Subgroup analysis of different T-cell subsets after BMT.

<p><b>A)/B</b>) Engraftment of CD4(+)/GFP(+) and CD8(+)/GFP(+) T-cells [G/l]: CD4(+)/GFP(+) (upper plots) and CD8(+)/GFP(+) T-cells (lower plots) were analysed by flow cytometry analysis 2, 4 and 6 weeks after BM transplantation. A lower percentage of CD4(+)/GFP(+) and CD8(+)/GFP(+) T-cells could be detected in gp130^loxPloxP recipients that were transplanted with GFP(+)gp130^ΔMx donor BM. Displayed are example flow cytometry plots for a recipient mouse of wildtype donor BM (A) as well as for a recipient animal of gp130 deficient BM (B) 2 weeks after BMT. <b>C)/D</b>) Engraftment of CD19(+)/GFP(+) B-cells [G/l]: CD19(+)/GFP(+) B-cells derived from GFP(+)gp130^ΔMx donor BM (Fig. 2D) engrafted decelerated compared to GFP(+)gp130^loxPloxP donor BM (Fig. 2C) 2 and 4 weeks after BM transplantation. Shown are example flow cytometry plots 2 weeks after BMT. <b>E)/F</b>) Engraftment of CD11b(+)/GFP(+) cells [G/l]: 2 weeks after BM transplantation the percentage of CD11b(+)/GFP(+) cells was lower in gp130^loxP/loxP recipients transplanted with GFP(+)gp130^ΔMx donor BM compared to controls. Displayed are example flow cytometry plots for 2 weeks transplanted mice having received wildtype (E) or gp130 deficient (F) BM respectively.</p

Dissection of intracellular gp130 signalling pathways.

<p><b>A</b>) Number of total white blood cells (WBC) [G/l] after BMT: Displayed are the total white blood cell counts after BM transplantation at the indicated time points (2, 4 and 6 weeks). Less total white blood cells could be detected in gp130^loxP/loxP mice that were transplanted with GFP(+)gp130^ΔMxSTAT BM. Transplantation of GFP(+)gp130^ΔMxRas donor BM did not result in any significant difference concerning the total WBC count. <b>B</b>) Delayed engraftment of CD45(+)/GFP(+) white blood cells (WBC) [G/l] after BMT is STAT-dependent: Displayed are the CD45(+)/GFP(+) (donor derived) cells after BM transplantation at the indicated time points (2, 4 and 6 weeks). Whereas transplantation of GFP(+)gp130^ΔMxRas donor BM into gp130^loxP/loxP animals did not lead to a delayed engraftment of CD45(+)/GFP(+) cells, transplantation of gp130^ΔMxSTAT into gp130^loxP/loxP resulted in a significant decrease of CD45(+)/GFP(+) cells. <b>C</b>) Defective Ras and STAT signalling in donor mice leads to thrombocytopenia: Depicted are the platelet counts 2, 4 and 6 weeks after BMT. Transplantation of GFP(+)gp130^ΔMxRas as well as GFP(+)gp130^ΔMxSTAT donor BM led to a significant thrombocytopenia 2 weeks after BM transplantation. <b>D</b>) STAT-deficiency in donor mice leads to anaemia after BM transplantation: Depicted are the haemoglobin values 2, 4 and 6 weeks after BM transplantation with a significant anaemia in gp130^loxP/loxP recipients transplanted with GFP(+)gp130^ΔMxSTAT donor BM 4 weeks after BMT. [**p<0.01, ***p<0.001].</p

Subgroup analysis of different T-cell subsets after BMT.

<p><b>A</b>) Engraftment of CD4(+)/GFP(+) T-cells [G/l]: CD4(+)/GFP(+) T-cells were analysed by flow cytometry analysis 2, 4 and 6 weeks after BM transplantation. A significant lower number of CD4(+)/GFP(+) T-cells could be detected in gp130^loxPloxP recipients that were transplanted with GFP(+)gp130^ΔMxSTAT donor BM. Transplantation of GFP(+)gp130^ΔMxRas donor BM resulted in the same number of CD4(+)/GFP(+) T-cells as transplantation of gp130^loxP/loxP BM into gp130^loxP/loxP littermates. <b>B</b>) Engraftment of CD8(+)/GFP(+) T-cells [G/l]: The absolute number of CD8(+)/GFP(+) T-cells was determined by flow cytometry analysis 2, 4 and 6 weeks after BM transplantation. The number of CD8(+)/GFP(+) T-cells was significantly lower in wildtype mice that were transplanted with GFP(+)gp130^ΔMxSTAT 2 and 4 weeks after BMT. <b>C</b>) Engraftment of CD19(+)/GFP(+) B-cells [G/l]: CD19(+)/GFP(+) B-cells derived from GFP(+)gp130^ΔMxSTAT donor BM engrafted into recipient mice with significant differences at the 2 and 4 week time point. Gp130^loxP/loxP recipients of GFP(+)gp130^ΔMxRas donor BM also displayed a somewhat delayed engraftment 2 weeks after BMT but recovered faster as shown 4 and 6 weeks after BMT. <b>D</b>) Engraftment of CD11b(+)/GFP(+) cells [G/l]: Transplantation of GFP(+)gp130^ΔMxRas donor BM into gp130^loxP/loxP resulted in a significant increase of CD11b(+)/GFP(+) cells at all indicated time points (2, 4, 6 weeks) after BMT. GFP(+)gp130^ΔMxSTAT donor BM led to the same number of CD11b(+)/GFP(+) cells as the transplantation of GFP(+)gp130^ΔMx donor BM. [*p<0.05, **p<0.01].</p

Survival analysis after BM transplantation.

<p>A) Survival curve after BMT of 1×10⁶ donor cells (6 mice per group): Recipient mice of all different donor genotypes survived BMT of 1×10⁶ donor cells. B) Survival curve after BMT of 2×10⁵ donor cells (8 mice per group): gp130^ΔMxSTAT transplanted mice showed a 75% survival after BMT with 2×10⁵ donor cells. All the other animals survived BMT to 100%. C) Survival curve after BM transplantation using the amount of 5×10⁴ donor cells (6 mice per group): Transplantation of gp130^loxP/loxP donor BM into gp130^loxP/loxP recipient mice led to a survival rate of 100%. 75% of gp130^loxP/loxP recipients of gp130^ΔMxRas donor BM survived the experiment. However, if gp130^loxP/loxP recipients were transplanted with gp130^ΔMx BM, they survived in 33%. Finally, transplantation of gp130^ΔMxSTAT donor BM led to 100% mortality with no (0%) surviving recipient mice. [*p<0.05].</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

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

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

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