Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The scavenger receptor CD36 downmodulates the early inflammatory response while enhancing bacterial phagocytosis during pneumococcal pneumonia.

doi: 10.4049/jimmunol.1202270

Figure Lengend Snippet: FIGURE 1. AMs and respiratory epithelial cells express CD36, and S. pneumoniae induces pulmonary CD36 expression. (A and B) FACS staining for CD36 on MH-S (A) and MLE-12 (B) cells. Black fill depicts CD36-stained cells, and open fill represents isotype control Ab. (C) CD36 IHC was conducted from uninfected lungs of WT and CD362/2 mice. The negative control represents Ag-retrieved lungs without incubation with Ab. Original magnification 3 100. (D) WT mice (n = 6 per time point) were intranasally inoculated with 105 CFU S. pneumoniae, and after indicated time points CD36 lung transcript levels were evaluated. (E–J) WT AMs (E, G, H) or respiratory epithelial cells (F, I, J) were treated with heat-killed S. pneumoniae for indicated time points, and CD36 RT-PCR (E, F) and CD36 FACS (G–J) were conducted. (K, L) WT mice (n = 4 per time point) were intranasally inoculated with 105 CFU S. pneumoniae, and CD36 expression was evaluated on AMs from BAL after indicated time points. Depicted is the percentage of CD45+/F4/80+/CD11c+/ CD11b2 cells (AMs) positive for CD36. Representative FACS plots are shown. Data presented are mean 6 SEM, and data are representative of duplicate experiments. *p , 0.05 versus time point 0.

Article Snippet: Cell treatments and cytokine and chemokine ELISAs Cells were plated at 106/ml in 96-well dishes in RPMI supplemented with 3% heat-inactivated FCS and 1% pen/strep and stimulated with 23 107/ml heat-killed S. pneumoniae (ATCC 6303) or 10 mg/ml S. aureus or S. pneumoniae LTA or 1 mg/ml LPS for 6 h. For CD36-blocking experiments, cells were pretreated with CD36-blocking Ab (clone JC63.1) or isotype control for 30 min, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments involving PC-BSA, cells were pretreated with PCBSA or BSA for 1 h, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments studying the effects of EO6 on cytokine synthesis, heat-killed S. pneumoniae (ATCC 6303) or E. coli were incubated with 25 mg/ml EO6 or isotype control Ab for 1 h prior to addition to cells at 2 3 107/ml for 6 h. Following cell treatments, supernatant was harvested and stored at220 ̊C until assays were performed.

Techniques: Expressing, Staining, Control, Negative Control, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The scavenger receptor CD36 downmodulates the early inflammatory response while enhancing bacterial phagocytosis during pneumococcal pneumonia.

doi: 10.4049/jimmunol.1202270

Figure Lengend Snippet: FIGURE 2. CD36 suppresses S. pneumoniae–medi- ated inflammation in vitro in AMs and respiratory ep- ithelial cells. (A, B) WT primary respiratory epithelial cells (A) or AMs (B) seeded at 106/ml were treated with 2 3 107 CFU/ml S. pneumoniae for 6 h with and without pretreatment with a CD36 blocking or isotype control Ab, and levels of the indicated cytokines were evaluated in the supernatant, using ELISA. (C) WT or CD362/2 AMs were treated with 2 3 107 CFU/ml heat-killed S. pneumoniae for 6 h, and levels of TNF-a and MIP-2 were evaluated in the supernatant. (D) WT or CD362/2 AMs were treated with 2 3 107 CFU/ml heat-killed S. pneumoniae or 10 mg/ml S. aureus LTA for 6 h, and levels of TNF-a were evaluated in the supernatant. All data presented are mean 6 SEM and representative of duplicate experiments. *p , 0.05 versus isotype at the identical concentration (A, B) or WT.

Article Snippet: Cell treatments and cytokine and chemokine ELISAs Cells were plated at 106/ml in 96-well dishes in RPMI supplemented with 3% heat-inactivated FCS and 1% pen/strep and stimulated with 23 107/ml heat-killed S. pneumoniae (ATCC 6303) or 10 mg/ml S. aureus or S. pneumoniae LTA or 1 mg/ml LPS for 6 h. For CD36-blocking experiments, cells were pretreated with CD36-blocking Ab (clone JC63.1) or isotype control for 30 min, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments involving PC-BSA, cells were pretreated with PCBSA or BSA for 1 h, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments studying the effects of EO6 on cytokine synthesis, heat-killed S. pneumoniae (ATCC 6303) or E. coli were incubated with 25 mg/ml EO6 or isotype control Ab for 1 h prior to addition to cells at 2 3 107/ml for 6 h. Following cell treatments, supernatant was harvested and stored at220 ̊C until assays were performed.

Techniques: In Vitro, Blocking Assay, Control, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The scavenger receptor CD36 downmodulates the early inflammatory response while enhancing bacterial phagocytosis during pneumococcal pneumonia.

doi: 10.4049/jimmunol.1202270

Figure Lengend Snippet: FIGURE 3. S. pneumoniae binds to CD36 via its PC, which suppresses inflammation. (A) WT BMDMs seeded at 1.6 3 106/ml were incubated with S. pneumoniae or E. coli (MOI 100) that were pretreated with either isotype (KLH) or EO6 Ab for 2 h at 4˚C, and bacterial binding was assessed using FACS. (B) WT or CD362/2 BMDMs seeded at 1.6 3 106/ml were incubated with FITC-labeled S. pneumoniae that had been pretreated with EO6 Ab (MOI 100) for 2 h at 4˚C, and bacterial binding was assessed using FACS. (C) WTAMs seeded at 106/ml were pretreated with either isotype or an EO6 Ab and incubated with 2 3 107 CFU/ml heat-killed S. pneumoniae or E. coli for 6 h, and levels of MIP-2 were evaluated in the su- pernatant. (D) WT or CD362/2 AMs seeded at 106/ml were pretreated with either PC-BSA or BSA and incu- bated with 2 3 107 CFU/ml heat-killed S. pneumoniae for 6 h, and levels of TNF-a were evaluated in the super- natant. (E) WT and CD362/2 BMDMs were incubated with heat-killed S. pneumoniae, 10 mg/ml S. pneumoniae LTA (R6 strain), or 1 mg/ml LPS for 6 h, and levels of MIP-2 were evaluated in the supernatant. All data pre- sented are mean 6 SEM and representative of duplicate experiments (A, B, E). *p , 0.05 versus isotype (A, C) or S. pneumoniae (B).

Article Snippet: Cell treatments and cytokine and chemokine ELISAs Cells were plated at 106/ml in 96-well dishes in RPMI supplemented with 3% heat-inactivated FCS and 1% pen/strep and stimulated with 23 107/ml heat-killed S. pneumoniae (ATCC 6303) or 10 mg/ml S. aureus or S. pneumoniae LTA or 1 mg/ml LPS for 6 h. For CD36-blocking experiments, cells were pretreated with CD36-blocking Ab (clone JC63.1) or isotype control for 30 min, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments involving PC-BSA, cells were pretreated with PCBSA or BSA for 1 h, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments studying the effects of EO6 on cytokine synthesis, heat-killed S. pneumoniae (ATCC 6303) or E. coli were incubated with 25 mg/ml EO6 or isotype control Ab for 1 h prior to addition to cells at 2 3 107/ml for 6 h. Following cell treatments, supernatant was harvested and stored at220 ̊C until assays were performed.

Techniques: Incubation, Binding Assay, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The scavenger receptor CD36 downmodulates the early inflammatory response while enhancing bacterial phagocytosis during pneumococcal pneumonia.

doi: 10.4049/jimmunol.1202270

Figure Lengend Snippet: FIGURE 4. CD36 suppresses early S. pneumoniae– mediated inflammation in vivo. (A–F) WT and CD362/2

Article Snippet: Cell treatments and cytokine and chemokine ELISAs Cells were plated at 106/ml in 96-well dishes in RPMI supplemented with 3% heat-inactivated FCS and 1% pen/strep and stimulated with 23 107/ml heat-killed S. pneumoniae (ATCC 6303) or 10 mg/ml S. aureus or S. pneumoniae LTA or 1 mg/ml LPS for 6 h. For CD36-blocking experiments, cells were pretreated with CD36-blocking Ab (clone JC63.1) or isotype control for 30 min, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments involving PC-BSA, cells were pretreated with PCBSA or BSA for 1 h, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments studying the effects of EO6 on cytokine synthesis, heat-killed S. pneumoniae (ATCC 6303) or E. coli were incubated with 25 mg/ml EO6 or isotype control Ab for 1 h prior to addition to cells at 2 3 107/ml for 6 h. Following cell treatments, supernatant was harvested and stored at220 ̊C until assays were performed.

Techniques: In Vivo

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The scavenger receptor CD36 downmodulates the early inflammatory response while enhancing bacterial phagocytosis during pneumococcal pneumonia.

doi: 10.4049/jimmunol.1202270

Figure Lengend Snippet: FIGURE 5. CD36 contributes to S. pneumoniae clearance in vivo but has no effects on mortality. (A) WT and CD362/2 mice (n = 7) were intranasally in- oculated with 105 CFU S. pneumoniae, and at 44 h post infection pulmonary bacterial load was evaluated. (B) WT or CD362/2 AMs seeded at 1.6 3 106/ml were incubated with FITC-labeled S. pneumoniae (MOI 100), and phagocytosis was assessed using FACS. (C and D) WT and CD362/2 mice (n = 12–13) were in- tranasally inoculated with (C) 105 or (D) 5 3 103 CFU S. pneumoniae, and survival was monitored for 10 d. Data in (A) and (B) are mean 6 SEM. *p , 0.05 versus WT.

Article Snippet: Cell treatments and cytokine and chemokine ELISAs Cells were plated at 106/ml in 96-well dishes in RPMI supplemented with 3% heat-inactivated FCS and 1% pen/strep and stimulated with 23 107/ml heat-killed S. pneumoniae (ATCC 6303) or 10 mg/ml S. aureus or S. pneumoniae LTA or 1 mg/ml LPS for 6 h. For CD36-blocking experiments, cells were pretreated with CD36-blocking Ab (clone JC63.1) or isotype control for 30 min, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments involving PC-BSA, cells were pretreated with PCBSA or BSA for 1 h, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments studying the effects of EO6 on cytokine synthesis, heat-killed S. pneumoniae (ATCC 6303) or E. coli were incubated with 25 mg/ml EO6 or isotype control Ab for 1 h prior to addition to cells at 2 3 107/ml for 6 h. Following cell treatments, supernatant was harvested and stored at220 ̊C until assays were performed.

Techniques: In Vivo, Infection, Incubation, Labeling