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Principal Investigator: CALVIN J KUO
Organization: STANFORD UNIVERSITY
Fiscal Year: 2022
Award: $393,500
Funding agency: National Institute of Diabetes and Digestive and Kidney Diseases
ABSTRACT
The COVID-19 pandemic, engendered by the novel coronavirus SARS-CoV-2, is a grave threat to public health,
with lung infection and respiratory failure. However, the intestine is also targeted by SARS-CoV-2, as many
patients present with GI symptoms and the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is
abundantly expressed by intestinal epithelium. COVID-19 mortality is strongly associated with systemic
inflammation as in “cytokine storm”, fostering attempts at therapeutic immunomodulation, and raising a critical
need for in vitro human systems modeling SARS-CoV-2-induced immune cell interactions with intestinal
epithelium. Conventional 3D organoid cultures (“enteroids”) allow intestinal epithelial SARS-CoV-2 infection but
unfortunately omit immune cells. Here we generate a holistic intestinal in vitro model of SARS-CoV-2 infection
using air-liquid interface (ALI) organoids containing both epithelium and infiltrating immune cells en bloc without
artificial reconstitution. The complex intestinal immune system of ALI intestinal organoids contains various innate
and adaptive immune cells, highly diverse T cell receptor (TCR) and B cell receptor (BCR) repertoire, and plasma
B cell-derived antibody transcripts. Importantly, immune components in these ALI organoids respond efficiently
to epithelial damage and ALI intestinal organoids are highly susceptible to bacterial and viral infections.
Here, we utilize this unique ALI organoid technology with integrated immune components to
explore sequelae of SARS-CoV-2 infection. Aim 1 establishes and optimizes BSL3 SARS-CoV-2 infection of
ALI intestinal organoids, exploiting a novel eversion method to relocate the apical aspect of ACE2-expressing
cells to the external surface, allowing survey of SARS-CoV-2 infection of different regions of small intestine and
colon. The time course of tissue-resident SARS-CoV-2-induced cross-talk between intestinal epithelium and
immune cells is unknown, as hindered by lack of human experimental systems. Thus, Aim 2 performs a scRNA-
seq and CyTOF study of SARS-CoV-2-induced immune responses within ALI organoids to (1) create a network
model of the temporal propagation of immunity and bidirectional communication between epithelium and immune
cells and (2) perform therapeutic testing against nodal vulnerabilities, correlating against clinical status (naïve,
convalescent, immunized, cross-reactive coronavirus). Current epithelial organoid systems also do not allow
study of adaptive immunity. Aim 3 thus recapitulates SARS-CoV-2 adaptive immune responses by co-culturing
ALI intestinal organoids with newly developed human lymph node organoids. Within such SARS-CoV-2-infected
co-cultures we correlate adaptive immune responses with clinical status including prior cross-reactive
coronavirus infection and SARS-CoV-2 naïve, convalescent and ultimately immunized states. Overall, we
leverage collaboration from Mark Davis (human LN culture) and Catherine Blish and Scott Boyd (SARS-CoV-2)
to create human in vitro systems modeling SARS-CoV-2-induced innate and adaptive immunity, with relevance
for pathogenesis investigations and therapeutics testing.
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patient><COVID-19 positive patient><COVID-19 public health crisis><COVID-19 related death><COVID-19 related fatality><COVID-19 related mortality><COVID-19 virus><COVID19 associated death><COVID19 associated fatality><COVID19 associated mortality><COVID19 crisis><COVID19 death><COVID19 epidemic><COVID19 fatality><COVID19 global health crisis><COVID19 global pandemic><COVID19 health crisis><COVID19 induced death><COVID19 induced fatality><COVID19 induced mortality><COVID19 infection><COVID19 mortality><COVID19 pandemic><COVID19 patient><COVID19 positive patient><COVID19 public health crisis><COVID19 related death><COVID19 related fatality><COVID19 related mortality><COVID19 virus><Cancers><Cell Body><Cell Communication><Cell Interaction><Cell-to-Cell Interaction><Cells><Checkpoint inhibitor><Clinical><Co-culture><CoV emergence><CoV-2><CoV2><Cocultivation><Coculture><Coculture Techniques><Collaborations><Colon><Common Cold><Communication><Complex><Convalescence><Coronaviridae><Coronaviridae Infections><Coronavirus><Coronavirus Infections><Data><Diarrhea><Emesis><Epithelial><Epithelial Cells><Event><Fostering><Glutelin><Gluten><Human><Immune><Immune checkpoint inhibitor><Immune infiltrates><Immune response><Immune system><Immunes><Immunity><Immunization><Immunize><Immunologic Model><Immunologic Sensitization><Immunologic Stimulation><Immunological Models><Immunological Sensitization><Immunological Stimulation><Immunological response><Immunomodulation><Immunostimulation><In Vitro><Incubators><Infection><Inflammation><Innate Immune Response><Innate Immunity><Intervention><Intervention Strategies><Intestinal><Intestines><Investigation><Liquid substance><Lung><Lung Respiratory System><Lung diseases><Lung infections><Lymph Node Reticuloendothelial System><Lymph node proper><Lymphatic nodes><MHC Receptor><Major Histocompatibility Complex Receptor><Malignant Neoplasms><Malignant 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coronavirus 2><Severe acute respiratory syndrome coronavirus 2 epidemic><Severe acute respiratory syndrome coronavirus 2 infection><Severe acute respiratory syndrome coronavirus 2 pandemic><Severe acute respiratory syndrome related corona virus 2><Small Intestines><Specificity><Surface><Survey Instrument><Surveys><Suspension Culture><Suspension substance><Suspensions><Symptoms><System><T cell infiltration><T cell tumor trafficking><T-Cell Antigen Receptors><T-Cell Receptor><T-Cells><T-Lymphocyte><Technology><Testing><Therapeutic><Time><Tissue Model><Tissue Preservation><Tissues><Tonsil><Transcript><Tumor-infiltrating immune cells><Type 2 Angiotensin II Receptor><Variant><Variation><Viral Diseases><Virus Diseases><Virus Replication><Vomiting><Wuhan coronavirus><adaptive immune response><adaptive immunity><allergic/immunologic body system><allergic/immunologic organ system><angiotensin converting enzyme 2><angiotensin converting enzyme II><bacteria infection><bacterial 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multiplication><virus-induced disease>