Genetic variants associated with increased risk for IA and probable pathogenetic mechanism(s) of susceptibility.

<p>SNP – single nucleotide polymorphism; OR – odds ratio; HSCT – hematopoietic stem cell transplantation; D – donor; R – recipient; RAGE – receptor for advanced glycation end products; CXCL – chemokine (C-X-C motif) ligand; MBL – mannose-binding lectin; MASP – MBL-associated serine protease; PLG – plasminogen; TLR – toll-like receptor; TNFR – tumor necrosis factor receptor; n.a. – not available; VNTR – variable number of tandem repeats.</p>1<p>For patients that underwent HSCT, the source of the variant (donor, recipient, or both) associated with susceptibility to IA is indicated.</p>2<p>The controls for the association reported were not hematological patients, but healthy subjects of comparable Dutch ancestry.</p>3<p>Association with increased susceptibility to IA was observed for a haplotype comprising SNPs in the IL-1 gene cluster, namely <i>IL1A</i> rs1800587/<i>IL1B</i> rs16944/<i>IL1RN</i> VNTR 86-bp(n), but not for single loci.</p>4<p>Association with increased susceptibility to IA was observed for the absence of the ACC haplotype in rs1800896, rs1800871, and rs1800872, respectively.</p>5<p>‘MBL-low genotypes’ correspond to a group of genotypes denoted by letters (O/O and LXA/O). LX represents an MBL promoter haplotype comprising SNPs of the MBL2 gene at positions −550 (H/L) and −221 (Y/X), known to influence transcription rates and to result in low concentrations of serum MBL. Nonsynonymous variants are collectively named O (including amino acid replacements at codons 52, 54, or 57) and cause a reduction of the MBL levels due to impaired assembly of MBL monomers into functional oligomers. A indicates the wild type.</p>6<p>Association results regard the comparisons DD <i>vs.</i> DN and DD <i>vs.</i> NN, respectively.</p>7<p>Association results regard R80T, N248S and S249P, and R80T or N248S and S249P, respectively.</p>8<p>Association with increased susceptibility to IA was observed for a <i>TLR4</i> haplotype (termed S4) that included both D299G and T399I.</p

Proposed model of the effect of rs5743836 in B-lymphocyte activation and proliferation.

<p>TLR9 activation by CpG DNA induces the production of IL-6, which upon binding to its receptor, promotes translocation of STAT3 to the nucleus. This transcription factor can bind to and trans-activate promoters containing STAT3 binding elements, thus initiating a specific transcriptional program. In cells harboring the TC genotype of rs5743836, STAT3 will bind to a new IL-6 RE site, created by the T/C substitution within the <i>TLR9</i> promoter. As a result, <i>TLR9</i> expression increases in response to IL-6. Therefore, in these cells, a loop of TLR9/IL-6 signaling amplification is created, leading to a deregulation in B-cell activation and proliferation upon CpG stimuli. Of note, the IL-6 needed to generate this loop may be TLR9-independent.</p

IL-6 increases the activity of the <i>TLR9</i> promoter carrying the C allele of rs5743836.

<p>(A) <i>In silico</i> analysis of the fragment of the <i>TLR9</i> promoter containing the T/C substitution using the TESS interface. L_a, log-likelihood score, L_q, a measure of the goodness-of-fit of the DNA sequence to the consensus binding motif. (B) Luciferase reporter assay of Raji B cells transfected with plasmid vectors containing the luciferase gene under the control of a 3.2 kb fragment of the promoter sequence carrying the T or C allele. After transfection, cells were left untreated or stimulated with IL-6 (n = 3). (C) IL-6 dose-dependent stimulation of either TT (n = 27) or TC (n = 25) PBMCs. Cells were left untreated or stimulated with increasing doses of recombinant IL-6. <i>TLR9</i> mRNA expression levels were determined by real-time PCR. (D) IL-6 secretion by of either TT (n = 21) or TC (n = 17) PBMCs. IL-6 in untreated cells was quantified by ELISA. (E) <i>TLR9</i> expression in TT (n = 21) or TC (n = 17) PBMCs in IL-6-neutralizing conditions. Cells were cultured with an anti-IL-6 antibody and <i>TLR9</i> mRNA expression was determined by real-time PCR. Results shown are the mean ± SD.</p

Human PBMCs carrying the TC genotype of rs5743836 stimulated via TLR9 show increased transcription of <i>TLR9</i> and enhanced B-cell proliferation.

<p>(A) TC PBMCs secrete higher amounts of IL-6 upon activation of TLR9 with CpG ODN 2006. IL-6 production was quantified by ELISA in the supernatants of TT (n = 22) or TC (n = 17) PBMCs following culture with CpG. (B) TLR9 activation of TC cells by CpG ODN 2006 increases <i>TLR9</i> gene expression. Quantification of <i>TLR9</i> mRNA expression in either TT (n = 24) or TC (n = 27) PBMCs cultured with: CpG ODN 2006 (0.05 µM); CpG ODN 2006 and anti-IL-6 antibody; CpG ODN 2006 and the TLR9 antagonist CpG ODN TTAGGG in a 1∶2 ratio. (C) Proliferation of CD19⁺ cells was assessed by CFSE dilution in PBMCs with different rs5743836 genotypes, TT (n = 40) and TC (n = 33), left untreated or stimulated with: CpG ODN 2006 (0.05 µM); CpG ODN 2006 and anti-IL-6 antibody; CpG ODN 2006 and CpG ODN TTAGGG in a 1∶2 ratio. Results shown are the means ± SD.</p

Genotype distributions and association test results of SNPs in the <i>PARK2</i> gene among BU patients and age- and gender-matched healthy controls.

<p>Genotype distributions and association test results of SNPs in the <i>PARK2</i> gene among BU patients and age- and gender-matched healthy controls.</p

Haploview pairwise analysis of Linkage Disequilibrium (LD) between NOD2 gene SNPs (A) and PARK2 gene SNPs (B).

<p>The r2 colour scheme was used. Measures of r2 = 0 are represented in white (not significant); 0</p

General characteristics of Buruli ulcer (BU) patients and healthy controls.

<p>General characteristics of Buruli ulcer (BU) patients and healthy controls.</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

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