LDP treatment affected the early microbiota
Study 1 design (Figure 1A): Nine-week-old wild-type (WT) female and male C57BL/6 mice were randomly paired (1:1) and co-housed for 4 d (one estrous cycle for house mouse). After co-housing, females was separated from males and fed solely and pregnancies and due dates were speculated according to the body weight changes. Several days before birth, pups were exposed to LDP (10 mg/L, ~1.5 mg/kg body weight)3 through their mothers or drinking water until 30 d of age (LDP, F and P groups) or not (Ctr group). At 12d, 16d, 20d, 24d and 28d of age, the pups were gavaged with sterilized pre-reduced PBS (Ctr and LDP groups), and fecal microbiota (F group, pooled fecal samples from Ctr mice with a concentration ~ 0.3 g feces/mL PBS) or probiotics (P group, a bacterial mixture of Lactobacillus bulgaricus and L. rhamnosus GG (LGG) at ~108/mL for each bacterium). For 12 day and 16 day mice, 50 μL and 100 μL liquid were gavaged, respectively; for the rest, 150 μL was gavaged. Pups with a bodyweight near the average level were selected for experiments before the first gavage. The mice in each group came from at least three dams and housed in at least 2 cages (n = 2–3 per cage) to avoid the cage-effect. Pups were separated from their mothers at 21 d of age and female and male pups were housed together. At 30 d of age (2 d after the last gavage), mice were killed and bacterial and tissue samples were collected for analysis.
As female and male pups were housed together throughout the 30 day experiment in Study 1, their GM composition was analyzed together here. Quantitative PCR (qPCR) using 16S universal or ITS1 primers20 (Table S1) showed no significant differences in the bacterial counts or fungal loads of the intestinal samples among all groups, suggesting that the overall microbial loads were not influenced by LDP (Figures S1A and S1B). Nonetheless, significant differences were found in the bacterial compositions of the ileal mucosal (p < 0.01, Adonis test) and colonic luminal (P < 0.001,Adonis test) samples of the LDP and Ctr mice, as shown by weighted UniFrac distance measurements (Figures 1B and 1C). Notably, there was no significant difference between Ctr and F and P mice (Figures 1B and 1C), suggesting that both FMT and probiotics treatments efficiently restored the overall bacterial composition of these two intestinal regions. Similar trends were also found in the ileal lumen and colonic mucosa as shown by the difference of PC1 in Figures S1F to S1I.
We then analyzed the influence of LDP on OTUs based on Linear discriminant analysis Effect Size (LEfSe). LDP treatment mainly resulted in a reduction in Lactobacillus and Candidatus Arthromitus (segmented filamentous bacteria (SFB)) in all examined intestinal regions (Figures 1D and S1C–S1E). It is also worth noting that a more broad difference was observed between the ileal samples of the LDP and Ctr mice than that in colonic samples (Figures 1D and S1C–S1E).
Next, we evaluated the efficiency of FMT and probiotics on restoring these two bacterial taxa. Based on the 16S rDNA sequencing data, FMT efficiently restored the intestinal levels of both SFB and Lactobacillus, while the probiotics treatment, as expected, only restored Lactobacillus (Figures 1E and 1F). Moreover, three days after every gavage (i.e., one day before the next gavage) and at the end of (30 d) the study 1, the fecal and (or) intestinal levels of SFB and two treating probiotics (L. bulgaricus and LGG) were also determined by qPCR using species-specific primers (Table S1) and similar results were obtained as compared to the 16S rDNA sequencing data (Figure S1J). These results indicate that at least during the treatment period (from 12 d to 30 d of age), the intestinal SFB and (or) Lactobacillus levels were consistently affected by LDP, FMT and probiotic treatment.
In conclusion, LDP disturbed the intestinal bacterial composition, including a significant reduction in SFB and Lactobacillus, which was fully (for FMT) or partially (for probiotic) restored by several FMT and probiotic treatments.
LDP treatment persistently dampened intestinal IgA response
Early-life high-dose antibiotic treatment has been shown to transiently inhibit the intestinal IgA response in mice 17-18. As intestinal SIgA is closely related to the development of MetS 21, we then sought to determine the influence of early-life LDP on intestinal IgA response. There were no significant difference in the fecal SIgA among all 14 d and 21 d age of mice, suggesting that the passive SIgA received from their mothers via breast milk was not influenced by LDP (Figure S2A). Nonetheless, in both 30-d-old male and female pups, when mice were able to actively generate intestinal SIgA 6, LDP tended to decrease the SIgA levels in ileal, caecal and colonic contents, which was (partially) restored by FMT and probiotic (Figures S2B, S2C, S2E, and S2F). However, because the small number (n = 2) of female pups in Ctr group of Study 1, we were not able to obtain statistically significant comparison results.
To further verify as well as determine the long-term effects of LDP on intestinal IgA response, we performed mouse Study 2 (Figure 2A). In this study, all the LDP, FMT, and probiotic treatments during early life were the same to Study 1, but the experimental period was extended to 25 weeks. And as a western diet (45% energy from lard) has been shown to accelerate the metabolic effects of LDP 3, the diet of mice was changed to a western diet at 6 weeks of age. In addition, a high-dose penicillin [HDP, 1.5 g/L (according to the recommended dose of penicillin for children 22) in drinking water from 17 to 24 d of age] group was added to study the difference in intestinal IgA response between therapeutic and sub-therapeutic antibiotics. At 30 d of age, parts of pups were killed for the analysis of serum and intestinal (S)IgA and IgA-related lymphocytes. At 5, 10, 16, and 25 weeks of age, feces were collected to analyze the SIgA levels (Figure 2A).
Consistent to the previous studies 17-18, a decrease in fecal SIgA upon transient HDP treatment was found in 5- and 10-week-old mice (Figure 2B). This effect was disappeared, however, in 16- and 25-week-old mice (Figure 2B). Nonetheless, transient LDP reduced fecal SIgA throughout the 25-week experiment, showing a more persistent influence on SIgA than HDP (Figure 2A). Determination of caecal SIgA at 30 d and 25 weeks of age also obtained consistent results (Figure 2C). The reduction in intestinal SIgA upon LDP treatment was fully restored by FMT but only partially by probiotics (Figure 2A and 2C). In addition to the intestinal SIgA, a moderate reduction in serum IgA was observed in LDP and HDP mice at 30 d of age as compared to that in Ctr, F and P mice, which, however, was disappeared at 25 weeks of age (Figure S2D).
Determination of the ileal IgA-producing antibody-secreting cells obtained consistent results (Figures 2D–2G and S3A–3D). In 30-d-old females, LDP decreased the absolute numbers of IgA+ plasma cells (PCs, IgA+ B220-) within the distal small intestine lamina propira (SLP) and Peyer’s patches (PPs) (Figures 2D and 2F). A reduction in the numbers of IgA+ B cells (IgA+ B220+) was also found in SLP but not PPs (Figures 2D and 2F). The inhibition of IgA+ antibody-secreting cells (ASCs) within SLP (but not PPs) by LDP was also observed in 25-week-old mice, suggesting a persistent effect (Figures 2E and 2G). The inhibition of IgA-producing ASCs by LDP was also fully or partially restored by FMT and probiotics (Figures 2D–2E). With respect to LDP, a stronger inhibition of IgA+ ASCs within SLP and PPs by HDP was observed in 30-d-old females; however, this inhibition was transient and disappeared in 25-week-old females (Figures 2D–2G). Similar results were also obtained from males (Figures S3A–S3D).
We then sought to determine the possible cause(s) contributing to the differences in SIgA production between LDP and HDP. Previous studies have shown that high-dose antibiotic treatment around weaning persistently reduced the ileal regulatory T cells (Tregs) in mice 23, a subgroup of which, RORγ+ Tregs, have been shown to inhibit the generation of intestinal SIgA 24. Consistent to these results, we found that HDP reduced the SLP Tregs at both 30 d and 25 weeks of ages, while LDP had no significant influence on SLP Tregs (Figures S3E–S3L). Further determination of RORγ+ Tregs subgroup obtained similar results (Figures S3E–S3L). These results suggest that a reduction in Tregs, especially RORγ+ Tregs, may therefore counteract the long-term inhibition of HDP on SIgA production 24.
Conclusively, with respect to the relatively short-term inhibition of intestinal IgA response by HDP, transient LDP during early life persistently dampened intestinal IgA response and reduced IgA production, which could be lasted for at least for 25 weeks. Moreover, this LDP-induced effect on intestinal IgA response could be (partially) restored by several times of FMT or probiotic treatments.
The inhibition of intestinal IgA response by LDP was GM- and early life-dependent
As intestinal IgA response is greatly dependent on the colonization and composition of GM 25-26, we then sought to determine the correlation between LDP-induced changes in GM and inhibition of intestinal IgA response.
We firstly examined the in vitro IgA-inducing capabilities of the antigens derived from the feces of 21-d-old mice in Study 1 as previously described 7, 27. Briefly, antigens separated from the feces through centrifugation were normalized and co-cultured with ileum tissue samples (containing no visible PPs) obtained from 8-week-old SPF C57/BL6 mice to study the effects of these antigens on ileal IgA production. After 2 d of cultivation, the ileum tissue produced significantly more IgA when co-cultured with antigens derived from the feces of Ctr and F mice than from LDP mice (Figure 3A). A similar trend was evident for the antigens from the feces of P mice compared to the LDP mice but did not reach significance (P = 0.094). Moreover, the mRNA expression of the Jchain and IL-6 of the ileum tissue was also higher, while tumor necrosis factor (ligand) superfamily, member 13 (also known as APRIL) and 13b (also known as BAFF) were not influenced, when cultured with antigens derived from the feces of Ctr and F mice than those from the LDP mice (Figure 3B), suggesting that Ctr and F antigens may increase IgA production through enhancing the survival and activity of IgA+ PCs 19.
These results suggest that LDP inhibit the intestinal immune response to GM, RNA-sequencing of the ileum from 30-d-old mice in Study 1 confirmed these results. Several biological functions related to IgA production and immune response to microbes that were downregulated in the LDP mice compared to the Ctr, F, and P groups, including PI3K signaling in B lymphocytes, B cell receptor signaling, dendritic cell maturation, and Th1 and Th17 pathways (Figure 3D).
To further determine the correlation between LDP-induced changes in GM and IgA production, we transferred fecal microbiota from 30-d-old mice in Study 1 to even-aged male germ free (GF) mice, and measured the fecal SIgA levels in recipients. Three weeks post-transfer, LDP recipients (LDPR) exhibited significantly lower fecal SIgA levels compared to that of Ctr recipients (CtrR) and F recipients (FR), as well as the P recipients (PR) (Figure 3C). The difference in the fecal SIgA levels of the LDPR, CtrR, FR, and PR lasted 6 weeks after the transfer (Figure 2C). However, contrary to the donors, the fecal SIgA differences disappeared 12 weeks after the transfer (Figure 3C), suggesting that the time factor (early life) also play an important role in the prolonged influence of LDP-induced GM alteration on IgA responses. Moreover, no significant difference in serum IgA 12 weeks after transfer was observed among all recipients (Figure 3C).
Together, these results indicated that LDP-induced changes in GM mediated the inhibition of intestinal IgA response and that early life is critical for the long-lasting effects of LDP on intestinal IgA response.
LDP induced persistent changes in the ileal microbiota in a SIgA-dependent manner
As intestinal SIgA is a master controller of the GM, we then sought to determine the effects of LDP-induced decrease in SIgA on GM. To this end, we performed Study 3 (Figure 4A) using SIgA-deficient (Pigr-/-) mice generated by Pigr+/- females and males. The LDP, FMT and probiotic treatments were the same to Study 1 but the experimental period was extended to 25 weeks. All mice were transferred to a western diet (45% energy from lard) at 6 weeks of age and lasted to the end of the experiment to accelerate the effects of LDP 3. In addition, due to the insufficient number of male Pigr-/- pups, only female Pigr-/- pups were selected for the experiment at 10 days of age.
We firstly analyzed the fecal, serum, and caecal (S)IgA levels. Before weaning, no significant difference in fecal SIgA was observed between WT (Pigr+/+ and Pigr+/-) and Pigr-/- pups while higher fecal SIgA levels were found in WT pups after weaning (Figure S4A). In 25-week-old mice, there was no significant difference in serum IgA among all groups in both WT and Pigr-/- mice (Figure S4B). However, a moderate (P = .065) increase in serum IgA was found in Pigr-/- as compared to WT females (Figure S4D). In consistent to the results found in Study 1, LDP decreased the caecal SIgA levels in 25-week-old WT mice, which was (partially) restored by FMT and probiotics (Figure S4C). In addition, no significant difference in serum and fecal (S)IgA was found between the WT mice of two genotypes (Pigr+/+ and Pigr+/-) (Figure S4E).
We then analyzed the influence of LDP on GM in Study 3. No significant difference in fecal bacterial composition was observed between 21-d-old Pigr-/- mice and their WT littermates (Figure S4G), suggesting that the starting GM was not influenced by the genotypes. At 25 weeks of age, WT LDP females showed a significantly different bacterial composition in the ileal but not colonic mucosa compared to WT Ctr females (Figures 4B and S4H, Adonis test), which was in line with previous study showing a more closely interaction between SIgA and ileal microbiota 28. Similar difference was found for WT males but did not reach significance (P = 0.082) (Figure 4C, Adonis test). Moreover, FMT (moderately) restored this LDP-induced changes in ileal mucosal microbiota in WT mice (Figures 4B and 4C). In contrast to WT mice, no significant difference in the ileal bacterial composition were found among all groups in Pigr-/- mice (Figure 4D, Adonis test), suggesting that intestinal SIgA may mediate this long-term effects of LDP on ileal microbiota.
To further verify this assumption, we performed mouse Study 4 using B cell-deficient (μMT) mice (Figure 4A). Briefly, pan B cells (including PCs) isolated from the spleens, PPs, mesenteric lymph nodes and ileal and colonic lamina propria of 30 day LDP-treated (LDP, F, and P groups) or -free (Ctr) WT SPF mice were purified and adoptively transferred to even-aged μMT mice. After transfer, mice were housed 2–3/cages (2 cages for each group) to avoid the cage-effect. Twelve weeks after the transfer, recipients were killed for GM analysis. B cells were successfully colonized in the recipients, as considerable SIgA was detected in the caecal contents except for recipients received PBS treatment (Figure S4F). As expected, we found a moderate (P = 0.143, Adonis test) difference in the ileal microbiota between Ctr and LDP recipients while a significant difference was found between LDP and F recipients (Figure 4E, Adonis test). In addition, PBS recipients showed a significant difference in the ileal microbiota compared to all other B cell-received recipients (Figure 4E, Adonis test).
Collectively, we demonstrated that transient LDP during early life can induce persistent changes in the ileal microbiota in an intestinal SIgA-dependent manner, which could be (partially) restored by FMT and probiotics.
LDP-induced reduction of SIgA increased bacterial encroachment and adipose inflammation
Intestinal SIgA can bind to the microbial antigens, preventing bacterial encroachment and adhesion to intestinal epithelial cells (IECs) as well as the translocation of bacteria and their metabolites, such as lipopolysaccharides (LPS) and flagellin, therefore protecting hosts from inflammation 19. We then sought to investigate the influences of LDP-induced reduction in intestinal SIgA on bacterial encroachment and inflammation in 25-week-old mice from Study 3.
As expected, confocal microscopy, using mucus-preserving Carnoy fixation 29, showed that WT LDP females exhibited reduced distance between the bacteria and IECs, and showed exaggerated bacterial translocation into the in the distal ileum as compared to Ctr females, which was restored in FMT and P females (Figures 5A and 5B). An enhanced bacterial translocation was observed in Pigr-/- mice than WT mice while no significant difference was observed among all Pigr-/- mice (Figures S5C and S5D). In consistent to this, a decrease in IgA+ bacteria in ileum was observed in LDP females as compared to that in Ctr, F, and P females (Figures 5C and 5D). This compromised control of bacteria by SIgA was accompanied with an increase in serum LPS in WT but not Pigr-/- LDP females as compared to that in Ctr, F, and P females with identical genotypes (Figure 5E), while no significant difference in serum TNF-α and IL-6 was observed among all groups (Figures S5M and S5N).
We then determined the influence of LDP on adipose inflammation, a key factor involved in the development of insulin resistance 30. A significant increase in macrophage infiltration in visceral adipose tissue, an indicator of adipose inflammation, was found in WT LDP females compared to that in WT Ctr females, as determined by immunostaining of F4/80 (Figures 5F and 5G). Gene expression analysis using qPCR also demonstrated increases in the mRNA expression of TNF-α and IL-6 in the visceral adipose tissue of WT LDP females than that of WT Ctr, F, and P females (Figures S6E and S6F). This LDP-induced adipose inflammation was disappeared in Pigr-/- females (Figure S6).
With respect to WT females, similar influences of LDP and FMT treatments on bacterial encroachment and adipose inflammation were observed in WT males, while a moderate influence of probiotics was found (Figures S5 and S6). Collectively, transient exposure to LDP during early life caused increases in ileal bacterial encroachment and adipose inflammation in a SIgA-dependent manner, which could be (partially) counteracted by FMT and probiotic treatments.
Dysbiosis and bacterial encroachment enhanced the development of inflammation and MetS
Previous studies have shown that LDP can enhance the development of MetS, however, the mechanism(s) and the therapies were poorly determined 3, 16. Considering an essential role of intestinal SIgA in controlling the GM and MetS 21, and the above results showing that intestinal SIgA mediated the LDP-induced increases in inflammation, a key factor contributing to MetS, we speculated that LDP-induced decreases in intestinal SIgA may also mediated the enhancement of MetS. To verify these assumptions, we measured the parameters related to MetS, including body weight (BW), fat mass (rate), serum hormones and glucose, and insulin sensitivity, in 25-week-old WT and Pigr-/- mice in Study 3.
LDP enhanced the development of western diet-induced MetS in WT females, including increases in BW, fat and adipocyte tissues masses, adipocytes size, hepatic triglyceride, and serum glucose, insulin, and leptin levels (Figures 6A–6F, S7C and S7D). In consistent to the elevated fast serum glucose and insulin, glucose and insulin tolerance tests also showed impaired glucose tolerance and insulin sensitivity in LDP females as compared to that in Ctr females (Figures S7G and S7H). In addition, LDP decreased the serum levels of peptide YY (PYY), an GM-related anti-obesity peptide secreted by L cells in the gastrointestinal tract 31; while increased serum insulin-like growth factor-1 (IGF-1), a hormone secreted by the liver and intestine that can promote growth and preadipocyte proliferation and is also closely linked to obesity and the GM 32-33 (Figure 6G). Similar effects of LDP on the metabolism were also observed in WT males (Figures 6 and S7). Moreover, FMT and probiotic treatments counteracted the LDP-effect on the development of MetS to different degrees: a more profound influence was found upon FMT treatment compared to probiotics (Figures 6 and S7).
Lastly, in contrast to the WT mice, no significant difference in these parameters was found among all groups in their female Pigr-/- littermates, demonstrating an indispensible role of intestinal SIgA in mediating LDP-induced effects on the development of MetS (Figure S8).