Antimicrobial peptide LL-37 increases rhinovirus-induced interferon β expression in human airway epithelial cells through a Ca2+-dependent mechanism

Abstract

The human cathelicidin LL-37 is active against both bacteria and viruses, but it also shows immunomodulatory properties. Here, we assess the impact of LL-37 on viral signaling in human airway epithelial BEAS-2B cells infected with the respiratory pathogen rhinovirus (RV). We show that LL-37 (4 μM) enhances RV-induced expression of interferon β (IFNβ) transcript and reduces viral-load. LL-37-evoked potentiation of RV-stimulated IFNβ does not involve up-regulation of the classical viral TLR3, MDA5 and RIG-I receptors. Moreover, the LL-37-induced stimulation of IFNβ expression in the presence of RV is abolished by chloroquine, an inhibitor of endosomal acidification. Interestingly, RV + LL-37-induced stimulation of IFNβ is observed in the absence but not in the presence of the Ca²⁺ chelating agent EGTA, indicating that Ca²⁺ is critical for this effect. Indeed, we demonstrate that LL-37 increases intracellular [Ca²⁺] in cells loaded with the fluorescent Ca²⁺ indicator Fluo-4 AM. Furthermore, we reveal that treatment with RV in combination with the Ca²⁺ ionophore A23187 promotes IFNβ expression, showing the importance of Ca²⁺. In conclusion, we demonstrate that LL-37 acts in synergy with RV to enhance IFNβ expression and that this effect involves LL-37-induced increase in intracellular [Ca²⁺].

Keywords: Cathelicidin; Endosome; Host defense peptide; Innate immunity; Interferons; Rhinovirus.

Fig. 1

LL-37 potentiates RV-induced IFNβ mRNA expression and attenuates RV-load in BEAS-2B cells. (A–D) Cells were pre-incubated with LL-37 (4 μM) for 1 h before RV (0.15 MOI) was included. After 1 h with RV, cells were incubated for 24 (A, D) and 48 h (B, C) in the continuous presence or absence of LL-37. Expression of IFNβ and RV transcripts were determined by qPCR (A, D). (A) Effects of LL-37 on RV-induced IFNβ mRNA expression. (B) IFNβ protein concentration in cell supernatants was determined with ELISA and normalized to total protein contents in each sample. (C) IFNβ immunoreactivity in cell supernatants was analyzed with dot blot applying PBS as blank. (D) Effects of LL-37 on RV-load. DMSO (0.1 %) was included as vehicle control. Values are presented as means ± SEM. ∗ represents P < 0.05. ns = not significant vs. vehicle control. Statistical significance was determined with Student’s two-tailed paired t-test for single comparisons between two groups and one-way ANOVA followed by Holm-Sidak’s post-hoc analysis for multiple comparisons as appropriate.

Fig. 2

LL-37 has no effect on RV-induced viral receptor expression in BEAS-2B cells. (A–C) Cells were pre-incubated with LL-37 (4 μM) for 1 h before RV (0.15 MOI) was included. After 1 h with RV, cells were incubated for 24 h in the continuous presence or absence of LL-37. (A) TLR3, (B) MDA5 and (C) RIG-I transcript expressions were determined by qPCR. DMSO (0.1 %) was included as vehicle control. Values are presented as means ± SEM. ∗ represents P < 0.05 vs. vehicle control. ns = not significant vs. vehicle control. Statistical significance was determined with one-way ANOVA followed by Holm-Sidak’s post-hoc analysis for multiple comparisons as appropriate.

Fig. 3

Stimulation with LL-37 increases intracellular Ca²⁺ concentration in BEAS-2B cells. (A, B) Cells were loaded with the fluorescent Ca²⁺ indicator dye Fluo-4 AM and stimulated with or without LL-37 (4 μM) in Ca²⁺-containing (2.5 mM) HEPES solution. (A) Fluorescence was assessed continuously every 5 min for 40 min in a fluorescence microplate reader, and the time curve plotted and normalized to background fluorescence before LL-37 or vehicle was introduced. (B) Fluorescence determined at the endpoint (40 min) and compared to vehicle control. The fluorescence signal was normalized to fluorescence in control cells for each of the 4 independent experiments. DMSO (0.1 %) was included as vehicle control. Values are presented as means ± SEM. ∗ represents P < 0.05 vs. vehicle control determined with Student’s two-tailed unpaired t-test for single comparisons between two groups as appropriate.

Fig. 4

LL-37 potentiates RV-induced IFNβ expression in the absence but not in the presence of EGTA. (A) Effects of the endosome acidification inhibitor chloroquine on LL-37-induced IFNβ transcript expression. BEAS-2B cells were pre-incubated with LL-37 (4 μM) + chloroquine (2 μg/ml) for 1 h before RV (0.15 MOI) was included. After 1 h with RV, cells were incubated for 24 h in the continuous presence or absence of LL-37 + chloroquine. (B) Effects of LL-37 on IFNβ mRNA expression in the absence or presence of EGTA. BEAS-2B cells were pre-incubated with LL-37 (4 μM) in the absence or presence of 0.5 mM EGTA for 1 h before RV (0.15 MOI) was included. After 1 h with RV, cells were incubated for 24 h in the continuous presence or absence of LL-37 and EGTA. (C) Stimulation with the Ca²⁺ ionophore A23187 (10 μM) in combination with RV for 24 h potentiates RV-stimulated IFNβ transcript expression in BEAS-2B cells. A23187 was introduced 1 h prior to RV. After 1 h with RV (0.15 MOI), cells were incubated for 24 h in the continuous presence or absence of A23187. DMSO and PBS (0.1 % for both) were included as vehicle controls. Values are presented as means ± SEM. ∗, ∗∗ and ∗∗∗∗ represent P < 0.05, P < 0.01 and P < 0.0001 determined with one-way ANOVA followed by Holm-Sidak’s post-hoc analysis for multiple comparisons as appropriate. ns = not significant.

Supplemental Fig. 1

RV and RV + LL37 have no effects on total protein levels. Total protein concentration in BEAS-2B cell lysates was determined at 48 h using a total protein assay. ns = not significant vs. vehicle control. Please, see legend to Fig. 1 for further information about experimental protocol and statistics.

Supplemental Fig. 2

Stimulation with LL-37 for 40 min increases intracellular Ca²⁺ concentration. The complete data set of fluorescence in 4 independent experiments with 8 replicates (culture wells) for each experiment (n = 32 in each group). ∗∗∗∗ represents P < 0.0001. Please, see legend to Fig. 3 for further information about experimental protocol and statistics.