Fig 2: K. pneumoniae exploits KpEVs to evade macrophage‐mediated immune clearance and facilitate hepatic colonization. (A) Schematic workflow of in vivo analysis of K. pneumoniae‐infected mice. The mice were intravenously administered KpEVs (1 × 1010 particles) 24 h before K. pneumoniae infection (oral infection, 5 × 107 bacteria). The survival of each mouse was monitored daily (Experiment 1). Two days after infection, the mice were sacrificed, and the cecum and liver were harvested (Experiment 2). (B) Survival analysis of K. pneumoniae‐infected mice following the administration of KpEVs. P‐values were determined using the log‐rank test. (C) Bacterial counts in the cecal mucosa and liver of each mouse with or without KpEVs administration 2 days post‐infection. Samples were compared using Student's t‐test, with **p < 0.01, NS, not statistically significant. (D) Immunofluorescence staining for the detection of CD206 in the livers of K. pneumoniae‐infected 55‐week‐old mice following intravenous administration of KpEVs. The scale bar is 50 µm. (E and F) Immunostaining analysis for the detection of iNOS in F4/80+ cells in the liver of K. pneumoniae‐infected mice following KpEV administration. The percentage of iNOS‐positive F4/80+ cells was calculated using the ImageJ analysis software. (G) MPO activity (chlorination activity and peroxidation activity) of liver homogenates from each mouse. Samples were compared using one‐way ANOVA with Tukey's HSD analysis for multiple comparisons and the Student's t test, with *p < 0.05 and **p < 0.01. NS: not statistically significant.

Fig 3: KpEVs induces a distinctive immunosuppressive phenotype in macrophages. KpEVs (2 × 107 particles/well, corresponding to 100 vesicles per cell) were applied to BMDMs for 15 h. (A) Flow cytometric analysis for the detection of BMDM development using anti‐F4/80 and anti‐CD11b antibodies. (B and C) Flow cytometric analysis of the proportion of CD206+ cells within the BMDM population using an anti‐CD206 antibody. The percentage of CD206+ cells was calculated using the FlowJo software. (D) Measurement of the phagocytic activity of KpEVs‐treated BMDMs using FluoSpheres beads. The mean fluorescence intensity of BMDMs that had taken up FluoSphere beads was calculated using the FlowJo software. (E‐H) BMDMs (2 × 105 cells) were stimulated with KpEVs (2 × 107 particles) for 15 h, followed by infection with K. pneumoniae (2 × 107 bacteria, MOI 100) for 1.5 h. Cells were then incubated in DMEM containing gentamicin (100 µg/mL) for 6 h, after which culture supernatants or BMDMs were collected. IL‐1β production in K. pneumoniae‐infected BMDMs following KpEVs treatment (E). Western blot analysis for the detection of cleaved caspase‐1 (p20) released from K. pneumoniae‐infected BMDMs following KpEVs treatment (F). Detection of LDH release from K. pneumoniae‐infected BMDMs after KpEVs treatment (G). Western blot analysis for the detection of cleaved GSDMD (GSDMD‐N) released from K. pneumoniae‐infected BMDMs following KpEVs treatment (H). Samples were compared using one‐way ANOVA with Tukey's HSD analysis for multiple comparisons and the Student's t test, with *p < 0.05 and **p < 0.01. NS: not statistically significant.

Fig 4: KpEVs migrate and accumulate in the liver via the bloodstream and induce CD206+ macrophages. (A) Representative images of in vivo imaging analysis of mice with or without intravenous administration of KpEVs using the IVIS system. The mice were intravenously administered KpEVs (1 × 1010 particles). (B) Three representative images of IVIS analysis of the livers of mice with or without intravenous administration of KpEVs. (C) The intensities of DiR fluorescence of KpEVs in the liver were analyzed using Living Image software 4.7.4. (D) Immunostaining analysis to detect KpEVs using an anti‐GFP antibody in the livers of mice with or without intravenous administration of GFP‐labeled KpEVs. The scale bar is 10 µm. (E and F) Immunofluorescence staining for the detection of CD206 in the livers of KpEVs‐administered 15‐week‐old mice without K. pneumoniae infection. The scale bar is 50 µm. The percentage of CD206+ cells among F4/80+ cells was calculated using ImageJ analysis software. (G) Schematic workflow of in vivo localization analysis of KpEVs. The mice were orally administered KpEVs (1 × 109 particles × 3 times). (H) Three representative images of in vivo imaging analysis of mice with or without oral administration of KpEVs using an IVIS system. (I) Three representative images of IVIS imaging analysis in the liver of mice with or without oral administration of KpEVs. (J) The intensities of DiR fluorescence of KpEVs in the liver were analyzed using Living Image software 4.7.4. (K) Immunofluorescence analysis for the detection of KpEVs using an anti‐GFP antibody in the liver of mice with or without oral administration of GFP‐labelled KpEVs. The scale bar is 50 µm. Samples were compared using the Student's t‐test, with **p < 0.01.