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1.
Nat Protoc ; 16(11): 5171-5192, 2021 11.
Article in English | MEDLINE | ID: covidwho-1500485

ABSTRACT

Human epithelial organoids-3D spheroids derived from adult tissue stem cells-enable investigation of epithelial physiology and disease and host interactions with microorganisms, viruses and bioactive molecules. One challenge in using organoids is the difficulty in accessing the apical, or luminal, surface of the epithelium, which is enclosed within the organoid interior. This protocol describes a method we previously developed to control human and mouse organoid polarity in suspension culture such that the apical surface faces outward to the medium (apical-out organoids). Our protocol establishes apical-out polarity rapidly (24-48 h), preserves epithelial integrity, maintains secretory and absorptive functions and allows regulation of differentiation. Here, we provide a detailed description of the organoid polarity reversal method, compatible characterization assays and an example of an application of the technology-specifically the impact of host-microbe interactions on epithelial function. Control of organoid polarity expands the possibilities of organoid use in gastrointestinal and respiratory health and disease research.


Subject(s)
Cell Differentiation , Gastrointestinal Tract , Organoids , Animals , Cell Culture Techniques , Epithelial Cells/cytology , Mice
2.
mBio ; 11(3)2020 06 23.
Article in English | MEDLINE | ID: covidwho-612678

ABSTRACT

It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 "don't eat me" signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Mycobacterium tuberculosis Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents.IMPORTANCE Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile.


Subject(s)
Betacoronavirus/immunology , CD47 Antigen/metabolism , Immunomodulation/immunology , Receptors, Pattern Recognition/immunology , A549 Cells , Adaptive Immunity/immunology , Animals , CD47 Antigen/genetics , Cell Line, Tumor , Cytokines/immunology , Female , Humans , Immunity, Innate/immunology , Lymphocytic choriomeningitis virus/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mycobacterium tuberculosis/immunology , SARS-CoV-2 , Up-Regulation/immunology
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