Fig 1.
T5KO mice are protected from intestinal inflammation and associated metabolic syndrome when colonized with Altered Schaedler Flora.
WT and T5KO C57BL/6 animals, both males and females, were born from mice colonized with the Altered Schaedler Flora and maintained in isolators. At 12 weeks of age, animals were euthanized. A. Final body weight. B. Fat pad weight. C. 5 h fasting blood glucose concentration. D. Spleen weight. E. Colon weight. F. Colon length. G. Caecum weight. H. Liver weight. I. Fecal Lcn2 levels at week 4 and week 12 of age. J. Fecal lipopolysaccharide (LPS) levels at week 4 and week 12 of age. K. Fecal flagellin (FliC) levels at week 4 and week 12 of age. L-N. Colonic pro-inflammatory cytokine-encoding genes were quantified by qRT-PCR (L, IL-6; M, CXCL-1; N, Lcn2). O. Hematoxylin & eosin staining was performed on colonic sections and used for the determination of histopathological scores. Data are the means +/- S.E.M. (n = 3–6). Significance was determined using t-test (* indicates p<0.05).
Fig 2.
WT and T5KO mice exhibit similar ASF community structure.
WT and T5KO mice, both males and females, were born from ASF-colonized mice and maintained in isolators. Feces were collected at weeks 6, 8 and 10 of age. A. Total bacterial load. B. Clostridium sp. (ASF 356) relative values. C. Lactobacillus intestinalis (ASF 360) relative values. D. Lactobacillus murinus (ASF 361) relative values. E. Mucispirillum shaedleri (ASF 457) relative values. F. Eubacterium plexicaudatum (ASF 492) relative values. G. Pseudoflavonifractor sp. (ASF 500) relative values. H. Clostridium sp. (ASF 502) relative values. I. Parabacteroides goldsteinii (ASF 519) relative values. J-L Principal coordinates analysis of the microbiota composition using beta diversity analysis of the Euclidean distance at 6 (J), 8 (K) and 10 (L) weeks of age. n = 7–9. Significance was determined using two-way group ANOVA corrected for multiple comparisons with a Bonferroni test (# indicates statistical significance), or Permanova for clustering analysis.
Fig 3.
ASF community structure differs between T5KO and WT mice following AIEC colonization.
Four-week old offspring of ASF-colonized WT and T5KO mice were removed from the isolator, placed in isolated ventilated cages and inoculated with AIEC reference strain LF82 placed in drinking water for two weeks, followed by return to autoclaved water. Feces were collected at weeks 4, 6, 10 and 12 of age. A. Total bacterial load. B. E. coli relative values. C. Clostridium sp. (ASF 356) relative values. D. Lactobacillus intestinalis (ASF 360) relative values. E. Lactobacillus murinus (ASF 361) relative values. F. Mucispirillum shaedleri (ASF 457) relative values. G. Eubacterium plexicaudatum (ASF 492) relative values. H. Pseudoflavonifractor sp. (ASF 500) relative values. I. Clostridium sp. (ASF 502) relative values. J. Parabacteroides goldsteinii (ASF 519) relative values. K-M Principal coordinates analysis of the microbiota composition using beta diversity analysis of the Euclidean distance at 4 (J), 6 (K) and 10 (L) weeks of age. n = 8–13. Significance was determined using two-way group ANOVA corrected for multiple comparisons with a Bonferroni test (# indicates statistical significance), or Permanova for clustering analysis.
Fig 4.
AIEC LF82 colonization alters microbiota composition in T5KO, but not WT, ASF mice.
Four-week old offspring of ASF-colonized WT and T5KO mice were remove from the isolator, placed in isolated ventilated cages and inoculated with AIEC reference strain LF82 placed in drinking water for two weeks, followed by return to autoclaved water. Feces were collected at weeks 4, 6, 8, 10 and 12 of age. A. Total bacterial load. B. E. coli relative values. C. Clostridium sp. (ASF 356) relative values. D. Lactobacillus intestinalis (ASF 360) relative values. E. Lactobacillus murinus (ASF 361) relative values. F. Mucispirillum shaedleri (ASF 457) relative values. G. Eubacterium plexicaudatum (ASF 492) relative values. H. Pseudoflavonifractor sp. (ASF 500) relative values. I. Clostridium sp. (ASF 502) relative values. J. Parabacteroides goldsteinii (ASF 519) relative values. K-L Principal coordinates analysis of the microbiota composition using beta diversity analysis of the Euclidean distance in WT ASF (K) and T5KO ASF (L) animals infected or not with AIEC reference strain LF82. n = 8–13. Significance was determined using Permanova for clustering analysis.
Fig 5.
AIEC colonization induces modest increase in low-grade inflammation and adiposity in ASF-T5KO mice.
Four-week old offspring of ASF-colonized WT and T5KO mice were removed from the isolator, placed in isolated ventilated cages and inoculated with AIEC reference strain LF82 placed in drinking water for two weeks, followed by return to autoclaved water. At 12 weeks of age, animals were removed from this isolator and euthanized. A. Fecal Lcn2 levels at week 4 and week 10 of age. B. Fecal lipopolysaccharide (LPS) levels at week 4 and week 10 of age. C. Fecal flagellin (FliC) levels at week 4 and week 10 of age. D. Final body weight. E. Fat pad weight. F. 5 h fasting blood glucose concentration. G. Spleen weight. H. Colon weight. I. Colon length. J. Caecum weight. K. Liver weight. L-N. Colonic pro-inflammatory cytokine-encoding genes were quantified by qRT-PCR (L, IL-6; M, CXCL-1; N, Lcn2). O. Hematoxylin & eosin staining was performed on colonic sections and used for the determination of histopathological scores. Data are the means +/- S.E.M. (n = 3–6). Significance was determined using t-test (* indicates p<0.05). Data are the means +/- S.E.M. (n = 3–6). Data in A, B and C combine both male and female animals. Significance was determined using t-test (* indicates p<0.05).
Table 1.
Primers used in the study [27].
Flagellated bacteria were determined based on [30]. N.A., not applicable.