An estrogen-independent and IL-1-dependent pathway controls vulvovaginal candidiasis through combined IL-17/IL-22 signaling

Abstract

Vulvovaginal candidiasis (VVC), caused by the commensal pathobiont Candida albicans, affects >75% of women, marring quality of life and incurring significant health costs. Estrogen (E2) activity is tightly linked to VVC susceptibility, and preclinical models employ E2 to establish vaginal colonization. Unlike most forms of candidiasis, VVC is not considered to be a condition of immune compromise. Rather, VVC is characterized by high levels of PMNs and inflammatory cytokines that drive immunopathology but fail to clear the fungus. The role of the Type 17 pathway in this condition is controversial. Th17 signature profiles are upregulated in vaginal tissue during VVC in mice and humans. However, loss of individual Th17 components by gene deletion or anti-cytokine administration does not predispose to disease. Here, we reveal an IL-1/Type 17-driven arm of immunity that operates to control C. albicans in the vaginal mucosa independently of estrogen. Il1r ^-/- mice subjected to VVC bore high vaginal loads, accompanied by reduced IL-17A/F and IL-22 expression and suppressed PMN influx. Although mice lacking IL-17, IL-22/IL-22R or IL-23 individually exhibited normal susceptibility to VVC, mice lacking receptors for both cytokines (Il17raIl22ra1 ^-/-) had high and persistent fungal loads, with increased vaginal tissue damage and elevated IL-1α/β levels. Thus, IL-1R serves as a master regulator of protective Type 17 responses, and moreover IL-1 signaling alone is insufficient to control fungal colonization. Interestingly, Il1r ^-/- and Il17raIl22ra1 ^-/- mice showed high fungal colonization in the absence of exogenous estrogen, and this susceptibility persisted even when mice were given progesterone to prevent estrus. Together, these data reveal an estrogen-independent pathway of vaginal antifungal host defense mediated by combinatorial actions of IL-17 and IL-22 and governed by upstream IL-1R signaling.

Keywords: Candida albicans; IL-1; Th17; cytokine signaling; fungal immunity; mucosal candidiasis; vaginitis.

Fig 1.. IL-1R signals control vaginal immunity to VVC

a. VVC model b, c. Mice administered E2 on day −3 and day 4 were infected vaginally with C. albicans. Fungal burdens in WT (C57BL/6, n=8-9), Il1r^−/− (n=8) or Il1b^−/− (n=9) mice on day 3 and 7. Data are geometric mean + geometric SD and significance determined by two-sided Mann-Whitney U test. d. Representative vaginal histology day 3 p.i. Green arrows denote C. albicans hyphae. Black arrows indicate PMNs e. PMN counts in VLF of indicated mice on day 3 and 7. Data are mean ± SEM, analyzed by two-sided Student’s t-test. F. Differentially expressed genes comparing whole vaginal tissue of WT versus Il1r^−/− mice at day 3 (adjusted p < 0.05, Student’s t-test and Benjamini and Hochberg’s analysis). Type 17 cytokines and their downstream effectors in blue.

Figure 2.. Combined loss of IL-17R and IL-22R signaling increases VVC susceptibility.

a. Fungal loads in VLF of E2-treated Il23a^−/− mice (n=5-9) mice on days 3 and 7 post-infection. b. Cytokine mRNA levels in whole vaginal tissue of Il23a^−/− mice by qPCR, relative to Gapdh and normalized to sham. c, d. Fungal loads in VLF from Il22ra^−/− (n=6-11) and Il17raIl22ra1^−/− mice (n=15-16). Data in a, c, and d are geometric mean ± geometric SD and data in b are mean ± SEM; two-tailed unpaired Mann-Whitney test (a, c, and d) and two-tailed unpaired Student’s t-test (b).

Figure 3.. IL-17/22-driven signaling dampens inflammation and tissue damage.

Fungal loads in VLF of E2-treated mice on days 3 and 7 post-infection. a. LDH activity in VLF on days 3 and 7. b. PMNs in VLF determined by cytology. c, Representative PAS and H&E staining of vaginal tissue on day 3 p.i.. Green arrows denote C. albicans hyphae. Black arrows indicate PMNs d. Indicated cytokine/chemokine levels were determined by Luminex. Data in a, b, and d are mean ± SEM; two-tailed unpaired Student’s t-test (a, d) and two-tailed unpaired Mann-Whitney test (b).

Figure 4.. IL-17/22 receptor deficiency drives VVC susceptibility in the absence of E2 conditioning.

a-d. Indicated mice were administered sesame oil (SO) vehicle (Veh). Left: fungal loads in VLF assessed on days 3 and 7 (Il23a^−/− n=8-9; Il22ra1^−/− n=10-11; Il1r^−/− n= 9; Il17raIl22ra1^−/− n=14-21). Right: % of mice with detectable fungal load over the indicated time course. e. PMN counts on day 3. f. LDH activity in VLF on day 3 and 7. g. Cytokine levels in VLF on day 3. Data in a-d, and h show geometric mean ± geometric SD and data in f and g show mean ± SEM; two-tailed unpaired Mann-Whitney test (a-d: left, e), Clearance data analyzed by Mantel-Cox and two-tailed unpaired Student’s t-test (f, g).

Figure 5.. Sex hormone dynamics in Il17raIl22ra1^−/− mice.

a. Hormone predominance and cell populations in VLF during the murine estrus cycle, image adapted from Ref. b. Estrus cycle tracking by PAP staining of VLF over 12 days. Each color/symbol represents an individual mouse. D: diestrus, M: metestrus, E: estrus, P: proestrus/estrus. c. Serum hormone levels at proestrus or estrus (n=4). d. Representative VLF imaging after E2 or P4 treatment at day −1. e. Fungal loads in VLF on days 3 and 7 post-infection after sesame oil (Vehicle) (n=7), estrogen (E2) (n=6-7), or progesterone (P4) (n=11-13) given on days −3 and 4 relative to infection. f. LDH activity in VLF on days 3 and 7. Data in c and f are mean ± SEM; two-tailed unpaired Student’s t-test and data in e are geometric mean ± geometric SD with Kruskal-Wallis test with Dunn’s multiple comparisons.