ABSTRACT
Background. COVID-19-associated pulmonary aspergillosis (CAPA) is a severe superinfection with the fungus Aspergillus frequently affecting critically ill COVID-19 patients. Pathophysiological insight, key to improve diagnostic and immunomodulatory therapeutic options, is lacking. Methods. We performed single-cell RNA sequencing (scRNA-seq) on 37 bronchoalveolar lavage (BAL) samples from 37 critically ill COVID-19 patients. Three groups were defined: patients who did not develop aspergillosis (COVID-19-only, n=22), CAPA patients with sampling < 5 days after CAPA diagnosis (early CAPA, n=6) and CAPA patients with sampling 5-11 days after CAPA diagnosis (late CAPA, n=9). All CAPA patients had probable/proven CAPA according to the 2020 ECMM/ISHAM consensus criteria. Additionally, we assessed neutrophil extracellular trap (NET) levels in a separate cohort of 33 biobanked COVID-19-only BAL samples and 24 early CAPA samples. Results. A total of 69008 cells passed quality filtering. CAPA patients had significantly lower BAL neutrophil proportions than COVID-19-only patients, particularly in early CAPA (Fig. 1A). Pseudotime inference revealed two neutrophil trajectories: a regular maturation trajectory, and a trajectory giving rise to "hybrid" neutrophils which express genes encoding proteins with antigen-presenting functions (Fig. 1B). The latter trajectory was dominant in CAPA patients (Fig. 1C). NETosis analyses revealed significantly higher levels of citrullinated histone H3 DNA complexes (H3Cit-DNA) in CAPA patients (Fig. 2A). This explains the low CAPA BAL neutrophil proportions, as neutrophils that underwent NETosis are no longer detected via scRNA-seq. CAPA patients with the lowest H3Cit-DNA levels had significantly decreased survival rates (Fig. 2B). Panel (A): BALF neutrophil proportions as analyzed by single-cell RNA sequencing using the Seurat R package are significantly lower in CAPA patients compared to COVID-19-only patients. Patients with early CAPA have significantly lower BALF neutrophil proportions than patients with late CAPA. Macrophage/monocyte and epithelial cell proportions are reciprocally increased in CAPA patients compared to COVID-19-only patients. P-values shown for differences between the pooled CAPA patients and the COVID-19-only patients. P-values were calculated using a generalized linear model correcting for age, Charlson Comorbidity Index at hospital admission, and administration of corticosteroids (prednisone equivalent dose 20 mg or higher) within 48 hours of BALF sampling. Panel (B): Two trajectories are defined using pseudotime inference calculated using the Slingshot R package: a trajectory dominant in COVID-19-only patients with regular maturation of progenitor neutrophils, and a trajectory dominant in CAPA patients with maturation towards a 'hybrid neutrophil' state, with neutrophils expression genes encoding proteins with functions in antigen presentation. Subsequently, the hybrid neutrophil proportion is significantly higher in CAPA patients compared to COVID-19-only patients, and is significantly higher in patients with early CAPA than those with late CAPA. The mature neutrophil proportion is reciprocally reduced in CAPA patients. P-values shown for differences between the pooled CAPA patients and the COVID-19-only patients. P-values were calculated using a generalized linear model correcting for age, Charlson Comorbidity Index at hospital admission, and administration of corticosteroids (prednisone equivalent dose 20 mg or higher) within 48 hours of BALF sampling. Panel (A): Myeloperoxidase (MPO) DNA levels were analyzed as measure for general NET-formation, while citrullinated histone H3 bound DNA (H3Cit-DNA) levels were analyzed as more specific PAD4-dependent NET-formation, in BALF samples from early CAPA and COVID-19-only patients. A trend towards higher MPO-DNA levels was found in early CAPA patients, while H3Cit-DNA levels were significantly higher in early CAPA compared to COVID-19-only patients. P-values calculated using Mann-Whitney U test. Panel (B): Kaplan-Meier analy is of patients with NETosis analyses, divided in early CAPA and COVID-19-only patients and subdivided according to H3Cit-DNA levels (cut-off at 20000 ng/mL for early CAPA and at 8000 ng/mL for COVID-19-only). Log-rank test was used to compare survival distributions. For the comparison early CAPA (low H3Cit-DNA) versus early CAPA (high H3Cit-DNA), the log-rank p-value was 0.033. Conclusion. CAPA patients display extremely high levels of released NETs in the lower respiratory tract, associated with a shift from the normal neutrophil maturation process towards "hybrid neutrophil" formation, probably upon encountering the fungus. In contrast to high NETosis contributing to mortality in severe COVID-19, CAPA patients likely require these NETs to survive aspergillosis. BAL NET levels hold promise as a tool to guide diagnosis, prognosis and treatment in these patients.
ABSTRACT
BACKGROUND AND AIMS: Acute kidney injury is common in patients infected with the novel coronavirus SARS-CoV-2. Predominant findings in case series of kidney biopsies include acute tubular injury and collapsing podocytopathy. We performed single-cell RNA sequencing on kidney biopsy of a patient with COVID19-associated Henoch-Schönlein vasculitis, to investigate the underlying molecular changes. METHOD: A 46-year-old woman presented with cutaneous vasculitis, arthritis, fever and microscopic hematuria. SARS-CoV-2 PCR on nasopharyngeal swab turned positive. Despite quick spontaneous resolution of symptoms, hematuria persisted and proteinuria increased in the next weeks. Subsequent kidney biopsy showed IgA nephropathy. Kidney biopsy was dissociated into a single-cell suspension and RNA was sequenced. 6126 kidney cells passed quality filters. Publicly available single cell sequencing data of 3 healthy kidney samples were integrated to allow comparison. The skin biopsy, performed at the initial presentation, was stained for the SARS-CoV-2 spike protein and the ACE2 protein using immunohistochemistry. RESULTS: Unsupervised clustering analysis of kidney identified 12 distinct cell types (Figure 1). T-lymphocytes were significantly enriched in COVID19 associated IgA nephropathy (16.7% versus 0.5%, 1.2% and 4.1% in healthy kidney, IgA nephropathy/ healthy kidney ratio of relative % of T-/NK-cell clusters of 8.5), with a deviation towards CD8 lymphocytes and NK(T) cells (Figure 2). NK cells were solely present in IgA nephropathy compared to healthy kidney (1.5% versus 0% in all healthy kidneys). Several genes involved in immune activation, oxidative stress and injury were upregulated in podocytes and mesangial cells. For example, one of the genes upregulated in podocytes was macrophage migration inhibitory factor (MIF) which is known to be involved in podocyte injury and mesangial sclerosis. In endothelial cells pathways involved in NK cell immunity, antigen presentation, interferon gamma signaling, and viral entry were upregulated. In T lymphocytes pathways of antigen presentation and T cell cytotoxicity were enriched. In the skin biopsy, immunohistochemistry was positive for SARS-CoV-2 spike protein inside inflammatory cells, while the ACE2 receptor was positive in the same inflammatory cells, as well as inside endothelial cells. CONCLUSION: Although both innate and adaptive immunity are considered to be involved in IgA nephropathy, our single cell sequencing data demonstrates that mainly T-lymphocytes, especially CD8 cells and NK cells, are enriched in COVID19 associated IgA nephropathy. Further elucidation of the involved pathways and the T cell receptor is planned. Interestingly, the SARS-CoV-2 virus could be identified inside the inflammatory cells in the skin in the context of cutaneous vasculitis, suggesting a direct pathologic effect.
ABSTRACT
Epidemiological and clinical reports indicate that SARS-CoV-2 virulence hinges upon the triggering of an aberrant host immune response, more so than on direct virus-induced cellular damage. To elucidate the immunopathology underlying COVID-19 severity, we perform cytokine and multiplex immune profiling in COVID-19 patients. We show that hypercytokinemia in COVID-19 differs from the interferon-gamma-driven cytokine storm in macrophage activation syndrome, and is more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels paired with deep-immune profiling shows that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Antigen presenting machinery expression is also reduced in critical disease. Furthermore, we report that neutrophils contribute to disease severity and local tissue damage by amplification of hypercytokinemia and the formation of neutrophil extracellular traps. Together our findings suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity.