Immediate myeloid depot for SARS-CoV-2 in the human lung

In the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, epithelial populations in the distal lung expressing Angiotensin-converting enzyme 2 (ACE2) are infrequent, and therefore, the model of viral expansion and immune cell engagement remains incompletely understood. Using human lungs to investigate early host-viral pathogenesis, we found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations. Human alveolar macrophages (AMs) reliably expressed ACE2 allowing both spike-ACE2-dependent viral entry and infection. In contrast to Influenza A virus, SARS-CoV-2 infection of AMs was productive, amplifying viral titers. While AMs generated new viruses, the interferon responses to SARS-CoV-2 were muted, hiding the viral dissemination from specific antiviral immune responses. The reliable and veiled viral depot in myeloid cells in the very early phases of SARS-CoV-2 infection of human lungs enables viral expansion in the distal lung and potentially licenses subsequent immune pathologies

Stem cell derived organoids and RNA virus pathogenesis

RNA viruses, including SARS-CoV-2, pose a dire threat to human health around the globe. Single-stranded RNA viruses target a wide range of organs, causing a diverse set of clinical symptoms. Here, Hepatitis C virus (HCV) in the Flaviviridae family, which targets the liver, and SARS-CoV-2 in the Coronaviridae family, which primarily targets lung cells, are studied in stem cell models. However, in vitro studies have been limited by the lack of robust laboratory model systems. The recent development of organoids and other stem cell-derived systems has enabled studies with tractable, biologically relevant, genetically diverse human systems to understand viral replication and innate immune response. Other in vitro models lack cellular polarity and have a dysregulated immune response while naturally susceptible in vivo models such as non-human primates are often not accessible for many laboratories. SARS-CoV-2 is the human pathogenic coronavirus causing COVID-19. While the primary target for the virus is lung epithelial cells, symptoms can be found across multiple organ systems including the gut, heart, and brain. The need to study the pathogenesis of SARS-CoV-2 in different cell types became clear early in the COVID-19 pandemic. Here, we used iPSC-derived cardiomyocytes to uncover sarcomeric fragmentation as a potential mechanism for cardiac-related symptoms during or after COVID-19 infection. To understand the effects of SARS-CoV-2 and viral variants that arose during the pandemic, an adult stem cell-derived airway organoid model was used. We found that organoids naturally had low levels of ACE2 receptor expression and supported low levels of SARS-CoV-2 infection. Overexpression of ACE2 significantly increased the percentage of cells that got productively infected. Single cell RNA-sequencing showed that cells had high expression of interferon-stimulated genes as well as of interferon beta and lambda. HCV infects hepatocytes and in 70% of cases leads to hepatocellular carcinoma if left untreated. While direct-acting antivirals (DAAs) have been a breakthrough in HCV treatment, they are expensive and do not prevent reinfection. Vaccination which stimulates T cell responses is essential for reducing HCV incidence, and an in vitro system which is susceptible to HCV and can interact with T cells is critically missing. Here, we develop a new system to coculture primary liver organoids derived from HCV+ patients with cytotoxic T cells recognizing a specific HCV epitope. Using quantitative time course microscopy, organoid viability is successfully tracked after peptide pulsing, with organoids expressing the HCV peptide dying at significantly higher rates than organoids without the peptide. Collectively, these studies take advantage of novel organoid technology to bring insight into the pathogenesis of two important viral infections: SARS-CoV-2 and HCV. We expect our studies to be impactful in the future by therapeutically addressing cardiac comorbidities in COVID-19 and finding vaccines for HCV

Modeling Multi-organ Infection by SARS-CoV-2 Using Stem Cell Technology.

SARS-CoV-2, the virus causing the current COVID-19 pandemic, primarily targets the airway epithelium and in lungs can lead to acute respiratory distress syndrome. Clinical studies in recent months have revealed that COVID-19 is a multi-organ disease causing characteristic complications. Stem cell models of various organ systems-most prominently, lung, gut, heart, and brain-are at the forefront of studies aimed at understanding the role of direct infection in COVID-19 multi-organ dysfunction

Modeling Multi-organ Infection by SARS-CoV-2 Using Stem Cell Technology.

SARS-CoV-2, the virus causing the current COVID-19 pandemic, primarily targets the airway epithelium and in lungs can lead to acute respiratory distress syndrome. Clinical studies in recent months have revealed that COVID-19 is a multi-organ disease causing characteristic complications. Stem cell models of various organ systems-most prominently, lung, gut, heart, and brain-are at the forefront of studies aimed at understanding the role of direct infection in COVID-19 multi-organ dysfunction

Modelling T-cell immunity against hepatitis C virus with liver organoids in a microfluidic coculture system.

Hepatitis C virus (HCV) remains a global public health challenge with an estimated 71 million people chronically infected, with surges in new cases and no effective vaccine. New methods are needed to study the human immune response to HCV since in vivo animal models are limited and in vitro cancer cell models often show dysregulated immune and proliferative responses. Here, we developed a CD8+ T cell and adult stem cell liver organoid system using a microfluidic chip to coculture 3D human liver organoids embedded in extracellular matrix with HLA-matched primary human T cells in suspension. We then employed automated phase contrast and immunofluorescence imaging to monitor T cell invasion and morphological changes in the liver organoids. This microfluidic coculture system supports targeted killing of liver organoids when pulsed with a peptide specific for HCV non-structural protein 3 (NS3) (KLVALGINAV) in the presence of patient-derived CD8+ T cells specific for KLVALGINAV. This demonstrates the novel potential of the coculture system to molecularly study adaptive immune responses to HCV in an in vitro setting using primary human cells

Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. It proves fatal for one percent of those infected. Neurological symptoms, which range in severity, accompany a significant proportion of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized primary human cortical tissue and stem cell-derived cortical organoids. We find significant and predominant infection in cortical astrocytes in both primary and organoid cultures, with minimal infection of other cortical populations. Infected astrocytes had a corresponding increase in reactivity characteristics, growth factor signaling, and cellular stress. Although human cortical cells, including astrocytes, have minimal ACE2 expression, we find high levels of alternative coronavirus receptors in infected astrocytes, including DPP4 and CD147. Inhibition of DPP4 reduced infection and decreased expression of the cell stress marker, ARCN1. We find tropism of SARS-CoV-2 for human astrocytes mediated by DPP4, resulting in reactive gliosis-type injury

Influence of Glycosylation Inhibition on the Binding of KIR3DL1 to HLA-B*57:01

<div><p>Viral infections can affect the glycosylation pattern of glycoproteins involved in antiviral immunity. Given the importance of protein glycosylation for immune function, we investigated the effect that modulation of the highly conserved HLA class I <i>N</i>-glycan has on KIR:HLA interactions and NK cell function. We focused on HLA-B*57:01 and its interaction with KIR3DL1, which has been shown to play a critical role in determining the progression of a number of human diseases, including human immunodeficiency virus-1 infection. 721.221 cells stably expressing HLA-B*57:01 were treated with a panel of glycosylation enzyme inhibitors, and HLA class I expression and KIR3DL1 binding was quantified. In addition, the functional outcomes of HLA-B*57:01 <i>N</i>-glycan disruption/modulation on KIR3DL1ζ⁺ Jurkat reporter cells and primary human KIR3DL1⁺ NK cells was assessed. Different glycosylation enzyme inhibitors had varying effects on HLA-B*57:01 expression and KIR3DL1-Fc binding. The most remarkable effect was that of tunicamycin, an inhibitor of the first step of <i>N</i>-glycosylation, which resulted in significantly reduced KIR3DL1-Fc binding despite sustained expression of HLA-B*57:01 on 721.221 cells. This effect was paralleled by decreased activation of KIR3DL1ζ⁺ Jurkat reporter cells, as well as increased degranulation of primary human KIR3DL1⁺ NK cell clones when encountering HLA-B*57:01-expressing 721.221 cells that were pre-treated with tunicamycin. Overall, these results demonstrate that <i>N</i>-glycosylation of HLA class I is important for KIR:HLA binding and has an impact on NK cell function.</p></div

Glycosylation inhibitor screening and titration: (A) Median fluorescence intensity (MFI) of Bw4 staining of untransfected 221 cells (221) and HLA-B*57:01 transfected 221 cells (B57) treated with a panel of glycosylation inhibitors (n = 2) (B) MFI of KIR-Fc staining of untransfected 221 cells (221) and HLA-B*57:01 transfected 221 cells (B57) treated with a panel of glycosylation inhibitors (n = 2)

<p>Glycosylation inhibitor screening and titration: (A) Median fluorescence intensity (MFI) of Bw4 staining of untransfected 221 cells (221) and HLA-B*57:01 transfected 221 cells (B57) treated with a panel of glycosylation inhibitors (n = 2) (B) MFI of KIR-Fc staining of untransfected 221 cells (221) and HLA-B*57:01 transfected 221 cells (B57) treated with a panel of glycosylation inhibitors (n = 2)</p

<i>N</i>-glycosylation inhibition increases HLA-B*57:01 surface expression while abrogating KIR3DL1-Fc binding: Anti-HLA-Bw4 antibody staining (A) and KIR3DL1-Fc staining (B) was performed on 221-HLA-B*57:01 cells (221-B57) treated with TUN (+T), CSP (+C), or PBS and untransduced 221 cells (221).

<p>Representative histograms are on left-sided panels and data representing five technical replicates are presented as bar graphs on right-sided panels.</p