Polarised human alveolar epithelia enable identification of dichloroacetate as an effective drug against respiratory viruses (preprint)

Respiratory viral infections are a significant cause of morbidity and mortality worldwide. The COVID-19 pandemic has highlighted the lack of chemotherapeutic tools available for fighting emerging viruses and the need to focus on preclinical models that better recapitulate human disease. We performed a comparative analysis of inhibitors of the PI3K/AKT/mTOR pathway, which is involved in virus-induced metabolic reprogramming, since strategies aimed at identifying cellular targets could serve to combat diverse viruses and hamper the development of resistance. Tests were performed in two human cell lines, MRC5 lung fibroblasts and Huh7 hepatoma cells, and the results showed that the inhibitors had markedly different effects on energy metabolism and antiviral activity. Thus, dichloroacetate (DCA) has potent antiviral activity against HCoV-229E in MRC5 cells but not in Huh7 cells, suggesting that the screening model is more critical than previously assumed. DCA was then tested in polarized human alveolar epithelia in air-liquid interface, a 3D model used to study respiratory infections. DCA reduced the viral progeny of HCoV-229E, SARS-CoV-2 and respiratory syncytial virus by 2-3 orders of magnitude, and it was effective even when applied once infection had been established. Although DCA has previously been shown to be effective against other viruses, suggesting that it could be a broad-spectrum antiviral, our experiments reinforce the need to use physiologically appropriate disease models to screen antiviral compound. 

The logic of coronavirus infection: revealing the heterogeneity of disease progression and treatment outcomes in COVID patients (preprint)

Pneumonia caused by coronavirus infection is a self-limiting disease. Its progression and prognosis are highly heterogeneous among people of different ages, genders, and living with different life styles. Such heterogeneity also exists in treatment outcomes of different patients. Various physiological and pathological factors, such as renewal of pulmonary cell, number of entry receptor and viral replication, have been identified linking to the development of the disease. However, it is still unclear how these factors collectively establish a causal relationship in the course of disease progression. In this study, we built a mechanistic model to explain the dynamics of infection and progression of coronavirus disease. We modeled how the interaction of pulmonary cells determine the dynamics of disease progression by characterizing the temporal dynamics of viral load, infected and health alveolar cells, and dysfuctional alveolar cells. The viral and cellular dynamics captured different stages of clinical manifestations in individual patient during disease progression: the incubation period, mild symptom period, and severe period. We further simulated clinical interference at different stages of disease progression. The results showed that some medical interventions show no improvement either in reducing the recovery rate nor shortening the recovery time. Our theoretical framework may provide a mechanistic explanation at the systems level for the progression and prognosis of coronavirus disease as well as other similar respiratory tract diseases.

Targeting G9a translational mechanism of SARS-CoV-2 pathogenesis for multifaceted therapeutics of COVID-19 and its sequalae (preprint)

By largely unknown mechanism(s), SARS-CoV-2 hijacks the host translation apparatus to promote COVID-19 pathogenesis. We report that the histone methyltransferase G9a noncanonically regulates viral hijacking of the translation machinery to bring about COVID-19 symptoms of hyperinflammation, lymphopenia, and blood coagulation. Chemoproteomic analysis of COVID-19 patient peripheral mononuclear blood cells (PBMC) identified enhanced interactions between SARS-CoV-2-upregulated G9a and distinct translation regulators, particularly the N6-methyladenosine (m6A) RNA methylase METTL3. These interactions with translation regulators implicated G9a in translational regulation of COVID-19. Inhibition of G9a activity suppressed SARS-CoV-2 replication in human alveolar epithelial cells. Accordingly, multi-omics analysis of the same alveolar cells identified SARS-CoV-2-induced changes at the transcriptional, m6A-epitranscriptional, translational, and post-translational (phosphorylation or secretion) levels that were reversed by inhibitor treatment. As suggested by the aforesaid chemoproteomic analysis, these multi-omics-correlated changes revealed a G9a-regulated translational mechanism of COVID-19 pathogenesis in which G9a directs translation of viral and host proteins associated with SARS-CoV-2 replication and with dysregulation of host response. Comparison of proteomic analyses of G9a inhibitor-treated, SARS-CoV-2 infected cells, or ex vivo culture of patient PBMCs, with COVID-19 patient data revealed that G9a inhibition reversed the patient proteomic landscape that correlated with COVID-19 pathology/symptoms. These data also indicated that the G9a-regulated, inhibitor-reversed, translational mechanism outperformed G9a-transcriptional suppression to ultimately determine COVID-19 pathogenesis and to define the inhibitor action, from which biomarkers of serve symptom vulnerability were mechanistically derived. This cell line-to-patient conservation of G9a-translated, COVID-19 proteome suggests that G9a inhibitors can be used to treat patients with COVID-19, particularly patients with long-lasting COVID-19 sequelae.

Biological Effects of Corticosteroids on Pneumococcal Pneumonia in Mice and Humans (preprint)

Background: Streptococcus pneumoniae is the most common bacterial cause of community acquired pneumonia and the acute respiratory distress syndrome (ARDS). Some clinical trials have demonstrated a beneficial effect of corticosteroid therapy in community acquired pneumonia, COVID-19, and ARDS, but the mechanisms of this benefit remain unclear. The objective of this study was to investigate the effects of corticosteroids on the pulmonary biology of pneumococcal pneumonia in an observational cohort of mechanically ventilated patients and in a mouse model of bacterial pneumonia with Streptococcus pneumoniae. Methods: We studied gene expression with lower respiratory tract transcriptomes from a cohort of mechanically ventilated patients and in mice. We also carried out comprehensive physiologic, biochemical, and histological analyses in mice to identify the mechanisms of lung injury in Streptococcus pneumoniae with and without adjunctive steroid therapy. Results: Transcriptomic analysis identified pleiotropic effects of steroid therapy on the lower respiratory tract in critically ill patients with pneumococcal pneumonia, findings that were reproducible in mice. In mice with pneumonia, dexamethasone in combination with ceftriaxone reduced (1) pulmonary edema formation, (2) alveolar protein permeability, (3) proinflammatory cytokine release, (4) histopathologic lung injury score, and (5) hypoxemia but did not increase bacterial burden. Conclusions: The gene expression studies in patients and in the mice support the clinical relevance of the mouse studies, which replicate several features of pneumococcal pneumonia and steroid therapy in humans. In combination with appropriate antibiotic therapy in mice, treatment of pneumococcal pneumonia with steroid therapy reduced hypoxemia, pulmonary edema, lung permeability, and histologic criteria of lung injury, and also altered inflammatory responses at the protein and gene expression level. The results from these studies provide evidence for the mechanisms that may explain the beneficial effects of glucocorticoid therapy in patients with community acquired pneumonia from Streptococcus Pneumoniae.

Cytoarchitecture of SARS-CoV-2 infected hamster lungs by X-ray phase contrast tomography: imaging workflow and classification for drug testing (preprint)

X-ray Phase Contrast Tomography (XPCT) based on wavefield propagation has been established as a high resolution three-dimensional (3D) imaging modality, suitable to reconstruct the intricate structure of soft tissues, and the corresponding pathological alterations. However, for biomedical research, more is needed than 3D visualisation and rendering of the cytoarchitecture in a few selected cases. First, the throughput needs to be increased to cover a statistically relevant number of samples. Second, the cytoarchitecture has to be quantified in terms of morphometric parameters, independent of visual impression. Third, dimensionality reduction and classification are required for identification of effects and interpretation of results. To address these challenges, we here design and implement a novel integrated and high throughput XPCT imaging and analysis workflow for 3D histology, pathohistology and drug testing. Our approach uses semi-automated data acquisition, reconstruction and statistical quantification. We demonstrate its capability for the example of lung pathohistology in Covid-19. Using a small animal model, different Covid-19 drug candidates are administered after infection and tested in view of restoration of the physiological cytoarchitecture, specifically the alveolar morphology. To this end, we then use morphometric parameter determination followed by a dimensionality reduction and classification based on optimal transport. This approach allows efficient discrimination between physiological and pathological lung structure, thereby providing quantitative insights into the pathological progression and partial recovery due to drug treatment. Finally, we stress that the XPCT image chain implemented here only used synchrotron radiation for validation, while the data used for analysis was recorded with laboratory µCT radiation, more easily accessible for pre-clinical research.

Could SP-A and SP-D Serum Levels Predict COVID-19 Severity? (preprint)

Given the various clinical manifestations that characterize COVID-19, the scientific community is constantly searching for biomarkers with prognostic value. SP-A and SP-D collectins play a crucial role in ensuring proper alveolar function and an alteration of their serum levels have been reported in several pulmonary diseases characterized by ARDS and pulmonary fibrosis. Considering that such clinical manifestations can also occur during SARS-CoV-2 infection, we wondered if these collectins could act as prognostic markers. In this regard, serum levels of SP-A and SP-D were measured by enzyme immunoassay in patients with SARS-CoV-2 infection (n=51) at admission (T0) and after 7 days (T1) and compared with healthy donors (n=11). SP-D increased in COVID-19 patients compared to healthy controls during the early phases of infection, while a significant reduction was observed at T1. Stratifying SARS-CoV-2 patients according to disease severity, increased serum SP-D levels were observed in severe compared to mild patients. In the light of these results SP-D, but not SP-A, seems to be an eligible marker of COVID-19 pneumonia and the early detection of SP-D serum levels could be crucial for a preventive clinical management

Bilateral spontaneous pneumothorax as a late complication of COVID-19, a case report (preprint)

The first case of sever pneumonia caused by coronavirus (COVID-19) was detected in Wuhan, China in December 2019 and spread rapidly around the world. Pneumothorax has been reported as an uncommon complication following COVID-19 infection which caused by alveolar rupture, air leakage and interstitial emphysema after alveolar damages. Bilateral pneumothorax is also an uncommon life threatening complication induced by COVID-19 which has to be considered in patients present with late sudden dyspnea after Coronavirus infection. In this case report, we are presenting a patient with mild COVID-19 pneumonia with a left massive pneumothorax following a late sudden dyspnea in third week of the disease, during the hospitalization, in addition to aggravating the respiratory condition, right pneumothorax was also added.

Whole Pulmonary Lavage: Treating Pulmonary Alveolar Proteinosis At The Time of COVID Pandemic (preprint)

Pulmonary alveolar proteinosis (PAP) is a rare syndrome due to increased production or decreased clearance of surfactant in alveoli and terminal bronchi that cause hypoxemic respiratory insufficiency. Here we present a patient with past medical history of PAP whose disease was exacerbated by superimposed COVID-19 pneumonia. He underwent whole pulmonary lavage (WPL). Evaluation of the viral count of the first and the last lavage of the left lung showed viral load in the alveolar space dropped by approximately 10-folds, however the magnitude of the viral load was substantial in both lavage samples. Whole pulmonary lavage may be used as a treatment option on patients with COVID pneumonia superimposed on a pulmonary alveolar proteinosis (PAP) exacerbation.

The Diagnostic And Therapeutic Value Of Bronchoscopy In COVID-19 Patients On Veno-venous Extracorporeal Membrane Oxygenator (ECMO) Support: A Single Center Experience. (preprint)

Introduction: Mechanically ventilated patients COVID-19 patients on veno-venous extracorporeal membrane oxygenation (VV-ECMO) support often require bronchoscopy for pulmonary toilet. However, bronchoscopy in these patients may lead to tracheobronchial bleeding from instrumentation and vial aerosolization. The aim of this study was to assess the indications, benefits, and complications of bronchoscopy in critically ill patients with COVID-19 on VV-ECMO. Methods: This was a single center observational cohort study comprising of adults with COVID-19 infection that required mechanical ventilation and VV-ECMO from January 1, 2019 to November 1, 2021 and needed bronchoscopy. The primary outcome was improvement in patient outcome defined as either in improvement in PaO2 levels or VV-ECMO parameters 6 hours after the procedure. Secondary outcomes included microbiological data from the BAL samples. Mann-Whitney U and χ2 tests were used to compare continuous and categorical variables, respectively. Wilcoxon rank sum test for comparing correlated non-parametric continuous data. The median difference was calculated using the Hodges-Lehman estimator. Results: A total of 89 bronchoscopies were performed in 44 patients with COVID-19 on VV-ECMO. Median (IQR) PaO2 was 64 (57-75) mmHg prior to bronchoscopy, whereas it was mildly improved to 70 (58-89) mmHg, 6 hours after the procedure [Hodges-Lehman median difference (95% CI): 4.5 (2.0 – 8.0) mm Hg, p <0.01]. There was no significant difference in VV-ECMO parameters before and after the procedure. 10 patients had different microorganisms in broncheo-alveolar lavage that were not diagnosed with tracheal aspirate. No patient developed new bleeding post bronchoscopy requiring interruption of anticoagulation. No proceduralist reported testing positive for COVID-19 up to 2 weeks post bronchoscopy. Conclusions: Bronchoscopy is a feasible and relatively safe procedure in COVID-19 patients on VV-ECMO and might be beneficial in select patients to improve oxygenation and tailor antibiotic therapy. Larger studies are required to evaluate the overall impact on patient’s recovery with serial bronchoscopies.

Transcriptome Heterogeneity in COVID-19-induced Acute Respiratory Distress Syndrome (preprint)

COVID-19 is a major etiology of acute respiratory distress syndrome (ARDS). The biological phenotypes and underlying mechanisms in COVID-19-induced ARDS are not fully understood. Bronchoalveolar lavage fluid (BALF) cells and clinical data were collected from patients with COVID-19-induced ARDS. Principal component analysis of genome-wide expression data obtained from bulk RNA sequencing of BALF cells subgrouped COVID-19-induced ARDS patients. Moreover, comparing transcriptome profiles between the subgroups showed two biological phenotypes, illustrated by up- and down-regulation of interferon (IFN) responses, despite no significant differences in clinical characteristics including onset and outcomes. In the low-IFN phenotype, in contrast to the high-IFN phenotype, the TLR-MyD88-IFN regulatory factor (IRF) 5 and cGAS-STING1 axes related to type Ⅰ IFN and the IRF8-interleukin (IL)-12-STAT4 and IRF1-IL-15-DNAX-activation protein 10 axes related to type Ⅱ IFN were inactivated at the transcriptional level, together with the PERK-C/EBP homologous protein axis and the IL-10-hemoglobin scavenger receptor CD163 axis. The pathogenesis of ARDS in the low-IFN phenotype was illustrated by damage to type II alveolar epithelial cells due to increased viral replication by reduced antiviral response, cytotoxicity, and apoptotic signaling and impaired free hemoglobin catabolism. Our data uncovered heterogeneous IFN responses, the underlying mechanisms, and related pathogenesis in COVID-19-induced ARDS.

Screening of Candida spp. in wastewater in Brazil: Workflow for monitoring as strategy of One Health approach (preprint)

The increase in global temperature, caused by the release of greenhouse gases, favors the pathogenic potential of fungi that, by adapting to higher temperatures in the environment, developed tolerance to the temperatures of mammals. Fungal diseases are frequently associated to poverty and, consequently, precarious conditions of hygiene and sanitation, extremely impaired by COVID-19 pandemics. Additionally, COVID-19 patients can develop a diffuse alveolar damage with severe inflammatory exudation. Dexamethasone, a corticosteroid largely used in the treatment of COVID-19, promotes an immunosuppression profile that facilitates the infection by opportunistic fungi, as Candida spp. In this work, we analyzed the prevalence of Candida yeasts in wastewater samples collected for tracking viral genetical material during COVID-19 pandemic. For this, yeasts obtained were identified by polyphasic taxonomy. Subsequently, the production of biofilm and hydrolytic enzymes, which are well-known virulence attributes, were investigated. Our results showed that all Candida spp. were able to form biofilm and had moderate activity to produce hydrolytic enzymes. We also proposed a workflow for monitoring wastewater with the use of Colony PCR in instead of conventional PCR, since this technique is fast, cheap inexpensive and reliable, improving an accurate on taxonomy identification of yeast in environmental samples, contributing to environmental monitoring as part of the One Health approach.

SOX9-regulated matrix proteins predict poor outcomes in patients with COVID-19 and pulmonary fibrosis (preprint)

Pulmonary fibrosis is an increasing and major cause of death worldwide. Understanding the cellular and molecular mechanisms underlying the pathophysiology of lung fibrosis may lead to urgently needed diagnostic and prognostic strategies for the disease. SOX9 is a core transcription factor that has been associated with fibrotic disease, however its role and regulation in acute lung injury and/or fibrosis have not been fully defined. In this study we apply a hypothesis based approach to uncover unique SOX9-protein signatures associated with both acute lung injury and fibrotic progression. Using in vivo models of lung injury in the presence or absence of SOX9, our study shows SOX9 is essential to the damage associated response of alveolar epithelial cells from an early time-point in lung injury. In parallel, as disease progresses, SOX9 is responsible for regulating tissue damaging ECM production from pro-fibrotic fibroblasts. In determining the in vivo role of SOX9 we identified secreted ECM components downstream of SOX9 as markers of acute lung injury and fibrosis. To underscore the translational potential of our SOX9-regulated markers, we analysed serum samples from acute COVID19, post COVID19 and idiopathic pulmonary fibrosis (IPF) patient cohorts. Our hypothesis driven SOX9-panels showed significant capability in all cohorts at identifying patients who had poor disease outcomes. This study shows that SOX9 is functionally critical to disease in acute lung injury and pulmonary fibrosis and its regulated pathways have diagnostic, prognostic and therapeutic potential in both COVID19 and IPF disease.

Neuropsychiatric sequelae in an experimental model of post-COVID syndrome in mice (preprint)

The global impact of the COVID-19 pandemic has been unprecedented, and presently, the world is facing a new challenge known as Post-COVID syndrome (PCS). Current estimates suggest that more than 65 million people are grappling with PCS, encompassing several manifestations, including pulmonary, musculoskeletal, metabolic, and neuropsychiatric sequelae (cognitive and behavioral). The mechanisms underlying PCS remain unclear. The present study aimed to: (i) comprehensively characterize the acute effects of pulmonary inoculation of the betacoronavirus MHV-A59 in immunocompetent mice at clinical, cellular, and molecular levels; (ii) examine potential acute and long-term pulmonary, musculoskeletal, and neuropsychiatric sequelae induced by the betacoronavirus MHV-A59; and to (iii) assess sex-specific differences. Male and female C57Bl/6 mice were initially inoculated with varying viral titers (3x103 to 3x105 PFU/30 L) of the betacoronavirus MHV-A59 via the intranasal route to define the highest inoculum capable of inducing disease without causing mortality. Further experiments were conducted with the 3x104 PFU inoculum. Mice exhibited an altered neutrophil/lymphocyte ratio in the blood in the 2nd and 5th day post-infection (dpi). Marked lung lesions were characterized by hyperplasia of the alveolar walls, infiltration of polymorphonuclear leukocytes (PMN) and mononuclear leukocytes, hemorrhage, increased concentrations of CCL2, CCL3, CCL5, and CXCL1 chemokines, as well as high viral titers until the 5th dpi. While these lung inflammatory signs resolved, other manifestations were observed up to the 60 dpi, including mild brain lesions with gliosis and hyperemic blood vessels, neuromuscular dysfunctions, anhedonic-like behavior, deficits in spatial working memory, and short-term aversive memory. These musculoskeletal and neuropsychiatric complications were exclusive to female mice and were prevented after ovariectomy. In summary, our study describes for the first time a novel sex-dependent model of PCS focused on neuropsychiatric and musculoskeletal disorders. This model provides a unique platform for future investigations regarding the effects of acute therapeutic interventions on the long-term sequelae unleashed by betacoronavirus infection.

Integrated histopathology, spatial and single cell transcriptomics resolve cellular drivers of early and late alveolar damage in COVID-19 (preprint)

The most common cause of death due to COVID-19 remains respiratory failure. Yet, our understanding of the precise cellular and molecular changes underlying lung alveolar damage is limited. Here, we integrate single cell transcriptomic data of COVID-19 donor lungs with spatial transcriptomic data stratifying histopathological stages of diffuse alveolar damage (DAD). We identify changes in cellular composition across progressive DAD, including waves of molecularly distinct macrophages and depleted epithelial and endothelial populations throughout different types of tissue damage. Predicted markers of pathological states identify immunoregulatory signatures, including IFN-alpha and metallothionein signatures in early DAD, and fibrosis-related collagens in organised DAD. Furthermore, we predict a fibrinolytic shutdown via endothelial upregulation of SERPINE1/PAI-1. Cell-cell interaction analysis revealed macrophage-derived SPP1/osteopontin signalling as a key regulator during early DAD. These results provide the first comprehensive, spatially resolved atlas of DAD stages, highlighting the cellular mechanisms underlying pro-inflammatory and pro-fibrotic pathways across alveolar damage progression.

Antiviral innate immune memory in alveolar macrophages following SARS-CoV-2 infection. (preprint)

Pathogen encounter results in long-lasting epigenetic imprinting that shapes diseases caused by heterologous pathogens. The breadth of this innate immune memory is of particular interest in the context of respiratory pathogens with increased pandemic potential and wide-ranging impact on global health. Here, we investigated epigenetic imprinting across cell lineages in a disease relevant murine model of SARS-CoV-2 recovery. Past SARS-CoV-2 infection resulted in increased chromatin accessibility of type I interferon (IFN-I) related transcription factors in airway-resident macrophages. Mechanistically, establishment of this innate immune memory required viral pattern recognition and canonical IFN-I signaling and augmented secondary antiviral responses. Past SARS-CoV-2 infection ameliorated disease caused by the heterologous respiratory pathogen influenza A virus. Insights into innate immune memory and how it affects subsequent infections with heterologous pathogens to influence disease pathology could facilitate the development of broadly effective therapeutic strategies.

Proximal immune-epithelial progenitor interactions drive chronic tissue sequelae post COVID-19 (preprint)

The long-term health effects of SARS-CoV-2, termed Post-Acute Sequelae of COVID-19 (PASC), are quickly evolving into a major public health concern, but the underlying cellular and molecular etiology remain poorly defined. There is growing evidence that PASC is linked to abnormal immune responses and/or poor organ recovery post-infection. However, the exact processes linking non-resolving inflammation, impaired tissue repair, and PASC are still unclear. In this report, we utilized a cohort of respiratory PASC patients with viral infection-mediated pulmonary fibrosis and a clinically relevant mouse model of post-viral lung sequelae to investigate the pathophysiology of respiratory PASC. Using a combination of imaging and spatial transcriptomics, we identified dysregulated proximal interactions between immune cells and epithelial progenitors unique to respiratory PASC but not acute COVID-19 or idiopathic pulmonary fibrosis (IPF). Specifically, we found a central role for lung-resident CD8+ T cell-macrophage interactions in maintaining Krt8hi transitional and ectopic Krt5+ basal cell progenitors, and the development of fibrotic sequelae after acute viral pneumonia. Mechanistically, CD8+ T cell derived IFN-{gamma} and TNF stimulated lung macrophages to chronically release IL-1{beta}, resulting in the abnormal accumulation of dysplastic epithelial progenitors in fibrotic areas. Notably, therapeutic neutralization of IFN-{gamma} and TNF, or IL-1{beta} after the resolution of acute infection resulted in markedly improved alveolar regeneration and restoration of pulmonary function. Together, our findings implicate a dysregulated immune-epithelial progenitor niche in driving respiratory PASC and identify potential therapeutic targets to dampen chronic pulmonary sequelae post respiratory viral infections including SARS-CoV-2.

Integrated Organ Immunity: Antigen-specific CD4-T cell-derived IFN-γ induced by BCG imprints prolonged lung innate resistance against respiratory viruses (preprint)

Bacille Calmette-Guerin (BCG) vaccination can confer non-specific protection against heterologous pathogens. However, the underlying mechanisms remain mysterious. Here, we show that mice immunized intravenously with BCG exhibited reduced weight loss and/or improved viral clearance when challenged with SARS-CoV-2 and influenza. Protection was first evident between 14 - 21 days post vaccination, and lasted for at least 42 days. Remarkably, BCG induced a biphasic innate response in the lung, initially at day 1 and a subsequent prolonged phase starting at ~15 days post vaccination, and robust antigen-specific Th1 responses. MyD88-dependent TLR signaling was essential for the induction of the innate and Th1 responses, and protection against SARS-CoV-2. Depletion of CD4+ T cells or IFN-{gamma} activity prior to infection obliterated innate activation and protection. Single cell and spatial transcriptomics revealed CD4-dependent expression of interferon-stimulated genes (ISGs) in myeloid, type II alveolar and lung epithelial cells. Thus, BCG elicits "integrated organ immunity" where CD4+ T cells act on local myeloid and epithelial cells to imprint prolonged antiviral innate resistance.

Phenotyping the virulence of SARS-CoV-2 variants in hamsters by digital pathology and machine learning (preprint)

SARS-CoV-2 has continued to evolve throughout the COVID-19 pandemic, giving rise to multiple variants of concern (VOCs) with different biological properties. As the pandemic progresses, it will be essential to test in near real time the potential of any new emerging variant to cause severe disease. BA.1 (Omicron) was shown to be attenuated compared to the previous VOCs like Delta, but it is possible that newly emerging variants may regain a virulent phenotype. Hamsters have been proven to be an exceedingly good model for SARS-CoV-2 pathogenesis. Here, we aimed to develop robust quantitative pipelines to assess the virulence of SARS-CoV-2 variants in hamsters. We used various approaches including RNAseq, RNA in situ hybridization, immunohistochemistry, and digital pathology, including software assisted whole section imaging and downstream automatic analyses enhanced by machine learning, to develop methods to assess and quantify virus-induced pulmonary lesions in an unbiased manner. Initially, we used Delta and Omicron to develop our experimental pipelines. We then assessed the virulence of recent Omicron sub-lineages including BA.5, XBB, BQ.1.18, BA.2 and BA.2.75. We show that in experimentally infected hamsters, accurate quantification of alveolar epithelial hyperplasia and macrophage infiltrates represent robust markers for assessing the extent of virus-induced pulmonary pathology, and hence virus virulence. In addition, using these pipelines, we could reveal how some Omicron sub-lineages (e.g., BA.2.75) have regained virulence compared to the original BA.1. Finally, to maximise the utility of the digital pathology pipelines reported in our study, we developed an online repository containing representative whole organ histopathology sections that can be visualised at variable magnifications (https://covid-atlas.cvr.gla.ac.uk). Overall, this pipeline can provide unbiased and invaluable data for rapidly assessing newly emerging variants and their potential to cause severe disease.

Expansion of profibrotic monocyte-derived alveolar macrophages in patients with persistent respiratory symptoms and radiographic abnormalities after COVID-19 (preprint)

As many as 10-30% of the over 760 million survivors of COVID-19 develop persistent symptoms, of which respiratory symptoms are among the most common. To understand the cellular and molecular basis for respiratory PASC, we combined a machine learning-based analysis of lung computed tomography (CT) with flow cytometry, single-cell RNA-sequencing analysis of bronchoalveolar lavage fluid and nasal curettage samples, and alveolar cytokine profiling in a cohort of thirty-five patients with respiratory symptoms and radiographic abnormalities more than 90 days after infection with COVID-19. CT images from patients with PASC revealed abnormalities involving 73% of the lung, which improved on subsequent imaging. Interstitial abnormalities suggestive of fibrosis on CT were associated with the increased numbers of neutrophils and presence of profibrotic monocyte-derived alveolar macrophages in BAL fluid, reflecting unresolved epithelial injury. Persistent infection with SARS-CoV-2 was identified in six patients and secondary bacterial or viral infections in two others. These findings suggest that despite its heterogenous clinical presentations, respiratory PASC with radiographic abnormalities results from a common pathobiology characterized by the ongoing recruitment of neutrophils and profibrotic monocyte-derived alveolar macrophages driving lung fibrosis with implications for diagnosis and therapy.

Prolonged exposure to lung-derived cytokines is associated with inflammatory activation of microglia in patients with COVID-19 (preprint)

Neurological impairment is the most common finding in patients with post-acute sequelae of COVID-19. Furthermore, survivors of pneumonia from any cause have an elevated risk of dementia. Dysfunction in microglia, the primary immune cell in the brain, has been linked to cognitive impairment in murine models of dementia and in humans. Here, we report a transcriptional response in human microglia collected from patients who died following COVID-19 suggestive of their activation by TNF- and other circulating pro-inflammatory cytokines. Consistent with these findings, the levels of 55 alveolar and plasma cytokines were elevated in a cohort of 341 patients with respiratory failure, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. While peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, cumulative cytokine exposure was higher in patients with COVID-19. Corticosteroid treatment, which has been shown to be beneficial in patients with COVID-19, was associated with lower levels of CXCL10, CCL8, and CCL2 - molecules that sustain inflammatory circuits between alveolar macrophages harboring SARS-CoV-2 and activated T cells. These findings suggest that corticosteroids may break this cycle and decrease systemic exposure to lung-derived cytokines and inflammatory activation of microglia in patients with COVID-19.