Mechanical ventilation affects respiratory microbiome of COVID-19 patients and its interactions with the host

Understanding the pathology of COVID-19 is a global research priority. Early evidence suggests that the respiratory microbiome may be playing a role in disease progression, yet current studies report contradictory results. Here, we examine potential confounders in COVID-19 respiratory microbiome studies by analyzing the upper (n=58) and lower (n=35) respiratory tract microbiome in well-phenotyped COVID-19 patients and controls combining microbiome sequencing, viral load determination, and immunoprofiling. We found that time in the intensive care unit and the type of oxygen support, both of which are associated to additional treatments such as antibiotic usage, explained the most variation within the upper respiratory tract microbiome, while SARS-CoV-2 viral load had a reduced impact. Specifically, mechanical ventilation was linked to altered community structure, lower species- and higher strain-level diversity, and significant shifts in oral taxa previously associated with COVID-19. Single-cell transcriptomic analysis of the lower respiratory tract of mechanically ventilated COVID-19 patients identified specific oral bacteria, different to those observed in controls. These oral taxa were found physically associated with proinflammatory immune cells, which showed higher levels of inflammatory markers. Overall, our findings suggest confounders are driving contradictory results in current COVID-19 microbiome studies and careful attention needs to be paid to ICU stay and type of oxygen support, as bacteria favored in these conditions may contribute to the inflammatory phenotypes observed in severe COVID-19 patients.

Discriminating Mild from Critical COVID-19 by Innate and Adaptive Immune Single-cell Profiling of Bronchoalveolar Lavages

How innate and adaptive lung immune responses to SARS-CoV-2 synchronize during COVID-19 pneumonitis and regulate disease severity is poorly established. To address this, we applied single-cell profiling to bronchoalveolar lavages from 44 patients with mild or critical COVID-19 versus non-COVID-19 pneumonia as control. Viral RNA-tracking delineated the infection phenotype to epithelial cells, but positioned mainly neutrophils at the forefront of viral clearance activity during COVID-19. In mild disease, neutrophils could execute their antiviral function in an immunologically controlled fashion, regulated by fully-differentiated T-helper-17 (TH17)-cells, as well as T-helper-1 (TH1)-cells, CD8+ resident-memory (TRM) and partially-exhausted (TEX) T-cells with good effector functions. This was paralleled by orderly phagocytic disposal of dead/stressed cells by fully-differentiated macrophages, otherwise characterized by anti-inflammatory and antigen-presenting characteristics, hence facilitating lung tissue repair. In critical disease, CD4+ TH1- and CD8+ TEX-cells were characterized by inflammation-associated stress and metabolic exhaustion, while CD4+ TH17- and CD8+ TRM-cells failed to differentiate. Consequently, T-cell effector function was largely impaired thereby possibly facilitating excessive neutrophil-based inflammation. This was accompanied by impaired monocyte-to-macrophage differentiation, with monocytes exhibiting an ATP-purinergic signalling-inflammasome footprint, thereby enabling COVID-19 associated fibrosis and worsening disease severity. Our work represents a major resource for understanding the lung-localised immunity and inflammation landscape during COVID-19.

Multiplex protein profiling of bronchoalveolar lavage in idiopathic pulmonary fibrosis and hypersensitivity pneumonitis.

CONTEXT: Idiopathic pulmonary fibrosis (IPF) and chronic hypersensitivity pneumonitis (HP) are diffuse parenchymal lung diseases characterized by a mixture of inflammation and fibrosis, leading to lung destruction and finally death. AIMS: The aim of this study was to compare different pathophysiological mechanisms, such as angiogenesis, coagulation, fibrosis, tissue repair, inflammation, epithelial damage, oxidative stress, and matrix remodeling, in both disorders using bronchoalveolar lavage (BAL). METHODS: at diagnosis, patients underwent bronchoscopy with BAL and were divided into three groups: Control (n = 10), HP (n = 11), and IPF (n = 11), based on multidisciplinary approach (clinical examination, radiology, and histology): Multiplex searchlight technology was used to analyze 25 proteins representative for different pathophysiological processes: Eotaxin, basic fibroblast growth factor (FGFb), fibronectin, hepatocyte growth factor (HGF), interleukine (IL)-8, IL-12p40, IL-17, IL-23, monocyte chemotactic protein (MCP-1), macrophage-derived chemokine (MDC), myeloperoxidase (MPO), matrix metalloproteinase (MMP)-8, MMP-9, active plasminogen activating inhibitor 1 (PAI-1), pulmonary activation regulated chemokine (PARC), placental growth factor (PlGF), protein-C, receptor for advanced glycation end products (RAGE), regulated on activation normal T cells expressed and secreted (RANTES), surfactant protein-C (SP-C), transforming growth factor-ß1 (TGF-ß1), tissue inhibitor of metalloproteinase-1 (TIMP-1), tissue factor, thymic stromal lymphopoietin (TSLP), and vascular endothelial growth factor (VEGF). RESULTS: All patients suffered from decreased pulmonary function and abnormal BAL cell differential compared with control. Protein levels were increased in both IPF and HP for MMP-8 (P = 0.022), MMP-9 (P = 0.0020), MCP-1 (P = 0.0006), MDC (P = 0.0048), IL-8 (P = 0.013), MPO (P = 0.019), and protein-C (P = 0.0087), whereas VEGF was decreased (P = 0.0003) compared with control. HGF was upregulated in HP (P = 0.0089) and active PAI-1 was upregulated (P = 0.019) in IPF compared with control. Differences in expression between IPF and HP were observed for IL-12p40 (P = 0.0093) and TGF-ß1 (P = 0.0045). CONCLUSIONS: Using BAL, we demonstrated not only expected similarities but also important differences in both disorders, many related to the innate immunity. These findings provide new clues for further research in both disorders.