IL-1 signaling inhibits Trichophyton rubrum conidia development and modulates the IL-17 response in vivo

Abstract

Dermatophytosis are one of the most common fungal infections in the world. They compromise keratinized tissues and the main etiological agent is Trichophyton rubrum. Macrophages are key cells in innate immunity and prominent sources of IL-1β, a potent inflammatory cytokine whose main production pathway is by the activation of inflammasomes and caspase-1. However, the role of inflammasomes and IL-1 signaling against T.rubrum has not been reported. In this work, we observed that bone marrow-derived macrophages produce IL-1β in response to T.rubrum conidia in a NLRP3-, ASC- and caspase-1-dependent fashion. Curiously, lack of IL-1 signaling promoted hyphae development, uncovering a protective role for IL-1β in macrophages. In addition, mice lacking IL-1R showed reduced IL-17 production, a key cytokine in the antifungal defense, in response to T.rubrum. Our findings point to a prominent role of IL-1 signaling in the immune response to T.rubrum, opening the venue for the study of this pathway in other fungal infections.

Keywords: ASC, Apoptosis-associated Speck-like protein containing a CARD; BMDM, bone marrow derived macrophages; CFU, colony-forming units; IL, interleukin; IL-1 signaling; IL-17; NLR; NLRP3 inflammasom; NLRP3, NOD-like receptor family, pyrin domain containing 3; NOD-like receptor; Trichophyton rubrum; WT; dermatophytosis; fungal infections; innate immunity; wild-type.

Figure 1.

BMDMs produce IL-1β in response to T.rubrum conidia but succumb to hyphae growth. LPS-primed BMDMs were incubated with T.rubrum conidia for 4, 6, 8 and 10 h (no fungi in control wells). (A) Interaction outcome was analyzed by optical microscopy (1000×) and arrows indicate fungal structures. (B) IL-1β levels quantified in the culture supernatants expressed as mean ± SEM. Two-way ANOVA and Bonferroni posttest: ***P < 0.001. Cytokine data are pooled from 3 independent experiments and microscopy photos taken from one experiment (representative of 3 independent experiments) are shown.

Figure 2.

NLRP3 and ASC deficiency reduces IL-1β secretion, but only lack of ASC interferes in phagocytosis. LPS-primed NLRP3^−/− or ASC^−/− BMDMs were incubated with T.rubrum conidia for 4, 6, 8 and 10 h (no fungi in control wells). (A) NLRP3^−/− Interaction outcome and (B) ASC^−/− Interaction outcome analyzed by optical microscopy (1000×). Arrows indicate fungal structures. (C) IL-1β production by NLRP3^−/− BMDMs and (D) IL-1β levels produced by ASC^−/− BMDMs. (E) ASC^−/− BMDMs were incubated with T.rubrum conidia for 4 h and conidia internalization was counted by optical microscopy. Results expressed as mean ± SEM. Two-way ANOVA and Bonferroni post test: *P < 0.05; **P < 0.01; ***P < 0.001. Cytokine data pooled from 2 independent experiments performed, in triplicate wells each, are shown. Microscopy photos taken from one experiment (representative of 2 independent experiments triplicate wells each) are shown.

Figure 3.

Caspase-1/-11 absence accelerates hyphae development and reduces IL-1β secretion. LPS-primed caspase-1/-11^−/− BMDMs were incubated with T.rubrum conidia for 4, 6, 8 and 10 h (no fungi in control wells). (A) Interaction outcome was analyzed by optical microscopy (1000×) and arrows indicate fungal structures. (B) IL-1β levels quantified in the culture supernatants expressed as mean ± SEM. Two-way ANOVA and Bonferroni posttest: ***P < 0.001. Cytokine data are pooled from 2 independent experiments performed in triplicate wells each. Microscopy photos taken from one experiment (representative of 2 independent experiments, triplicate wells each) are shown.

Figure 4.

Impaired IL-1 signaling leads to faster hyphae development and reduced IL-1β production. LPS-primed IL-1R^−/− BMDMs were incubated with T.rubrum conidia for 4, 6, 8 and 10 h (no fungi in control wells). (A) Interaction outcome was analyzed by optical microscopy (1000×) and arrows indicate fungal structures. (B) IL-1β levels quantified in the culture supernatants. Two-way ANOVA and Bonferroni posttest: *P < 0.05; ***P < 0.001. (C) Fungal loads in WT and IL-1R^−/− BMDMs determined by CFU assay. Data expressed as mean ± SEM. Cytokine and CFU data were pooled from 2 independent experiments performed in triplicate wells each. Microscopy photos taken from one experiment (representative of 2 independent experiments, triplicate wells each) are shown.

Figure 5.

T.rubrum affects preferentially the liver but not the spleen. Animals were infected i.P. with T.rubrum conidia (5 × 10⁶/ animal) and fungal burden were determined 7 and 14 days post infection (dpi). (A) Fungal burden in Liver. (B) Fungal burden in Spleen. Unpaired t-test: no significance found. Data pooled from 3 to 4 independent experiments (groups of 3 animals in each experiment) expressed as mean ± SEM.

Figure 6.

IL-1R^−/− mice show defective IL-17 production in response to T.rubrum. Animals were infected i.p. with T.rubrum conidia (5 × 10⁶/ animal) and cytokines levels (IL-1β; IFN-γ; IL-4 and IL-17) were determined in liver homogenates (A) 7 and (B) 14 days post infection (dpi). Two-way ANOVA and Bonferroni posttest: *P < 0.05; ***P < 0.001. Data, expressed as mean ± SEM, were pooled from 2 to 4 independent experiments (groups of 3 animals in each experiment).

Figure 7.

Proposed model for the role of IL-1β in response to T.rubrum. Macrophages phagocytose T.rubrum, leading to the activation of NLRP3 inflammasome and, consequently, to IL-1β production. By your turn, IL-1β, signaling through IL-1R, delays fungal development into hyphae inside the macrophages and also helps to shape the IL-17 response in vivo.