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. 2013 Jan;94(Pt 1):40-49.
doi: 10.1099/vir.0.045013-0. Epub 2012 Oct 10.

The human cathelicidin LL-37 inhibits influenza A viruses through a mechanism distinct from that of surfactant protein D or defensins

Shweta Tripathi  1 , Tesfaldet Tecle  1 , Anamika Verma  1 , Erika Crouch  2 , Mitchell White  1 , Kevan L Hartshorn  1
Affiliations

The human cathelicidin LL-37 inhibits influenza A viruses through a mechanism distinct from that of surfactant protein D or defensins

Shweta Tripathi et al. J Gen Virol. 2013 Jan.

Abstract

LL-37, the only human cathelicidin, is a cationic antimicrobial peptide with antibacterial and antifungal activity. LL-37 is released from neutrophil granules and produced by epithelial cells. It has been implicated in host defence against influenza A virus (IAV) in recent studies. We now demonstrate dose-related neutralizing activity of LL-37 against several seasonal and mouse-adapted IAV strains. The ability of LL-37 to inhibit these IAV strains resulted mainly from direct effects on the virus, since pre-incubation of virus with LL-37 was needed for optimal inhibition. LL-37 bound high-density lipoprotein (HDL), and pre-incubation of LL-37 with human serum or HDL reduced its antiviral activity. LL-37 did not inhibit viral association with epithelial cells as assessed by quantitative RT-PCR or confocal microscopy. This finding contrasted with results obtained with surfactant protein D (SP-D). Unlike collectins or human neutrophil defensins (HNPs), LL-37 did not induce viral aggregation under electron microscopy. In the electron microscopy studies, LL-37 appeared to cause disruption of viral membranes. LL-37 had additive antiviral activity when combined with other innate inhibitors like SP-D, surfactant protein A and HNPs. Unlike HNPs, LL-37 did not bind SP-D significantly. These findings indicate that LL-37 contributes to host defence against IAV through a mechanism distinct from that of SP-D and HNPs.

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Figures

Fig. 1.
Fig. 1.
Binding of LL-37 to IAV or S. aureus and neutralization of IAV by LL-37. (a) Binding assessed by solid-phase ELISA. Results are means±sem of four separate experiments. Binding of LL-37 to IAV (Phil82 strain), S. aureus and HDL was significantly greater than binding to BSA at all concentrations tested. (b–d) Neutralization assessed using the fluorescent focus assay for detection of viral nucleoprotein. All results are means±sem of four or more separate experiments. The Phil82 IAV strain was used. (b) Effect of pre-incubation of Phil82 IAV with LL-37 versus scrambled LL-37 (sLL-37). (c) Effects of varying the time of addition of LL-37. Using all methods, LL-37 reduced viral infectivity significantly compared with the control, but inhibition was significantly less when cells were incubated with LL-37 either before or after infection than when the virus was pre-incubated with LL-37. (d) Effects of HDL, apolipoprotein A1 or the EGFR-signalling inhibitor AG1478 on antiviral activity of LL-37. *, P<0.05 compared with binding to BSA in (a) and compared with infectivity of untreated virus in (b) and (c). **, P<0.05 compared with the control, sLL-37 (b), pre-incubation of cells with LL-37 or delayed addition of LL-37 (c) as assessed by ANOVA. #, HDL reduced antiviral activity of LL-37 significantly as assessed by ANOVA.
Fig. 2.
Fig. 2.
Inhibition of infectivity of IAV strains by LL-37 or CRAMP in HBTE cells or MDCK cells. These experiments were performed as in Fig. 1(b). (a) Effects of LL-37 or sLL-37 on replication of Phil82 in HTBE cells. sLL-37 caused increased viral replication at the lowest concentration tested, but reduced replication at the highest concentrations tested. LL-37 inhibited viral replication at all concentrations tested, and its effects were significantly greater than those of sLL-37 as compared by ANOVA. (b) Inhibition of three mouse-adapted IAV strains by LL-37 in MDCK cells. In this set of experiments, sLL-37 did not inhibit replication of the WSN strain significantly. (c) CRAMP inhibits replication of Phil82 and PR-8 in MDCK cells. Results are means±sem of four or more experiments. *, P<0.05 compared with controls; **, P<0.005 compared with controls [in (b) and (c), * and ** apply to all viruses tested].
Fig. 3.
Fig. 3.
Effects of LL-37 on viral interactions with A549 cells under confocal microscopy. Alexa Fluor 594-labelled Phil82 IAV was incubated with control buffer, LL-37 or SP-D followed by incubation of A549 cell monolayers with the virus. The virus appears red; cell nuclei were stained with DAPI 350 and appear blue and cell membranes were labelled with WGA–Oregon green 488. Results are representative of four experiments. Virus alone is shown in the upper panels. The left column of images was taken at the wavelength of IAV alone (red); the right panel of images combines wavelengths of virus, cell membranes and nuclei. No consistent alteration in the pattern of virus binding to cells was seen with LL-37; however, SP-D caused formation of large viral aggregates. The concentration of LL-37 used was 8 µg ml−1, but similar results were obtained with 4 and 16 µg ml−1. The concentration of SP-D used was 2 µg ml−1.
Fig. 4.
Fig. 4.
Effects of LL-37 on viral replication as assessed by qPCR. IAV (strain Phil82) was pre-incubated with LL-37 as in Fig. 1(b) and the virus was then incubated with A549 cells for 45 min. The cell supernatant was then harvested and the cells were washed several times prior to preparing cell homogenates. In (a), cell supernatants and cell homogenates were obtained after the 45 min period of initial infection with IAV. RNA was isolated and qPCR was performed. Results are expressed as mean no. of fluorescent foci (FFC) ml−1. The mean FFC ml−1 in control samples for (a) was 190 846 for supernatant and 6216 for cell-associated virus. Results shown are percentages of control copy numbers for samples treated with LL-37. There were no significant differences in viral RNA quantities between control and LL-37-treated samples in (a). (b) RNA was isolated from cells and supernatants after 24 h of infection. In this case, the control FFC ml−1 was 2645 and 158 584, respectively, for supernatant and cells (i.e. cell-associated virus increased markedly in cells compared with 45 min samples). LL-37 at concentrations ≥2 µg ml−1 decreased viral RNA significantly in supernatants and cells in (b). (c) SP-D caused significant reductions in cell-associated viral RNA at both 45 min and 24 h after infection. The quantities of RNA at both time points were reduced significantly at SP-D concentrations of 50 ng ml−1 or higher. All results are means±sem of four or more separate experiments. *, P<0.05 compared with controls.
Fig. 5.
Fig. 5.
LL-37 does not cause viral aggregation. (a) Viral aggregation assessed by measuring increases in light scattering through the viral suspension induced by either LL-37 or SP-D. SP-D induced viral aggregation strongly, as reported previously, but LL-37 did not. LL-37 also did not alter the aggregating effects of SP-D significantly (SP-D+LL-37). All results are means±sem of seven separate experiments. (b) Effects of LL-37 or defensins on appearance of virus under EM. Phil82 IAV (Phil) was pre-incubated with LL-37 (64 µg ml−1), HNP-1 (40 µg ml−1) or RC2 (40 µg ml−1) for 30 min and evaluated by EM. Images in the upper panel were taken at lower magnification and used the Phil82 IAV strain. This panel shows the lack of viral aggregation induced by LL-37 compared with controls. Aggregation was also not evident using lower concentrations of LL-37 (not shown). The effects of HNP-1 and RC2 are shown for comparison. The experiment shown is representative of six experiments using various concentrations of LL-37 (2–64 µg ml−1). The lower panels show higher-power views of either Phil82 or PR-8 virus particles treated with control buffer or LL-37. Note the intact hair-like haemagglutinin spikes on the surface of the control viruses. The Phil82 sample was treated with LL-37 (32 µg ml−1) and shows viral membrane breaks and blebbing. The PR-8 sample treated with LL-37 (16 µg ml−1) shows marked disruption of particle morphology. These results are representative of three or more experiments.
Fig. 6.
Fig. 6.
Interactions of LL-37 with SP-D. In (a), LL-37 or HNP-2 were coated on ELISA plates and binding of SP-D was tested. SP-D showed significant dose-related binding to HNP-2 but not to LL-37. In (b), viral neutralization was assessed in the presence of LL-37 alone or LL-37 combined with 50 ng SP-D ml−1. The combination of LL-37 with 4 µg SP-D ml−1 caused significantly greater reduction in infectivity than either LL-37 or SP-D alone. Similar results were obtained using combinations of LL-37 with 100 ng SP-D ml−1 (not shown). Results are means±sem of four experiments. *, P<0.05 compared with controls; **, P<0.005 compared with controls.
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