Intranasal Introduction of Fc-Fused Interleukin-7 Provides Long-Lasting Prophylaxis against Lethal Influenza Virus Infection

Abstract

Influenza A virus (IAV) infection frequently causes hospitalization and mortality due to severe immunopathology. Annual vaccination and antiviral drugs are the current countermeasures against IAV infection, but they have a limited efficacy against new IAV variants. Here, we show that intranasal pretreatment with Fc-fused interleukin-7 (IL-7-mFc) protects mice from lethal IAV infections. The protective activity of IL-7-mFc relies on transcytosis via neonatal Fc receptor (FcRn) in the lung and lasts for several weeks. Introduction of IL-7-mFc alters pulmonary immune environments, leading to recruitment of T cells from circulation and their subsequent residency as tissue-resident memory-like T (TRM-like) cells. IL-7-mFc-primed pulmonary TRM-like cells contribute to protection upon IAV infection by dual modes. First, TRM-like cells, although not antigen specific but polyclonal, attenuate viral replication at the early phase of IAV infection. Second, TRM-like cells augment expansion of IAV-specific cytotoxic T lymphocytes (CTLs), in particular at the late phase of infection, which directly control viruses. Thus, accelerated viral clearance facilitated by pulmonary T cells, which are either antigen specific or not, alleviates immunopathology in the lung and mortality from IAV infection. Depleting a subset of pulmonary T cells indicates that both CD4 and CD8 T cells contribute to protection from IAV, although IL-7-primed CD4 T cells have a more prominent role. Collectively, we propose intranasal IL-7-mFc pretreatment as an effective means for generating protective immunity against IAV infections, which could be applied to a potential prophylaxis for influenza pandemics in the future.

Importance: The major consequence of a highly pathogenic IAV infection is severe pulmonary inflammation, which can result in organ failure and death at worst. Although vaccines for seasonal IAVs are effective, frequent variation of surface viral proteins hampers development of protective immunity. In this study, we demonstrated that intranasal IL-7-mFc pretreatment protected immunologically naive mice from lethal IAV infections. Intranasal pretreatment with IL-7-mFc induced an infiltration of T cells in the lung, which reside as effector/memory T cells with lung-retentive markers. Those IL-7-primed pulmonary T cells contributed to development of protective immunity upon IAV infection, reducing pulmonary immunopathology while increasing IAV-specific cytotoxic T lymphocytes. Since a single treatment with IL-7-mFc was effective in the protection against multiple strains of IAV for an extended period of time, our findings suggest a possibility that IL-7-mFc treatment, as a potential prophylaxis, can be developed for controlling highly pathogenic IAV infections.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Figures

FIG 1

Protective effect of IL-7–mFc against lethal influenza virus infection. (A) Mice (BALB/c, n = 8 mice per group) were treated with 1 μg of IL-7–mFc via the indicated routes. At 14 days after the treatment, the mice were infected with 3 LD50 of H5N2. (B) Mice (BALB/c, n = 6 mice per group) received the indicated dose of IL-7–mFc, IL-7, or PBS intranasally and then were infected with 3 LD50 of H5N2 14 days later. (C) C57BL/6 and FcRn−/− mice (n = 8 mice per group) were administered PBS or 1 μg of IL-7–mFc intranasally. At 14 days after treatment, the mice were infected with 3 LD50 of H5N2 virus. (D) The mice (BALB/c, n = 11 mice per group) received 1 μg of IL-7–mFc intranasally at the indicated time points prior to the infection. All groups of mice were then simultaneously infected with 3 LD50 of H5N2 at day 0. The survival rate was analyzed by the Kaplan-Meier method. The results are representative of more than two independent experiments with similar results. ††, P < 0.01 by log-rank test.

FIG 2

Protective effect of IL-7–mFc against infection with other strains of IAV. The mice (BALB/c, n = 6 mice per group) were infected with a lethal dose of A/PR/8/34 H1N1 (A), A/California/04/09 H1N1 (B), or A/Philippines/2/82 H3N2 (C) at 14 days after intranasal IL-7–mFc treatment. The average body weight loss is shown as a percentage of initial weight at the time of infection (mean ± standard error of the mean [SEM]). The survival rates were analyzed by the Kaplan-Meier method. The results are representative of two independent experiments with similar results. †, P < 0.05; ††, P < 0.01 (by log rank test).

FIG 3

Effect of intranasal IL-7–mFc pretreatment on pulmonary T cells and viral titer in the BALF after IAV infection. BALB/c mice were treated with IL-7–mFc intranasally and infected with 3 LD50 of H5N2 after 14 days. (A) Absolute numbers of T cells from total lung homogenates at 3 dpi and 7 dpi were measured. (B) Frequencies of the CD62Llow CD44high population of CD8 and CD4 T cells were analyzed at 7 dpi. (C) Absolute numbers of pulmonary B and NK cells were measured at 3 dpi and 7 dpi. (D) H5N2-specific IgG titers in the BALF and serum were analyzed at 7 dpi. (E and F) Virus titers (E) and relative expression of H5N2 NS-1 mRNA (normalized by housekeeping gene L32) in total lung homogenates (F) were analyzed at 3 dpi and 7 dpi. The bars indicate the mean ± SEM for 6 mice per group. Representative data from more than two independent experiments are shown. *, P < 0.05; **, P < 0.01 (by Student's t test). White bars, PBS control group; black bars, IL-7–mFc-treated group.

FIG 4

Effect of IL-7–mFc on IAV-induced pulmonary pathology. BALB/c mice were treated intranasally with IL-7–mFc and then infected with 3 LD50 of H5N2 after 14 days. (A) H&E staining of lung sections at a magnification of ×50 (left) and total inflammatory scores (right) at 7 dpi. Scale bars, 1 mm. (B) Total protein concentration in the BALF at 7 dpi. (C) Levels of inflammatory cytokines and chemokines in the BALF at 3 dpi and 7 dpi. (D) Numbers of neutrophils, monocytes, and macrophages analyzed by flow cytometry at 3 dpi and 7 dpi. The bars indicate the mean ± SEM for 8 mice per group. Representative data from more than three independent experiments are shown. *, P < 0.05; **, P < 0.01 (by Student's t test). H&E, hematoxylin and eosin. White bars, PBS control group; black bars, IL-7–mFc-treated group.

FIG 5

Effect of intranasal IL-7–mFc pretreatment on protection against IAV infection in BALB/c-nude mice. BALB/c-nude and WT BALB/c mice were treated with PBS or IL-7–mFc and infected with 3 LD50 of H5N2. (A) Weight loss and survival. Average body weight loss is shown as the mean ± SEM for 10 mice per group, and the survival rate was analyzed by the Kaplan-Meier method. The data are representative of two independent experiments. †, P < 0.05; ††, P < 0.01 (by log rank test). (B) The levels of inflammatory cytokines and chemokines in the BALF were analyzed by ELISA. (C) The numbers of pulmonary neutrophils were analyzed at 7 dpi by flow cytometry. The results are expressed as the mean ± SEM for 8 mice per group. The data are representative of two independent experiments with similar results. *, P < 0.05; **, P < 0.01 (by Student's t test). White bars, PBS-treated WT mice; black bars, IL-7–mFc-treated WT mice; white bars with horizontal lines, PBS-treated BALB/c-nude mice; white bars with vertical lines, IL-7–mFc-treated BALB/c-nude mice.

FIG 6

Involvement of CD4 T cells in IL-7–mFc-mediated regulation of immunopathology after lethal IAV infection. (A) Mice (BALB/c, n = 18) were treated with IL-7–mFc and infected with 3 LD50 of H5N2 after 14 days. Then, 200 μg of anti-CD4, anti-CD8, anti-TCRγδ, or isotype MAb was injected i.p. four times at −1 dpi, 0 dpi, 1 dpi, and 4 dpi. Weight loss and survival curves were analyzed by combining two independent experiment with 9 mice per group. (B) Intracellular cytokine staining for HA529–543-specific IFN-γ production of CD8 T cells from total lung homogenates was analyzed at 7 dpi. HA-specific IFN-γ production was calculated as the difference in IFN-γ+ CD8 T cells between groups with HA529–543 and no stimulation. Representative dot plots (left) and summary graphs (right) are shown. **, P < 0.01 by Student's t test. White bars, PBS control; black bars, IL-7–mFc-treated group. (C to E) Levels of inflammatory cytokines and chemokines in BALF (C), number of pulmonary neutrophils (D), and HA529–543-specific IFN-γ production by CD8 T cells (E) in mice treated with IL-7–mFc after administration of anti-CD4 and IgG control at 7 dpi. The results are representative of three independent experiments and are expressed as the mean ± SEM for 8 mice per group. **, P < 0.01 by one-way ANOVA with Bonferroni's posttest. White bars, IL-7–mFc with IgG control; white bars with horizontal lines, IL-7–mFc with IgG control; black bars, IL-7–mFc with anti-CD4 MAb treatment. (F) Mice (BALB/c, n = 6 per group) were intranasally treated with IL-7–mFc and infected with a lethal dose of H5N2 after 14 days. Mice were treated with anti-CD4 MAb at 1 day, 4 days, or 7 days after IAV infection. Weight loss and survival rates are shown. The data are representative of two independent experiments. ††, P < 0.01 by log rank test.