IL-22 and IDO1 Affect Immunity and Tolerance to Murine and Human Vaginal Candidiasis

<div><p>The ability to tolerate <i>Candida albicans</i>, a human commensal of the gastrointestinal tract and vagina, implicates that host defense mechanisms of resistance and tolerance cooperate to limit fungal burden and inflammation at the different body sites. We evaluated resistance and tolerance to the fungus in experimental and human vulvovaginal candidiasis (VVC) as well as in recurrent VVC (RVVC). Resistance and tolerance mechanisms were both activated in murine VVC, involving IL-22 and IL-10-producing regulatory T cells, respectively, with a major contribution by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1). IDO1 was responsible for the production of tolerogenic kynurenines, such that replacement therapy with kynurenines restored immunoprotection to VVC. In humans, two functional genetic variants in <i>IL22</i> and <i>IDO1</i> genes were found to be associated with heightened resistance to RVVC, and they correlated with increased local expression of IL-22, IDO1 and kynurenines. Thus, IL-22 and IDO1 are crucial in balancing resistance with tolerance to <i>Candida</i>, their deficiencies are risk factors for RVVC, and targeting tolerance via therapeutic kynurenines may benefit patients with RVVC.</p></div

Vaginal candidiasis in IL-22- or IDO1-deficient mice.

<p>C57BL/6, IL-22- or IDO1-deficient mice (<i>n</i> = 6) were intravaginally inoculated with 5×10⁶<i>C. albicans</i> blastoconidia. (<b>A</b>) Periodic acid-Schiff-staining of vaginal sections and inflammatory cell recruitment in vaginal fluids (May–Grünwald Giemsa staining in the insets) at different days post-infection (dpi). Representative images of histology sections and vaginal fluids were acquired with a 40× and 100× objective respectively. Scale bars, 100 µm. (<b>B</b>) Polymorphonuclear cells (PMNs) quantification in the vaginal fluids. PMNs were identified by nuclear morphology and enumerated per field at ×100 magnification. Each point represents an individual mouse, and horizontal bar indicates the means. **<i>P</i><0.01 and ***<i>P</i><0.001, IDO1- or IL-22-deficient <i>vs.</i> wild-type mice at the indicated days. (<b>C</b>) <i>S100a8</i> and <i>S100a9</i> mRNA expression in vaginal tissue by real-time RT-PCR. The mRNA-normalized data were expressed as relative mRNA in IDO1- or IL-22-deficient <i>vs.</i> wild-type mice. *<i>P</i><0.05 and **<i>P</i><0.01, IDO1- or IL-22-deficient <i>vs.</i> wild-type mice at the indicated days. (<b>D</b>) Levels of calprotectin during vaginal candidiasis. **<i>P</i><0.01, IDO1- or IL-22-deficient <i>vs.</i> wild-type mice. (<b>E</b>) Vaginal fungal burden in mice at different dpi. CFU were quantified by culturing serial dilutions of vaginal fluids (VF) from each mouse and expressed as Log₁₀ CFU/100 µl VF ± s.e.m. *<i>P</i><0.05, IDO1- or IL-22-deficient <i>vs.</i> wild-type mice at the days indicated. Data are pooled or representative (histology) from four independent experiments. (<b>F</b>) Cytokine levels (pg/mg, cytokine/total proteins for each sample) in the vaginal fluids (at 3 dpi for IL-17F and IL-17A). Results represent mean cytokine levels (± s.e.m.) from samples pooled from three experiments (<i>n</i> = 4–6 total samples per group). *<i>P</i><0.05, IDO1- or IL-22-deficient <i>vs.</i> wild-type mice. (<b>G</b>) Levels of IL-22 (pg/mg, cytokine/total proteins) and (<b>H</b>) fungal growth (at 3 dpi) in mice treated with 300 µg of mAb neutralizing IL-22 or isotype control mAb (None) given intraperitoneally the day of and 1 day after the primary infection. (<b>I</b>) Fungal growth (at 3 dpi) in mice treated intravaginally with rIL-22 or PBS (None) the day of and 1 and 2 days after the infection. Pooled data from two experiments (<i>n</i> = 6). *<i>P</i><0.05 and ***<i>P</i><0.001, treated <i>vs.</i> untreated mice. N.S., not significant.</p

IDO1 and kynurenines mediate tolerance in murine VVC.

<p>(<b>A</b>) IDO1 protein and (<b>B</b>) gene expression in the vagina of C57BL/6 mice (<i>n</i> = 4) intravaginally infected with <i>C. albicans</i>. Proteins in vaginal cell lysates (3 dpi) were visualized by western blotting with rabbit polyclonal IDO1 specific antibody. Scanning densitometry was done on a Scion Image apparatus. Western blots out of 2 independent experiments and corresponding pixel density ratio normalized against β-tubulin. <i>Ido1</i> mRNA expression [normalized to mRNA of naïve (dpi 0) mice] in vaginal tissue (RT-PCR) at different dpi. (<b>C</b>) Relative concentrations of kynurenines (Kyn) and (<b>D</b>) kynurenine-to-tryptophan (Kyn/Trp) ratio in vaginal fluids at different dpi. Pooled results from 3 different experiments. *<i>P</i><0.05, IDO1-deficient <i>vs.</i> C57BL/6 mice at the days indicated. N.S., not significant. (<b>E</b>) Vaginal fungal growth (Log₁₀ CFU/100 µl VF ± s.e.m.) at different dpi in mice (<i>n</i> = 6) treated intraperitoneally with a mixture of l-kynurenine, 3-hydroxykynurenine and 3-hydroxyanthranilic acid or PBS (None). Pooled data from 3 different experiments.*<i>P</i><0.05, treated <i>vs.</i> untreated mice at the days indicated. N.S., not significant. (<b>F</b>) Periodic acid-Schiff-stained vaginal sections and inflammatory cell recruitment in vaginal fluids (May–Grünwald Giemsa staining in the insets) acquired with a 40× and 100× objective, respectively, at 21 dpi. Scale bars, 100 µm. Representative image from 3 experiments. (<b>G</b>) Cytokine levels (pg/mg, cytokine/total proteins for each sample, at 21 dpi) in the vaginal fluids of mice treated as above. Pooled data from 3 different experiments. *<i>P</i><0.05, treated <i>vs.</i> untreated (None) mice. (<b>H</b>) Vaginal immunohistochemistry of naïve or infected mice at 3 days after re-challenge. Double staining was done with anti-IL-10-FITC and polyclonal rabbit to FoxP3 followed by anti-rabbit TRITC. Cell nuclei were stained with DAPI (blue). Representative pictures (out of 2 experiments) were taken with a 20× objective. Scale bars, 50 µm.</p

Functional genetic variants in <i>IL22</i> and <i>IDO1</i> genes influence susceptibility to RVVC.

<p>(<b>A</b>) Frequencies (%) of the different genotypes for single nucleotide polymorphisms (SNPs) in <i>IL22</i> (rs2227485), <i>IDO1</i> (rs3808606) and <i>DECTIN1</i> (rs16910526, Y238X) genes among controls (<i>n</i> = 263) and patients with RVVC (<i>n</i> = 145). (<b>B</b>) Cytokines (pg/mg, cytokine/total proteins) and (<b>C</b>) calprotectin levels (mean values ± s.e.m.) in the vaginal fluids of women bearing the CC, CT or TT genotypes at rs2227485 in <i>IL22</i>. (<b>D</b>) Cytokine levels (pg/mg, cytokine/total proteins, mean values ± s.e.m.) in the vaginal fluids of women bearing the CC, CT or TT genotypes at rs3808606 in <i>IDO1</i>. Data in B, C and D indicate mean values from measurements using samples obtained from at least 10 different women for each genotype, assessed in quadruplicates. (<b>E</b>) IDO protein expression in cells from the vaginal fluids of women bearing the CC or TT genotypes at rs3808606 in <i>IDO1</i>. Western blots of a representative sample out of 10 with similar results are shown along with the corresponding pixel density ratio normalized against β-tubulin. (<b>F</b>) Kynurenine-to-tryptophan (Kyn/Trp) ratios in vaginal fluids of women bearing the CC or TT genotypes at rs3808606 in <i>IDO1</i>. Pooled data from vaginal fluids obtained from at least 10 different women for each genotype. *<i>P</i><0.05, TT <i>vs.</i> CC genotypes.</p

Vaginal candidiasis in IL-17A- or IL-17F-deficient mice.

<p>(<b>A</b>) Vaginal fungal burden in C57BL/6, IL-17A- or IL-17F-deficient mice (<i>n</i> = 6) intravaginally inoculated with 5×10⁶<i>C. albicans</i> blastospores. CFU were quantified by culturing serial dilutions of vaginal fluid (VF) from each mouse at different days post-infection (dpi) and expressed as Log₁₀ CFU/100 µl VF ± s.e.m. Pooled data from 3 experiments. (<b>B</b>) Histological analysis of periodic acid-Schiff-stained vaginal sections and inflammatory cell recruitment in vaginal fluids (May–Grünwald Giemsa staining in the insets) at 3 dpi. Representative images (out of 3 experiments) of histology sections and vaginal fluids were acquired with a 40× and 100× objective respectively. Scale bars, 100 µm. (<b>C</b>) Polymorphonuclear cells (PMNs) quantification in the vaginal fluids at different dpi. PMNs were identified by nuclear morphology and enumerated per field at ×100 magnification. Each point represents an individual mouse, and horizontal bar indicates the means. (<b>D</b>) Cytokine levels (pg/mg, cytokine/total proteins for each sample) in the vaginal fluids of naïve or infected (3 dpi) mice. Data are pooled or representative (histology) from 3 independent experiments. * <i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, knockout <i>vs.</i> wild-type mice. N.S., not significant.</p

Treg cells control the Th1/Th17 cell balance in murine VVC.

<p>(<b>A</b>) IFN-γ and IL-17A production (pg/mg, cytokine/total proteins for each sample) in the vagina fluids of mice (<i>n</i> = 6) intravaginally inoculated with 5×10⁶<i>C. albicans</i> blastoconidia and re-infected 3 weeks later. Assays were done at 3 days after the primary infection (infected) or re-challenge. Pooled data from 5 experiments. (<b>B</b>) Vaginal immunohistochemistry of re-infected mice. Staining was done with anti-IFN-γ-PE and anti-IL-17A-PE antibody. Cell nuclei were stained with DAPI (blue). Representative pictures (out of 2 experiments) were taken with a 20× objective. Scale bars, 200 µm. (<b>C</b>) Expression (RT-PCR) of transcript factor genes in purified CD4⁺ T cells from the draining lymph nodes. Assays were done at 3 days after the primary infection (infected) or re-challenge. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001, knockout <i>vs.</i> C57BL/6 mice at 3 days after re-challenge and re-challenged <i>vs.</i> infected mice. Control immunostaining performed on the vaginas from naïve mice revealed an increased expression of IL-17A in IL-17RA- and IL-10-deficient mice and of IFN-γ in IL-10-deficient mice. (<b>D</b>) Fungal growth in C57BL/6 or IL-10-deficient mice (<i>n</i> = 6) inoculated with 5×10⁶<i>C. albicans</i> blastospores. Fungal growth was quantified in the vaginal fluids at different days post-infection (dpi) and expressed as Log₁₀ CFU/100 µl VF ± s.e.m. (<b>E</b>) Polymorphonuclear cells (PMNs) quantification in the vaginal fluids at different dpi. PMNs were identified by nuclear morphology and enumerated per field at ×100 magnification. Pooled data from 3 experiments. *<i>P</i><0.05 and **<i>P</i><0.01, 3 or 21 <i>vs.</i> 0 dpi.</p

Immunity and tolerance to VVC occurs through distinct innate recognition receptors.

<p>(<b>A</b>) In vivo kinetics of fungal growth in the vagina of C57BL/6, MyD88- or TRIF- deficient mice (<i>n</i> = 6), inoculated intravaginally with 5×10⁶<i>C. albicans</i> blastospores. Fungal growth was quantified at different dpi and expressed as Log₁₀ CFU/100 µl VF ± s.e.m. Pooled data from 4 experiments. *<i>P</i><0.05, MyD88- or TRIF-deficient <i>vs.</i> wild-type mice. (<b>B</b>) Periodic acid-Schiff-stained vaginal sections and inflammatory cell recruitment in vaginal fluids (May–Grünwald Giemsa staining in the insets) at 3 days after the primary infection or after re-challenge. Representative images of histology sections and vaginal fluids (out of 4 experiments) were acquired with a 40× and 100× objective respectively. Scale bars, 100 µm. (<b>C</b>) Fungal growth at 3 dpi in mice (<i>n</i> = 6) after the primary infection or re-challenge. Pooled data from 4 experiments. *<i>P</i><0.05 and **<i>P</i><0.01, Re-challenged <i>vs.</i> not re-challenged (infected) mice. (<b>D</b>) Cytokine levels (pg/mg, cytokine/total proteins for each sample, at 3 dpi) in the vaginal fluids. Pooled data from 4 experiments.*<i>P</i><0.05 Knockout <i>vs.</i> C57BL/6 mice. (<b>E</b>) IFN-γ, IL-17A and IL-10 production (pg/mg, cytokine/total proteins) in the vaginal fluids of mice (<i>n</i> = 6) at 3 days after the primary infection (infected) or re-challenge. Pooled data from 4 experiments.*<i>P</i><0.05, Knockout <i>vs.</i> C57BL/6 mice at 3 days after re-challenge and re-challenged <i>vs.</i> infected mice. (<b>F</b>) Fungal growth in C57BL/6 and TLR-deficient mice (<i>n</i> = 6) at different dpi. Pooled data from 3 experiments. *<i>P</i><0.05, TLR-deficient <i>vs.</i> wild-type mice. (<b>G</b>) Fungal growth (at 3 dpi) in mice (<i>n</i> = 6) re-challenged 3 weeks after the primary infection. Pooled data from 3 experiments. *<i>P</i><0.05, WT <i>vs.</i> KO mice.</p

Characteristics of peptides selected from the constant region of antibodies.

a<p>cleavage probability;</p>b<p>0: hydrophobic, *: polar, +, and −: positively charged and negatively charged residues;</p>c<p>conserved amino acids in human Ig and different organisms;</p>d<p>conserved amino acids only in human Ig;</p><p>pI, isoelectric point; M.M., molecular mass (dalton); Tryp, trypsin; Chym, chymotrypsin.</p

Characterization of N10K conformational transition.

<p>A. Far-UV CD spectra of N10K (100 µM) in water, recorded as a function of time: time 0 (^______); 1 h (— — ); 3 h (- - -); 5 h (— - — -); 7 h (— - - — - -); 20 h (– – –); 6 days (• • •); 20 days (— • — •). B. Interaction of N10K (1 mM) with <i>C. albicans</i> (5×10⁷ cell/ml) after 150 min incubation (^______) using a 0.1 mm path lenght cell, 20°C. Fresh N10K in water (— —); after 150 min incubation (— - — -); after 570 min incubation (- - -). The samples in water were prepared taking aliquots of 100 µM from a 1.8 mM stock water solution and were collected with a 1 mm cell. For the sake of comparison of these CD profiles with the one obtained in the presence of <i>C. albicans</i>, the signal intensity of the water CD spectra has been divided by 10 and the ordinate kept in θ (mdeg). C. Electron micrograph of N10K prepared diluting 1∶5 the stock water solution stored at 4°C for 20 days. D. Electron micrograph of negative control peptide prepared in the same conditions.</p

<i>In vitro</i> fungicidal activity of the selected Fc-peptides against fungal strains.

*<p>% inhibitory activity at 100 µg/ml in comparison to the control growth (water);</p>**<p>EC₅₀, half maximal effective concentration;</p><p>n.d., not determined.</p