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. 2016 Mar 1;310(5):L415-25.
doi: 10.1152/ajplung.00398.2015. Epub 2015 Dec 18.

Estrogenic compounds reduce influenza A virus replication in primary human nasal epithelial cells derived from female, but not male, donors

Jackye Peretz  1 , Andrew Pekosz  2 , Andrew P Lane  3 , Sabra L Klein  4
Affiliations

Estrogenic compounds reduce influenza A virus replication in primary human nasal epithelial cells derived from female, but not male, donors

Jackye Peretz et al. Am J Physiol Lung Cell Mol Physiol. .

Abstract

Influenza causes an acute infection characterized by virus replication in respiratory epithelial cells. The severity of influenza and other respiratory diseases changes over the life course and during pregnancy in women, suggesting that sex steroid hormones, such as estrogens, may be involved. Using primary, differentiated human nasal epithelial cell (hNEC) cultures from adult male and female donors, we exposed cultures to the endogenous 17β-estradiol (E2) or select estrogen receptor modulators (SERMs) and then infected cultures with a seasonal influenza A virus (IAV) to determine whether estrogenic signaling could affect the outcome of IAV infection and whether these effects were sex dependent. Estradiol, raloxifene, and bisphenol A decreased IAV titers in hNECs from female, but not male, donors. The estrogenic decrease in viral titer was dependent on the genomic estrogen receptor-2 (ESR2) as neither genomic ESR1 nor nongenomic GPR30 was expressed in hNEC cultures and addition of the genomic ER antagonist ICI 182,780 reversed the antiviral effects of E2. Treatment of hNECs with E2 had no effect on interferon or chemokine secretion but significantly downregulated cell metabolic processes, including genes that encode for zinc finger proteins, many of which contain estrogen response elements in their promoters. These data provide novel insights into the cellular and molecular mechanisms of how natural and synthetic estrogens impact IAV infection in respiratory epithelial cells derived from humans.

Keywords: SERMs; antiviral defenses; estradiol; respiratory disease; zinc finger proteins.

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Figures

Fig. 1.
Fig. 1.
17β-estradiol (E2) inhibits infectious virus production in human nasal epithelial cell (hNEC) from female, but not male, donors. hNEC cultures from female (A) or male (B) donors were pretreated with E2 (0.1, 1, and 10 nM) or vehicle for 24 h in the basolateral media and then infected with influenza A virus [IAV; multiplicity of infection (MOI) = 0.1 50% tissue culture infectious dose (TCID50)/cell] via the apical membrane. Supernatants were collected every 24 h postinfection (hpi) and virus titers were analyzed by TCID50 assay. The limit of detection is indicated by a dotted line. The data represent means ± SE. *P < 0.05, E2-treated cultures were significantly different from sex-matched vehicle controls based on a multivariate analysis of variance (MANOVA) followed by planned comparisons, with n = 15 hNEC wells per treatment group.
Fig. 2.
Fig. 2.
Extended E2 pretreatment and E2 treatment postinfection inhibits infectious virus production in hNECs from female donors. hNEC cultures from females were pretreated with E2 (10 nM) for 72 h before IAV infection (A) or treated with E2 (10 nM) 24 h after infection with IAV (B) (MOI = 0.1 TCID50/cell). Supernatants were collected every 24 hpi and virus titers were analyzed by TCID50 assay. The limit of detection is indicated by a dotted line. The data represent means ± SE. *P < 0.05, E2-treated cultures were significantly different from vehicle controls based on a MANOVA followed by planned comparisons, with n = 12 hNEC wells per treatment group.
Fig. 3.
Fig. 3.
The estrogen receptor antagonist ICI 182,780 reverses the effects of E2 on virus titers in hNECs from females. hNEC cultures from female donors were pretreated with E2 (10 nM), ICI (100 nM), E2+ ICI, or vehicle for 24 h in the basolateral media and then infected with IAV (MOI = 0.1 TCID50/cell) via the apical membrane. Supernatants were collected every 24 hpi and virus titers were analyzed by TCID50 assay. The limit of detection is indicated by a dotted line. The data represent means ± SE. *P < 0.05, E2-treated cultures were significantly different from vehicle controls based on a MANOVA followed by planned comparisons, with n = 12 hNEC wells per treatment group.
Fig. 4.
Fig. 4.
E2 stimulates the expression of ESR2, but not ESR1, mRNA in hNECs from female donors only. hNEC cultures from female and male donors were pretreated with E2 (10 nM) or vehicle for 24 h in the basolateral media and infected with IAV (MOI = 0.1 TCID50/cell) via the apical membrane, and after 24 h, the cells were collected for analysis of ESR1 (A) and ESR2 (B) expression. In hNECs from females only, the kinetics of mRNA expression of ESR1 (C) and ESR2 (D) was analyzed in cultures collected at 24 and 48 hpi. The fold change of ESR1 and ESR2 mRNA was determined by the ΔΔCt method using 18sRNA as the reference gene. The data represent means ± SE with the stippled line indicating the normalized expression level of genes in vehicle-treated, mock-infected cultures. *P < 0.05, significant difference from the mock vehicle control by one-way ANOVA and Tukey post hoc tests, with n = 3–5 hNEC wells per treatment group per sex.
Fig. 5.
Fig. 5.
hNEC cultures derived from female donors express ESR2, but not ESR1, protein. hNEC cultures from female donors were pretreated with E2 (10 nM) or vehicle for 24 h in the basolateral media and then infected with IAV (MOI = 0.1 TCID50/cell) via the apical membrane. After 24 h, the cells were lysed and subjected to Western blot analysis for ESR1, ESR2, and ACTB measurements (A). Protein isolated from MCF-7 cells was used as a positive control for estrogen receptor expression. Quantification of ESR2 relative to ACTB was quantified by ImageJ analyses (B).
Fig. 6.
Fig. 6.
E2 treatment reduces metabolic processes in IAV-infected hNECs from females. hNEC cultures from female donors were pretreated with E2 (10 nM) or vehicle for 24 h in the basolateral media and then infected with IAV (MOI = 0.1 TCID50/cell) via the apical membrane. At 24 and 48 hpi, cells were harvested, and RNA was isolated and spotted on Affymetrix Human GeneST2.0 arrays. Data from infected hNECs were normalized to uninfected hNECs within the same treatment group and ANOVAs in Partek Genomics Suite version 6.0 were used to determine significant differences between E2- and vehicle-treated, IAV-infected hNECs. The Venn diagram (A) illustrates the number of differentially expressed genes at 24 and 48 hpi. Gene ontology (B) was used to group genes differentially expressed between E2- and vehicle-treated, IAV-infected hNECs at 24 hpi into functional hierarchies based on enrichment score, with scores >3 indicating significant differences. C: heat map was created using unbiased hierarchical clustering to represent the largest family of differentially expressed genes [i.e., genes that encode zinc finger proteins (ZFP)] between E2- and vehicle-treated, IAV-infected hNEC cultures at 24 hpi.
Fig. 7.
Fig. 7.
Identification of putative estrogen response elements (EREs) in ZFP gene promoters. A: Dragon ERE Finder version 3.0 (http://datam.i2r.a-star.edu.sg/ereV3/) was used to identify putative EREs in the promoters of ZFP genes. The WebLogo sequence generator was used to develop a graphical representation of the ERE nucleic acid sequence conservation (GGTCA-nnn-TGACC) among the identified ZFP genes (6). B: cells were collected for analysis by real time RT-PCR to validate the ZNF91, ZMYM6, and ZFAND4 mRNA expression levels. Fold change of the target genes was determined by the ΔΔCt method using 18sRNA as the reference gene. The data represent means ± SE. *P < 0.05, significant differences compared with vehicle based on Student's t-tests at each time point, with n = 4–5 hNEC wells per treatment group.
Fig. 8.
Fig. 8.
Select estrogenic compounds with affinity for ESR2 reduce virus titers in hNECs from female donors. hNEC cultures from female donors were pretreated with vehicle and either selective estrogen receptor modulators (A), including clomiphene citrate, ospemifene, and raloxifene or bisphenol (BPA) (B) for 24 h in the basolateral media and infected with IAV (MOI = 0.1 TCID50/cell) via the apical membrane. Supernatants were collected every 24 hpi and virus titers were analyzed by TCID50. The limit of detection is indicated by a dotted line. The data represent means ± SE. *P < 0.05, significant difference between vehicle-treated hNECs and raloxifene-treated (A) or BPA-treated (B) based on one-way ANOVAs and Tukey post hoc tests, with n = 9 hNEC wells per treatment group.
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