Fig 1.
Type I IFN responses during L. pneumophila infection are mediated by the cGAS/STING pathway.
(A-C) WT and Tmem173-/- mouse BMDMs were left untreated or stimulated with 1 ug/ml L. pneumophila DNA (JR32 DNA) or 5 ug/ml 2´3-cGAMP (A) or were infected with L. pneumophila JR32 WT and 130b WT, or mutant strains deficient for dotA or sdhA at MOI 10 for 6 h (B, C). Expression of Ifnb (A, B) or Irg1 (C) was measured by qRT-PCR. (D-G) WT and cGAS-deficient BMDMs were stimulated with L. pneumophila DNA or 2´3-cGAMP or infected with L. pneumophila JR32 WT, and expression of Ifnb and Irg1 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 L. pneumophila JR32 WT or instilled with PBS as control (H-J). Ifnb and Irg1 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 p < 0.05 were considered significant.
Fig 2.
The cGAS/STING axis contributes to the production of pro-inflammatory cytokines during L. pneumophila infection.
(A-F) WT, Tmem173-/- and cGas-/- BMDMs were infected for 6 h with L. pneumophila WT at MOI 10 and relative cytokine expression was determined by qRT-PCR. (G-J) Cytokine protein concentrations in whole lung homogenates from L. pneumophila-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 p < 0.05 were considered significant.
Fig 3.
Endogenous HAQ STING is strongly impaired in mounting a type I IFN and proinflammatory cytokine responses against Legionella infection or stimulation with DNA or CDNs.
(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 L. pneumophila 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 IFNB (A), IL1B (B), IL6 (C) and TNFA (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.
Fig 4.
L. pneumophila infection and stimulation with DNA or cGAMP induce weak cGAS-dependent type I IFN responses in THP-1 cells.
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 L. pneumophila (B). IFNB 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.
Fig 5.
Endogenous R232H STING is partly deficient in sensing bacterial CDN but responds normally to Legionella infection or stimulation with DNA.
(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 L. pneumophila 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 IFNB (A), IL1B (B), and IL6 (C) and TNFA (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.
Fig 6.
STING contributes to the antibacterial defense in mice infected with L. pneumophila.
WT, cGAS- and STING-deficient mice were intranasally infected with 1×106 L. pneumophila 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.
Table 1.
Distribution of TMEM173/STING HAQ and R232H in German patients and healthy controls.
Table 2.
Distribution of TMEM173/STING HAQ and R232H in patients and healthy controls from Netherlands cohort.