Primary Swine Respiratory Epithelial Cell Lines for the Efficient Isolation and Propagation of Influenza A Viruses.

Influenza virus isolation from clinical samples is critical for the identification and characterization of circulating and emerging viruses. Yet efficient isolation can be difficult. In these studies, we isolated primary swine nasal and tracheal respiratory epithelial cells and immortalized swine nasal epithelial cells (siNEC) and tracheal epithelial cells (siTEC) that retained the abilities to form tight junctions and cilia and to differentiate at the air-liquid interface like primary cells. Critically, both human and swine influenza viruses replicated in the immortalized cells, which generally yielded higher-titer viral isolates from human and swine nasal swabs, supported the replication of isolates that failed to grow in Madin-Darby canine kidney (MDCK) cells, and resulted in fewer dominating mutations during viral passaging than MDCK cells.IMPORTANCE Robust in vitro culture systems for influenza virus are critically needed. MDCK cells, the most widely used cell line for influenza isolation and propagation, do not adequately model the respiratory tract. Therefore, many clinical isolates, both animal and human, are unable to be isolated and characterized, limiting our understanding of currently circulating influenza viruses. We have developed immortalized swine respiratory epithelial cells that retain the ability to differentiate and can support influenza replication and isolation. These cell lines can be used as additional tools to enhance influenza research and vaccine development.

Production of amphiregulin and recovery from influenza is greater in males than females.

BACKGROUND: Amphiregulin (AREG) is an epidermal growth factor that is a significant mediator of tissue repair at mucosal sites, including in the lungs during influenza A virus (IAV) infection. Previous research illustrates that males of reproductive ages experience less severe disease and recover faster than females following infection with IAV. METHODS: Whether males and females differentially produce and utilize AREG for pulmonary repair after IAV infection was investigated using murine models on a C57BL/6 background and primary mouse and human epithelial cell culture systems. RESULTS: Following sublethal infection with 2009 H1N1 IAV, adult female mice experienced greater morbidity and pulmonary inflammation during the acute phase of infection as well as worse pulmonary function during the recovery phase of infection than males, despite having similar virus clearance kinetics. As compared with females, AREG expression was greater in the lungs of male mice as well as in primary respiratory epithelial cells derived from mouse and human male donors, in response to H1N1 IAVs. Internalization of the epidermal growth factor receptor (EGFR) was also greater in respiratory epithelial cells derived from male than female mice. IAV infection of Areg knock-out (Areg-/-) mice eliminated sex differences in IAV pathogenesis, with a more significant role for AREG in infection of male compared to female mice. Deletion of Areg had no effect on virus replication kinetics in either sex. Gonadectomy and treatment of either wild-type or Areg-/- males with testosterone improved the outcome of IAV as compared with their placebo-treated conspecifics. CONCLUSIONS: Taken together, these data show that elevated levels of testosterone and AREG, either independently or in combination, improve resilience (i.e., repair and recovery of damaged tissue) and contribute to better influenza outcomes in males compared with females.

Evaluation of the innate immune responses to influenza and live-attenuated influenza vaccine infection in primary differentiated human nasal epithelial cells.

The host innate immune response to influenza virus is a key determinant of pathogenic outcomes and long-term protective immune responses against subsequent exposures. Here, we present a direct contrast of the host responses in primary differentiated human nasal epithelial cell (hNEC) cultures following infection with either a seasonal H3N2 influenza virus (WT) or the antigenically-matched live-attenuated vaccine (LAIV) strain. Comparison of the transcriptional profiles obtained 24 and 36h post-infection showed that the magnitude of gene expression was greater in LAIV infected relative to that observed in WT infected hNEC cultures. Functional enrichment analysis revealed that the antiviral and inflammatory responses were largely driven by type III IFN induction in both WT and LAIV infected cells. However, the enrichment of biological pathways involved in the recruitment of mononuclear leukocytes, antigen-presenting cells, and T lymphocytes was uniquely observed in LAIV infected cells. These observations were reflective of the host innate immune responses observed in individuals acutely infected with influenza viruses. These findings indicate that cell-intrinsic type III IFN-mediated innate immune responses in the nasal epithelium are not only crucial for viral clearance and attenuation, but may also play an important role in the induction of protective immune responses with live-attenuated vaccines.

The M2 protein of live, attenuated influenza vaccine encodes a mutation that reduces replication in human nasal epithelial cells.

The influenza A virus components of the live, attenuated influenza vaccine (LAIV) encode the HA and NA gene segments from a circulating virus strain and the remaining gene segments from the cold-adapted master donor virus, A/Ann Arbor/6/1960 (H2N2). The master donor virus imparts at least three phenotypes: temperature-sensitivity (ts), attenuation (att), and cold-adaption (ca). The genetic loci responsible for the att and ts phenotypes of LAIV were mapped to PB1, PB2, and NP by reverse genetics experiments using immortalized cell lines. However, some in vivo studies have demonstrated that the M segment, which acquired an alanine (Ala) to serine (Ser) mutation at M2 position 86 during cold adaption - a mutation found in no other influenza A virus strain - contributes to the att phenotype. Prior studies have shown this region of the M2 cytoplasmic tail to be critical for influenza virus replication. Using reverse genetics, we demonstrate that certain amino acid substitutions at M2 positions 83 and 86 alter the replication of influenza A/Udorn/307/72 (H3N2). Importantly, substitution of a Ser at M2 position 86 reduces A/Udorn/307/72 replication in differentiated primary human nasal epithelial cell (hNECs) cultures, but does not considerably affect replication in MDCK cells. When a Ser was substituted for Ala at M2 86 in LAIV, the virus replicated to higher titers and with faster kinetics in hNEC cultures, implicating this amino acid change as contributing to LAIV attenuation. Increased replication also resulted in increased production of IFN-λ. These data indicate the LAIV associated Ser mutation at M2 position 86 contributes to the att phenotype and is associated with a differential regulation of interferon in LAIV infection.

Progesterone-Based Therapy Protects Against Influenza by Promoting Lung Repair and Recovery in Females.

Over 100 million women use progesterone therapies worldwide. Despite having immunomodulatory and repair properties, their effects on the outcome of viral diseases outside of the reproductive tract have not been evaluated. Administration of exogenous progesterone (at concentrations that mimic the luteal phase) to progesterone-depleted adult female mice conferred protection from both lethal and sublethal influenza A virus (IAV) infection. Progesterone treatment altered the inflammatory environment of the lungs, but had no effects on viral load. Progesterone treatment promoted faster recovery by increasing TGF-ß, IL-6, IL-22, numbers of regulatory Th17 cells expressing CD39, and cellular proliferation, reducing protein leakage into the airway, improving pulmonary function, and upregulating the epidermal growth factor amphiregulin (AREG) in the lungs. Administration of rAREG to progesterone-depleted females promoted pulmonary repair and improved the outcome of IAV infection. Progesterone-treatment of AREG-deficient females could not restore protection, indicating that progesterone-mediated induction of AREG caused repair in the lungs and accelerated recovery from IAV infection. Repair and production of AREG by damaged respiratory epithelial cell cultures in vitro was increased by progesterone. Our results illustrate that progesterone is a critical host factor mediating production of AREG by epithelial cells and pulmonary tissue repair following infection, which has important implications for women's health.