Journal: PLoS Pathogens

Article Title: Innate immune memory through TLR2 and NOD2 contributes to the control of Leptospira interrogans infection

doi: 10.1371/journal.ppat.1007811

Figure Lengend Snippet: 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 peritoneal cells for cytometry or ex vivo stimulation. Cells were plated, and adherent cells were 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. B) Flow cytometry analysis showing the percentage of macrophages (CD11b + /F4/80 + ) recovered in peritoneal lavages from treated (CL429) or control (PBS) mice. The plot corresponds to 2 independent experiments pooled with n = 5 mice in each group. C) Representative phase contrast microscopy image of adherent peritoneal cells seeded in 96-well plates at 3 h post-collection (non stimulated (NS) cells). D) Pro-inflammatory cytokine (IL-6, IL-1β) and chemokine (RANTES, KC) production determined by ELISA 24 h post-stimulation in the supernatant of peritoneal cells. Data are representative of 4 independent experiments (n = 4 to 6 mice per group, treated individually). E) Nitric oxide (NO) production in peritoneal cells 24 h after stimulation assessed by the Griess reaction. Data are representative of 4 independent experiments (n = 4 to 6 mice per group, treated individually). F) Peritoneal macrophages were exposed to bioluminescent L . interrogans serovar Manilae MFlum1 strain at MOI of 100. Bioluminescence was measured 24 h post-stimulation in 100 μL culture supernatant to assess the number of live leptospires. Data are representative of 2 independent experiments ( n = 5). Statistical analysis was performed by 2-way ANOVA comparing PBS vs CL429 for each stimulation. For macrophage phenotyping (B) and survival (F), statistical analysis was performed by the unpaired t -test. * p -value < 0.05; ** p -value < 0.01; *** p -value < 0.001.

Article Snippet: Cells supernatants were kept at -20°C until the cytokine dosage, which was performed using ELISA Duo-set kits from R&D Systems for mouse IL-1β, RANTES, IL-6, KC and human IL-6, according to the instructions provided by the supplier.

Techniques: Injection, Cytometry, Ex Vivo, Flow Cytometry, Control, Microscopy, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: Innate immune memory through TLR2 and NOD2 contributes to the control of Leptospira interrogans infection

doi: 10.1371/journal.ppat.1007811

Figure Lengend Snippet: A) Chronogram of the experiment. Rag-2γc 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 peritoneal cells for ex vivo stimulation. Cells were plated and exposed for 24 h to E . coli LPS (100 ng/mL) or Leptospira interrogans serovar Manilae, Fiocruz or Verdun, at MOI of 100. B) Representative phase contrast microscopy image of non stimulated peritoneal cells seeded in 96-well plates at 3 h post-collection. C) Pro-inflammatory cytokine (IL-6) and chemokine (RANTES and KC) production determined by ELISA in the supernatant of cells 24 h post-stimulation. Data are representative of 2 independent experiments (n = 6 mice per group, treated individually). Statistical analysis was performed by the unpaired t -test comparing PBS vs CL429. D) Nitric oxide (NO) production in peritoneal cells at 24 h after stimulation assessed by the Griess reaction. Data are representative of 2 independent experiments (n = 6 mice per group, treated individually). Statistical analysis was performed by 2-way ANOVA comparing PBS vs CL429 for each secondary stimulation. * p -value < 0.05; ** p -value < 0.01; *** p -value < 0.001.

Article Snippet: Cells supernatants were kept at -20°C until the cytokine dosage, which was performed using ELISA Duo-set kits from R&D Systems for mouse IL-1β, RANTES, IL-6, KC and human IL-6, according to the instructions provided by the supplier.

Techniques: Injection, Ex Vivo, Microscopy, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: Innate immune memory through TLR2 and NOD2 contributes to the control of Leptospira interrogans infection

doi: 10.1371/journal.ppat.1007811

Figure Lengend Snippet: 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 < 0.05; ** p -value < 0.01; *** p -value < 0.001.

Article Snippet: Cells supernatants were kept at -20°C until the cytokine dosage, which was performed using ELISA Duo-set kits from R&D Systems for mouse IL-1β, RANTES, IL-6, KC and human IL-6, according to the instructions provided by the supplier.

Techniques: Injection, Cytometry, Staining, Ex Vivo, Derivative Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Produced, Microscopy, Infection

Journal: PLoS Pathogens

Article Title: Innate immune memory through TLR2 and NOD2 contributes to the control of Leptospira interrogans infection

doi: 10.1371/journal.ppat.1007811

Figure Lengend Snippet: A) Chronogram of the experiment. C57/BL6 WT mice were injected IP with 200 μL PBS containing 2.5% DMSO (vehicle) or 25 μg CL429 8 weeks before the collection of peritoneal cells for ex vivo stimulation. Cells were plated and exposed for 24 h to E . coli LPS (100 ng/mL) or live L . interrogans serovar Manilae, Fiocruz or Verdun, at MOI of 100. B) Flow cytometry analysis of the peritoneal lavages showing the proportions of (CD11b +/ F4/80 + ) macrophages from CL429-treated and PBS-treated mice 8 weeks post-treatment. C) Representative phase contrast microscopy images of non stimulated peritoneal cells from PBS- and CL429-treated cells seeded in 96-well plates 3 h post-collection. D) Nitric oxide (NO) production in peritoneal cells 24 h after stimulation assessed by the Griess reaction. E) Pro-inflammatory cytokines (IL-1ß, IL6) and chemokine (KC) production determined by ELISA 24 h post-stimulation in the supernatant of peritoneal cells. Data are representative of 2 experiments in (n = 3 or 4) mice. Statistical analysis was performed by 2-way ANOVA comparing PBS vs CL429 for each stimulation. For macrophage phenotyping (B), statistical analysis was performed by the unpaired t -test. * p -value < 0.05; ** p -value < 0.01; *** p -value < 0.001.

Article Snippet: Cells supernatants were kept at -20°C until the cytokine dosage, which was performed using ELISA Duo-set kits from R&D Systems for mouse IL-1β, RANTES, IL-6, KC and human IL-6, according to the instructions provided by the supplier.

Techniques: Injection, Ex Vivo, Flow Cytometry, Microscopy, Enzyme-linked Immunosorbent Assay

Journal: ACS Pharmacology & Translational Science

Article Title: Lipopolymer/siRNA Nanoparticles Targeting the Signal Transducer and Activator of Transcription 5A Disrupts Proliferation of Acute Lymphoblastic Leukemia

doi: 10.1021/acsptsci.4c00336

Figure Lengend Snippet: Figure 2. Proinflammatory cytokine response to LPNPs in human PBMCs. (A) Schematic diagram (created with BioRender.com) of PBMC isolation from peripheral blood of human healthy volunteers and treatment with positive controls or LPNPs at a regular ratio of 7.5:1.0 (R7.5, w/ w) and a higher ratio of 12.0:1.0 (R12.0, w/w). After 24 h of treatment, cell pellets were collected for RT-qPCR, and culture supernatants were collected for ELISA. (B,D,F,H) Proinflammatory cytokine (IL-6, IFN-γ, and TNF-α) mRNA levels in PBMCs by positive controls (PMA/IO/PMA +IO) and siRNAs alone (60 nM CsiRNA/siSTAT5A) (B), PEI-A alone or complexed with siRNAs (D), PEI-B alone or complexed with siRNA (F), and PEI-C alone or complexed with siRNAs (H). (C,E,G,I) Protein (IL-6, IFN-γ, and TNF-α) levels in culture supernatants in PBMCs by positive controls (PMA/IO/PMA+IO), siRNAs alone (60 nM CsiRNA/siSTAT5A) (C), PEI-A alone or complexed with siRNAs (E), PEI-B alone or complexed with siRNAs (G), and PEI-C alone or complexed with siRNAs (I). UT: untreated cells.

Article Snippet: Validated DUO ELISA kits (cat. no. DY210-05: human TNF-α, cat. no. DY206-05: human IL-6, cat. no. DY285B-05: human IFN-γ, and cat. no. DY008B: DuoSet ELISA ancillary reagent kit 2) were obtained from R&D Systems.

Techniques: Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay