Figure 1.
Gut microbiota comparison of inoculated mice.
Principal Component Analysis (PCA) plot based on DGGE profiles of 16S rRNA gene PCR derived amplicons of feces samples collected from mice in the inoculation experiment at nine weeks of age. Germ-free (GF) mice were inoculated at one week of age (red balls) or at three weeks of age (yellow balls) with a bacteria suspension made from caecal content of specific pathogen-free (SPF) mice from Taconic Farms (blue balls illustrate different aliquots of the suspension). Their gut microbiota was compared with SPF mice raised in our barrier (green balls). ANOVA based on Principal Component (PC) 1 explaining 14.7% of the variance confirmed a significant difference between mice inoculated at three weeks of age and all other mice (p<0.01). No significant differences of PC values were found between ex-GF mice inoculated at three weeks of age and the suspension aliquots used to inoculate these mice with. DGGE: Denaturing Gradient Gel Electrophoresis.
Figure 2.
Gut microbiota comparison of co-housed mice.
Principal Component Analysis (PCA) plot based on DGGE profiles of 16S rRNA gene PCR derived amplicons of feces samples collected from mice in the co-housing experiment at nine weeks of age. Germ-free (GF) mice were conventionalized at one week of age (red balls) or at three weeks of age (yellow balls) by co-housing them with specific pathogen-free (SPF) mice (green balls). No differences in gut microbiota composition were detected between the SPF and co-housed ex-GF mice. DGGE: Denaturing Gradient Gel Electrophoresis.
Figure 3.
Microbial inoculation at three weeks of age results in pro-inflammatory tuning of the immune system.
Cytokines were measured in mesenteric lymph node (MLN) cell culture supernatants and analyzed by use of FlowCytomix Multiplex Th1/Th2 10plex. MLN were isolated from specific pathogen-free (SPF) mice (SPF), germ-free (GF) mice (GF) and ex-GF mice inoculated with a caecal microbiota suspension at one (1W) or three weeks (3W) of age. The lymphocytes were stimulated for 24 hrs with 1 µg/mL lipopolysaccharide (LPS) from Escherichia coli O26:B6 before the supernatants were used for analysis. Only cytokines with a significant difference among groups are illustrated. Error bars represent the SEM. * represents p<0.05 compared with SPF mice.
Figure 4.
A germ-free postnatal period affects long-term immune-regulatory homeostasis.
Percentages of regulatory T cells (CD4+FoxP3+) and tolerogenic dendritic cells (CD103+CD11c+) in cells isolated from spleen (SPL) and mesenteric lymph node (MLN) were determined by flow cytometry. Specific Pathogen Free mice (SPF), germ-free mice (GF) and ex-GF mice inoculated with a caecal microbiota suspension or co-housed with SPF mice at one (1W) or three weeks (3W) of age are illustrated. Error bars represent the SEM. * represents p<0.05, ** <0.01, *** <0.001 compared with SPF mice.
Figure 5.
A germ-free postnatal period increases serum regulatory cytokines.
Analyses of serum cytokines were performed by ELISA. Serum was extracted from Specific Pathogen Free (SPF) mice, germ-free (GF) mice and ex-GF mice co-housed at one (1W) or three weeks (3W) of age with SPF mice. Error bars represent the SEM. * represents p<0.05 compared with SPF mice.
Figure 6.
A germ-free postnatal period alters systemic mononuclear cell populations.
Distribution of cell populations in spleen (A) and mesenteric lymph node (B) sampled from mice in the co-housing experiment were measured by flow cytometry. Germ-free (GF) mice were conventionalized at one week of age (1W) or at three weeks of age (3W) by co-housing them with Specific Pathogen Free mice (SPF) raised in our barrier. A group of mice remained germ-free (GF). Percentages of NK cells (CD3−CD49b+), NKT cells (CD3+CD49b+), CD69+ and CD103+ T cells (CD3+CD4+) and IFN-γ producing T cells (CD3+CD4+) are illustrated. Lymphocytes were cultivated for 4 hrs with 50 ng/mL PMA and 750 ng/mL Ionomycin (Sigma-Aldrich) in the presence of BD GolgiStop before cells were harvested and intracellular IFN-γ stained. The mean is illustrated. * represents p<0.05, ** <0.01, *** <0.001 compared with SPF mice. PMA: Phorbol 12-Myristate 13-Acetate.