Changes in intestinal gene expression and microbiota composition during late pregnancy are mouse strain dependent

www.nature.com/scientificreports OPEN Received: 4 December 2017 Accepted: 20 June 2018 Published: xx xx xxxx Changes in intestinal gene expression and microbiota composition during late pregnancy are mouse strain dependent Marlies Elderman1,2, Floor Hugenholtz1,3, Clara Belzer1,3, Mark Boekschoten1,4, Bart de Haan2, Paul de Vos1,2 & Marijke Faas2,5 Hormones and placental factors are thought to underlie the maternal immunological changes during pregnancy. However, as several intestinal microbiota are linked to immune modulations, we hypothesized that the intestinal microbiota are altered during pregnancy in favor of species associated with pregnancy associated immune modulations. We studied the fecal microbiota composition (MITchip) and intestinal and peripheral immune cells (microarray and flow cytometry) in pregnant and non-pregnant C57BL/6 and BALB/c mice. Pregnancy influenced intestinal microbiota diversity and composition, however in a mouse strain dependent way. Pregnant BALB/c mice had, among others, a relative higher abundance of Lactobacillus paracasei et rel., Roseburia intestinalis et rel. and Eubacterium hallii et rel., as compared to non-pregnant BALB/c mice, while the microbiota composition in B6 mice hardly changed during pregnancy. Additionally, intestinal immunological pathways were changed during pregnancy, however again in a mouse strain dependent way. Correlations between various bacteria and immunological genes were observed. Our data do support a role for the microbiome in changing immune responses in pregnancy. However, other factors are also involved, such as for instance changes in SCFA or changes in sensitivity to bacteria, since although immunological changes are observed in B6 mice, hardly any changes in microbiota were found in this strain. Follow up studies are needed to study the exact relationship between these parameters. Pregnancy is associated with immunological adaptations of the mother in order to tolerate and support the development of the semi-allogeneic fetus. Changes in both in the innate and the specific immune response are reported during pregnancy. A decrease in the T helper 1 (Th1)/T helper 2 (Th2) ratio1,2 and in natural killer (NK) cells, and an increase in regulatory T cell (Treg) numbers3,4 has been found during pregnancy. It is known that peripheral immune responses are influenced by the placenta by direct contact with fetal tissue in the placenta, due to circulation of the immune cells through the placenta, or due to contact with soluble products produced by the placenta, such as cytokines, micro-particles and exosomes5–7. However, recent studies showed that the intestinal microbiome changes during pregnancy, which may also underlie the immunological changes during pregnancy8. Modulation of immune cells can be induced by microbiota species present in the intestinal lumen9–12. Segmented filamentous bacteria for example showed to be able to induce the development of intestinal T helper 17 (Th17) cells13, while several bacteria species from the genus Clostridium have been shown to promote the accumulation of intestinal Tregs9,10. Additionally, Lactobacillus plantarum has been shown to enhance the number of peripheral Tregs12. One of the mechanisms by which bacteria can modulate immune cells is by producing short chain fatty acids (SCFA), such as butyrate, which has been shown to induce the differentiation of Tregs in the colon14. This is particularly true for Clostridium species from the clusters IV and XIVa9. However, whether 1 Top Institute Food and Nutrition, Wageningen, The Netherlands. 2Division of Medical Biology, department of Pathology and Medical Biology, University of Groningen, Groningen, The Netherlands. 3Laboratory of Microbiology, Wageningen University and Research, Wageningen, The Netherlands. 4Division of Human Nutrition, Wageningen University and Research, Wageningen, The Netherlands. 5Department of Obstetrics and Gynaecology, University of Groningen and University Medical Centre Groningen, Groningen, The Netherlands. Correspondence and requests for materials should be addressed to M.E. (email: ) SCIENTIFIC REPOrtS | (2018) 8:10001 | DOI:10.1038/s41598-018-28292-2 1 www.nature.com/scientificreports/ intestinal bacteria are also involved in changes in peripheral immune cells during pregnancy, and whether intestinal immune cells are involved in this, is unknown. In this study we investigated a possible relationship between microbiota and the immunological adaptations at the end of pregnancy in mice. To this end, we studied the effect of pregnancy on the microbiota composition and the effect of pregnancy on both intestinal immune cells and peripheral immune cells with a microarray (colon tissue) and flow cytometry (spleen and mesenteric lymph nodes (MLN)). We combined the transcriptomics data and the microbiota data and performed a bio-mathematical analysis, in order to find bacterial species, which may potentially be related to pregnancy induced differences in intestinal immunity. Non-pregnant mice served as control. We used two different mouse strains (C57BL/6 and BALB/c) that were shown to have different microbiota profiles and intestinal immunological responses15,16, to study the consequences of a different pre-pregnancy starting point in the intestinal microbiota-host interactions. Results Intestinal microbiota composition changed during pregnancy, but in a mouse strain dependent way. We determined the microbiota composition of non-pregnant and pregnant mice in both strains using the phylogenetic microarray, the mouse intestinal tract Chip (MITChip). We determined the richness (number of unique species) and Shannon diversity (calculation between richness and evenness) of the microbiota composition. The data was analyzed with a Two-way ANOVA (TWA), to assess if there was an interaction between pregnancy and strain on the microbiota composition in the colon. A Bonferroni post-test was performed when interaction was found, to determine whether the effect of pregnancy was the same in both mouse strains. Overall, pregnancy decreased both microbiota diversity and richness (TWA, p < 0.05) (Fig. 1A,B), but the ratio of Firmicutes/Bacteroidetes was not influenced by pregnancy (Fig. 1C). Redundancy analysis showed that the total variation in microbiota composition explained by the variables genotype and pregnancy is 26.8%. Pregnancy explained 12.5% of the variance in microbiota composition (Fig. 1D). However, pregnancy-induced differences in microbiota at species like level were mainly observed in BALB/c mice. In B6 mice, pregnancy did not affect the microbiota at species like level. Pregnant BALB/c mice had a significantly relative higher abundance of, among others, Allobaculum et rel., Lactobacillus salivarius et rel., Lactobacillus plantarum et rel., Unclassified Clostridiales XVI, Clostridium perfringens et rel., Lactobacillus paracasei et rel., and Roseburia intestinalis et rel., as compared to no (...truncated)