Fig 2: Bacterial load and granulocyte marker MPO in the mouse lung on day 7 of challenge infection with various serovars of C. trachomatis after i.n. vaccination with adjuvanted 5cVAC. In the short-term protection model (Figure 1a), mice were either pre-treated with c-di-AMP-adjuvanted 5cVAC, the adjuvant alone, or buffer, as indicated. The mice were challenged with C.tr. serovar E, D, L2, or A (marked by boxed letters to the left) to check for cross-serovar protection (a1–d1, a2–d2). On day 7 of chlamydial challenge (i.e., week 8 in Figure 1a), the amount of viable, infectious Chlamydia (left panels, mean ± standard deviation of log10 (IFU)) and the level of MPO (right panels, mean ± standard deviation of log10)) were determined in lung homogenate of the pretreated animals surviving that period (n = 6–10 per group for serovar E, D and L2, n = 4–7 for serovar A—as indicated by the number of symbols in the figure). Mock infection: n = 8. LOD = limit of detection; n.d. = not determined; ns = not significant. The performed statistical analysis is described in Section 2. * and *** indicate statistical significances with p < 0.05 and <0.001, respectively. The exact size of each group can be found in Supplement Table S2.

Fig 3: Body weight, clinical score, bacterial load, granulocyte marker MPO, TNF-a, and IFN-? in lung challenge infection with C. trachomatis E in the short-term protection model after i.n. vaccination with c-di-AMP-adjuvanted two-component 2cVAC. In this modified short-term protection model (see Figure 1a), vaccinated mice received only two of the five components contained in 5cVAC (2cVAC: PmpD and Ctad1) plus c-di-AMP by the i.n. route (blue triangles). Animals pretreated with adjuvant, or buffer, respectively, served as negative controls. Per group, n = 8–9 differently pretreated 15-week-old animals were infected i.n. with 1.3 × 106 IFU of C.tr. E. On each of the following days, body weight ((a), in % as mean ± standard deviation) and clinical score ((b), median ± interquartile range) were assessed. On day 7 after i.n. challenge infection, the surviving animals (also indicated by the number of symbols in the figure) were sacrificed for determination of the amount of viable, infectious Chlamydia ((c), mean ± standard deviation of log10 (IFU); n = 6–9), and the levels (mean ± standard deviation of log10; n = 7–9) of MPO (d), TNF-a (e), and IFN-? (f) in the lung homogenate. Mock infection: n = 4 for bacterial load, n = 8 for other parameters. For better comparability, data obtained in a previous, otherwise identical experiment with i.n. 5cVAC pretreatment are also depicted (red triangles). LOD = limit of detection; ns = not significant. The performed statistical analysis is described in Section 2. *,**,*** indicate statistical significances with p < 0.05, <0.01, and <0.001, respectively. Statistical significance (p < 0.05) between the 5cVAC and the adjuvant/buffer control groups is marked with # in the panels (a,c,d–f). The exact size of each group can be found in Supplement Table S2.

Fig 4: Mice infected i.n. with C.tr. serovars D, E, and L2 show lower myeloperoxidase (MPO) concentrations in lung homogenate after overcoming a primary lung infection with C.tr. serovar E. Animals previously i.n. infected with C.tr. genital serovar E or mock material (n = 6–7) were infected a second time with the C.tr. serovars D, E or L2 7 weeks after the primary infection. Mice infected twice with mock material (n = 4) served as negative control; the MPO-level of all individual control animals was infection with C.tr. serovars D, E or L2, mice were painlessly sacrificed and the level of MPO (mean ± SD of log10(IFU)) was determined in lung homogenate. LOD: Limit of detection. The performed statistical analysis is described in Section 2. ** indicates statistical significances between the previously mock-infected animal groups and groups initially infected with C.tr. E with p < 0.01.

Fig 5: Decreased Hedgehog signaling results in increased sensitivity to DSS-induced colitis. (A) Experimental set up: Villin-CreERT2-Ihh+/+ (Ihh+/+, n = 10) and Villin-CreERT2-Ihhfl/fl animals (Ihh?, n = 10), or Gli1-CreERT2-ZsGreen-Smo+/+ (Smo+/+, n = 11) and Gli1-CreERT2-ZsGreen-Smofl/fl animals (Smo?, n = 10) were injected intraperitoneally with 1 mg tamoxifen for 5 consecutive days. Animals were provided with drinking water containing 2% DSS for 6 or 7 days, starting 7 days after induction of recombination. Experiments were performed in 2 separate mouse facilities. For both mouse models, (B) the relative weight to the weight at the start of DSS administration is shown, (C) disease and histologic scores were determined as described in the Materials and Methods section, (D) MPO levels and IL10 protein levels were determined in colon lysates and are shown as corrected for total protein content, and serum amyloid A levels were measured in the serum at death. (E) Representative images of H&E-stained sections of the colons of each mouse model. Scale bars: 50 µm. Bars represent means, error bars represent SEM. *P < .05, **P < .01, ***P < .001.