Fig 1: Gas6 represses the invasion of K. pneumoniae into Caco-2 cells by increasing the expression of ZO-1 and occludin.(A) Administration of Gas6 recombinant protein to Caco-2 cells to analyze the expression of Axl, ZO-1, and occludin. Addition of Gas6 recombinant protein to the apical surface (left scheme) or the basolateral side (right scheme) of Caco-2 cells. (B) Western blot analysis to detect Axl, Gas6, ZO-1, and occludin in Caco-2 cells treated with human Gas6 recombinant protein from the apical or basolateral sides. Each western blotting image represents three independent replicates (n = 3). (C) Western blot analysis was performed to detect Axl, Gas6, ZO-1, and occludin. Gas6 recombinant protein (1 μg) was added to the apical side of Caco-2 cells grown in a Transwell co-culture system for 3 h prior to K. pneumoniae infection. Each western blotting image represents three independent replicates (n = 3). (D) Gas6 recombinant protein (1 μg) was added to Caco-2 cells grown on the Transwell insert for 3 h prior to K. pneumoniae infection. The cells were immunostained with an anti-ZO-1 antibody or an anti-occludin antibody, and an anti-E-cadherin antibody. Each image has four independent replicates. Scale bar = 20 μm. (E) Fluorescence intensity per pixel of anti-ZO-1 antibody, an anti-occludin antibody, and anti-E-cadherin antibody were measured by ImageJ analysis software. Each dot represents four independent replicates (n = 4 or 5 per group). Data are presented as the mean ± SD. NS: not significant, **p < 0.01. p values were calculated by the Student’s t test. (F) Gas6 recombinant protein (1 μg) was added to Caco-2 cells grown on the Transwell insert for 3 h prior to K. pneumoniae infection. The cells were immunostained with an anti-Klebsiella pneumoniae antibody and an anti-E-cadherin antibody. Each image has four independent replicates. Scale bar = 20 μm. (G) Fluorescence intensity per pixel of anti-Klebsiella pneumoniae antibody were measured by the ImageJ analysis software. Each dot represents five independent replicates (n = 5 per group). Data are presented as the mean ± SD. **p < 0.01. p values were calculated by the Student’s t test.
Fig 2: Gas6 released by macrophages that recognized K. pneumoniae infection is co-localized with Axl tyrosine kinase receptor on the epithelial cells.(A) Cytokine array analysis of the K. pneumoniae infection model based on the Transwell insert co-culture system consisting of Caco-2 cells and RAW264.7 macrophages. Quantification of Axl signals using a laser scanner. Each dot represents four independent replicates (n = 4 per group). Data are presented as the mean ± SD. **p < 0.01. p values were calculated by the Student’s t test. (B and C) Secretion of Axl (B) and Gas6 (C) by Caco-2 cells or RAW264.7 macrophages. Culture supernatants or lysates of K. pneumoniae were added to Caco-2 cells or RAW264.7 macrophages for 12 h. The culture media were then collected for ELISA analysis of Axl or Gas6 levels. Each dot represents six independent replicates (n = 6 per group). Data are presented as the mean ± SD. **p < 0.01. p values were calculated by one way analysis of variance. (D) K. pneumoniae-infected Caco-2 cells grown on a Transwell insert in the presence or absence of RAW264.7 macrophages were immunostained with anti-Gas6 and anti-Axl antibodies. Each image has six independent replicates. Scale bar = 50 μm. (E) Fluorescence intensity per pixel of anti-Axl antibody (left panel) or an anti-Gas6 antibody (right panel) were measured by the ImageJ analysis software. Each dot represents six independent replicates (n = 6 per group). Data are presented as the mean ± SD. **p < 0.01. p values were calculated by one way analysis of variance.
Fig 3: Gas6/Axl signaling in Caco-2 cells inhibits K. pneumoniae invasion by enhancing the expression of tight junction proteins.(A) Western blotting was performed to detect the expression of Axl, Gas6, ZO-1, and occludin in Caco-2 cells infected with K. pneumoniae in the presence of an Axl inhibitor (R428) or an anti-Gas6 antibody. Prior to K. pneumoniae infection, cells were treated for 3 h with Axl inhibitor (R428; 20 nM) or 1 μg of anti-Gas6 antibody. Each western blotting image represents three independent replicates (n = 3). (B) Western blotting signal intensity was analyzed by ImageJ software. Each dot represents three independent replicates (n = 3). Data are presented as the mean ± SD. **p < 0.01. p values were calculated by one way analysis of variance. (C) Axl inhibitor (R428; 20 nM) or 1 μg of anti-Gas6 antibody were added to Caco-2 cells grown on the insert in the presence of RAW264.7 macrophages for 3 h prior to K. pneumoniae infection. Next, Caco-2 cells were immunostained with an anti-ZO-1 antibody and an anti-occludin antibody. Each image has four (group of Caco-2 cells/RAW264.7 cells stained with an anti-ZO-1 antibody) or five independent replicates. Scale bar = 50 μm. (D) Fluorescence intensity per pixel of anti-ZO-1 antibody (left panel) or an anti-occludin antibody (right panel) were measured by the ImageJ analysis software. Each dot represents four or five independent replicates (n = 4 or 5 per group). Data are presented as the mean ± SD. **p < 0.01. p values were calculated by one way analysis of variance. (E) Axl inhibitor (R428; 20 nM) or 1 μg of anti-Gas6 antibody were added to Caco-2 cells grown on the insert in the presence or absence of RAW264.7 macrophages for 3 h prior to K. pneumoniae infection. Next, Caco-2 cells were immunostained with an anti-Klebsiella pneumoniae antibody. Each image has six independent replicates. Scale bar = 50 μm. (F) Fluorescence intensity per pixel of K. pneumoniae were measured by the ImageJ analysis software. Each dot represents six independent replicates (n = 6 per group). Data are presented as the mean ± SD. **p < 0.01. p values were calculated by one way analysis of variance. (G) Bacterial counts within Caco-2 cells treated with an Axl inhibitor (R428) or an anti-Gas6 antibody in the presence of BMDMs. Prior to K. pneumoniae infection, Caco-2 cells were treated for 3 h with Axl inhibitor (R428; 20 nM) or 1 μg of anti-Gas6 antibody. Cells were lysed with PBS/1% Triton X-100, and the lysate was plated on LB agar. The number of CFU was counted after 24 h incubation. Each dot represents six independent replicates (n = 6 per group). Data are represented as mean ± SD. **p < 0.01. p values were calculated by one way analysis of variance.
Fig 4: Gas6 and Axl expression in the cecal mucosa during K. pneumoniae infection decreases with aging.(A and B) Sections of cecal mucosa (A) or liver (B) were from mice aged 15 or 57 weeks at 2 days after infection with K. pneumoniae ATCC43816 pmCherry and immunostained with an anti-Gas6 antibody and an anti-Axl antibody. Each image has six independent replicates. Scale bar = 50 μm. (C) Detection of Axl, Gas6, ZO-1, and occludin by western blotting. Cecum and liver tissues were collected, homogenized, and analyzed using an anti-Axl antibody, an anti-Gas6 antibody, an anti-ZO-1 antibody, or an anti-occludin antibody. Each lane of western blotting images represents the protein from an individual mouse (mouse number = 5 (cecum) or 4 (liver) per group). Western blotting analysis represents three independent replicates. (D) Western blotting signal intensities were analyzed by ImageJ software. Each dot represents the value collected from an individual mouse (mouse number = 5 (cecum) or 4 (liver) per group). Data are presented as the mean ± SD. NS: not significant, *p < 0.05, **p < 0.01. p values were calculated by the Student’s t test. (E) Linear correlation between Gas6 expression and Axl expression in the cecum or liver of mice aged 15 (red circles) or 57 (blue circles) weeks infected with K. pneumoniae ATCC43816 pmCherry. Each dot represents an ELISA measured value of each protein sample collected from an individual mouse (mouse number = 5 per group). r > 0.70 and p < 0.05. (F) The population of CD11b+F4/80+ macrophages in the intestinal mucosa of young (14-week-old) and old (56-week-old) mice was examined by staining with anti-F4/80 and anti-CD11b antibodies. The percentage of F4/80-positive/CX3CR1-negative cells in 14-week-old and 56-week-old mice was 29.5% and 8.87%, respectively. Data are representative of four mice per group.
Fig 5: Gas6 prevents systemic infection by orally infecting K. pneumoniae in elderly mice.(A) Treatment scheme used to analyze the effect of Gas6 recombinant protein on the susceptibility of 57-week-old mice to infection by K. pneumoniae. Antibiotics were administered 4 weeks before administration of Gas6 recombinant protein. Gas6 recombinant protein (125 μg protein/kg) was administered intraperitoneally to 57-week-old mice three times every 24 h prior to bacterial infection. Survival was monitored daily (Experiment A). At 2 days post-infection, the mice were sacrificed, and the cecum and liver were harvested (Experiment B). (B) Effect of Gas6 recombinant protein on survival of 57-week-old mice infected with K. pneumoniae ATCC43816 pmCherry. Each mouse was orally inoculated with K. pneumoniae ATCC43816 pmCherry (5 × 107 bacteria). Each dot was represented from an individual mouse (n = 6 per group). p values were determined using the log-rank test. (C) Bacterial counts in cecal mucosa, liver, and cecal contents were determined 2 days post-infection. Each tissue and cecal contents were homogenized in PBS. The homogenates were plated on LB agar containing 400 μg/mL ampicillin and the number of CFU was counted. Each dot represents the value from an individual mouse (n = 5 or 6 per group). Data are presented as the mean ± SD. *p < 0.05, **p < 0.01. p values were calculated by one way analysis of variance. (D) Detection of F4/80 by western blotting. Cecal mucosa were collected, homogenized, and analyzed by western blotting with anti-F4/80 antibodies. Each lane of western blotting images represents the protein from an individual mouse (mouse number = 5 per group). Western blotting image represents three independent replicates. (E) Western blotting signal intensities were analyzed by ImageJ software. Each dot represents the protein sample collected from an individual mouse (mouse number = 5 per group). Data are presented as the mean ± SD. ** p < 0.01. p values were calculated by one way analysis of variance. (F) Sections of cecal mucosa were from mice aged 15-week, 57-week, or Gas6 administered 57-week-old mice at 2 days after infection with K. pneumoniae ATCC43816 pmCherry and immunostained with an anti-Gas6 antibody and an anti-Axl antibody. Each image has six independent replicates. Scale bar = 50 μm. (G) Fluorescence intensity was analyzed by ImageJ software. Each dot represents the value from an individual mouse (n = 6 per group). Data are presented as the mean ± SD. **p < 0.01. p values were calculated by one way analysis of variance. (H) Detection of Axl, Gas6, ZO-1, and occludin by western blotting. Cecal mucosa were collected, homogenized, and analyzed by western blotting with anti-Axl, anti-Gas6, anti-ZO-1, or anti-occludin antibodies. Each lane of western blotting images represents the protein from an individual mouse (mouse number = 5 per group). Western blotting image represents three independent replicates. (I) Western blotting signal intensities were analyzed by ImageJ software. Each dot represents the value collected from an individual mouse (mouse number = 5 per group). Data are presented as the mean ± SD. NS: not significant, *p < 0.05, **p < 0.01. p values were calculated by one way analysis of variance.
Supplier Page from Abcam for Mouse Axl ELISA Kit