17β-estradiol protects females against influenza by recruiting neutrophils and increasing virus-specific CD8 T cell responses in the lungs

Abstract

17β-Estradiol (E2) treatment limits the pathology associated with pulmonary diseases caused by pathogens, allergens, and asthma, partly by reducing the production of proinflammatory cytokines and chemokines. To test the hypothesis that E2 protects against influenza A virus (IAV) infection by altering the recruitment and activity of innate immune cells and T cells, chemokine concentrations were measured and innate and adaptive immune cells were enumerated from the lungs of E2- and placebo-treated ovariectomized female C57BL/6 mice following infection. Females treated with E2 experienced less morbidity but had similar lung virus titers to placebo-treated females. Females treated with E2 had lower induction of CCL2 but higher CCL3 and CXCL1 responses in their lungs than placebo-treated females. Pulmonary recruitment of neutrophils, NK cells, macrophages, and dendritic cells was increased following infection, but only neutrophil numbers were greater in E2-treated than placebo-treated females. Neutrophils enhance the responses of influenza virus-specific CD8 T cells to promote virus clearance and improve the outcome of infection. Total numbers of virus-specific CD8 T cells were not altered by treatment with E2, but the proportion of gamma interferon (IFN-γ)- and tumor necrosis factor alpha (TNF-α)-producing, virus-specific CD8 T cells was increased. Neutrophil depletion in E2-treated females increased morbidity, reduced pulmonary production of chemoattractants for neutrophils, and reduced IFN-γ production by virus-specific CD8 T cells. Neutrophils mediate both inflammation and tissue repair during IAV infection and are regulated by E2 to improve the outcome of influenza in females.

Importance: Severe influenza is associated with excessive inflammation that leads to tissue damage. We demonstrate that estradiol (E2) is a potent anti-inflammatory hormone that reduces the severity of influenza A virus infection in females. Treatment of female C57BL/6 mice with E2 does not affect virus replication but rather alters the production of chemokines, pulmonary recruitment of neutrophils, and the cytokine responses of virus-specific CD8 T cells to protect females against severe influenza.

FIG 1

Treatment with 17β-estradiol (E2) protects females against IAV infection. Adult C57BL/6 female mice were ovariectomized and treated with placebo (Ovx) or exogenous E2 (Ovx+E2) followed by intranasal inoculation with influenza A virus (IAV). The correlation between absolute uterine horn mass (i.e., the biomarker for circulating E2) and plasma E2 concentrations in placebo-treated (n = 24/treatment) and E2-treated (n = 24/treatment) females was quantified and included both IAV-infected and uninfected females (A). Placebo-treated (n = 79 total) and E2-treated (n = 76 total) females were monitored daily for changes in body mass for 7 days p.i. (B). Infectious virus titers (C) were measured in lungs removed from infected females 7 days p.i. The dotted line in panel C represents the limit of detection for the assay. (D) Variation in disease tolerance between placebo-treated (n = 20) and E2-treated (n = 28) females, measured as the change in body mass relative to virus titers in the lungs at 7 days p.i. Uninfected females were assigned a virus titer value of 2.5 log₁₀ TCID₅₀/ml, which is the limit of detection for the assay. Data represent means ± standard errors of the means (SEM). Significant differences between ovariectomized placebo- and 17β-estradiol-treated females are represented by asterisks (P < 0.05).

FIG 2

Exogenous 17β-estradiol (E2) treatment alters chemokine responses in the lungs during IAV infection. Adult C57BL/6 female mice were ovariectomized and treated with placebo (Ovx) or exogenous E2 (Ovx+E2) followed by intranasal inoculation with medium alone or influenza A virus (IAV). Cell-free supernatants from homogenates of lungs removed from females at day 7 p.i. were used to quantify CCL2 (monocyte chemoattractant protein 1 [MCP-1]) (A), CCL3 (macrophage inflammatory protein 1α [MIP-1α]) (B), CCL5 (RANTES) (C), and CXCL1 (KC) (D) relative to samples from uninfected females (n = 5 to 10/treatment). Data represent means ± SEM. Significant differences between ovariectomized placebo- and 17β-estradiol-treated females are represented by asterisks (P < 0.05).

FIG 3

Treatment with 17β-estradiol (E2) increases the influx of neutrophils into the lungs during IAV infection. Adult C57BL/6 female mice were ovariectomized and treated with placebo (Ovx) or exogenous E2 (Ovx+E2) followed by intranasal inoculation with medium alone (mock) or influenza A virus (IAV). Neutrophil populations were defined as CD11b⁺ CD11c⁻ MHC-II⁻ Ly6g⁺ (A and B). The total number of neutrophils was enumerated from whole lungs of placebo- and E2-treated females on several days p.i. (C). Bars represent means ± SEM. Significant differences between placebo- and E2-treated females, as determined by post hoc analyses, are represented by an asterisk (P < 0.05; n = 7 or 8/treatment).

FIG 4

Exogenous 17β-estradiol (E2) treatment does not affect CD4 T cell populations in the lungs. Adult C57BL/6 female mice were ovariectomized and treated with placebo (Ovx) or exogenous E2 (Ovx+E2) followed by intranasal inoculation with influenza A virus (IAV). At day 7 p.i., the total number of CD4 T cells (A), the proportion of cytokine-expressing CD4 T cells in response to influenza virus antigen (D^bHA_211–255 and D^bNP_311–325) stimulation ex vivo (B), and the total number of FoxP3-expressing CD4 T cells (C) were analyzed in the lungs. Bars represent means ± SEM (n = 8/treatment).

FIG 5

Exogenous 17β-estradiol (E2) treatment increases cytokine production from influenza virus-specific CD8 T cells in the lungs. Adult C57BL/6 female mice were ovariectomized and treated with placebo (Ovx) or exogenous E2 (Ovx+E2) followed by intranasal inoculation with influenza A virus (IAV). At day 7 p.i., the total number of CD8 T cells (A), the number of D^bNP_366–374-specific CD8 T cells (B), the median fluorescence intensity (MFI) for cytokine production (C), and the proportion of cytokine-expressing CD8 T cells in response to influenza virus antigen (D^bNP_366–374) stimulation ex vivo (D) were analyzed in the lungs. Bars represent means ± SEM. Significant differences between ovariectomized placebo and E2-treated females are represented by asterisks (P < 0.05; n = 8/treatment).

FIG 6

Depletion of neutrophils reverses the protective effects of 17β-estradiol (E2) on responses to IAV in females. All adult C57BL/6 female mice were ovariectomized and treated with placebo (Ovx) or exogenous E2 (Ovx+E2) followed by intranasal inoculation with medium alone or influenza A virus (IAV). At days 3 and 5 p.i., placebo- and E2-treated mice were administered anti-Ly6g antibody (clone 1A8) to deplete neutrophils or an isotype control (rat IgG2a). At day 7 p.i., the total number of lung neutrophils (A) was assessed. Percent changes in the body mass of placebo-treated (B) or E2-treated (C) females administered anti-Ly6g, the isotype control (Iso), or no antibody (no Ab) (n = 9 to 20/group) were monitored for 7 days p.i. Infectious virus titers (D) were measured by TCID₅₀, and chemokine concentrations (E and F) were measured by CBA or ELISA in homogenates from lungs removed 7 days p.i and analyzed relative to concentrations from mock-infected females from the same treatment group (n = 7 or 8/treatment). The dotted line in panel D represents the limit of detection for the assay. The total number of lung CD8 T cells (G) or D^bNP_366–374-specific CD8 T cells (H) and the proportion of IFN-γ-expressing CD8 T cells in response to influenza virus antigen (D^bNP_366–374) stimulation ex vivo (I) were assessed (n = 7 or 8/treatment). Bars represent means ± SEM. Significant differences between E2-treated females administered anti-Ly6g or the isotype control are represented by * (P < 0.05), and significant differences between E2-treated females administered anti-Ly6g or no Ab are indicated by # (P < 0.05).