Infection of Primary Nasal Epithelial Cells Grown at an Air-Liquid Interface to Characterize Human Coronavirus-Host Interactions

Summary

The nasal epithelium is the primary barrier site encountered by all respiratory pathogens. Here, we outline methods to use primary nasal epithelial cells grown as air-liquid interface (ALI) cultures to characterize human coronavirus-host interactions in a physiologically relevant system.

Abstract

Three highly pathogenic human coronaviruses (HCoVs) - SARS-CoV (2002), MERS-CoV (2012), and SARS-CoV-2 (2019) - have emerged and caused significant public health crises in the past 20 years. Four additional HCoVs cause a significant portion of common cold cases each year (HCoV-NL63, -229E, -OC43, and -HKU1), highlighting the importance of studying these viruses in physiologically relevant systems. HCoVs enter the respiratory tract and establish infection in the nasal epithelium, the primary site encountered by all respiratory pathogens. We use a primary nasal epithelial culture system in which patient-derived nasal samples are grown at an air-liquid interface (ALI) to study host-pathogen interactions at this important sentinel site. These cultures recapitulate many features of the in vivo airway, including the cell types present, ciliary function, and mucus production. We describe methods to characterize viral replication, host cell tropism, virus-induced cytotoxicity, and innate immune induction in nasal ALI cultures following HCoV infection, using recent work comparing lethal and seasonal HCoVs as an example¹. An increased understanding of host-pathogen interactions in the nose has the potential to provide novel targets for antiviral therapeutics against HCoVs and other respiratory viruses that will likely emerge in the future.

Introduction

Seven human coronaviruses (HCoVs) have been identified to date and cause a range of respiratory diseases². The common or seasonal HCoVs (HCoV-NL63, -229E, -OC43, and -HKU1) are typically associated with upper respiratory tract pathology and cause an estimated 10%-30% of common cold cases annually. Though this is the typical clinical phenotype associated with the common HCoVs, these viruses can cause more significant lower respiratory tract disease in at-risk populations, including children, older adults, and immunocompromised individuals^3,4. Three pathogenic HCoVs have emerged and cause....

Protocol

The use of nasal specimens was approved by the University of Pennsylvania Institutional Review Board (protocol # 800614) and the Philadelphia VA Institutional Review Board (protocol # 00781).

1. Infection of nasal ALI cultures

NOTE: Acquisition of clinical specimens, as well as growth and differentiation of nasal ALI cultures, is outside the scope of this paper. Specific methods for culturing primary nasal epithelial cells can be found in recently published works utilizing these cultures^18,22,23. The below....

Results

The representative figures are partially adapted from data that can be found in the manuscript Otter et al.¹. Nasal ALI cultures derived from four or six donors were infected with one of four HCoVs (SARS-CoV-2, MERS-CoV, HCoV-NL63, and HCoV-229E) according to the protocols described above, and the average apically shed viral titers for each virus are depicted in Figure 1A. While all four of these HCoVs replicate productively in nasal ALI cultures, SARS-CoV-2 and HCoV-.......

Discussion

The methods detailed here describe a primary epithelial culture system in which patient-derived nasal epithelial cells are grown at an air-liquid interface and applied to the study of HCoV-host interactions. Once differentiated, these nasal ALI cultures recapitulate many features of the in vivo nasal epithelium, including a heterogeneous cellular population with ciliated, goblet, and basal cells represented, as well as intact mucociliary function with robustly beating cilia and mucus secretion. This heterogeneou.......

Materials

Name	Company	Catalog Number	Comments
Alexa Fluor secondary antibodies (488, 594, 647)	Invitrogen	Various
BSA (bovine serum albumin)	Sigma-Aldrich	A7906
cOmplete mini EDTA-free protease inhibitor	Roche	11836170001
Cytotoxicity detection kit	Roche	11644793001
DMEM (Dulbecco's Modified Eagle Media)	Gibco	11965-084
DPBS (Dulbecco's Phosphate Buffered Saline)	Gibco	14190136
DPBS + calcium + magnesium	Gibco	14040-117
Endohm-6G measurement chamber	World Precision Instruments	ENDOHM-6G
Epithelial cell adhesion marker (EpCAM; CD326)	eBiosciences	14-9326-82
Epithelial Volt/Ohm (TEER) Meter (EVOM)	World Precision Instruments	300523
FBS (Fetal Bovine Serum)	HyClone	SH30071.03
FV10-ASW software for imaging	Olympus	Version 4.02
HCoV-NL63 (Human coronavirus, NL63)	BEI Resources	NR-470
HCoV-NL63 nucleocapsid antibody	Sino Biological	40641-V07E
Hoescht stain	Thermo Fisher	H3570
Laemmli sample buffer (4x)	BIO-RAD	1610747
LLC-MK2 cells	ATCC	CCL-7	To titrate HCoV-NL63
MERS-CoV (Human coronavirus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), EMC/2012)	BEI Resources	NR-44260
MERS-CoV nucleocapsid antibody	Sino Biological	40068-MM10
MUC5AC antibody	Sigma-Aldrich	AMAB91539
Olympus Fluoview confocal microscope	Olympus	FV1000
Phalloidin-iFluor 647 stain	Abcam	ab176759
PhosStop easy pack (phosphatase inhibitors)	Roche	PHOSS-RO
Plate reader	Perkin Elmer	HH34000000	Any plate reader or ELISA reader is sufficient; must be able to read absorbance at 492 nm
RIPA buffer (50 mM Tris pH 8; 150 mM NaCl; 0.5% deoxycholate; 0.1% SDS; 1% NP40)	Thermo Fisher	89990	Can prep in-house or purchase
RNeasy Plus Kit	Qiagen	74134
SARS-CoV-2 (SARS-Related Coronavirus 2, Isolate USA-WA1/2020)	BEI Resources	NR-52281
SARS-CoV-2 nucleocapsid antibody	Genetex	GTX135357
Triton-X 100	Fisher Scientific	BP151100
Type IV β- tubulin antibody	Abcam	ab11315
VeroCCL81 cells	ATCC	CCL-81	To titrate MERS-CoV
VeroE6 cells	ATCC	CRL-1586	To titrate SARS-CoV-2