data_sheet_4_Toll-Interleukin 1 Receptor Domain-Containing Adaptor Protein 180L Single-Nucleotide Polymorphism Is Associated With Susceptibility to Recurrent Pneumococcal Lower Respiratory Tract Infections in Children.docx

<p>Lower respiratory tract infections (LRTI) are often caused by Streptococcus pneumoniae (Spn) and can be recurrent in 8% of children older than 2 years of age. Spn is recognized by pattern-recognition receptors (PRRs) of the innate immune system, in particular toll-like receptors (TLRs) 2 and 4. To assess whether a defect somewhere along this TLR signaling pathway increases susceptibility to recurrent pneumococcal LRTI, we conducted a prospective case–control study with 88 healthy individuals and 45 children with recurrent LRTI aged 2–5 years old. We examined cell surface expression of TLR2 and TLR4, as well as eight genetic variants of these receptors or associated co-receptors TLR1 and TLR6. Interleukin-6 production was measured after whole blood stimulation assays with specific agonists and heat-killed Spn. Our findings reveal that single-nucleotide polymorphisms within toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) alone or in combination with TLR1 N248S, TLR1 I602S, or TLR6 S249P polymorphisms contributes to various degree of susceptibility to recurrent pneumococcal LRTI in children by modulating the inflammatory response. In that respect, carriage of the TIRAP S180L heterozygous trait increases the likelihood to protect against pneumococcal LRTI, whereas children carrying the mutant homozygous TIRAP 180L polymorphism might be more likely susceptible to recurrent pneumococcal LRTI.</p

table_1_Toll-Interleukin 1 Receptor Domain-Containing Adaptor Protein 180L Single-Nucleotide Polymorphism Is Associated With Susceptibility to Recurrent Pneumococcal Lower Respiratory Tract Infections in Children.docx

<p>Lower respiratory tract infections (LRTI) are often caused by Streptococcus pneumoniae (Spn) and can be recurrent in 8% of children older than 2 years of age. Spn is recognized by pattern-recognition receptors (PRRs) of the innate immune system, in particular toll-like receptors (TLRs) 2 and 4. To assess whether a defect somewhere along this TLR signaling pathway increases susceptibility to recurrent pneumococcal LRTI, we conducted a prospective case–control study with 88 healthy individuals and 45 children with recurrent LRTI aged 2–5 years old. We examined cell surface expression of TLR2 and TLR4, as well as eight genetic variants of these receptors or associated co-receptors TLR1 and TLR6. Interleukin-6 production was measured after whole blood stimulation assays with specific agonists and heat-killed Spn. Our findings reveal that single-nucleotide polymorphisms within toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) alone or in combination with TLR1 N248S, TLR1 I602S, or TLR6 S249P polymorphisms contributes to various degree of susceptibility to recurrent pneumococcal LRTI in children by modulating the inflammatory response. In that respect, carriage of the TIRAP S180L heterozygous trait increases the likelihood to protect against pneumococcal LRTI, whereas children carrying the mutant homozygous TIRAP 180L polymorphism might be more likely susceptible to recurrent pneumococcal LRTI.</p

data_sheet_3_Toll-Interleukin 1 Receptor Domain-Containing Adaptor Protein 180L Single-Nucleotide Polymorphism Is Associated With Susceptibility to Recurrent Pneumococcal Lower Respiratory Tract Infections in Children.docx

<p>Lower respiratory tract infections (LRTI) are often caused by Streptococcus pneumoniae (Spn) and can be recurrent in 8% of children older than 2 years of age. Spn is recognized by pattern-recognition receptors (PRRs) of the innate immune system, in particular toll-like receptors (TLRs) 2 and 4. To assess whether a defect somewhere along this TLR signaling pathway increases susceptibility to recurrent pneumococcal LRTI, we conducted a prospective case–control study with 88 healthy individuals and 45 children with recurrent LRTI aged 2–5 years old. We examined cell surface expression of TLR2 and TLR4, as well as eight genetic variants of these receptors or associated co-receptors TLR1 and TLR6. Interleukin-6 production was measured after whole blood stimulation assays with specific agonists and heat-killed Spn. Our findings reveal that single-nucleotide polymorphisms within toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) alone or in combination with TLR1 N248S, TLR1 I602S, or TLR6 S249P polymorphisms contributes to various degree of susceptibility to recurrent pneumococcal LRTI in children by modulating the inflammatory response. In that respect, carriage of the TIRAP S180L heterozygous trait increases the likelihood to protect against pneumococcal LRTI, whereas children carrying the mutant homozygous TIRAP 180L polymorphism might be more likely susceptible to recurrent pneumococcal LRTI.</p

data_sheet_2_Toll-Interleukin 1 Receptor Domain-Containing Adaptor Protein 180L Single-Nucleotide Polymorphism Is Associated With Susceptibility to Recurrent Pneumococcal Lower Respiratory Tract Infections in Children.docx

<p>Lower respiratory tract infections (LRTI) are often caused by Streptococcus pneumoniae (Spn) and can be recurrent in 8% of children older than 2 years of age. Spn is recognized by pattern-recognition receptors (PRRs) of the innate immune system, in particular toll-like receptors (TLRs) 2 and 4. To assess whether a defect somewhere along this TLR signaling pathway increases susceptibility to recurrent pneumococcal LRTI, we conducted a prospective case–control study with 88 healthy individuals and 45 children with recurrent LRTI aged 2–5 years old. We examined cell surface expression of TLR2 and TLR4, as well as eight genetic variants of these receptors or associated co-receptors TLR1 and TLR6. Interleukin-6 production was measured after whole blood stimulation assays with specific agonists and heat-killed Spn. Our findings reveal that single-nucleotide polymorphisms within toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) alone or in combination with TLR1 N248S, TLR1 I602S, or TLR6 S249P polymorphisms contributes to various degree of susceptibility to recurrent pneumococcal LRTI in children by modulating the inflammatory response. In that respect, carriage of the TIRAP S180L heterozygous trait increases the likelihood to protect against pneumococcal LRTI, whereas children carrying the mutant homozygous TIRAP 180L polymorphism might be more likely susceptible to recurrent pneumococcal LRTI.</p

data_sheet_1_Toll-Interleukin 1 Receptor Domain-Containing Adaptor Protein 180L Single-Nucleotide Polymorphism Is Associated With Susceptibility to Recurrent Pneumococcal Lower Respiratory Tract Infections in Children.docx

<p>Lower respiratory tract infections (LRTI) are often caused by Streptococcus pneumoniae (Spn) and can be recurrent in 8% of children older than 2 years of age. Spn is recognized by pattern-recognition receptors (PRRs) of the innate immune system, in particular toll-like receptors (TLRs) 2 and 4. To assess whether a defect somewhere along this TLR signaling pathway increases susceptibility to recurrent pneumococcal LRTI, we conducted a prospective case–control study with 88 healthy individuals and 45 children with recurrent LRTI aged 2–5 years old. We examined cell surface expression of TLR2 and TLR4, as well as eight genetic variants of these receptors or associated co-receptors TLR1 and TLR6. Interleukin-6 production was measured after whole blood stimulation assays with specific agonists and heat-killed Spn. Our findings reveal that single-nucleotide polymorphisms within toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) alone or in combination with TLR1 N248S, TLR1 I602S, or TLR6 S249P polymorphisms contributes to various degree of susceptibility to recurrent pneumococcal LRTI in children by modulating the inflammatory response. In that respect, carriage of the TIRAP S180L heterozygous trait increases the likelihood to protect against pneumococcal LRTI, whereas children carrying the mutant homozygous TIRAP 180L polymorphism might be more likely susceptible to recurrent pneumococcal LRTI.</p

Endogenous HAQ STING is strongly impaired in mounting a type I IFN and proinflammatory cytokine responses against <i>Legionella</i> infection or stimulation with DNA or CDNs.

<p>(A-D) PBMCs from healthy volunteers (N = 4 for WT and N = 4 for HAQ) were isolated by density gradient centrifugation. 7 d after isolation cells were infected for 6 h with <i>L</i>. <i>pneumophila</i> at MOIs 10 and 50 or stimulated for the same period with 1 and 5 ug/ml 2´-3´cGAMP or either bacterial or synthetic DNA at a concentration of 0.2 or 1 ug/ml. RNA was isolated and the expression of <i>IFNB</i> (A), <i>IL1B</i> (B), <i>IL6</i> (C) and <i>TNFA</i> (D) was determined by qRT-PCR. Data are shown as the RQ of specified mRNAs. Data represent the mean ± SEM of 4 independent experiments carried out in triplicates. Differences were assessed with the Mann-Whitney U Test. Comparisons with a p < 0.05 were considered significant.</p

The cGAS/STING axis contributes to the production of pro-inflammatory cytokines during <i>L</i>. <i>pneumophila</i> infection.

<p>(A-F) WT, <i>Tmem173</i>^<i>-/-</i> <i>and cGas</i>^<i>-/-</i> BMDMs were infected for 6 h with <i>L</i>. <i>pneumophila</i> WT at MOI 10 and relative cytokine expression was determined by qRT-PCR. (G-J) Cytokine protein concentrations in whole lung homogenates from <i>L</i>. <i>pneumophila</i>-infected mice were quantified by sandwich ELISA. Data are shown as mean ± SEM. (A-F) Data representative of 3 to 4 independent experiments carried out in duplicates. (G-J) Data representative of 6 o 7 mice per group. Data were analyzed through the Mann-Whitney U Test. Comparisons with a <i>p</i> < 0.05 were considered significant.</p

Type I IFN responses during <i>L</i>. <i>pneumophila</i> infection are mediated by the cGAS/STING pathway.

<p>(A-C) WT and <i>Tmem173</i>^-/- mouse BMDMs were left untreated or stimulated with 1 ug/ml <i>L</i>. <i>pneumophila</i> DNA (JR32 DNA) or 5 ug/ml 2´3-cGAMP (A) or were infected with <i>L</i>. <i>pneumophila</i> JR32 WT and 130b WT, or mutant strains deficient for <i>dotA</i> or <i>sdhA</i> at MOI 10 for 6 h (B, C). Expression of <i>Ifnb</i> (A, B) or <i>Irg1</i> (C) was measured by qRT-PCR. (D-G) WT and cGAS-deficient BMDMs were stimulated with <i>L</i>. <i>pneumophila</i> DNA or 2´3-cGAMP or infected with <i>L</i>. <i>pneumophila</i> JR32 WT, and expression of <i>Ifnb</i> and <i>Irg1</i> was quantified by qRT-PCR (D-F) or production of IP-10 was measured by ELISA (G). (H-N) WT, STING- and cGAS-deficient mice were intranasally infected with 1×10⁶ <i>L</i>. <i>pneumophila</i> JR32 WT or instilled with PBS as control (H-J). <i>Ifnb</i> and <i>Irg1</i> expression in the lungs was assessed 48 (H, I) or 144 h p.i. (K-N) by qRT-PCR, or IP-10 production was measured at 48 h (J). Data are represented as the relative quantification (RQ) of specified mRNAs. Data are shown as the mean + SEM of three to four independent experiments, measured in technical duplicates (Fig. 1A-G) or 6 to 7 mice per group (Fig. H-N). Analyses were performed through the Mann-Whitney U Test. Comparisons with a <i>p</i> < 0.05 were considered significant.</p

Distribution of <i>TMEM173/STING</i> HAQ and R232H in German patients and healthy controls.

<p>Distribution of <i>TMEM173/STING</i> HAQ and R232H in German patients and healthy controls.</p

<i>L</i>. <i>pneumophila</i> infection and stimulation with DNA or cGAMP induce weak cGAS-dependent type I IFN responses in THP-1 cells.

<p>WT THP-1 or cGAS-/- THP-1 clones A5 and B5 were allowed differentiation prior to stimulation with either cGAMP or synthetic DNA (A) or infection with two different strains of <i>L</i>. <i>pneumophila</i> (B). <i>IFNB</i> expression was determined by qRT-PCR. Data represent mean ± SEM of 2 independent experiments carried out in duplicates. Analyses were performed by employing the Mann-Whitney U Test. Comparisons with a p < 0.05 were considered significant.</p