CL429 treatment leads to a systemic enhanced antileptospiral response.

A) Chronogram of the experiment. C57BL/6 WT mice were injected IP with 200 μL PBS containing 2.5% DMSO (vehicle) or 25 μg CL429 2 and 1 weeks before the collection of splenic and bone marrow cells for cytometry staining, macrophage differentiation and ex vivo stimulation. Splenic cells were plated and exposed for 24 h to 100 ng/mL E. coli LPS or live Leptospira interrogans serovar Manilae, Fiocruz or Verdun at a MOI of 100. Bone marrow cells were differentiated into bone marrow-derived macrophages (BMMs) for 7 days, plated and stimulated with LPS or live leptospires. B) Interferon(IFN)-γ IFN production in the supernatant of splenic cells 24 h post-stimulation assessed by ELISA. Data correspond to 2 independent experiments pooled with n = 4 mice per group, treated individually. C) Flow cytometry analysis of IFN-γ produced by splenic NK and T cells at 24 h post-stimulation. Data correspond to one experiment with n = 6 female mice. D) Flow cytometry analysis of BMMs 7 days post-differentiation of bone marrow cells (upper panel) and representative microscopy images of BMMs showing no major morphological differences in BMMs from PBS- or CL429-treated mice (lower panel). E) Pro-inflammatory cytokine (IL-6) and chemokine (RANTES and KC) profiles determined by ELISA on the supernatant of cells 24 h post-stimulation. F) Nitric oxide (NO) production by BMMs at 24 h after stimulation assessed by the Griess reaction. G) Survival of L. interrogans serovar Manilae bioluminescent strain MFlum1 in contact with BMMs assessed by bioluminescence measurement in culture 24 h post-infection. (D-G) Data are representative of 2 independent experiments with n = 5 mice per group, treated individually. Statistical analysis was performed by the unpaired t-test comparing PBS vs CL429 for each secondary stimulation. * p-value p-value p-value < 0.001.