Human cathelicidin production by the cervix

Abstract

hCAP18/LL-37 is the sole human cathelicidin; a family of host defence peptides with key roles in innate host defence. hCAP18/LL-37 is expressed primarily by neutrophils and epithelial cells, but its production and function in the lower genital tract is largely uncharacterised. Despite the significant roles for cathelicidin in multiple organs and inflammatory processes, its impact on infections that could compromise fertility and pregnancy is unknown. The aim of this study was to investigate cathelicidin production, regulation and function in the cervix. hCAP18/LL-37 was found to be present in cervicovaginal secretions collected from women in the first trimester of pregnancy and to be expressed at significantly higher levels in samples from women with alterations in vaginal bacterial flora characteristic of bacterial vaginosis. In endocervical epithelial cell lines, expression of the gene encoding hCAP18/LL-37 (CAMP) was not affected by TLR agonists, but was found to be up-regulated by both 1, 25 hydroxyvitamin D3 and 25 hydroxyvitamin D3. However, no association was found between serum levels of vitamin D and hCAP18/LL-37 concentrations in cervicovaginal secretions (n = 116). Exposure to synthetic LL-37 had a pro-inflammatory effect on endocervical epithelial cell lines, increasing secretion of inflammatory cytokine IL-8. Together these data demonstrate inducible expression of hCAP18/LL-37 in the female lower reproductive tract in vivo and suggest the capacity for this peptide to modulate host defence to infection in this system. Further investigation will elucidate the effects of hCAP18/LL-37 on the physiology and pathophysiology of labour, and may lead to strategies for the prevention of infection-associated preterm birth.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Cervical hCAP18/LL-37 expression.

(A) Correlation between cervicovaginal hCAP18/LL-37 and Myeloperoxidase (r = 0.1; n = 77; p = 0.3). (B) Representative western blot of hCAP18/LL-37 expression in cervicovaginal secretions. The 18 kDa hCAP18 and the 5–10 kDa cleaved peptide LL-37 were detected in cervicovaginal secretions.

Figure 2. hCAP18/LL-37 expression in cervicovaginal secretions of women with and without Bacterial Vaginosis.

hCAP18/LL-37 concentration in women with normal flora, intermediate vaginal flora and bacterial vaginosis. Data presented as median ± interquartile range. **, p

Figure 3. Effect of TLR agonists on CAMP and DEFB4 expression in endocervical (END E6/E7) and ectocervical (ECT E6/E7) cell lines.

Cells cultured for 24(250 ng/ml), POLY (I:C) (5 ng/ml), Rec FLA-ST (100 ng/ml) and FSL-1 (100 ng/ml), and untreated controls. (A) END E6/E7 CAMP expression (n = 3), (B) ECT E6/E7 CAMP expression (n = 3), (C) END E6/E7 DEFB4 expression (n = 3), (D) ECT E6/E7 DEFB4 expression (n = 3). Data presented as mean fold change relative to control ± SEM. **, ***, P<0.01, 0.001 respectively compared with control. (One-way ANOVA with Dunnett's post-test).

Figure 4. Effect of vitamin D on CAMP and DEFB4 expression in endocervical (END E6/E7) and ectocervical (ECT E6/E7) cell lines.

Cells treated for 24(control), 3 nM, 30 nM or 100 nM of 25(OH) vitamin D3 or 1,25 (OH) vitamin D3. (A) END E6/E7 CAMP expression (n = 3), (B) ECT E6/E7 CAMP expression (n = 3), (C) END E6/E7 DEFB4 expression (n = 3), (D) ECT E6/E7 DEFB4 expression (n = 3). Data presented as mean fold change relative to control ± SEM. ##, ###, p<0.01, 0.001 respectively compared to 25 (OH) vitamin D3 control. *, **, p<0.05, 0.01 compared to 1,25(OH) vitamin D control3. (One-way ANOVA with Dunnett's post-test).

Figure 5. Effect of 25(OH) vitamin D3 on CYP27B1 expression in endocervical (END E6/E7) and ectocervical (ECT E6/E7) cell lines.

Cells treated for 24(control), 3 nM, 30 nM or 100 nM 25(OH) vitamin D3. (A) End E6/E7 cells (n = 3), (B) Ect E6/E7 cells (n = 3). Data presented as mean fold change relative to control ± SEM (error bars). *, **, **, p<0.05, 0.01, 0.001 respectively compared with control (One-way ANOVA with Dunnett's post-test).

Figure 6. Relationship between 25(OH) vitamin D and hCAP18/LL37 in endocervical (END E6/E7) and ectocervical (ECT E6/E7) cell lines.

Correlation between serum 25(OH) vitamin D and CVS hCAP18/LL-37 (r = −0.13; n = 122; p = 0.14). Data presented as scatter plots and divided in to deficient 75 nmol/l. Data analysed by Pearson's Correlation.

Figure 7. Effect of LL-37 receptor antagonists on IL-8 secretion in endocervical (END E6/E7) and ectocervical (ECT E6/E7) cell lines.

END E6/E7 cells were pre-treated for 30 minutes with or without antagonists before being cultured for 24 hours with scrambled LL-37 (25 µg/ml) and LL-37 (25 µg/ml). (A) END E6/E7 treated with PTX (GPCR antagonist, 200 ng/ml), (B) ECT E6/E7 cells treated with PTC, (C) END E6/E7 cells treated with WRW4 (FPR2 antagonist, 10 µM), (D) ECT E6/E7 cells treated with WRW4, (E) END E6/E7 cells treated with KN-62 (P2X7R antagonist, 10 µM), (F) ECT E6/E7 cells treated with KN-62, (G) END E6/E7 cells treated with oATP (P2X7R antagonist, 100 µM) and (H) ECT E6/E7 cells treated with oATP. Data presented as mean concentration ± SEM (error bars). *, **, ***, p<0.05, 0.01, 0.001 respectively. (2way ANOVA with multiple comparison tests).