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

Endogenous R232H STING is partly deficient in sensing bacterial CDN but responds normally to <i>Legionella</i> infection or stimulation with DNA.

<p>(A-D) PBMCs from healthy volunteers (N = 3 for WT and N = 3 for R232H) were isolated by density gradient centrifugation. 7 d after isolation cells were infected for 6 h with <i>L</i>. <i>pneumophila</i> at MOI 10 or stimulated for the same period with 1 ug/ml 2´-3´cGAMP, Rp-c-diAMPSS, cGMP or either bacterial DNA at a concentration of 1 ug/ml. RNA was isolated and the expression of <i>IFNB</i> (A), <i>IL1B</i> (B), and <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 3 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

STING contributes to the antibacterial defense in mice infected with <i>L</i>. <i>pneumophila</i>.

<p>WT, cGAS- and STING-deficient mice were intranasally infected with 1×10⁶ <i>L</i>. <i>pneumophila</i> WT and the bacterial loads in the lungs were assessed 144 h p.i. Data represent mean ± SEM of 6–13 mice per group. Comparisons were performed with the Mann-Whitney U Test. Comparisons with p < 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