Cathelicidin host defence peptide augments clearance of pulmonary Pseudomonas aeruginosa infection by its influence on neutrophil function in vivo

Abstract

Cathelicidins are multifunctional cationic host-defence peptides (CHDP; also known as antimicrobial peptides) and an important component of innate host defence against infection. In addition to microbicidal potential, these peptides have properties with the capacity to modulate inflammation and immunity. However, the extent to which such properties play a significant role during infection in vivo has remained unclear. A murine model of acute P. aeruginosa lung infection was utilised, demonstrating cathelicidin-mediated enhancement of bacterial clearance in vivo. The delivery of exogenous synthetic human cathelicidin LL-37 was found to enhance a protective pro-inflammatory response to infection, effectively promoting bacterial clearance from the lung in the absence of direct microbicidal activity, with an enhanced early neutrophil response that required both infection and peptide exposure and was independent of native cathelicidin production. Furthermore, although cathelicidin-deficient mice had an intact early cellular inflammatory response, later phase neutrophil response to infection was absent in these animals, with significantly impaired clearance of P. aeruginosa. These findings demonstrate the importance of the modulatory properties of cathelicidins in pulmonary infection in vivo and highlight a key role for cathelicidins in the induction of protective pulmonary neutrophil responses, specific to the infectious milieu. In additional to their physiological roles, CHDP have been proposed as future antimicrobial therapeutics. Elucidating and utilising the modulatory properties of cathelicidins has the potential to inform the development of synthetic peptide analogues and novel therapeutic approaches based on enhancing innate host defence against infection with or without direct microbicidal targeting of pathogens.

Figure 1. Exogenous LL-37 enhances pulmonary clearance of P. aeruginosa.

Wild type C57Bl/6 mice were weighed, then inoculated with 3×10⁷ cfu of P. aeruginosa PAO1 or PBS and 10 µg LL-37 peptide or PBS by intranasal delivery. a) Immediately after inoculation of all mice, a subset (called 0 hr; n = 3 per group) were culled and their lungs homogenised (60 minutes after initial inoculation), or b–f) 6 or 24 hours after inoculation mice were re-weighed and culled, and their lungs were lavaged before homogenisation. BALF and lung homogenates were serially diluted, plated and incubated overnight at 37°C before bacterial colonies were counted and corrected for volume. Mean PAO1 cfu +/− SEM in the lung homogenate (a, c & e) or BALF (d & f) for infected animals (n≥9 per condition) are displayed. No bacteria were detected in samples from uninfected mice. b) Data show mean percentage weight loss +/− SEM. For statistical analyses bacterial counts were normalised by logarithmic transformation. Analyses were conducted using 2 way ANOVA with Bonferroni's post tests; * p<0.05, ** p<0.01.

Figure 2. Exogenous LL-37 promotes an early neutrophil response to P. aeruginosa.

Wild type C57Bl/6 mice were inoculated with 3×10⁷ cfu of P. aeruginosa PAO1 or PBS and 10 µg LL-37 peptide or PBS by intranasal delivery. At 6 hours (a, b & e) or 24 hours (c, d & f) after inoculation mice were culled and their lungs were lavaged. BALF was cytocentrifuged and differential counts were conducted for neutrophils (a–d) and monocytes (e & f). Data show Tukey box and whiskers plots for infected (a, c, e & f) (n≥9 per condition) and uninfected (b & d) animals (n≥5 per condition). Analyses were conducted using the Mann Whitney test; * p<0.05. ND denotes “not detected”.

Figure 3. P. aeruginosa, but not exogenous LL-37, induces pulmonary cytokine responses.

Wild type C57Bl/6 mice were inoculated with 3×10⁷ cfu of P. aeruginosa PAO1 and 10 µg LL-37 peptide or PBS by intranasal delivery. At 6 hours (a–e) or 24 hours (b–j) after inoculation, mice were culled and their lungs were lavaged. BALF was centrifuged to remove cells and levels of TNF (a, f), IL-6 (b, g), MIP-2 (c, h), KC (d, i) and MCP-1 (e, j) were determined. Data show Tukey box and whiskers plots for n≥9 animals per condition. Analyses were conducted using the Mann Whitney test.

Figure 4. Cathelicidin-deficient mice display impaired pulmonary clearance of P. aeruginosa.

Camp−/− mice and wild type controls were inoculated with 3×10⁷ cfu of P. aeruginosa PAO1 or PBS by intranasal delivery. At 6 or 24 hours after inoculation mice were culled and their lungs were lavaged before homogenisation. BALF and lung homogenates were serially diluted, plated and incubated overnight at 37°C before bacterial colonies were counted and corrected for volume. Mean PAO1 cfu +/− SEM in the lung homogenate (a & c) or BALF (b & d) for infected animals (n≥10 per condition) are displayed. No bacteria were detected in samples from uninfected mice. For statistical analyses bacterial counts were normalised by logarithmic transformation. Analyses were conducted using 2 way ANOVA with Bonferroni's post tests; * p<0.05.

Figure 5. Cathelicidin-deficient mice display impaired late neutrophil responses to P. aeruginosa.

Camp−/− mice and wild type controls were inoculated with 3×10⁷ cfu of P. aeruginosa PAO1 by intranasal delivery. At 6 hours (a & b) or 24 hours (c & d) after inoculation mice were culled and their lungs were lavaged. BALF was cytocentrifuged and differential counts were conducted for neutrophils (a & c) and monocytes (b & d). Data show Tukey box and whiskers plots for n≥8 animals per condition. Analyses were conducted using the Mann Whitney test; * p<0.05.

Figure 6. P. aeruginosa, but not cathelicidin sufficiency, induces pulmonary cytokine responses.

Camp−/− mice and wild type controls were inoculated with 3×10⁷ cfu of P. aeruginosa PAO1 by intranasal delivery. At 6 hours (a–e) or 24 hours (b–j) after inoculation, mice were culled and their lungs were lavaged. BALF was centrifuged to remove cells and levels of TNF (a, f), IL-6 (b, g), MIP-2 (c, h), KC (d, i) and MCP-1 (e, j) were determined. Data show Tukey box and whiskers plots for n≥8 animals per condition. Analyses were conducted using the Mann Whitney test.

Figure 7. Exogenous LL-37 enhances pulmonary clearance of P. aeruginosa in cathelicidin-deficient mice.

Camp−/− mice were inoculated with 3×10⁷ cfu of P. aeruginosa PAO1 or PBS and 10 µg LL-37 peptide or PBS by intranasal delivery. At 6 or 24 hours after inoculation mice were re-weighed and culled, and their lungs were lavaged before homogenisation. BALF and lung homogenates were serially diluted, plated and incubated overnight at 37°C before bacterial colonies were counted and corrected for volume. Mean PAO1 cfu +/− SEM in the lung homogenate (a & c) or BALF (b & d) for infected animals (n≥6 per condition at 6 hours and n≥10 per condition at 24 hours) are displayed. No bacteria were detected in samples from uninfected mice. For statistical analyses bacterial counts were normalised by logarithmic transformation. Analyses were conducted using 2 way ANOVA with Bonferroni's post tests; ** p<0.01, *** p<0.001.

Figure 8. Exogenous LL-37 promotes an early neutrophil response to P. aeruginosa in cathelicidin-deficient mice.

Camp−/− mice were inoculated with 3×10⁷ cfu of P. aeruginosa PAO1 and 10 µg LL-37 peptide or PBS by intranasal delivery. At 6 hours (a) or 24 hours (b) after inoculation mice were culled and their lungs were lavaged. BALF was cytocentrifuged and differential counts were conducted for neutrophils. Data show Tukey box and whiskers plots for n≥8 animals per condition. Analyses were conducted using the Mann Whitney test; * p<0.05.