Oropharynx

Palatine tonsils are secondary lymphoid organs (SLOs) representing the first line of immunological defense against inhaled or ingested pathogens. We generated an atlas of the human tonsil composed of >556,000 cells profiled across five different data modalities, including single-cell transcriptome, epigenome, proteome, and immune repertoire sequencing, as well as spatial transcriptomics. This census identified 121 cell types and states, defined developmental trajectories, and enabled an understanding of the functional units of the tonsil. Exemplarily, we stratified myeloid slan-like subtypes, established a BCL6 enhancer as locally active in follicle-associated T and B cells, and identified SIX5 as putative transcriptional regulator of plasma cell maturation. Analyses of a validation cohort confirmed the presence, annotation, and markers of tonsillar cell types and provided evidence of age-related compositional shifts. We demonstrate the value of this resource by annotating cells from B cell-derived mantle cell lymphomas, linking transcriptional heterogeneity to normal B cell differentiation states of the human tonsil.

COVID-19 remains a serious emerging global health problem, and little is known about the role of oropharynx commensal microbes in infection susceptibility and severity. Here, we present the oropharyngeal microbiota characteristics identified by shotgun metagenomic sequencing analyses of oropharynx swab specimens from 31 COVID-19 patients, 29 influenza B patients, and 28 healthy controls. Our results revealed a distinct oropharyngeal microbiota composition in the COVID-19 patients, characterized by enrichment of opportunistic pathogens such as Veillonella and Megasphaera and depletion of Pseudopropionibacterium, Rothia, and Streptococcus. Based on the relative abundance of the oropharyngeal microbiome, we built a microbial classifier to distinguish COVID-19 patients from flu patients and healthy controls with an AUC of 0.889, in which Veillonella was identified as the most prominent biomarker for COVID-19 group. Several members of the genus Veillonella, especially Veillonella parvula which was highly enriched in the oropharynx of our COVID-19 patients, were also overrepresented in the BALF of COVID-19 patients, indicating that the oral cavity acts as a natural reservoir for pathogens to induce co-infections in the lungs of COVID-19 patients. We also found the increased ratios of Klebsiella sp., Acinetobacter sp., and Serratia sp. were correlated with both disease severity and elevated systemic inflammation markers (neutrophil-lymphocyte ratio, NLR), suggesting that these oropharynx microbiota alterations may impact COVID-19 severity by influencing the inflammatory response. Moreover, the oropharyngeal microbiome of COVID-19 patients exhibited a significant enrichment in amino acid metabolism and xenobiotic biodegradation and metabolism. In addition, all 26 drug classes of antimicrobial resistance genes were detected in the COVID-19 group, and were significantly enriched in critical cases. In conclusion, we found that oropharyngeal microbiota alterations and functional differences were associated with COVID-19 severity.

Viral infection of the respiratory tract can be associated with propagating effects on the airway microbiome, and microbiome dysbiosis may influence viral disease. Here, we investigated the respiratory tract microbiome in coronavirus disease 2019 (COVID-19) and its relationship to disease severity, systemic immunologic features, and outcomes. We examined 507 oropharyngeal, nasopharyngeal, and endotracheal samples from 83 hospitalized COVID-19 patients as well as non-COVID patients and healthy controls. Bacterial communities were interrogated using 16S rRNA gene sequencing, and the commensal DNA viruses Anelloviridae and Redondoviridae were quantified by qPCR. We found that COVID-19 patients had upper respiratory microbiome dysbiosis and greater change over time than critically ill patients without COVID-19. Oropharyngeal microbiome diversity at the first time point correlated inversely with disease severity during hospitalization. Microbiome composition was also associated with systemic immune parameters in blood, as measured by lymphocyte/neutrophil ratios and immune profiling of peripheral blood mononuclear cells. Intubated patients showed patient-specific lung microbiome communities that were frequently highly dynamic, with prominence of Staphylococcus. Anelloviridae and Redondoviridae showed more frequent colonization and higher titers in severe disease. Machine learning analysis demonstrated that integrated features of the microbiome at early sampling points had high power to discriminate ultimate level of COVID-19 severity. Thus, the respiratory tract microbiome and commensal viruses are disturbed in COVID-19 and correlate with systemic immune parameters, and early microbiome features discriminate disease severity. Future studies should address clinical consequences of airway dysbiosis in COVID-19, its possible use as biomarkers, and the role of bacterial and viral taxa identified here in COVID-19 pathogenesis. IMPORTANCE COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of the respiratory tract, results in highly variable outcomes ranging from minimal illness to death, but the reasons for this are not well understood. We investigated the respiratory tract bacterial microbiome and small commensal DNA viruses in hospitalized COVID-19 patients and found that each was markedly abnormal compared to that in healthy people and differed from that in critically ill patients without COVID-19. Early airway samples tracked with the level of COVID-19 illness reached during hospitalization, and the airway microbiome also correlated with immune parameters in blood. These findings raise questions about the mechanisms linking SARS-CoV-2 infection and other microbial inhabitants of the airway, including whether the microbiome might regulate severity of COVID-19 disease and/or whether early microbiome features might serve as biomarkers to discriminate disease severity.

Epstein-Barr virus (EBV) colonizes secondary lymphoid tissues to establish persistent infection and is strongly associated with malignancy and autoimmunity. Our understanding of EBV infection biology is hindered by a lack of models that capture infected B cell activity in the lymphoid tissue microenvironment. We therefore developed an EBV human tonsil organoid model to evaluate key B cell states and antiviral responses, including after primary infection. EBV promoted B cell differentiation into germinal center-like phenotypes and transcriptomic analyses highlighted numerous B cell transcriptional programs unique to EBV-infected cells. B cell receptor repertoire analysis revealed that most EBV+ B cells underwent class switching but only rarely participated in somatic hypermutation. CD4 T cells, highly activated by organoid infection, limited EBV+ B cell outgrowth in both primary and secondary EBV infection. Our findings demonstrate human tonsil organoids as a physiologically relevant model to investigate key aspects of EBV immunity and pathogenesis.