Antiviral activity and increased host defense against influenza infection elicited by the human cathelicidin LL-37

Abstract

The extensive world-wide morbidity and mortality caused by influenza A viruses highlights the need for new insights into the host immune response and novel treatment approaches. Cationic Host Defense Peptides (CHDP, also known as antimicrobial peptides), which include cathelicidins and defensins, are key components of the innate immune system that are upregulated during infection and inflammation. Cathelicidins have immunomodulatory and anti-viral effects, but their impact on influenza virus infection has not been previously assessed. We therefore evaluated the effect of cathelicidin peptides on disease caused by influenza A virus in mice. The human cathelicidin, LL-37, and the murine cathelicidin, mCRAMP, demonstrated significant anti-viral activity in vivo, reducing disease severity and viral replication in infected mice to a similar extent as the well-characterized influenza virus-specific antiviral drug zanamivir. In vitro and in vivo experiments suggested that the peptides may act directly on the influenza virion rather than via receptor-based mechanisms. Influenza virus-infected mice treated with LL-37 had lower concentrations of pro-inflammatory cytokines in the lung than did infected animals that had not been treated with cathelicidin peptides. These data suggest that treatment of influenza-infected individuals with cathelicidin-derived therapeutics, or modulation of endogenous cathelicidin production may provide significant protection against disease.

Figure 1. LL-37 Protects Mice Against Influenza Virus Disease.

(A,B) Groups of 5 mice were inoculated with 10 MLD₅₀ of A/Puerto Rico/8/1934 influenza virus by the intranasal route on day 0. Mice were nebulized with 200 µl of saline (control), zanamivir (500 µg/ml), LL-37 peptide (500 µg/ml) or scrambled LL-37 control peptide (500 µg/ml) once daily from day -1 to day 7. Mouse body weight (A) and survival (B) was monitored daily up to 14 days post infection. Data represent mean values ± SEM, for three independent experiments. Statistical analysis was performed using Kaplan Meier with a Mantel-Cox (log rank) test. Survival curves obtained with Zanamivir and LL-37 treatments were significantly different (P≤0.001) compared to saline control treatment. There was no difference between saline treated and sLL-37 treated groups. (C) Groups of three mice (Female, 6–8 week old Balb/c) were inoculated with 10 MLD₅₀ of A/PR/8/34 virus intranasally on day 0. Mice were nebulized with 200 µl of saline (control) zanamivir (500 µg/ml), LL-37 peptide (500 µg/ml) or scrambled LL-37 control peptide (500 µg/ml) once daily from day -1 to day 2. Mice were euthanized on day 3 and viral titer in the lungs was assessed by plaque assay. Figure is representative of three independent experiments. Figure shows mean values ± SEM. Statistical analysis was performed using a Student t-test to compare virus infected animals with virus/peptide and virus/zanamivir treated animals (*P≤0.05).

Figure 2. Cathelicidins Show Species-Specific Antiviral Effects.

(A,B,D,E) Groups of 5 mice were infected with 10 MLD₅₀ of A/PR/8/34 influenza virus via intranasal administration on day 0. Mice were nebulized with 200 µl of saline (control), zanamivir (500 µg/ml), the murine cathelicidin mCRAMP (500 µg/ml) (A and B) or the porcine cathelicidin Protegrin-1 (500 µg/ml) (D and E) once daily from day -1 to day 7. Mouse body weight (A, D) and survival (B, E) was monitored daily up to 14 days post infection. Data represent mean values ± SEM, for three independent experiments (A and B) or one experiment (D and E). Statistical analysis was performed using Kaplan Meier with a Mantel-Cox (log rank) test. Survival curves obtained with Zanamivir and mCRAMP treatments were significantly different (P≤0.001) compared to saline control treatment. There was no difference between saline treated and Protegrin treated groups. (C, F) Groups of three mice were infected with 10 MLD₅₀ of A/PR/8/34 virus via intranasal administration on day 0. Mice were nebulized with 200 µl of saline (control), zanamivir (500 µg/ml), the murine cathelicidin mCRAMP (500 µg/ml) or the porcine cathelicidin Protegrin-1 (500 µg/ml) once daily from day -1 to day 2. Mice were euthanized on day 3 and viral titer in the lungs was assessed by plaque assay. Figure shows mean values ± SEM. Statistical analysis was performed using an unpaired t-test to compare virus infected animals with virus/peptide and virus/zanamivir treated animals (*P≤0.05, **P≤0.01, ***P≤0.001).

Figure 3. Cathelicidins Mediate Changes in Lung Cytokine Concentrations Following Influenza Virus Infection.

Groups of 5 mice were infected with 10 MLD₅₀ of A/PR/8/34 influenza virus via intranasal administration on day 0. Mice were nebulized with 200 µl of saline (control), or LL-37 peptide (500 µg/ml) once daily from day -1 to day 2. Mice were euthanized on day 3 and concentration of the indicated cytokines in the bronchoalveolar lavage (BAL) fluid were measured by BioPlex assay. Figures show mean values ± SEM. Statistical analysis was performed using an two-way analysis of variance (ANOVA) (*P≤0.05).

Figure 4. Cathelicidins show antiviral activity against influenza virus in vitro.

Influenza virus was pre-incubated with cathelicidin peptide or control peptide scrambled LL-37 (sLL-37) (A) at a range of concentrations as indicated for 1 hour at room temperature and a plaque formation assay was then performed to assess virus titer in MDCK-L cells in the presence of trypsin. Viruses used were A/PR/8/34 (H1N1) (A, B) or A/Udorn/307/72 (C). The antiviral activity of the cathelicidins LL-37 (A, C), mCRAMP (B) and Protegrin-1 (B) was assessed. Figures are representative of at least three independent experiments. Figures show mean values ± SEM. Statistical analysis was performed using an unpaired t-test to compare virus only titer with virus + peptide (*P≤0.05, ** P≤0.01).

Figure 5. D-Isomer cathelicidins inhibit influenza virus.

(A,B) Groups of 5 mice were infected with 10 MLD₅₀ of A/PR/8/34 influenza virus via intranasal administration on day 0. Mice were nebulized with 200 µl of saline (control), zanamivir (500 µg/ml), D-LL-37 peptide (500 µg/ml) or D-mCRAMP peptide (500 µg/ml) once daily from day -1 to day 7. Mouse weight and survival was monitored daily up to 14 days post infection. Data represent mean values ± SEM from n = 1 experiment. Statistical analysis was performed using Kaplan Meier with a Mantel-Cox (log rank) test. Survival curves obtained with Zanamivir, D-LL-37 and D-mCRAMP treatments were significantly different (P≤0.001) compared to saline control treatment. (C) Groups of 5 mice were infected with 10MLD₅₀ of A/PR/8/34 virus via intranasal administration on day 0. Mice were nebulized with 200 µl of saline (control), zanamivir (500 µg/ml), D-LL-37 peptide (500 µg/ml) or D-mCRAMP peptide (500 µg/ml) once daily from day -1 to day 2. Mice were euthanized on day 3 and viral titer in the lungs was assessed by plaque assay. Figure is representative of n = 3 independent experiments. Figure shows mean values ± SEM. Statistical analysis was performed using an unpaired t-test to compare virus infected animals with virus/peptide and virus/zanamivir treated animals (*P≤0.05). (D) The antiviral activity of the cathelicidins D-LL-37 and D-mCRAMP was assessed. Figure is representative of n = 3 independent experiments. Figure shows mean values ± SEM. Statistical analysis was performed using an unpaired t-test to compare PR/8 only titer with PR/8 + Peptide (*P≤0.05, ** P≤0.01).