Mannose receptor-derived peptides neutralize pore-forming toxins and reduce inflammation and development of pneumococcal disease (2020)
Figure 4

Figure 4. PLY-mediated bacterial invasion of the lung epithelium and intracellular bacterial survival are inhibited by MRC-1 peptides. (A) IL-8 released by human THP-1 macrophages stimulated with purified PLY, LLO or SLO (0.5 μg/ml) in the presence or absence of 100 μM peptides P2, P3 or control peptide CP2 for 18 h. Cholesterol (100 μM) was used as positive control to inhibit hemolysis. Data are mean ± s.e.m from three independent experiments. **** denotes P< 0.0001 by two-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.

(B) Schematic showing the cellular architecture of the 3D lung epithelial model.

(C) 3D volume images of the GFP-lung epithelial models at 1 h and 3 h post stimulation with 1 μg/ml PLY in the presence or absence of 100 μM peptide P2 or the control peptide CP2. Images are representative of two independent experiments with n=3 models/condition.

(D) Invasion of wild-type pneumococci T4 (TIGR4) or its isogenic PLY mutant T4Δply into the lung epithelial models (n=3/condition) in the presence or absence of 100 μM peptide P2 or the control peptide CP2 at 2h post infection was measured using CFU viability assay following gentamicin killing of extracellular bacteria. Anti-PLY was used as control to test the effect of blocking PLY. Data in d and e are mean ± s.e.m. of n=3 models/condition from two independent experiments. % bacterial entry = (bacteria uptaken/input) x100. ** denotes P < 0.01 by one-way ANOVA with Dunnett's post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.

(E) Human DCs were infected with type 4 and type 2 pneumococci, T4 and D39, respectively, at MOI of 10 in the presence or absence of 100 μM peptides, P2 or CP2, and intracellular bacteria were counted at 3 h post infection following gentamicin killing of extracellular bacteria. Cytochalasin D (0.5 mM) was used as negative control to inhibit phagocytosis. Anti-PLY was used as control to test the effect of blocking PLY. Data are mean ± s.e.m. of three independent experiments. **** denotes P < 0.0001 by two-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.

(F) DCs were infected with the unencapsulated pneumococcal strain T4R in the presence of 100 μM peptides, P2 or CP2 at MOI of 10 for 2 h. Immunofluorescence microscopy images show that in DCs treated with peptide P2 (but not the control peptide CP2), intracellular T4R (green) do not co-localize with MRC-1 (red), but with the autophagy protein LC3B (cyan). Images are representative of three independent experiments. Scale bars, 10 μm. In magnified images, scale bars, 5 μm. Arrows indicate regions of co-localization of intracellular T4R with MRC-1 and LC3B.

(G) Quantification of percentage of intracellular S. pneumoniae (n=50) in infected DCs that co-localize with MRC-1 and LC3B. Data are mean±s.e.m from two independent experiments. **** denotes P <0.0001 by two-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. P values are shown in Appendix Table S4.