The human cathelicidin LL-37 has antiviral activity against respiratory syncytial virus

Abstract

Respiratory syncytial virus is a leading cause of lower respiratory tract illness among infants, the elderly and immunocompromised individuals. Currently, there is no effective vaccine or disease modifying treatment available and novel interventions are urgently required. Cathelicidins are cationic host defence peptides expressed in the inflamed lung, with key roles in innate host defence against infection. We demonstrate that the human cathelicidin LL-37 has effective antiviral activity against RSV in vitro, retained by a truncated central peptide fragment. LL-37 prevented virus-induced cell death in epithelial cultures, significantly inhibited the production of new infectious particles and diminished the spread of infection, with antiviral effects directed both against the viral particles and the epithelial cells. LL-37 may represent an important targetable component of innate host defence against RSV infection. Prophylactic modulation of LL-37 expression and/or use of synthetic analogues post-infection may represent future novel strategies against RSV infection.

Figure 1. LL-37 has antiviral activity against RSV.

a) Sequences of LL-37, three 22-mer peptides (representing the N-terminal, central core and C-terminal regions) and scrambled LL-37 peptide, b) HEp-2 or c) 16HBE14o^- cells were infected with RSV (MOI = 0.005) and simultaneously treated with LL-37 or a scrambled control LL-37 (scrLL-37) at the concentrations indicated for 2 hours. Cells were washed and incubated for 24 hours at 37°C, and then the number of infected cells was quantified. d) HEp-2 cells were exposed to LL-37 and RSV (MOI = 0.005) simultaneously (“Simultaneous” condition), for 2 hours, or exposed for 2 hours to RSV that had been preincubated with LL-37 for 1 hour (“37°C pre-incubation” and “4°C pre-incubation” conditions), or exposed to RSV for 2 hours and washed before a 1 hour incubation with LL-37 (“Virus first” condition), or treated with LL-37 for 1 hour and washed before a 2 hour infection with RSV (“LL-37 first” condition). Every treatment was followed by a wash step. After 24 hours at 37°C, the number of infected cells was quantified. b, c & d) Infected cells were quantified using an immunoplaque assay and expressed as a percentage relative to untreated, RSV infected cells. Results are shown as mean +/− SEM of n = 3-5. For statistical analysis, RSV-positive foci counts were log transformed and analysed by one way ANOVA with Dunnett Multiple Comparison Post-tests to compare treatments to untreated infected controls * p<0.05, ** p<0.01, *** p<0.001.

Figure 2. LL-37 is protective against RSV-induced cell death.

HEp-2 cells were treated with LL-37 at 0–50 µg/ml, a scrambled control LL-37 at 50 µg/ml (Scr 50), or ribavirin (200 µM), in the presence (a & c) or absence (b) of simultaneous infection with RSV (MOI = 0.005). Cells were incubated for 2 hours at 37°C, washed and placed in fresh medium. In some wells LL-37 or ribavirin was added again at this point (c; “continuous treatment”). Cells were cultured at 37°C for 24 hours (a) or 5 days (b & c), after which the number of viable metabolically active cells was assessed using an MTT assay with measurement of optical density at 450 nm, expressed as a percentage of untreated infected (a & c) or untreated (b) cells. Results are shown as mean +/− SEM of n = 3-4. The data were analysed by 1-way ANOVAs with Dunnett Multiple Comparison Post-tests comparing peptide concentrations against the “no peptide” control (** p<0.01, *** p<0.001), and 2-way ANOVA with Bonferroni Post-tests comparing pre-treatment protocols versus continuous treatments (## p<0.01).

Figure 3. LL-37 has protective antiviral effects by acting both on viral particles and cells.

a) RSV (MOI = 0.05) was pre-mixed with LL-37, or a scrambled LL-37 control peptide (scrLL-37) at 25 µg/ml, followed immediately by a 1∶10 dilution that was used to infect HEp-2 cells for 2 hours or RSV (MOI = 0.005) was pre-mixed with LL-37 at 2.5 µg/ml before immediate use. b) HEp-2 cells were simultaneously treated for 2 hours with RSV (MOI = 0.005) and LL-37 (“sim LL-37” condition), or a scrambled LL-37 control peptide (“sim scrLL-37” condition), at 25 µg/ml, or were pre-treated with 25 µg/ml LL-37 for 1 hour, washed and then infected with RSV (MOI = 0.005, 2 hours) at 1, 3 or 24 hours after initial exposure to LL-37. Cells were washed and cultured for 24 hours at 37°C, then the number of infected cells was quantified using an immunoplaque assay and expressed as a percentage relative to untreated, RSV infected cells. Results are shown as mean +/− SEM of n≥3. For statistical analysis, RSV-positive foci counts were log transformed and analysed by a) two-way ANOVA with Bonferroni post-test to compare LL-37 treatment with appropriate control and the effects of dilution, or b) one way ANOVA with Dunnett Multiple Comparison Post-tests to compare treatments to untreated infected controls; ** p<0.01, *** p<0.001.

Figure 4. LL-37 is retained by epithelial cells and does not act via modulation of type I interferon production.

HEp-2 cells were treated with 25 µg/ml of LL-37 (a) or TAMRA-labelled LL-37 (b) for 1 hour, or left untreated. a) LL-37 treated cells were then subjected to repeated wash steps or left unwashed, before cell lysis and protein collection. The presence of LL-37 was assessed by western immunoblot. Data is representative of n = 3 independent experiments. b) TAMRA-labelled LL-37 treated cells were washed and then Hoechst staining was added before imaging by confocal microscopy. A merged brightfield/Hoechst/TAMRA-LL-37 image is shown, nuclei in blue and TAMRA-labelled LL-37 in red. Image is at 63× magnification. c) Vero cells and HEp-2 cells were infected with RSV (MOI = 0.005) and simultaneously treated with LL-37 or a scrambled control LL-37 (scrLL-37) at 25 µg/ml for 2 hours. Cells were washed, incubated for 24 hours at 37°C, then the number of infected cells was quantified using an immunoplaque assay and expressed as a percentage relative to untreated, RSV infected cells. Results are shown as mean +/− SEM of n = 3-5. For statistical analysis, RSV-positive foci counts were log transformed and analysed by one way ANOVA with Dunnett Multiple Comparison Post-tests to compare treatments to untreated infected controls *** p<0.001.

Figure 5. LL-37 inhibits the production of new viral particles and the spread of infection.

a) HEp-2 cells were simultaneously exposed to RSV with either 25 µg/ml LL-37 or scrambled LL-37 (scrLL-37) for 2 hours, washed and incubated in fresh media with (“continuous”), or without (“sim”) replacement of LL-37 or scrambled LL-37 at 25 µg/ml, at 37°C for 1 day or 3 days. Supernatants were then transferred in serial dilution to infect fresh HEp-2 cells and incubated for 2 hours, before these cells were incubated for 24 hours and assessed by immunoplaque assay, expressed as a percentage relative to those infected with supernatant from untreated, RSV infected cells. Results are shown as mean +/− SEM of n = 3. For statistical analysis, RSV-positive foci counts were log transformed and analysed by one way ANOVA with Dunnett Multiple Comparison Post-tests to compare treatments to untreated infected controls * p<0.05, *** p<0.001, b, c & d) HEp-2 cells were either infected with RSV alone (MOI = 0.0005) for 2 hours (b, panels 1 & 5), simultaneously exposed to RSV with either 25 µg/ml LL-37 (b, panels 2 & 6; c & d, “LL-37 sim”) or scrambled LL-37 (b, panels 4 & 8; c & d, “scrLL-37 sim”) for 2 hours, or incubated with RSV alone for 2 hours, then washed, before 25 µg/ml LL-37 was added for 1 hour (b, panels 3 & 7; c & d, “LL-37 virus first”). Subsequently, cells were washed and cultured in fresh media for 48 hours (b, panels 1–4; c) or 72 hours (b, panels 5–8; d) at 37°C, before immunoplaque assay. Extent of infection in different treatments was evaluated by light microscopy with infected cells staining dark (b). Images are representative of n = 3 independent experiments. Magnification ×40. c & d) Areas of infection on light micrographs were quantified from 4 random fields per sample from each of n = 3 independent experiments using Image J. Data represent +/− SEM of n = 3. Analysis was performed by one way ANOVA with Dunnett Multiple Comparison Post-tests to compare treatments to untreated infected controls ** p<0.01, *** p<0.001.

Figure 6. The antiviral activity of LL-37 is retained by a central truncated fragment.

HEp-2 cells were simultaneously treated with RSV (MOI = 0.005) and full length LL-37 or one of three 22-mer peptide fragments (all at 1 µg/ml, 10 µg/ml, and 25 µg/ml; see Figure 1a) for 2 hours before washing and 24 hours culture at 37°C. The number of infected cells was quantified using an immunoplaque assay and expressed as a percentage relative to untreated, infected cells. Results are shown as mean +/− SEM of n = 3. For statistical analysis, RSV-positive foci counts were log transformed and compared to no peptide treatment, analysed by 2-way ANOVA with Bonferroni Post-tests for specific peptide concentrations ** p<0.01, *** p<0.001.