Proteomic Characterization of Acute Kidney Injury in Patients Hospitalized with SARS-CoV2 Infection (preprint)

Background Acute kidney injury (AKI) is a known complication of COVID-19 and is associated with an increased risk of in-hospital mortality. Unbiased proteomics using biological specimens can lead to improved risk stratification and discover pathophysiological mechanisms. Methods Using measurements of ~4000 plasma proteins in two cohorts of patients hospitalized with COVID-19, we discovered and validated markers of COVID-associated AKI (stage 2 or 3) and long-term kidney dysfunction. In the discovery cohort (N= 437), we identified 413 higher plasma abundances of protein targets and 40 lower plasma abundances of protein targets associated with COVID-AKI (adjusted p <0.05). Of these, 62 proteins were validated in an external cohort (p <0.05, N =261). Results We demonstrate that COVID-AKI is associated with increased markers of tubular injury (NGAL) and myocardial injury. Using estimated glomerular filtration (eGFR) measurements taken after discharge, we also find that 25 of the 62 AKI-associated proteins are significantly associated with decreased post-discharge eGFR (adjusted p <0.05). Proteins most strongly associated with decreased post-discharge eGFR included desmocollin-2, trefoil factor 3, transmembrane emp24 domain-containing protein 10, and cystatin-Cindicating tubular dysfunction and injury. Conclusions Using clinical and proteomic data, our results suggest that while both acute and long-term COVID-associated kidney dysfunction are associated with markers of tubular dysfunction, AKI is driven by a largely multifactorial process involving hemodynamic instability and myocardial damage.

Mapping Systemic Inflammation and Antibody Responses in Multisystem Inflammatory Syndrome in Children (MIS-C) (preprint)

Initially, the global outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spared children from severe disease. However, after the initial wave of infections, clusters of a novel hyperinflammatory disease have been reported in regions with ongoing SARS-CoV-2 epidemics. While the characteristic clinical features are becoming clear, the pathophysiology remains unknown. Herein, we report on the immune profiles of eight Multisystem Inflammatory Syndrome in Children (MIS-C) cases. We document that all MIS-C patients had evidence of prior SARS-CoV-2 exposure, mounting an antibody response with normal isotype-switching and neutralization capability. We further profiled the secreted immune response by high-dimensional cytokine assays, which identified elevated signatures of inflammation (IL-18 and IL-6), lymphocytic and myeloid chemotaxis and activation (CCL3, CCL4, and CDCP1) and mucosal immune dysregulation (IL-17A, CCL20, CCL28). Mass cytometry immunophenotyping of peripheral blood revealed reductions of mDC1 and non-classical monocytes, as well as both NK- and T-lymphocytes, suggesting extravasation to affected tissues. Markers of activated myeloid function were also evident, including upregulation of ICAM1 and Fc{gamma}R1 in neutrophil and non-classical monocytes, well-documented markers in autoinflammation and autoimmunity that indicate enhanced antigen presentation and Fc-mediated responses. Finally, to assess the role for autoimmunity secondary to infection, we profiled the auto-antigen reactivity of MIS-C plasma, which revealed both known disease-associated autoantibodies (anti-La) and novel candidates that recognize endothelial, gastrointestinal and immune-cell antigens. All patients were treated with anti-IL6R antibody or IVIG, which led to rapid disease resolution tracking with normalization of inflammatory markers. One Sentence SummaryThis study maps the cellular and serological immune dysfunction underlying a novel pediatric inflammatory syndrome associated with SARS-CoV-2.

A Streamlined CyTOF Workflow To Facilitate Standardized Multi-Site Immune Profiling of COVID-19 Patients (preprint)

Mass cytometry (CyTOF) represents one of the most powerful tools in immune phenotyping, allowing high throughput quantification of over 40 single parameters at single-cell resolution. However, wide deployment of CyTOF-based immune phenotyping studies are limited by complex experimental workflows and the need for specialized CyTOF equipment and technical expertise. Furthermore, differences in cell isolation and enrichment protocols, antibody reagent preparation, sample staining and data acquisition protocols can all introduce technical variation that can potentially confound integrative analyses of large data-sets of samples processed across multiple labs. Here, we present a streamlined whole blood CyTOF workflow which addresses many of these sources of experimental variation and facilitates wider adoption of CyTOF immune monitoring across sites with limited technical expertise or sample-processing resources or equipment. Our workflow utilizes commercially available reagents including the Fluidigm MaxPar Direct Immune Profiling Assay (MDIPA), a dry tube 30-marker immunophenotyping panel, and SmartTube Proteomic Stabilizer, which allows for simple and reliable fixation and cryopreservation of whole blood samples. We validate a workflow that allows for streamlined staining of whole blood samples with minimal processing requirements or expertise at the site of sample collection, followed by shipment to a central CyTOF core facility for batched downstream processing and data acquisition. We further demonstrate the application of this workflow to characterize immune responses in a cohort of hospitalized COVID-19 patients, highlighting key disease-associated changes in immune cell frequency and phenotype.