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Establishing Human Lung Organoids and Proximal Differentiation to Generate Mature Airway Organoids

The lack of a robust in vitro model of the human respiratory epithelium hinders the understanding of the biology and pathology of the respiratory system. ...

The University of Hong Kong

Hong Kong Science and Technology Park

Severe acute respiratory syndrome (SARS) is a recently recognized condition of viral origin associated with substantial morbidity and mortality rates in adults. Little information is available on its radiologic manifestations in children.

Severe acute respiratory syndrome (SARS): chest radiographic features in children.

To elucidate the adaptive strategies of influenza A viruses (IAVs) to human, we proposed a computational approach to identify human-adaptive mutations in seasonal IAVs, which have not been analyzed comprehensively. We compared representative PB2 sequences of 1425 avian IAVs and 2176 human IAVs and identified a total of 42 human-adaptive markers, including 28 and 31 markers in PB2 proteins of seasonal viruses H1N1 and H3N2, respectively. Notably, this comprehensive list encompasses almost all the markers identified in prior computational studies and 21 novel markers including an experimentally verified mutation K526R, suggesting the predictive power of our method. The strength of our analysis derives from the enormous amount of recently available sequences as well as the recognition that human-adaptive mutations are not necessarily conserved across subtypes. We also utilized mutual information to profile the inter-residue coevolution in PB2 protein. A total of 35 and 46 coevolving site pairs are identified in H1N1 and H3N2, respectively. Interestingly, 13 out of the 28 (46.4%) identified markers in H1N1 and 16 out of the 31 (51.6%) in H3N2 are embraced in the coevolving pairs. Many of them are paired with well-characterized human-adaptive mutations, indicating potential epistatic effect of these coevolving residues in human adaptation. Additionally, we reconstructed the PB2 evolutionary history of seasonal IAVs and demonstrated the distinct adaptive pathway of PB2 segment after reassortment from H1 to H3 lineage. Our study may provide clues for further experimental validation of human-adaptive mutations and shed light on the human adaptation process of seasonal IAVs.

Large-scale sequence analysis reveals novel human-adaptive markers in PB2 segment of seasonal influenza A viruses.

Novel reassortant avian influenza H7N9 virus and pandemic 2009 H1N1 (H1N1pdm) virus cause human infections, while avian H7N2 and swine H1N1 virus mainly infect birds and pigs, respectively. There is no robust in vitro model for assessing the infectivity of emerging viruses in humans. Based on a recently established method, we generated long-term expanding 3D human airway organoids which accommodate four types of airway epithelial cells: ciliated, goblet, club, and basal cells. We report differentiation conditions which increase ciliated cell numbers to a nearly physiological level with synchronously beating cilia readily discernible in every organoid. In addition, the differentiation conditions induce elevated levels of serine proteases, which are essential for productive infection of human influenza viruses and low-pathogenic avian influenza viruses. We also established improved 2D monolayer culture conditions for the differentiated airway organoids. To demonstrate the ability of differentiated airway organoids to identify human-infective virus, 3D and 2D differentiated airway organoids are applied to evaluate two pairs of viruses with known distinct infectivity in humans, H7N9/Ah versus H7N2 and H1N1pdm versus an H1N1 strain isolated from swine (H1N1sw). The human-infective H7N9/Ah virus replicated more robustly than the poorly human-infective H7N2 virus; the highly human-infective H1N1pdm virus replicated to a higher titer than the counterpart H1N1sw. Collectively, we developed differentiated human airway organoids which can morphologically and functionally simulate human airway epithelium. These differentiated airway organoids can be applied for rapid assessment of the infectivity of emerging respiratory viruses to human.

Differentiated human airway organoids to assess infectivity of emerging influenza virus.

Glycine decarboxylase (GLDC) was prioritized as a candidate susceptibility gene to severe influenza in humans. The higher expression of GLDC derived from genetic variations may confer a higher risk to H7N9 and severe H1N1 infection. We sought to characterize GLDC as functional susceptibility gene that GLDC may intrinsically regulate antiviral response, thereby impacting viral replication and disease outcome. We demonstrated that GLDC inhibitor AOAA and siRNA depletion boosted IFNβ- and IFN-stimulated genes (ISGs) in combination with PolyI:C stimulation. GLDC inhibition and depletion significantly amplified antiviral response of type I IFNs and ISGs upon viral infection and suppressed the replication of H1N1 and H7N9 viruses. Consistently, GLDC overexpression significantly promoted viral replication due to the attenuated antiviral responses. Moreover, GLDC inhibition in H1N1-infected BALB/c mice recapitulated the amplified antiviral response and suppressed viral growth. AOAA provided potent protection to the infected mice from lethal infection, comparable to a standard antiviral against influenza viruses. Collectively, GLDC regulates cellular antiviral response and orchestrates viral growth. GLDC is a functional susceptibility gene to severe influenza in humans.

Identification and characterization of GLDC as host susceptibility gene to severe influenza.

Human respiratory syncytial virus (HRSV) infection is a significant cause of morbidity and mortality, particularly among the children and the elderly. Despite extensive efforts, there is currently no formally approved vaccine and effective antiviral options against HRSV infection are limited. The development of vaccines and antiviral strategies for HRSV was partly hampered by the lack of an efficient lethal mouse model to evaluate the efficacy of the candidate vaccines or antivirals. In this study, we established a lethal HRSV mouse model by consecutively passaging a clinical HRSV isolate, GZ08-0. GZ08-18 was isolated from mouse bronchioalveolar lavage fluids at the 50 passage of GZ08-0. Importantly, all GZ08-18-inoculated mice succumbed to the infection by day 7 post infection, whereas all GZ08-0-inoculated mice recovered from the infection. Subsequent investigations demonstrated that GZ08-18 replicated to a higher titer in mouse lungs, induced more prominent lung pathology, and resulted in higher expression levels of a number of key pro-inflammatory cytokines including IFN-γ, MIP-1α, and TNF-α in comparison to that of GZ08-0. The cyclophosphamide pretreatment rendered the mice more susceptible to a lethal outcome with less rounds of virus inoculation. Full genome sequencing revealed 17 mutations in GZ08-18, some of which might account for the dramatically increased pathogenicity over GZ08-0. In addition, by using ribavirin as a positive control, we demonstrated the potential application of this lethal mouse model as a tool in HRSV investigations. Overall, we have successfully established a practical lethal mouse model for HRSV with a mouse-adapted virus, which may facilitate future in vivo studies on the evaluation of candidate vaccines and drugs against HRSV.

Establishment of a lethal aged mouse model of human respiratory syncytial virus infection.

Viruses are obligate intracellular microbes that exploit the host metabolic machineries to meet their biosynthetic demands, making these host pathways potential therapeutic targets. Here, by exploring a lipid library, we show that AM580, a retinoid derivative and RAR-α agonist, is highly potent in interrupting the life cycle of diverse viruses including Middle East respiratory syndrome coronavirus and influenza A virus. Using click chemistry, the overexpressed sterol regulatory element binding protein (SREBP) is shown to interact with AM580, which accounts for its broad-spectrum antiviral activity. Mechanistic studies pinpoint multiple SREBP proteolytic processes and SREBP-regulated lipid biosynthesis pathways, including the downstream viral protein palmitoylation and double-membrane vesicles formation, that are indispensable for virus replication. Collectively, our study identifies a basic lipogenic transactivation event with broad relevance to human viral infections and represents SREBP as a potential target for the development of broad-spectrum antiviral strategies.

SREBP-dependent lipidomic reprogramming as a broad-spectrum antiviral target.

Human infection with Middle East respiratory syndrome coronavirus (MERS-CoV) poses an ongoing threat to public health worldwide. The studies of MERS patients with severe disease and experimentally infected animals showed that robust viral replication and intensive proinflammatory response in lung tissues contribute to high pathogenicity of MERS-CoV. We sought to identify pattern recognition receptor (PRR) signaling pathway(s) that mediates the inflammatory cascade in human macrophages upon MERS-CoV infection.

Activation of C-Type Lectin Receptor and (RIG)-I-Like Receptors Contributes to Proinflammatory Response in Middle East Respiratory Syndrome Coronavirus-Infected Macrophages.

A novel coronavirus-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-emerged in humans in Wuhan, China, in December 2019 and has since disseminated globally. As of April 16, 2020, the confirmed case count of coronavirus disease 2019 (COVID-19) had surpassed 2 million. Based on full-genome sequence analysis, SARS-CoV-2 shows high homology to SARS-related coronaviruses identified in horseshoe bats. Here we show the establishment and characterization of expandable intestinal organoids derived from horseshoe bats of the Rhinolophus sinicus species that can recapitulate bat intestinal epithelium. These bat enteroids are fully susceptible to SARS-CoV-2 infection and sustain robust viral replication. Development of gastrointestinal symptoms in some patients with COVID-19 and detection of viral RNA in fecal specimens suggest that SARS-CoV-2 might cause enteric, in addition to respiratory, infection. Here we demonstrate active replication of SARS-CoV-2 in human intestinal organoids and isolation of infectious virus from the stool specimen of a patient with diarrheal COVID-19. Collectively, we established the first expandable organoid culture system of bat intestinal epithelium and present evidence that SARS-CoV-2 can infect bat intestinal cells. The robust SARS-CoV-2 replication in human intestinal organoids suggests that the human intestinal tract might be a transmission route of SARS-CoV-2.

Infection of bat and human intestinal organoids by SARS-CoV-2.

Rapid accumulation of viral proteins in host cells render viruses highly dependent on cellular chaperones including heat shock protein 90 (Hsp90). Three highly pathogenic human coronaviruses, including MERS-CoV, SARS-CoV and SARS-CoV-2, have emerged in the past 2 decades. However, there is no approved antiviral agent against these coronaviruses. We inspected the role of Hsp90 for coronavirus propagation. First, an Hsp90 inhibitor, 17-AAG, significantly suppressed MERS-CoV propagation in cell lines and physiological-relevant human intestinal organoids. Second, siRNA depletion of Hsp90β, but not Hsp90α, significantly restricted MERS-CoV replication and abolished virus spread. Third, Hsp90β interaction with MERS-CoV nucleoprotein (NP) was revealed in a co-immunoprecipitation assay. Hsp90β is required to maintain NP stability. Fourth, 17-AAG substantially inhibited the propagation of SARS-CoV and SARS-CoV-2. Collectively, Hsp90 is a host dependency factor for human coronavirus MERS-CoV, SARS-CoV and SARS-COV-2. Hsp90 inhibitors can be repurposed as a potent and broad-spectrum antiviral against human coronaviruses.

Human coronavirus dependency on host heat shock protein 90 reveals an antiviral target.

Enteroviruses, such as EV-A71 and CVA16, mainly infect the human gastrointestinal tract. Human coronaviruses, including SARS-CoV and SARS-CoV-2, have been variably associated with gastrointestinal symptoms. We aimed to optimize the human intestinal organoids and hypothesize that these optimized intestinal organoids can recapitulate enteric infections of enterovirus and coronavirus. We demonstrate that the optimized human intestinal organoids enable better simulation of the native human intestinal epithelium, and that they are significantly more susceptible to EV-A71 than CVA16. Higher replication of EV-A71 than CVA16 in the intestinal organoids triggers a more vigorous cellular response. However, SARS-CoV and SARS-CoV-2 exhibit distinct dynamics of virus-host interaction; more robust propagation of SARS-CoV triggers minimal cellular response, whereas, SARS-CoV-2 exhibits lower replication capacity but elicits a moderate cellular response. Taken together, the disparate profile of the virus-host interaction of enteroviruses and coronaviruses in human intestinal organoids may unravel the cellular basis of the distinct pathogenicity of these viral pathogens.

Human Intestinal Organoids Recapitulate Enteric Infections of Enterovirus and Coronavirus.

SPINK6 was identified in human skin as a cellular inhibitor of serine proteases of the KLK family. Airway serine proteases are required to cleave hemagglutinin (HA) of influenza A viruses (IAVs) to initiate an infection in the human airway. We hypothesized that SPINK6 may inhibit common airway serine proteases and restrict IAV activation. We demonstrate that SPINK6 specifically suppresses the proteolytic activity of HAT and KLK5, HAT- and KLK5-mediated HA cleavage, and restricts virus maturation and replication. SPINK6 constrains the activation of progeny virions and impairs viral growth; and vice versa, blocking endogenous SPINK6 enhances HA cleavage and viral growth in physiological-relevant human airway organoids where SPINK6 is intrinsically expressed. In IAV-infected mice, SPINK6 significantly suppresses viral growth and improves mouse survival. Notably, individuals carrying the higher SPINK6 expression allele were protected from human H7N9 infection. Collectively, SPINK6 is a novel host inhibitor of serine proteases in the human airway and restricts IAV activation.

Article	Year
Severe acute respiratory syndrome (SARS): chest radiographic features in children. Pediatric radiology\| PubMed ID: 14624321	2004
Large-scale sequence analysis reveals novel human-adaptive markers in PB2 segment of seasonal influenza A viruses. Emerging microbes & infections\| PubMed ID: 29593225	2018
Differentiated human airway organoids to assess infectivity of emerging influenza virus. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 29891677	2018
Identification and characterization of GLDC as host susceptibility gene to severe influenza. EMBO molecular medicine\| PubMed ID: 30498026	2019
Establishment of a lethal aged mouse model of human respiratory syncytial virus infection. Antiviral research\| PubMed ID: 30503888	2019
SREBP-dependent lipidomic reprogramming as a broad-spectrum antiviral target. Nature communications\| PubMed ID: 30631056	2019
Activation of C-Type Lectin Receptor and (RIG)-I-Like Receptors Contributes to Proinflammatory Response in Middle East Respiratory Syndrome Coronavirus-Infected Macrophages. The Journal of infectious diseases\| PubMed ID: 31562757	2020
Infection of bat and human intestinal organoids by SARS-CoV-2. Nature medicine\| PubMed ID: 32405028	2020
Human coronavirus dependency on host heat shock protein 90 reveals an antiviral target. Emerging microbes & infections\| PubMed ID: 33179566	2020
Human Intestinal Organoids Recapitulate Enteric Infections of Enterovirus and Coronavirus. Stem cell reports\| PubMed ID: 33626333	2021
SPINK6 inhibits human airway serine proteases and restricts influenza virus activation. EMBO molecular medicine\| PubMed ID: 34826211	2022

Man Chun Chiu

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