derived N-butyloxycarbonyl hexapeptide (Box5) altered the lineage of AT2 cells. This study demonstrates that elevated PAI-1 regulates AT2 proliferation and differentiation via the Wnt5a/ß catenin cascades. PAI-1 could serve as autocrine signaling for lung injury repair.

Vasculopathy and Increased Vascular Congestion in Fatal COVID-19 and Acute Respiratory Distress Syndrome.

Rationale: The leading cause of death in coronavirus disease 2019 (COVID-19) is severe pneumonia, with many patients developing acute respiratory distress syndrome (ARDS) and diffuse alveolar damage (DAD). Whether DAD in fatal COVID-19 is distinct from other causes of DAD remains unknown. Objective: To compare lung parenchymal and vascular alterations between patients with fatal COVID-19 pneumonia and other DAD-causing etiologies using a multidimensional approach. Methods: This autopsy cohort consisted of consecutive patients with COVID-19 pneumonia (n = 20) and with respiratory failure and histologic DAD (n = 21; non-COVID-19 viral and nonviral etiologies). Premortem chest computed tomography (CT) scans were evaluated for vascular changes. Postmortem lung tissues were compared using histopathological and computational analyses. Machine-learning-derived morphometric analysis of the microvasculature was performed, with a random forest classifier quantifying vascular congestion (CVasc) in different microscopic compartments. Respiratory mechanics and gas-exchange parameters were evaluated longitudinally in patients with ARDS. Measurements and Main Results: In premortem CT, patients with COVID-19 showed more dilated vasculature when all lung segments were evaluated (P = 0.001) compared with controls with DAD. Histopathology revealed vasculopathic changes, including hemangiomatosis-like changes (P = 0.043), thromboemboli (P = 0.0038), pulmonary infarcts (P = 0.047), and perivascular inflammation (P < 0.001). Generalized estimating equations revealed significant regional differences in the lung microarchitecture among all DAD-causing entities. COVID-19 showed a larger overall CVasc range (P = 0.002). Alveolar-septal congestion was associated with a significantly shorter time to death from symptom onset (P = 0.03), length of hospital stay (P = 0.02), and increased ventilatory ratio [an estimate for pulmonary dead space fraction (Vd); p = 0.043] in all cases of ARDS. Conclusions: Severe COVID-19 pneumonia is characterized by significant vasculopathy and aberrant alveolar-septal congestion. Our findings also highlight the role that vascular alterations may play in Vd and clinical outcomes in ARDS in general.

A case report of pediatric systemic lupus erythematosus with diffuse alveolar hemorrhage following COVID-19 infection: Causation, association, or chance?

RATIONALE: Diffuse alveolar hemorrhage (DAH) is a rare manifestation of childhood systemic lupus erythematosus (SLE) that can be life-threatening. Several reports have linked previous or concurrent coronavirus disease (COVID-19) infections with a high prevalence of autoimmune and autoinflammatory disorders. PATIENT CONCERNS: We report a case of a 13-year-old female who presented with DAH due to SLE 2 months after a laboratory-confirmed severe COVID-19 infection. DIAGNOSES: The patient was diagnosed with DAH due to SLE 2 months after a laboratory-confirmed severe COVID-19 infection. INTERVENTIONS AND OUTCOMES: The patient was treated with intravenous methylprednisolone pulse, broad-spectrum antibiotics, and supportive measures. In addition, she received 6 sessions of plasma exchange and maintenance methylprednisolone therapy (2 mg/kg/day). The patient then improved and was discharged on prednisolone, hydroxychloroquine, and azathioprine. LESSONS: We suggest plasmapheresis be considered a treatment for SLE-associated DAH in the context of active disease when conventional treatment has failed to induce a rapid response. In addition, further studies are needed to assess the role of COVID-19 as an autoimmune disease trigger, particularly for SLE.

[Features of the cell composition of inflammatory infiltrate in different phases of diffuse alveolar lung damage with COVID-19]. / Osobennosti kletochnogo sostava vospalitel'nogo infil'trata v raznye fazy diffuznogo al'veolyarnogo povrezhdeniya legkikh pri COVID-19.

BACKGROUND: Acute respiratory distress syndrome (ARDS) with COVID-19 has a worse prognosis than ARDS with other diseases. Mortality from ARDS with COVID-19 is 26.0 - 61.5%, and due to other causes - 35.3-37.2%. OBJECTIVE: To find of the correlation between polymorphonuclear leukocytes (PMNs), lymphocytes, and macrophages in the cellular composition of the inflammatory infiltrate at different stages and phases of diffuse alveolar damage (DAD) with COVID-19, analyzing the autopsy material. MATERIAL AND METHODS: The lung tissue of 25 patients who died from ARDS with COVID-19 without a secondary bacterial or mycotic infection, another thanatologically significant pathology of the lungs, was studied. To study the cellular composition of the inflammatory infiltrate and the dynamics of its changes a double immunohistochemical analysis of the expression of antibodies to CD15, CD3, and CD68 was used. RESULTS: The inflammatory infiltrate and intraalveolar exudate in the exudative phase of DAD was represented by 56.8% of PMNs (CD15-positive cells; hereinafter - the average value of the percentage of positive cells to the total number of cells of the inflammatory infiltrate), 6.9% - lymphocytes (CD3-positive cells) and 19.5% macrophages (CD68-positive cells). In the early stage of the proliferative phase: 14.1% PMNs, 38.7% lymphocytes and 13.5% macrophages. In the late stage of the proliferative phase: 11.3% PMNs, 14.5% lymphocytes and 39.3% macrophages. CONCLUSIONS: In the exudative phase of DAD a statistically significant predominance of PMN was revealed, which could determine the main volume of lung damage and the severity of ARDS with COVID-19. In the early stage of the proliferative phase of DAD, a statistically significant change in the composition of the inflammatory infiltrate was revealed to compare with the exudative phase: a significant decrease in the content of PMNs relative to the total number of cells in the inflammatory infiltrate; an increase in the number of lymphocytes, which is probably associated with the start of organization and repair processes. In the late stage of the proliferative phase of DAD, compared with its early stage, was revealed a statistically significant increase in the number of macrophages in ratio.

Function of epithelial stem cell in the repair of alveolar injury.

Alveoli are the functional units of blood-gas exchange in the lung and thus are constantly exposed to outside environments and frequently encounter pathogens, particles and other harmful substances. For example, the alveolar epithelium is one of the primary targets of the SARS-CoV-2 virus that causes COVID-19 lung disease. Therefore, it is essential to understand the cellular and molecular mechanisms by which the integrity of alveoli epithelial barrier is maintained. Alveolar epithelium comprises two cell types: alveolar type I cells (AT1) and alveolar type II cells (AT2). AT2s have been shown to function as tissue stem cells that repair the injured alveoli epithelium. Recent studies indicate that AT1s and subgroups of proximal airway epithelial cells can also participate alveolar repair process through their intrinsic plasticity. This review discussed the potential mechanisms that drive the reparative behaviors of AT2, AT1 and some proximal cells in responses to injury and how an abnormal repair contributes to some pathological conditions.

[Pattern of mortality and pathomorphology patterns in lungs according to the materials of forensic medical examination of COVID-19 deceased (March-December 2020)]. / Struktura smertnosti i patomorfologicheskaya kharakteristika legkikh po materialam sudebno-meditsinskogo issledovaniya umershikh ot COVID-19 (mart­dekabr' 2020 g.).

The objective was to study the pattern of mortality and pathomorphological changes in lungs according to the forensic medical investigation of the corpses of the persons who died at the age of 37 to 60 years and older from COVID-19 outside healthcare facilities with no symptoms according to the catamnesis data. Eighty-nine autopsy samples taken from patients with COVID-19 confirmed by polymerase chain reaction (PCR) from the Thanatology Department of the Republican Center of Forensic Medicine for March-December 2020 were analyzed. Pathomorphological changes in the lungs corresponded to different phases of diffuse alveolar injury (DAI) development. During the exudative phase, edema, sloughing of the alveolar epithelium, presence of hyaline membranes, and thrombohemorrhagic events were predominant. In the proliferative phase, fibrin and fibrin-like deposits in alveoli, interalveolar septa thickening, excessive growth of conjunctive tissue in the interalveolar septa, pronounced fatty liver dystrophy, severe myocardium edema were observed.

Anatomopathological Aspects and Clinical Correlation of COVID-19: A Systematic Review.

OBJECTIVE: The objective of this systematic review was to analyze the main morphofunctional changes in the involvement of multiple organs in patients infected with SARS-CoV-2, correlating anatomopathological findings with the clinical picture. METHODS: The present study selected articles through electronic search of indexed journals in the PubMed and SciVerse Scopus databases, from December 2019 to May 2020, using the keywords "autopsy," "pathogenicity," and "COVID-19." Two hundred nine articles were identified, and the full texts of 18 articles were reviewed, 5 of them being selected for this review. RESULTS: The ACE2 receptor plays a role in introducing viral material into the cell, having high expression in type II alveoli. Histopathological analyzes of the lungs of patients with COVID-19 show that SARS-CoV-2 produces, in this organ, in addition to an inflammatory process, a diffuse alveolar damage (DAD), which can cause acute respiratory distress syndrome (ARDS). Macroscopically, the lungs become heavier, firmer, and redder. The clinical features of these patients are variable; the most common are respiratory symptoms associated with fever, myalgia, or fatigue. CONCLUSION: The observations points to the consensus that the lungs are the main targets of COVID-19, with morphological and functional changes of interest, including important sequels, and presenting diffuse alveolar damage as a substrate for an unfavorable outcome with ARDS. Changes in micro and macroscopic levels corroborate to the clinical progression of the disease and that these alterations are not specific, which ratify, in addition to the anatomopathological examination, a need to use the association of clinical and epidemiological data for diagnostic confirmation.

Abnormal respiratory progenitors in fibrotic lung injury.

Recent advances in single-cell RNA sequencing (scRNA-seq) and epithelium lineage labeling have yielded identification of multiple abnormal epithelial progenitor populations during alveolar type 2 (ATII) cell differentiation into alveolar type 1 (ATI) cells during regenerative lung post-fibrotic injury. These abnormal cells include basaloid/basal-like cells, ATII transition cells, and persistent epithelial progenitors (PEPs). These cells occurred and accumulated during the regeneration of distal airway and alveoli in response to both chronic and acute pulmonary injury. Among the alveolar epithelial progenitors, PEPs express a distinct Krt8+ phenotype that is rarely found in intact alveoli. However, post-injury, the Krt8+ phenotype is seen in dysplastic epithelial cells. Fully understanding the characteristics and functions of these newly found, injury-induced abnormal behavioral epithelial progenitors and the signaling pathways regulating their phenotype could potentially point the way to unique therapeutic targets for fibrosing lung diseases. This review summarizes recent advances in understanding these epithelial progenitors as they relate to uncovering regenerative mechanisms.

Studying the clinical, radiological, histological, microbiological, and immunological evolution during the different COVID-19 disease stages using minimal invasive autopsy.

The WHO defines different COVID-19 disease stages in which the pathophysiological mechanisms differ. We evaluated the characteristics of these COVID-19 disease stages. Forty-four PCR-confirmed COVID-19 patients were included in a prospective minimal invasive autopsy cohort. Patients were classified into mild-moderate (n = 4), severe-critical (n = 32) and post-acute disease (n = 8) and clinical, radiological, histological, microbiological and immunological data were compared. Classified according to Thoracic Society of America, patients with mild-moderate disease had no typical COVID-19 images on CT-Thorax versus 71.9% with typical images in severe-critical disease and 87.5% in post-acute disease (P < 0.001). Diffuse alveolar damage was absent in mild-moderate disease but present in 93.8% and 87.5% of patients with severe-critical and post-acute COVID-19 respectively (P = 0.002). Other organs with COVID-19 related histopathological changes were liver and heart. Interferon-γ levels were significantly higher in patients with severe-critical COVID-19 (P = 0.046). Anti-SARS CoV-2 IgG was positive in 66%, 40.6% and 87.5% of patients with mild-moderate, severe-critical and post-acute COVID-19 respectively (n.s.). Significant differences in histopathological and immunological characteristics between patients with mild-moderate disease compared to patients with severe-critical disease were found, whereas differences between patients with severe-critical disease and post-acute disease were limited. This emphasizes the need for tailored treatment of COVID-19 patients.

Channels and Transporters of the Pulmonary Lamellar Body in Health and Disease.

The lamellar body (LB) of the alveolar type II (ATII) cell is a lysosome-related organelle (LRO) that contains surfactant, a complex mix of mainly lipids and specific surfactant proteins. The major function of surfactant in the lung is the reduction of surface tension and stabilization of alveoli during respiration. Its lack or deficiency may cause various forms of respiratory distress syndrome (RDS). Surfactant is also part of the innate immune system in the lung, defending the organism against air-borne pathogens. The limiting (organelle) membrane that encloses the LB contains various transporters that are in part responsible for translocating lipids and other organic material into the LB. On the other hand, this membrane contains ion transporters and channels that maintain a specific internal ion composition including the acidic pH of about 5. Furthermore, P2X4 receptors, ligand gated ion channels of the danger signal ATP, are expressed in the limiting LB membrane. They play a role in boosting surfactant secretion and fluid clearance. In this review, we discuss the functions of these transporting pathways of the LB, including possible roles in disease and as therapeutic targets, including viral infections such as SARS-CoV-2.

SARS-CoV-2-triggered mast cell rapid degranulation induces alveolar epithelial inflammation and lung injury.

SARS-CoV-2 infection-induced hyper-inflammation links to the acute lung injury and COVID-19 severity. Identifying the primary mediators that initiate the uncontrolled hypercytokinemia is essential for treatments. Mast cells (MCs) are strategically located at the mucosa and beneficially or detrimentally regulate immune inflammations. In this study, we showed that SARS-CoV-2-triggered MC degranulation initiated alveolar epithelial inflammation and lung injury. SARS-CoV-2 challenge induced MC degranulation in ACE-2 humanized mice and rhesus macaques, and a rapid MC degranulation could be recapitulated with Spike-RBD binding to ACE2 in cells; MC degranulation altered various signaling pathways in alveolar epithelial cells, particularly, the induction of pro-inflammatory factors and consequential disruption of tight junctions. Importantly, the administration of clinical MC stabilizers for blocking degranulation dampened SARS-CoV-2-induced production of pro-inflammatory factors and prevented lung injury. These findings uncover a novel mechanism for SARS-CoV-2 initiating lung inflammation, and suggest an off-label use of MC stabilizer as immunomodulators for COVID-19 treatments.

Immunoreactivity of the SARS-CoV-2 entry proteins ACE-2 and TMPRSS-2 in murine models of hormonal manipulation, ageing, and cardiac injury.

Previous work indicates that SARS-CoV-2 virus entry proteins angiotensin-converting enzyme 2 (ACE-2) and the cell surface transmembrane protease serine 2 (TMPRSS-2) are regulated by sex hormones. However, clinical studies addressing this association have yielded conflicting results. We sought to analyze the impact of sex hormones, age, and cardiovascular disease on ACE-2 and TMPRSS-2 expression in different mouse models. ACE-2 and TMPRSS-2 expression was analyzed by immunostaining in a variety of tissues obtained from FVB/N mice undergoing either gonadectomy or sham-surgery and being subjected to ischemia-reperfusion injury or transverse aortic constriction surgery. In lung tissues sex did not have a significant impact on the expression of ACE-2 and TMPRSS-2. On the contrary, following myocardial injury, female sex was associated to a lower expression of ACE-2 at the level of the kidney tubules. In addition, after myocardial injury, a significant correlation between younger age and higher expression of both ACE-2 and TMPRSS-2 was observed for lung alveoli and bronchioli, kidney tubules, and liver sinusoids. Our experimental data indicate that gonadal hormones and biological sex do not alter ACE-2 and TMPRSS-2 expression in the respiratory tract in mice, independent of disease state. Thus, sex differences in ACE-2 and TMPRSS-2 protein expression observed in mice may not explain the higher disease burden of COVID-19 among men.