Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Platelets promote human macrophages-mediated macropinocytosis of Clostridioides difficile

doi: 10.3389/fcimb.2023.1252509

Figure Lengend Snippet: Interaction of platelets with C. difficile , monocytes and macrophages. (A–C) Washed Platelets (WP) from Healthy Donors (HD) were incubated with heat-inactivated C. difficile coupled to FITC ( CD H FITC) at ratios 1:1 and 1:10 for 2h. CD H FITC-WP complexes were detected by flow cytometry (A) and fluorescence microscopy (B, C) using anti-CD61 PE antibody. (A, B) are representative experiments. (B) Images on the right are magnifications corresponding to white boxes on the left. (C) Quantification of CD H FITC-associated platelets from fluorescent micrographs is shown. (D) WP from HD were incubated with live CD160 and live NAP1/BI/027 C. difficile strains coupled to FITC and with CD H FITC plus NAP1/BI/027 secretome ( CD H FITC+Sec) for 2h. In all cases, WP:bacteria ratios of 1:1 and 1:10 were tested and C. difficile -WP complexes were detected by flow cytometry. Representative histograms are shown. (E) Whole peripheral blood from HD was cultured in the presence or absence of CD H for 2h. Monocytes (Mo)-WP complexes were measured by flow cytometry using anti-CD14 Alexa Fluor 647 and anti-CD61 PE antibodies. (F) Macrophages (MΦs) and WP from HD were co-cultured in the presence or absence of CD H for 1h. MΦs-WP complexes were assessed by confocal microscopy after fixation, permeabilization and staining. The white boxes correspond to the magnified images on the right (representative experiment). White arrowheads point WP inside MΦs. DAPI (nuclei) is shown in blue, CD61 (platelets) in red and CD14 (macrophages) in purple. DIC: differential interference contrast. Scale bar: 10 μm (A, C, F) correspond to three individual donors in three independent experiments, (D) corresponds to four independent experiments, (E) corresponds to five individual donors in five independent experiments. (C) Bars represent the mean ± SEM. (E) Each dot corresponds to an individual healthy donor. (C) Mann-Whitney test, (E) Wilcoxon test. *p<0.05, ****p<0.0001.

Article Snippet: In vitro infection of cells was performed with vegetative live non-toxigenic CD160 (NR-43516, BEI Resources) or hypervirulent NAP1/BI/027 (Sanitary Bacteriology Service INEI-ANLIS, Dr. Carlos G. Malbrán, Argentina) C. difficile strains.

Techniques: Incubation, Flow Cytometry, Fluorescence, Microscopy, Bacteria, Cell Culture, Confocal Microscopy, Staining, MANN-WHITNEY

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Platelets promote human macrophages-mediated macropinocytosis of Clostridioides difficile

doi: 10.3389/fcimb.2023.1252509

Figure Lengend Snippet: Evaluation of endocytic pathways against C. difficile . A) Macrophages (MΦs) from Healthy Donors (HD) were cultured with endocytosis inhibitors (Citochalasin B (Cit B, actin polymerization inhibitor), Citochalasin D (Cit D, inhibitor of actin polymerization by ATP hydrolysis), Colchicine (Colch, microtubules polymerization inhibitor), Vincristine (Vincr, microtubules polymerization inhibitor), Nystatin (Nist, inhibitor of caveolae/lipid mediated endocytosis), Bafilomycin (Bafilo, autophagy inhibitor) or Amiloride (Amilo, macropinocytosis inhibitor)). After 30min, heat-inactivated C. difficile coupled to FITC ( CD H FITC) was added for 1h. CD H FITC internalization was assessed by flow cytometry (A, B) and confocal microscopy (C) . (A) Percentage of FITC positive cells (uptake). (B) Representative histograms. (C) Confocal micrographs of untreated (-) or Amiloride treated (Amilo) MΦs obtained after cell fixation, staining with anti-CD14 Alexa Fluor 647 antibody (macrophages) and DAPI (nuclei) counterstaining. The white boxes correspond to the magnified images on the right. White arrowheads point internalized CD H FITC. (D) MΦs were stimulated with CD H FITC in the presence or absence of Washed Platelets (WP) (ratio 1:10:100) for 1h. Amiloride was added 30 minutes before to block macropinocytosis. Percentage of macropinocytic cells was measured by flow cytometry. (E) Cells were fixated, permeabilized and stained with anti-CD14 Alexa Fluor 647 (macrophages) and anti-CD61 PE (platelets) antibodies. DAPI (nuclei) was employed as counterstain and confocal images were obtained. The white boxes correspond to the magnified images on the right (orthogonal views showing platelets (CD61) and internalized CD H FITC in macrophages (DIC/DAPI)). In the panoramic micrographs, white arrowheads point macrophages internalizing CD H. DAPI (nuclei) is shown in blue, CD61 (platelets) in red and CD14 (macrophages) in purple. DIC: differential interference contrast. Scale bar: 10 μm. (F–H) MΦs were stimulated with CD H FITC or CD H FITC plus NAP1/BI/027 secretome ( CD H FITC+Sec) or infected with live CD160 FITC (non-toxigenic) or NAP1/BI/027 FITC (hypervirulent) C. difficile strains as stated in (D) . (A–C, E) correspond to three individual donors in three independent experiments, (D) correspond to nine individual donors in 9 independent experiments. (F–H) correspond to four donors in independent experiments. (A) Violin plots, (D, F–H) Bars represent the mean ± SEM. (D) Each dot corresponds to an individual healthy donor. (A, D) Friedman test. (F–H) 2-way ANNOVA *p<0.05, **p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: In vitro infection of cells was performed with vegetative live non-toxigenic CD160 (NR-43516, BEI Resources) or hypervirulent NAP1/BI/027 (Sanitary Bacteriology Service INEI-ANLIS, Dr. Carlos G. Malbrán, Argentina) C. difficile strains.

Techniques: Cell Culture, Flow Cytometry, Confocal Microscopy, Staining, Blocking Assay, Infection