Figure 6.
Capture of LV encapsulated pneumococci by KCs. (A) Representative IVM images of the liver sinusoids of mice (n = 3) 10 min after i.v. infection with 5 × 107 CFU of isogenic TH870 derivatives producing the LV (type 14, 19F, and 23F) or HV (type 3, 6A, and 8) capsule variants (left). Scale bar, 20 μm. Bacterium-capture capacity of KCs are presented as KC-associated bacteria per FOV (right, n = 5–10 random fields). (B) IVM visualization of liver captured LV pneumococci in the KC-deficient mice (n = 3). Scale bar, 20 μm. Hepatic captured bacteria are quantified as numbers per FOV (right, n = 5–10 random fields). (C) Bacteremia kinetics of LV isogenic TH870 derivatives (type 14, 19F, and 23F) during the first 30 min after infection with 106 CFU in Clec4f-DTR mice were treated with (+) or without (−) DT. n = 5–10. (D) Proportional distribution of viable LV isogenic TH870 derivatives (type 14, 19F, and 23F) in the blood, liver, and spleen 10 min after infection in Clec4f-DTR mice pretreated with (+) or without (−) DT. n = 3–6. (E) Representative IVM images (left) showing the LV TH87014 outside (arrow) and inside (arrowhead) of KCs (blue) 20 min after i.v. inoculation of 108 CFU. Scale bar, 10 μm. Intracellular bacteria were detected by the activation of pH-sensitive dye pHrodo (red). The ratio of intra- and extracellular bacteria was quantified (right, n = 10 random fields). (F) Survival of KC-depleted mice after i.v. infection with 108 CFU of TH87014. n = 5. (G) Evasion of mouse KC capture by the HV capsule types of pneumococci. Freshly isolated primary KCs were used to test bacterial adherence with TH870 derivatives of representative LV (14, 19F, and 23F) and HV (3, 6A, and 8) capsule types. Bacterium-binding levels were calculated by dividing the KC-associated bacterial numbers to the total bacterial doses. n = 3. All mice were used in C57BL/6 background. Data were representative results (A, B, E, and G) or pooled (C, D, and F) from two independent experiments. Ordinary one-way ANOVA with Tukey’s multiple comparisons test (A), two-way ANOVA with Sidak’s (B and G) or Tukey’s (C) multiple comparisons test, log-rank test (F), **, P < 0.01, ****, P < 0.0001.

Capture of LV encapsulated pneumococci by KCs. (A) Representative IVM images of the liver sinusoids of mice (n = 3) 10 min after i.v. infection with 5 × 107 CFU of isogenic TH870 derivatives producing the LV (type 14, 19F, and 23F) or HV (type 3, 6A, and 8) capsule variants (left). Scale bar, 20 μm. Bacterium-capture capacity of KCs are presented as KC-associated bacteria per FOV (right, n = 5–10 random fields). (B) IVM visualization of liver captured LV pneumococci in the KC-deficient mice (n = 3). Scale bar, 20 μm. Hepatic captured bacteria are quantified as numbers per FOV (right, n = 5–10 random fields). (C) Bacteremia kinetics of LV isogenic TH870 derivatives (type 14, 19F, and 23F) during the first 30 min after infection with 106 CFU in Clec4f-DTR mice were treated with (+) or without (−) DT. n = 5–10. (D) Proportional distribution of viable LV isogenic TH870 derivatives (type 14, 19F, and 23F) in the blood, liver, and spleen 10 min after infection in Clec4f-DTR mice pretreated with (+) or without (−) DT. n = 3–6. (E) Representative IVM images (left) showing the LV TH87014 outside (arrow) and inside (arrowhead) of KCs (blue) 20 min after i.v. inoculation of 108 CFU. Scale bar, 10 μm. Intracellular bacteria were detected by the activation of pH-sensitive dye pHrodo (red). The ratio of intra- and extracellular bacteria was quantified (right, n = 10 random fields). (F) Survival of KC-depleted mice after i.v. infection with 108 CFU of TH87014. n = 5. (G) Evasion of mouse KC capture by the HV capsule types of pneumococci. Freshly isolated primary KCs were used to test bacterial adherence with TH870 derivatives of representative LV (14, 19F, and 23F) and HV (3, 6A, and 8) capsule types. Bacterium-binding levels were calculated by dividing the KC-associated bacterial numbers to the total bacterial doses. n = 3. All mice were used in C57BL/6 background. Data were representative results (A, B, E, and G) or pooled (C, D, and F) from two independent experiments. Ordinary one-way ANOVA with Tukey’s multiple comparisons test (A), two-way ANOVA with Sidak’s (B and G) or Tukey’s (C) multiple comparisons test, log-rank test (F), **, P < 0.01, ****, P < 0.0001.

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