Fig 2: 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 3: 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.

Fig 4: 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 5: WT and Tollip-/- Macrophage Cytokine Responses after TLR and NLR Stimulation.Bone marrow-derived macrophages (BMDM) were isolated from WT and Tollip-/- mice and stimulated with media, PAM3 (250ng/ml), a TLR 2/1 agonist, or LPS (10ng/ml), TLR4 agonist, for 24hr, and concentrations of A) TNF and B) IL-10 were measured from cellular supernatants. C) We stimulated mouse BMDM with LPS 100pg/ml for 2 hours, then stimulated overnight with C) media control or the NLRP3 ligand nigericin (100µg/ml) and D) NLRC4 ligand Salmonella flagellin (FliC, 100ng/ml) or bovine serum albumin (100ng/ml), coincubated with Lipofectamine 2000 to permit cytosolic translocation of each protein. IL-1ß concentrations from cellular supernatants were measured by ELISA. Error bars are means ± SEM; statistical analyses were performed by paired t-tests using Prism 7.0e. * p < 0.05, Student’s two-sided t-test. The experiment shown is representative of three independent experiments, each conducted in triplicate.