The Response of Human Alveolar Epithelial Cells in a Novel Tissue‐Equivalent Respiratory Model to H1N1 and H3N2 Influenza A Viruses
Authors: Gappa‐Fahlenkamp Heather , Bhowmick Rudra 
Published: November 2023

Influenza A virus (IAV), the major agent for primary viral pneumonia, is responsible for approximately 250,000 to 500,000 deaths worldwide annually, according to the World Health Organization. The pathogenic IAV strains cause major cytokine dysregulation, called the “cytokine storm”, leading to robust macrophage recruitment into the pulmonary alveoli. The more pathogenic strains of the virus may alter cytokine/chemokine production of the alveolar epithelial cells and alveolar macrophages leading to increased migration and differentiation of activated alveolar macrophages that drive an excessive inflammatory response. The molecular mechanisms involved in the interaction of virus‐infected resident lung cells, such as alveolar epithelial cells and alveolar macrophages, and transmigrating antigen presenting cell (APC) precursors that are recruited in response to viral infection have not been defined fully. There are few in vitro models that are currently available to study the pulmonary immune responses during IAV infections. Monolayers of cultured cells do not recapitulate the in vivo pulmonary physiology, which requires intercellular communication occurring within a three‐dimensional (3D) environment. We have developed a novel 3D tissue‐equivalent respiratory model (TERM) to study immune responses of infected alveolar epithelial cells (AECs), fibroblasts, macrophages and the transmigrating APC precursors across a pulmonary endothelium. Initially, human small airway epithelial cells (hSAECs) were grown on the surface of a thin, porous scaffold comprised of collagen and chitosan. The hSAECs were maintained at an air‐liquid interface for 14 days, after which they were infected with either pathogenic H1N1 or the mildly pathogenic H3N2 strains. Uninfected cells were included as controls. After infection, cytokine content in the conditioned media was measured by ELISA. The marker protein expression in the infected cells was measured by ELISA, flow cytometry, qRT‐PCR, and immunohistochemistry. Our results show that H1N1 infections yield a massive increase in cytokines and a significant change in hSAEC marker proteins, while H3N2 infections did not result in a marked change in such marker proteins. Our results clearly show that the TERM, containing the AECs, closely resembles the in vivo pulmonary physiology. Future work includes the use of the TERM to determine differential abilities of a pathogenic strain (H1N1) of influenza and a mildly pathogenic strain (H3N2) to drive key differences in the response of alveolar epithelial cells and macrophages that would result in a pathologic proinflammatory response or a protective antiviral immune response.Support or Funding InformationResearch reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number P20GM103648. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.