IL-37 dampens inflammasome activation in phagocytic cells.

<p>Alveolar macrophages and epithelial cells from naive mice and peripheral neutrophils were pre-exposed to recombinant IL-37 precursor for 8 hours before stimulation with live <i>Aspergillus</i> conidia for 2 hours. (<b>A</b>) Percent of phagocytosis and conidiocidal activity. (<b>B</b>) <i>Il1b</i> and <i>Nos2</i> mRNA expression by RT-PCR on total lung cells. Ct, control cells. None, <i>Aspergillus</i>-pulsed, untreated cells. (<b>C</b>) Activation of distinct intracellular kinases in RAW cells, using Proteome Profiler Array, pre-exposed to 100 ng/ml IL-37 for 8 hours before stimulation with live <i>Aspergillus</i> conidia for 30 min. Data are representative (Proteome Profiler Array) or pooled from two experiments. *P<0.05,**P<0.01, ***P<0.001, IL-37-stimulated <i>vs</i> unstimulated cells.</p

NLRP3-deficient mice exhibit reduced neutrophil recruitment and IL-1β production in pulmonary aspergillosis.

<p>C57BL/6 and <i>Nlrp3^−/−</i> mice were infected intranasally with <i>A. fumigatus</i> and treated with 1000 ng/mouse recombinant IL-37 precursor administered intraperitoneally 1 hour before the infection. (<b>A</b>) BAL fluid morphometry [number of total (T) cells and polymorphonuclear neutrophils (P). Values represent the mean±SD of three mice per group and are representative of 3 independent experiments]. (<b>B</b>) Lung histology (periodic acid-Schiff staining) and cell recruitment (insets). Scale bars, 100 µm and 25 µm, respectively. Arrows indicate neutrophils. (<b>C</b>) <i>Mpo</i> and <i>Cxcl2</i> mRNA expression by RT-PCR on total lung cells. (<b>D</b>) IL-1β production (ELISA on lung homogenates). Assays were done 3 days after the infection. Data are representative (histology) or pooled from three experiments. *P<0.05, <i>Nlrp3^−/− vs</i> C57BL/6 mice and treated <i>vs</i> untreated (None) mice. Naïve, uninfected and untreated mice.</p

IL-37 restrains inflammation in fungal allergy and <i>Cftr^−/−</i> mice.

<p>(<b>A</b>) Lung histology (PAS- and Masson's trichrome-stained sections, scale bars 100 and 25 (insets) µm); (<b>B</b>) hydroxyproline content (µg/lung); (<b>C</b>) expression of mucins (RT-PCR on total lung cells); (<b>D</b>) expression of cytokines and Th transcription factors in total lung cells from mice with ABPA and treated with IL-37. None, untreated mice. Naïve, uninfected and untreated mice. (<b>E</b>) Fungal growth (Log₁₀ CFU, mean±SD) in the lungs of <i>Cftr^−/−</i> mice infected intranasally with <i>A. fumigatus</i> and treated intraperitoneally with IL-37, at the dose of 1000 ng/mouse, 1 hour before the infection. (<b>F</b>) BAL fluid morphometry [number of total (T) cells and polymorphonuclear neutrophils (P) upon May Grunwald Giemsa staining]. (<b>G</b>) Lung histology (periodic acid-Schiff staining) and cell recruitment (insets). Scale bars, 100 µm and 25 µm in the insets; (<b>H</b>) <i>Mpo</i> and <i>Cxcl2</i> mRNA expression and (<b>I</b>) cytokine gene expression on total lung cells by RT-PCR, 3 days after the infection. Data are pooled from two experiments. *P<0.05, **P<0.01, treated <i>vs</i> untreated (None) mice. Naïve, uninfected and untreated mice.</p

Real-time murine PCR primers used in this study.

<p>Real-time murine PCR primers used in this study.</p

IL-37 reduces inflammatory cell recruitment in mice with inflammatory aspergillosis.

<p>C57BL/6 mice were infected intranasally (in) with <i>A. fumigatus</i> and pretreated one time with different doses IL-37 administered intraperitoneally (ip) at different times before the infection. Mice were assessed for: (<b>A</b>) fungal growth (Log₁₀ CFU, mean±SD) in the lungs at 1 and 3 days post-infection (dpi); (<b>B</b>) BAL fluid morphometry [number of total (T) cells and polymorphonuclear neutrophils (P) upon May Grunwald Giemsa staining. Values represent the mean±SD of three mice per group and are representative of 3 independent experiments]; (<b>C</b>) lung histology (periodic acid-Schiff and, in the inset, TUNEL staining). Red arrows indicate PMN and white arrows indicate increased deposition of DNA on lung parenchyma (in TUNEL-stained sections). Scale bars, 25 µm; (<b>D</b>) myeloperoxidase (<i>Mpo</i>), <i>Cxcl1</i> and <i>Cxcl2</i> mRNA expression by RT-PCR on total lung cells; (<b>E</b>) lung histology (periodic acid-Schiff staining, scale bars, 25 µm) and (<b>F</b>) <i>Mpo</i>, <i>Cxcl1</i> and <i>Cxcl2</i> mRNA expression (RT-PCR on total lung cells) in mice pretreated with IL-37 given ip or in, at different hours before the infection. (<b>G</b>) Numbers of CD11b/Gr1–positive cells were assessed by flow cytometry of total lung cells from LPS-treated mice. Data are representative (histology) or pooled from three experiments. *P<0.05,**P<0.01, treated <i>vs</i> untreated (None) mice. Naïve, uninfected and untreated mice.</p

IL-37 impairs inflammasome activation in mice with aspergillosis.

<p>C57BL/6 mice were infected intranasally with <i>A. fumigatus</i> and treated intraperitoneally with recombinant IL-37 precursor, at the indicated doses, 96, 48 and 1 hour before the infection. (<b>A</b>) NLRP3 expression in the lung by immunofluorescence staining with anti-CIAS1/Nlrp3 antibody. In the insets, positive staining of epithelial cells. Nuclei were counterstained with DAPI. Scale bars, 100 µm. (<b>B, E</b>) Gene expression on total lung cells by RT-PCR. (<b>C</b>) Cytokine production (ELISA) on lung homogenates. (<b>D</b>) Immunoblot analysis on whole lung lysates of IL-1β and Caspase 1 using rabbit specific antibodies and rabbit anti-actin. Goat anti-rabbit IgG-HRP were used as secondary antibody. Corresponding pixel density ratio was normalized against actin. Assays were done a day after the infection. Data are representative (immunoblotting) or pooled from three experiments. *P<0.05, **P<0.01, treated <i>vs</i> untreated (None) mice. Naïve, uninfected and untreated mice.</p

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

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-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

GAG protects mice from experimental DSS-induced colitis.

<p>(A) Body weight losses, (B) stool and histological score, (C) histology of colonic sections and (D) cytokine concentrations present in total colonic cells a day after the 7-day of DSS rest in C57BL/6 and <i>p47^phox−/−</i> (CGD) mice with or without GAG treatment. *P<0.05, GAG treated vs. untreated mice.</p