3D Human

Airway Spheroid Model

Precision medicine: an in vitro model for rhinovirus infections

 

Abstract

               Human rhinovirus (RV) is the most prevalence respiratory virus infection in hospitalized children. RV comprised three species, including RV-A, -B and -C. RV-A and RV-B are the classical RVs first isolated in 1950s, and these two species were also categorized into major and minor group according to their use of intercellular adhesion molecule 1 (ICAM-1) and low-density lipoprotein receptor (LDLR) as the cellular receptor, respectively. RV-C was identified in 2006 while its receptor, cadherin-related family member 3 (CDHR3) was found in 2015. RV-A and RV-B mainly cause self-limiting upper respiratory tract infection while RV-C has an extended niche in causing bronchiolitis, pneumonia and associated with wheezing illness and asthma exacerbation.

               The proposed establishment of the 3D human airway spheroid is a unique pseudostratified epithelium model which retains the self-renewal and expansion properties with physiological active cell composition. This makes long term culture and observation possible and fills the gap between cell line and patient studies to understand the RV pathogenesis.

               We aim to establish this technique in CUHK and utilize this model to assess the rhinovirus infection in terms of its tissue tropism, replication and possible tissue remodelling.

 

Background

               Respiratory disease is one of the main causes of death in China. The growing of smoker population and the worsen air pollution in terms of severity and frequency highlight the pressing needs in conducting research in respiratory diseases. The advancement in the prevention and treatment of respiratory disease are important to ensure the quality of life. Respiratory disease includes respiratory infectious diseases caused by microbes and it also refers to chronic respiratory morbidity including asthma, chronic obstructive pulmonary diseases (COPD) and lung cancer.

 

               With the mission in improving paediatric health, our group interests in better understand the respiratory viral infection in children, which is the top reason for hospitalization in Hong Kong. The biological interaction between virus infection and the chronic respiratory co-morbidity, such as the history of allergic rhinitis and asthma was partially addressed by cohort studies. However, the biological interplays have only been addressed using cell lines and differentiated cells. A systematically investigation using a physiological model that supports long-term observation deserved a research exploration. Our group is going to establish the 3D human airway spheroid model in CUHK and utilize it to assess the rhinovirus infection in terms of its tissue tropism, replication and possible tissue remodelling. 

 

               

               The human rhinovirus infection precedes as many as 50% of asthma exacerbations in children. (1) A higher prevalence of RVs was found in children with asthma exacerbations than those with controlled asthma. (2) Specifically, RV species A and C were more associated to childhood asthma exacerbations. (3) A local study conducted in 2009 found that 29.7% and 7% of the hospitalized children and adults were positive with RV infection in their nasopharyngeal samples and RV-C contributing 37% of the wheezing episodes. (4) It is in agreement with a study conducted in US that childhood asthma exacerbation is associated with RV-C. (5)

 

               Studying in vivo effects of RV pathogenesis is currently limited to scarce histopathological specimens ofhuman post-mortem airway tissue and limited resemblance of animal models. (6) It is incompletely understood how RV interacts with its host cells and unknown how RV remodels the airways and causing chronic outcomes. In addition, because of the lack of RV-C receptor expression in standard cell lines, (7,8) the isolation and propagation of such virus is challenging, and therefore making the basic research of RV-C incomplete. This lack of understanding seriously hampers the development of urgently needed RV therapeutics.

 

               The 3D human airway spheroid model takes its niche to provide a high throughput and standardized human airway model with individual patent’s characteristics retained. (9) The spheroid is derived from primary human tissue without any transformation. It recapitulates its 3D structure as in vivo and with the capacity to self-organize into a pseudostratified epithelium composed of all proximal airway epithelial cells found in vivo (Figure 1) with the presence of the physiological active goblet cell secreting mucus (Figure 1D), ciliated cell beating its cilia and basal cells (Figure 2D). The important differences of these spheroids and transwell differentiated cells are their indefinite expansion (for more than a year) and experimental manipulability (e.g. expression of lentiviral constructs, gene editing performed using CRISPR/Cas9).

 

               The spheroid model also captures the dynamics and individual variation in the patient mucosa due to genomic diversity. The rotavirus infection modelling and the antiviral therapy testing was performed in the intestinal organoid system, (10) and successfully applied in cystic fibrosis (9) and cancer studies. Hopefully, the establishment of the airway spheroid model can be used to as the novel experimental avenues to assess the responsiveness of RSV and RV infection to different potential therapeutics on an individual basis. We plan to work on the laboratory standard RSV and RV strains in the first stage and the patient-derived strains in the later stage.

               A series of preliminary experiments showed that the newly established human 3D airway spheroids can readily be infected with RFP-RSV (Figure 1E-G). With a comparison between the epithelium on the human bronchial explant culture and the 3D airway spheroid (Figure 2), both express the essential receptor CDHR3 for RV-C infection (Figure 2E and 2F). The airway spheroid would be an excellent in vitro surrogate for human RV infection in vivo.

 

               In this proposal, we will infect the 3D human airway spheroid with rhinovirus A, B and C and for the first time investigate viral spread and its direct effects on human respiratory epithelium.

 

Aim

               To establish the 3D human airway spheroid model and assess the rhinovirus infection in terms of its tissue tropism, replication and possible tissue remodelling.

 

Specific Objectives

1. Establish human 3D airway spheroid as a relevant model for human RVs infection.

2. Study the spreading mechanism of RV and its effect on human airway epithelium using time-lapse microscopy.

3. Compare the RV tissue tropism between 3D airway spheroid and human bronchial explant cultures

 

4. Examine the antiviral pathways important for RV replication and viral spread.

 

Research

Plan

Cell lines

1. Establish human 3D airway spheroid as a relevant model for human RVs infection.

2. Study the spreading mechanism of RV and its effect on human airway epithelium using time-lapse microscopy.

3. Compare the RV tissue tropism between 3D airway spheroid and human bronchial explant cultures

 

4. Examine the antiviral pathways important for RV replication and viral spread.

Virus Cultures

RESPIRATORY SYNCYTIAL VIRUS

               Using reverse genetics with the MP224 plasmid as described, (14) the red fluorescent protein (RFP) gene was inserted to the RSV. RFP was shown to be expressed in virus infected spheroid (Figure 1E and 1F). HEp-2 cells will be infected by RSV at a multiplicity of infection (MOI) of 0.1. Infected cells will be incubated at 37°C until extensive cytopathic effect (CPE) is induced at day 3 or 4. Medium will be removed, and fresh medium will be added. After an additional 3 to 4 h incubation at 37°C, the supernatant will be collected and cell debris will be pelleted by centrifugation. The supernatant will be filtered through a 0.2μM Filter-Stericup. Polyethylene glycol 6000 will be added to the supernatant at a final concentration of 10%, and stirred at 4°C for 2 h. The virus is collected by centrifugation at 4000 rpm for 30 min and resuspended in 1:10 of the original volume in DMEM + 10% FCS. The titer of virus stocks will be determined by virus titration in HEp2 cells.

RHINOVIRUS GENERATION & PROPAGATION

               For the construction of eGFP-RVs, a plasmid containing eGFP, pEGFP-C1 (Invitrogen, 6084-1) was purchased. The eGFP fragment franked by the RV protease 2A recognition site of RV-C15 was generated by PCR and will be generated for RV-A2, RV-B14 and RV-A16 (Table 1). We have done the eGFP-RV-C15 full-length infectious cDNA by a ligation with an eGFP fragment franked by RV-C15 2A recognition site 5’-CTCATCAGCTCAGCGGGACCGAGC-3’ synthesized by PCR using the BlpI restriction site during the visit of Dr. Lee Wai-ming (one of our Scientific Advisors in the Joint Research Laboratory) to our lab in March 2017. For the generation of eGFP inserted RV of A2, B14 and A16 types, the full-length infectious RV-A2, RV-B14 and RV-A16 cDNA in a cloning vector, pMJ3 (gifts from Dr Lee Wai-ming) (8) will be checked by restriction analysis, transformed in E. coli competent cells with ampicillin selection. The plasmid DNA will be extracted using miniprep spin column (QIAGEN) and eluted in water and underwent phenol/chloroform extraction, ethanol precipitation, washing and drying. The proteins and nucleotides within the extracted pellet will be cleaned by ammonium acetate and underwent plasmid linearization with Cla1 in CutSmart Buffer (NEB) at 37°C overnight. The digestion will be confirmed by electrophoresis and the linearized cDNA was subjected to in vitro transcription using MEGAscript T7 kit (Ambion). By preparing a 80% confluent H1-HeLa cell monolayer stably expressing the human CDHR3529Y in a 60mm petri dish, the viral RNA will be transfected into the cell using Lipofectamine 2000 as described. (13) CPE can be observed as early as 24 hour post transfection and the transfected cells will be subjected to two freeze-thaw cycles. The virus stock will be prepared by centrifugation at 10,000 room at 4°C. The supernatant of the virus stock will be inoculated into a 48 well plate of H1-HeLa cell expressing the human CDHR3529Y for further propagation, similar freeze-thaw cycles and centrifugation will be employed and the titer of the stock was quantified by viral titration assay. The first working stock will be sequenced again for the identity verification.

               The propagation of these RVs will be done by seeding CDHR3529Y H1-HeLa in T150 flasks and used at around 85% confluent. Medium will be removed and seed virus stock will be added at a multiplicity of infection of 0.05 in a 6 ml medium. Virus adsorption will be allowed for 2 hours at 33°C in a humidified 5% CO2 atmosphere incubator. The inoculated flask will be top up to 20ml with fresh medium. The progeny virus will be harvested when cytopathic effect was observed in 60% of cells, usually 3-5 days. The infected culture will be subjected to freeze-thaw cycles and centrifugation. The virus will be aliquoted into small volume and stored at -80°C for subsequent use.

VIRUS TITRATION

                HEp-2 and CDHR3529Y H1-HeLa cells will be seeded on 96-well tissue culture plates one day before the TCID50 assay. Cells will be washed once with PBS. Virus samples or culture supernatants will be titrated in serial half-log10 dilutions with the corresponding culture medium prior to the addition of the diluted virus to the cell plates in quadruplicate. The highest viral dilution leading to CPE in ~50% of inoculated wells was estimated using the Karber method.

 

3D Model

 Human lung tissue will be obtained from patients undergo lung resection in Prince of Wales Hospital. The tissue that will be used in the human 3D human airway spheroid model and the explant cultures will be the residual tissue from a pathological test. We will include three independent experiment with the preincubation of RV-B14 and RV-A16 with 0.59uM pleconaril (a capsid-binding inhibitor) (15) to block RV infection of 3D airway spheroids, as a proof-of-concept experiment and establishing them as the relevant model for human RV infection in vivo.

I. THE 3D HUMAN AIRWAY SPHEROID MODEL SYSTEM

               The 3D human airway spheroid model system derived from primary human lung tissue, were developed at Hubrecht Institute (Utrecht, the Netherlands, Figure 2). Briefly, the freshly acquired human lung tissue will be chopped into 1-3 mm3, and minced further using scalpels and washed one time in PBS. The lung fragment will be digested in collagenase with constant shaking for 2 h at 37°C. The cells in the digested tissue will be further dislodged by pipetting up and down. FBS will be added to stop the digestion and the cell suspension will pass through a 100 mm cell strainer followed by a centrifugation step. The red blood cell lysis buffer will be added to remove red blood cell and the pellet will be resuspended in matrigel (Trevigen) in the presence of culture medium supplemented with neuregulin-1b and other growth factors. The cell colonies in matrigel will be dropped into a 24 well culture plate.  Upon gel solidification, 0.5ml culture medium will be added and the spheroids will be maintained at 37⁰C at 5% CO2. Replace the medium for every 2-3 days. It will take approximately 14 days to the spheroid culture to reach confluence. For passaging, these spheroids will be disrupted by adding cold medium and vigorous pipetting into smaller fragments followed by centrifugation as described. (16) The resulting spheroid fragments will be resuspended in new matrigel and plated onto the culture plates in form of droplets.

               For infection, spheroids will be fragmented as described above and a part of the suspension will be trypsinized to generate single cell suspension with its cell number determined in the Coulter Counter (Beckman Coulter). The cell number will be used to estimate the total number of cells present in the spheroids and thus the calculation of the MOI. RFP-RSV and eGFP-RVs at an MOI of 1 in 0.4 ml will be added to the spheroid fragments and incubated for 4 h with occasional mixing in a water-jacketed 37°C incubator with 5% CO2. After that, the virus-inoculated spheroid will be washed using culture medium and resuspended into fresh matrigel and plated as described above for 14 days.

II. THE HUMAN BRONCHIAL & LUNG EXPLANT CULTURES

               Briefly, the human bronchial (Figure 3) and lung tissue explant cultures will be prepared as described (17) and inoculated with the RFP-RSV and eGFP-RV with the same input viral load, 106 TCID50/ml with a culture medium as negative control. The explant cultures will be inoculated by submerging in 1ml of virus stock for 1h at 33°C or 37°C. Explants will be washed 3 times with PBS to remove unbounded virus. The explants will be cultured for further 48h post infection (hpi). At 1, 24 and 48 hpi, the supernatant of the infected culture will be collected for viral load titration for viral kinetic study using HEp-2 cell for RSV and CDHR3529Y expressing H1-HeLa for RVs. Infected tissue will be subjected to in vivo imaging or fixed in 10% formalin for pathological investigations.

Post Infection Analysis

               We will characterize infected 3D airway spheroids histologically to identify infected cell types by the use antibodies of b-tubulin for ciliated cells (Figure 2A), MUC5AC for goblet cells (Figure 2B) and p63 for basal cells (Figure 2C and 2D). At the same time, the membrane protein and the nuclei of the 3D airway spheroid will be labelled by with fluorescent b-actenin and histone markers (Figure 1A). Therefore, the cell-cell junctions and nuclei of individual cells during live cell imaging can be seen together with the behaviour of RV infected cells and uninfected cells (apoptosis, migration, epithelial cell repair).

               To better understand the innate immune response of epithelium following RV infection, we will perform mRNA extraction and qPCR on infected vs uninfected 3D airway spheroids to identify the major antiviral gene responses and the major Th-2 or asthma driving genes expression (e.g. IFN-β, IL-4, IL-5, IL-8, IL-10, IL-13, IL-17, IL-25, IL-27, IL-28, IL-29, IL-31, IL-33, TLR3, TLR4, TLR7, TSLP, MDA5, CCL2, CCL11, CCL17, CCL20, CXCL10).

 

STATISTICAL ANALYSIS

               Data will be presented as Mean ± SEM. Comparisons between groups will be performed with Mann–Whitney test, except for the analysis of gene expression, which will be analyzed with one-way ANOVA (with data of normal distribution) using GraphPad Prism 6.0 for Mac. Differences will be considered significant at a P value less than 0.05.

 

Expected Delievables

  1. A biobank of 3D human airway spheroid

  2. A comparison the biological characteristics of RV of Species A in the minor and major group, Species B and Species C in their interaction with human airway epithelium.

  3. A reliable human platform for drug screening and possibly personalized medicine.

  4. An article in international referred journals and a presentation in an international conference.

  5. A PhD student will be trained.

 

Project Signifiance

                  A potential additional benefit from developing such complex engineered lung tissues is for disease modelling. Chronic lung diseases are distinguished by specific tissue remodelling processes and complex cell-cell interactions that are not easily recapitulated in typical cell culture systems. Therefore, we sought to develop an 3D airway spheroid culture system combining multiple lung cell types as both a step toward eventual regenerative approaches, and as a system to study disease-relevant cell-cell interactions and complex tissue remodelling processes.

 

Signifiance to CUHK

               UMC Utrecht has been using the 3D airway spheroid to study the RSV infection. CUHK will acquire this technique and will be able to apply this technology to address the fundamental biological question of the mechanism how rhinovirus infection changes airway epithelial function to induce one of the most frequent infection during infancy. In addition, with the collection of biological tissue from the Chinese population, because of the self-renewal property of this airway spheroid, CUHK will be able to build an airway spheroid biobank. We can further define the genomic constitution of these samples, e.g. the identification of single-nucleotide polymorphism (SNP) with risk allele towards specific respiratory diseases, and provide a clinically relevant platform for personalized medicine testing. This will speed up the selection of candidate compounds in which a randomized control trial might take years to tell. The result generated from this proposal would also foster multidisciplinary collaboration within CUHK.

 

Reference 

  1. Thumerelle C, Deschildre A, Bouquillon C, Santos C, Sardet A, Scalbert M, et al. Role of viruses and atypical bacteria in exacerbations of asthma in hospitalized children: a prospective study in the Nord-Pas de Calais region (France). Pediatr Pulmonol. 2003;35(2):75-82.

  2. Khetsuriani N, Kazerouni NN, Erdman DD, Lu X, Redd SC, Anderson LJ, et al. Prevalence of viral respiratory tract infections in children with asthma. J Allergy Clin Immunol. 2007;119(2):314-21.

  3. Khetsuriani N, Lu X, Teague WG, Kazerouni N, Anderson LJ, Erdman DD. Novel human rhinoviruses and exacerbation of asthma in children. Emerg Infect Dis. 2008;14(11):1793-6.

  4. Lau SK, Yip CC, Lin AW, Lee RA, So LY, Lau YL, et al. Clinical and molecular epidemiology of human rhinovirus C in children and adults in Hong Kong reveals a possible distinct human rhinovirus C subgroup. J Infect Dis. 2009;200(7):1096-103.

  5. Miller EK, Edwards KM, Weinberg GA, Iwane MK, Griffin MR, Hall CB, et al. A novel group of rhinoviruses is associated with asthma hospitalizations. J Allergy Clin Immunol. 2009;123(1):98-104 e1.

  6. Bartlett NW, Walton RP, Edwards MR, Aniscenko J, Caramori G, Zhu J, et al. Mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation. Nat Med. 2008;14(2):199-204.

  7. Ashraf S, Brockman-Schneider R, Gern JE. Propagation of rhinovirus-C strains in human airway epithelial cells differentiated at air-liquid interface. Methods Mol Biol. 2015;1221:63-70.

  8. Bochkov YA, Palmenberg AC, Lee WM, Rathe JA, Amineva SP, Sun X, et al. Molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus C. Nat Med. 2011;17(5):627-32.

  9. Dekkers JF, Berkers G, Kruisselbrink E, Vonk A, de Jonge HR, Janssens HM, et al. Characterizing responses to CFTR-modulating drugs using rectal organoids derived from subjects with cystic fibrosis. Sci Transl Med. 2016;8(344):344ra84.

  10. Yin Y, Bijvelds M, Dang W, Xu L, van der Eijk AA, Knipping K, et al. Modeling rotavirus infection and antiviral therapy using primary intestinal organoids. Antiviral Res. 2015;123:120-31.

  11. Ashraf S, Brockman-Schneider R, Bochkov YA, Pasic TR, Gern JE. Biological characteristics and propagation of human rhinovirus-C in differentiated sinus epithelial cells. Virology. 2013;436(1):143-9.

  12. Foxman EF, Storer JA, Fitzgerald ME, Wasik BR, Hou L, Zhao H, et al. Temperature-dependent innate defense against the common cold virus limits viral replication at warm temperature in mouse airway cells. Proc Natl Acad Sci U S A. 2015;112(3):827-32.

  13. Lee WM, Wang W, Bochkov YA, Lee I. Reverse genetics system for studying human rhinovirus infections. Methods Mol Biol. 2015;1221:149-70.

  14. Guerrero-Plata A, Casola A, Suarez G, Yu X, Spetch L, Peeples ME, et al. Differential response of dendritic cells to human metapneumovirus and respiratory syncytial virus. Am J Respir Cell Mol Biol. 2006;34(3):320-9.

  15. Ledford RM, Collett MS, Pevear DC. Insights into the genetic basis for natural phenotypic resistance of human rhinoviruses to pleconaril. Antiviral Res. 2005;68(3):135-8.

  16. Bartfeld S, Clevers H. Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori. J Vis Exp. 2015(105).

  17. Chan RW, Hemida MG, Kayali G, Chu DK, Poon LL, Alnaeem A, et al. Tropism and replication of Middle East respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an in-vitro and ex-vivo study. Lancet Respir Med. 2014;2(10):813-22.

 

Appendix

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The Chinese University of Hong Kong

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