Cesarean Section Induces Microbiota-Regulated Immune Disturbances in C57BL/6 Mice.

Epidemiological studies have shown that children born by cesarean section (CS) are at higher risk of developing chronic inflammatory diseases, and it has been suggested that a skewed gut microbial colonization process early in life and altered priming of the immune system are causative. The aim of this study was to clarify whether impaired regulatory immunity in CS-delivered C57BL/6 mice is dependent on gut microbiota (GM) disturbances. The GM of conventionally bred mice born by CS differed clearly from mice born by vaginal delivery. The proportion of regulatory T cells was reduced in mice born by CS, whereas the invariant NKT (iNKT) cell subset was increased compared with vaginal delivery mice. In addition, regulatory markers (Foxp3, Il10, Ctla4) and macrophage markers (Cd11c, Egr2, Nos2) were downregulated, whereas iNKT markers (Il4, Il15) were upregulated in ileum of CS-delivered mice. The GM of CS-delivered mice was sufficient to transfer the shifts in immunity associated with delivery mode when inoculated into germ-free mice. Feeding a prebiotic diet reestablished gene expression of intestinal immune markers and iNKT cells in CS mice but was not sufficient to restore the level of regulatory T cells. The results support that CS delivery is associated with microbiota-mediated shifts in regulatory immunity and, therefore, provide a basis for future microbiota-directed therapeutics to infants born by CS.

Low-gainer diet-induced obese microbiota transplanted mice exhibit increased fighting.

Weight gain variation is a great challenge in diet-induced obesity studies since low-gainer animals are of limited experimental value. The inbred C57BL/6 (B6) mice are frequently used models due to their genetic homogeneity and susceptibility to diet-induced obesity (DIO). The aim of this study is to investigate if the gut microbiota (GM) influences the fraction of low weight gainers in DIO studies. A total of 100 male B6 mice (donor population) were fed a high-fat diet for 14 weeks and divided into the study groups high gainer (HG) and low gainer (LG) based on their weight gain. Subsequently, fecal matter transplantation (FMT) was done on germ-free B6 mice with GM from HG and LG donors (FMT population). LG (13.35 ± 2.5 g) and HG (25.52 ± 2.0 g) animals were identified by the weight gain from week 1 to week 12. Interestingly, the start weight of the LG (20.36 ± 1.4 g) and HG (21.59 ± 0.7 g) groups differed significantly. Transplanting LG or HG fecal matter to germ-free mice resulted in significant differences in weight gain between HG and LG, as well as differences in serum leptin levels and epididymal fat pad weight. A clear LG-specific GM composition could not be distinguished by 16S rRNA gene amplicon sequencing. Surprisingly, significantly more fighting was recorded in LG groups of both donor populations and when transplanted to germ-free mice. The HG and LG phenotypes could be transferred to germ-free mice. The increased fighting in the LG group in both studies suggests not only that the tendency to fight can be transferred by FMT in these mice, but also that fighting should be prevented in DIO studies to minimize the number of LG animals.

Cesarean section induced dysbiosis promotes type 2 immunity but not oxazolone-induced dermatitis in mice.

Delivery by cesarean section (CS) is associated with an altered gut microbiota (GM) colonization and a higher risk of later chronic inflammatory diseases. Studies investigating the association between CS and atopic dermatitis (AD) are contradictive and often biased by confounding factors. The aim of this study was therefore to provide experimental evidence for the association between CS and AD in a mouse model and clarify the role of the GM changes associated with CS. It was hypothesized that CS-delivered mice, and human CS-GM transplanted mice develop severe dermatitis due to early dysbiosis. BALB/c mice delivered by CS or vaginally (VD) as well as BALB/c mice transplanted with GM from CS or VD human donors were challenged with oxazolone on the ear. The severity of dermatitis was evaluated by ear thickness and clinical and histopathological assessment which were similar between all groups. The immune response was assessed by serum IgE concentration, local cytokine response, and presence of immune cells in the draining lymph node. Both CS-delivered mice and mice inoculated with human CS-GM had a higher IgE concentration. A higher proportion of Th2 cells were also found in the CS-GM inoculated mice, but no differences were seen in the cytokine levels in the affected ears. In support of the experimental findings, a human cohort analysis from where the GM samples were obtained found that delivery mode did not affect the children's risk of developing AD. In conclusion, CS-GM enhanced a Th2 biased immune response, but had no effect on oxazolone-induced dermatitis in mice.

Effects of delivery mode on behavior in mouse offspring.

Cesarean section (CS) has been associated with an increased risk of mental disorders in the offspring. This could possibly be explained by an inadequate microbial colonization early in life with a consequential disturbed gut-brain interaction. To investigate the link between delivery mode and behavior and develop a suitable animal model for further research of the gut-brain axis, the aim of this study was to characterize the gut microbiota (GM) together with the behavioral response in various behavioral tests in CS-delivered mice. We hypothesized that mice delivered by CS would present with disturbances in normal physiological behavior possibly due to an inadequate microbial colonization. C57BL/6 mice delivered by CS or vaginal delivery (VD) were cross fostered and, as adults, observed for anxiety-related behavior in the open field test, social deficits in a sociability test and compulsive behavior in the marble burying test. GM was analyzed by 16S rRNA gene amplicon sequencing. The open field test showed that CS-delivered mice had a decreased activity and accelerated defecation compared to VD-delivered mice. In addition, CS-delivered female mice spend less time interacting with cage mates in the sociability test, whereas there was no effect of CS delivery on the average number of marbles buried. In conclusion, CS-delivered mice had a more pronounced anxiety-like behavior and showed less preference for sociability in female offspring.

Cesarean section increases sensitivity to oxazolone-induced colitis in C57BL/6 mice.

Children born by cesarean section (CS) have an increased risk of developing inflammatory bowel disease (IBD), possibly due to skewed microbial colonization during birth and consequently impaired bacterial stimulation of the developing immune system. The aim of this study was to investigate the association between CS and experimental colitis in a murine model of IBD. It was hypothesized that CS aggravates colonic inflammation due to a change in gut microbiota (GM) composition. C57BL/6 mice, delivered by CS or vaginal delivery (VD), were intra-rectally challenged with oxazolone at 8 weeks of age and monitored for colitis symptoms. The results showed that CS delivered mice experienced an increased body weight loss and colon weight, together with higher colonic concentrations of TNF-α and MPO compared with VD mice. Increased infiltration of inflammatory cells was present in CS delivered mice, as well as a downregulation in expression of the gut integrity genes occludin and tight junction protein 1 indicative of an impaired barrier function. The GM from CS delivered mice without colitis partly contributed to the increase in colitis symptoms when inoculated into germ-free recipient mice. In conclusion, CS increased sensitivity to oxazolone induced colitis in mice.

TL1A Aggravates Cytokine-Induced Acute Gut Inflammation and Potentiates Infiltration of Intraepithelial Natural Killer Cells in Mice.

BACKGROUND: The tumor necrosis factor alpha (TNFα)-homologous cytokine TL1A is emerging as a major player in intestinal inflammation. From in vitro experiments on human lymphocytes, TNF-like molecule 1A (TL1A) is known to activate a highly inflammatory lymphoid response in synergy with interleukin (IL)-12 and IL-18. Carriers of specific genetic polymorphisms associated with IL-12, IL-18, or TL1A signaling have increased Crohn's disease risk, and all 3 cytokines are upregulated during active disease. The study aim was to investigate whether the type 1-polarizing cytokines IL-12 and IL-18 could directly initiate intestinal pathology in mice and how TL1A would influence the resulting inflammatory response. METHODS: Conventional barrier-bred and germ-free mice were randomly allocated to different groups and injected twice with different combinations of IL-12, IL-18, and TL1A, and killed 3 days after the first injection. All treatment groups were co-housed and fed a piroxicam-supplemented chow diet. RESULTS: Intestinal pathology was evident in IL-12- and IL-18-treated mice and highly exacerbated by TL1A in both the colon and ileum. The cytokine-induced intestinal inflammation was characterized by epithelial damage, increased colonic levels of TNFα, IL-1ß, IFN-γ, and IL-6, and various chemokines along with gut microbiota alterations exhibiting high abundance of Enterobacteriaceae. Furthermore, the inflamed ileum and colon exhibited a TL1A-specific increased infiltration of intraepithelial natural killer cells co-expressing NKG2D and IL-18Ra and a higher frequency of unconventional T cells in the colonic epithelium. Upon cytokine injection, germ-free mice exhibited similar intraepithelial lymphoid infiltration and increased colonic levels of IFNγ and TNFα. CONCLUSIONS: This study demonstrates that TL1A aggravates IL-12- and IL-18-induced intestinal inflammation in the presence and absence of microbiota.

Sensitivity to oxazolone induced dermatitis is transferable with gut microbiota in mice.

Atopic Dermatitis (AD) has been associated with gut microbiota (GM) dysbiosis in humans, indicating a causative role of GM in AD etiology. Furthermore, the GM strongly correlates to essential disease parameters in the well-known oxazolone-induced mouse model of AD. Here, we demonstrate that it is possible to transfer both a high-responding and a low-responding AD phenotype with GM from conventional mice to germ-free mice. The mice inoculated with the high-responding GM had significantly higher clinical score, increased ear thickness, and increased levels of IL-1ß, TNFα, IL-4, IL-5, and IL-6 compared to the mice inoculated with the low-responding GM. The inter-individual variation was in general not affected by this increase in effect size. Germ-free mice induced with AD revealed a high disease response as well as high inter-individual variation indicating protective properties of certain microbial taxa in this model. This study underlines that the GM has a strong impact on AD in mouse models, and that the power of studies may be increased by the application of mice inoculated with a specific GM from high responders to increase the effect size.