Interspecies cathelicidin comparison reveals divergence in antimicrobial activity, TLR modulation, chemokine induction and regulation of phagocytosis

Abstract

Cathelicidins are short cationic peptides initially described as antimicrobial peptides, which can also modulate the immune system. Because most findings have been described in the context of human LL-37 or murine CRAMP, or have been investigated under varying conditions, it is unclear which functions are cathelicidin specific and which functions are general cathelicidin properties. This study compares 12 cathelicidins from 6 species under standardized conditions to better understand the conservation of cathelicidin functions. Most tested cathelicidins had strong antimicrobial activity against E. coli and/or MRSA. Interestingly, while more physiological culture conditions limit the antimicrobial activity of almost all cathelicidins against E. coli, activity against MRSA is enhanced. Seven out of 12 cathelicidins were able to neutralize LPS and another 7 cathelicidins were able to neutralize LTA; however, there was no correlation found with LPS neutralization. In contrast, only 4 cathelicidins enhanced DNA-induced TLR9 activation. In conclusion, these results provide new insight in the functional differences of cathelicidins both within and between species. In addition, these results underline the importance not to generalize cathelicidin functions and indicates that caution should be taken in extrapolating results from LL-37- or CRAMP-related studies to other animal settings.

Figure 1. Antibacterial activity of cathelicidins.

E. coli and MRSA (1 × 10⁶ CFU/ml) of were grown in MHB or DMEM ± FCS for 16 hours under constant shaking (200 RPM). Every 15 minutes the OD was measured. Growth delay was defined as the time needed for peptide-treated bacteria to grow above an OD of 0.6 compared to the control bacteria (no peptide added) (A). Cathelicidins were tested in different concentrations (0.31 μM, 0.63 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM) to determine the antimicrobial activity in MHB against E. coli (B) or MRSA (C) and in more physiological medium DMEM + FCS (D,E). Results are presented as average +/− SEM (N = 4).

Figure 2. Cathelicidin induced chemokine and cytokine release by RAW264.7 cells.

RAW264.7 cells were incubated for 2 h and 24 h with cathelicidins (0.08 μM, 0.31 μM, 1.25 μM, 5 μM, and 20 μM), after which the supernatants were harvested and tested for release of CCL2 at 2 h (A) and 24 h (B), CCL5 at 24 h (C) and CXCL10 at 24 h (D). Dotted line represents average cytokine release of control samples. Results are presented as average +/− SEM (N = 3). Statistical differences were determined by Two-way ANOVA with Bonferroni post-hoc test.

Figure 3. Effects of cathelicidins on TLR-2, -4, and -9 activation.

LPS (100 ng/ml) (A), LTA (1 μg/ml) (B) or ODN-1826 (2.5 nM) (C) was mixed with different cathelicidins (0.08 μM, 0.31 μM, 1.25 μM, 5 μM, and 20 μM) before addition to the RAW264.7 cells. Supernatants were collected after 2 hours (A,B) or 24 hours (C) incubation and TNFα expression was measured. The dotted line represents the average cytokine release of control samples. Results are presented as average +/− SEM (N = 3). Statistical differences were determined by Two-way ANOVA with Bonferroni post-hoc test.

Figure 4. Effects of cathelicidins on phagocytosis by RAW264.7 cells.

RAW264.7 cells were incubated with cathelicidins (0.31 μM, 1.25 μM, and 5 μM) and red fluorescent latex beads (10 beads to 1 cell) at 37 °C (energy dependent uptake) or on ice (non-specific adherence) for 30 minutes. Histograms show control (no peptide present) bead uptake at 37 °C (gray, filled) and 0 °C (black line) (A), or uptake in presence of different concentrations of indicated cathelicidins (B–D); 0 μM (red, filled), 0.31 μM (black line), 1.25 μM (green line) or 5 μM (blue line). Uptake was quantified by determining the MFI after correction for 0 °C control (E). Results are presented as average +/− SEM (N = 3). Statistical differences were determined by Two-way ANOVA with Bonferroni post-hoc test.