Innate immune factors in recurrent and persistent infections of the lower female genital tract

Sammanfattning: Background: Infections of the female genital tract are common and have severe impact on the quality of life and sexual health of affected women. The innate immune system is crucial and is involved in the defense of infections. Understanding innate immunity of the vaginal and cervical mucosa is important to enable development of future preventive and therapeutic strategies for genital infections. Objective: The aim of this thesis was to investigate innate immune factors in women with recurrent candida vulvovaginitis (RVVC) and high grade squamous intraepithelial lesions (HSIL) of the cervix, induced by human papilloma virus (HPV). This in order to contribute to the understanding of the innate immune response in these two common infections of the lower female genital tract. Material and Methods: Clinical examinations, measurements of intravaginal nitric oxide (NO) levels and vaginal biopsies were performed in 28 patients with RVVC and 31 healthy controls. Cervical biopsies and vaginal lavage were collected from 19 patients with HPV induced HSIL and 14 controls. Immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), western blot and reverse transcriptase real time polymerase chain reaction (PCR)) were used to identify and quantify inducible nitric oxide synthase (iNOS), antimicrobial proteins, cytokines and encoding genes. Adhesion and binding assays were performed to evaluate the adhesive capacity of candida and to demonstrate the binding between candida and the antimicrobial protein psoriasin. Transmission electron microscopy was conducted to measure the cell wall thickness in C. albicans affected by psoriasin. To evaluate a new treatment strategy for RVVC, the effect of chlorhexidine digluconate and fluconazole on C. albicans eradication and biofilm was investigated in RVVC and commensal strains, using the crystal violet method and viable count. Results: NO levels were significantly higher in patients during acute infection compared to controls. Levels decreased after fluconazole treatment but remained higher than in controls. Furthermore, increased expression of iNOS was observed in the epithelial basal layer in patients both before and after treatment. There were positive correlations between NO levels, clinical symptoms and examination scores. Findings indicated that C. albicans induces production of psoriasin during mucosal candida infection. Psoriasin was shown to interact with β-glucan in the candida cell wall and inhibit candida adhesion to surfaces. In HPV induced HSIL, psoriasin expression and protein levels increased significantly after surgical treatment and reached similar levels as in controls. The mRNA expression of the pro- inflammatory cytokine IL-8 was higher before treatment and restored to the same levels as in controls six months after lesions were excised. Chlorhexidine digluconate prevented new biofilm formation and reduced already established C. albicans biofilm. Moreover, the number of candida cells in both planktonic state and within the biofilm were significantly decreased. Although fluconazole reduced the growth of C. albicans, no effect was observed on biofilm or candida cells in the biofilm. Conclusions: Several factors of the innate immune system are involved in the local immune response to RVVC and cervical HPV induced HSIL. The mucosal inflammation during an acute episode of RVVC is well demonstrated by the pronounced increase of vaginal NO, also corresponding to intensity of symptoms. The AMP psoriasin was upregulated in RVVC and was found to have an anti-adhesive effect on C. albicans, a result contributing to the understanding of host-pathogen interaction during candida infections. Although fluconazole is the first line treatment of RVVC, the effect is not always satisfactory. According to our results, one explanation could be an inability of fluconazole to dissolve biofilm and eliminate candida cells within the biofilm. Instead local application of chlorhexidine digluconate might be an alternative prophylactic and treatment strategy that inhibits biofilm formation and eradicates C. albicans both in planktonic phase and within biofilm. Alterations in expression of antimicrobial peptides and pro-inflammatory markers in HPV induced cervical HSIL demonstrate activation of innate immunity also in premalignant lesions, however the importance of these results needs further exploration.