Fig 2: Intracellular L. pneumophila (Lp) Replication and Nonspecific Autophagy in WT and Tollip-/- Macrophages.A) Bone marrow-derived macrophages (BMDM) from WT and Tollip-/- mice were infected with flagellin-deficient L. pneumophila (JR32 strain) expressing the luminescent lux gene. Relative luminescence was measured over time. B) Representative images depicting LC3+ organelles (green) and DAPI-stained nuclei (blue) from control (EV) or THP-1 cells with TOLLIP deleted via CRISPR-Cas9 gene editing (TOLLIP-KO) in RPMI supplemented with 10% FCS (ctrl) or EBSS (starve) for 4 hours. C) Tukey plot of the number of autophagosomes counted from 100 cells in EV and TOLLIP-KO cells in nutrient-enriched or starvation medium after 4 hours. Box demonstrates median and interquartile range of values. * p < 0.05, Mann-Whitney U-test, data is representative of three independent experiments. D) Western blot of THP-1 cells comparing LC3-II expression with ß-actin as a control in TOLLIP-KO and control cell lines under nutrient-rich and starvation conditions, with or without chloroquine. E) Quantitative analysis of LC3-II expression, normalized to ß-actin, in TOLLIP-deficient cells in control and TOLLIP-KO cells. Data are expressed as mean +/- SD of three separate experiments grouped together. * p < 0.05 by Student’s t test.

Fig 3: Cytokine Responses from WT and Tollip-/- mice after L. pneumophila (Lp) aerosol infection.Mice were infected with 106 CFU WT Lp Philadelphia-1 strain and after 24 hours, cytokine concentrations were measured from bronchoalveolar (BAL) fluid and serum. A) BAL cytokine concentrations measured by ELISA from WT and Tollip-/- mice. * p < 0.05, Student’s two-sided t-test. B) BAL cytokines, measured by Luminex cytokine assay 24 hours after Lp aerosol infection. * p < 0.05, ** p < 0.01, *** p < 0.001, at 1% false discovery rate. Error bars represent +/- SEM. C) Serum cytokine concentrations measured by ELISA. * p < 0.05, Student’s two-sided t-test. Error bars represent +/- SEM. Data are representative of three independent experiments with N = 5 in each group.

Fig 4: WT and Tollip-/- Macrophage Cytokine Responses after L. pneumophila (Lp) Infection in vitro.Bone marrow-derived macrophages were isolated and infected with either negative control (mock), WT L. pneumophila (WT Lp), flagellin-deficient L. pneumophila (?flaA Lp), or dot-icm-locus-deficient L. pneumophila (?dotA Lp) on the JR32 background at MOI 10 overnight and A) TNF, B) IL-1ß, and C) IL-6 concentrations were measured from cellular supernatants. * p < 0.05, Student’s two-sided t-test. Experiment shown is representative of three independent experiments, each performed in triplicate. Error bars represent +/- SEM of mean.

Fig 5: TOLLIP-deficient mice demonstrate enhanced clearance of L. pneumophila (Lp) after aerosol infection.Mice were infected with approximately 106 CFU WT Lp Philadelphia-1 strain and at 24, 72, and 144 hours post infection, lungs were harvested. A) Bacterial counts were measured by CFU on BCYE agar plates. B) Total polymorphonuclear cell counts from bronchoalveolar fluid. C) Total mononuclear cells from bronchoalveolar fluid samples. D) Hematoxylin and eosin staining of representative sections of D) WT and E) Tollip-/- mouse lung 72hr after Lp aerosol infection. * p< 0.05, Student’s t-test, two sided. Error bars represent +/- SEM. N = 5 mice in each group at each time point. Data are representative of three independent experiments.