Low-dose penicillin exposure in early life decreases Th17 and the susceptibility to DSS colitis in mice through gut microbiota modification

Antibiotic exposure in early life can lead to a significant change of the gut microbiota and may contribute to later onset of inflammatory bowel disease (IBD). However, the relationship between early-life antibiotic treatment and IBD is ambiguous, according to contradicting results of epidemiologic studies. In the present study, we demonstrated that low-dose penicillin pre-treatment had a unique protective effect against mouse colitis induced by dextran sodium sulfate (DSS). Low-dose penicillin also suppressed the expression of pro-inflammatory cytokine IL-17 in various intestinal tissues, and decreased the amount of Th17 cells in small-intestine lamina propria. Neither metronidazole nor enrofloxacin had a similar effect. We further confirmed that low-dose penicillin could cause specific changes of the gut microbiota, especially the eradication of segmented filamentous bacteria (SFB). Mice without SFB inoculation showed no disparity when treated with penicillin or water. Taken together, the results showed that low-dose penicillin can achieve a highly specific manipulation of sensitive bacteria and interfere with development of intestinal immune system in early life. The study may further indicate the possibility of achieving a favorable immune state among a certain group of patients with IBD, or other autoimmune diseases, by fine-tuning the gut microbiota.


Low-dose penicillin treatment suppresses Il17 expression and ileal Th17 differentiation. Help
T (Th) cells play an important role in orchestrating inflammation in both chemically induced colitis and human IBD. The protective role of penicillin pre-treatment might be due to an immune fingerprint on T cell differentiation. So we next measured the gene expression of key cytokines of different Th cells in terminal ileum tissues, mesenteric lymph nodes (MLNs) and Peyer's patches (PPs). Several cytokines were expressed differently under treatments, but penicillin decreased Il17a mRNA level in all three tissues consistently while metronidazole and enrofloxacin didn't ( Fig. 2a). Il17f expression in terminal ileum was also down-regulated by penicillin ( Supplementary Fig. S1). IL-17 is a group of pro-inflammatory cytokines implicated in host defensive against infection. They are mainly produced by Th17 cells and these cells mostly differentiate in small-intestinal lamina propia (SI-LP) 16,17 . So we next examined if differentiation of SI-LP Th17 cells was interfered by penicillin. It turned out there were fewer Th17 cells residing in SI-LP after 2-week penicillin treatment ( Supplementary Fig. S2) and the disparity still existed one week after penicillin cessation (Fig. 2d). As CD4+ Th cells and Th1 cells remained rather intact (Fig. 2b,c and f), the lack of Th17 cells is not due to an over-all suppression of T cells but a specific inhibition.
Low-dose penicillin imposed transient and small changes to diversity and structure of fecal bacteria community. In order to understand the gut microbial profile under low-dose antibiotic pressure, we applied 16S rRNA gene analysis of fecal bacteria from mice treated with regular water, low-dose penicillin or metronidazole, the latter two with disparate inflammation phenotypes and Il17 expression. High-throughput sequencing produced 1842370 valid reads (with 48483 ± 14751 per sample) from 38 samples collected respectively at baseline, 2 weeks after antibiotic treatment and 1 week after treatment cessation (Fig. 3a). The reads were delineated into 688 operational taxonomy units (OTUs) at the similarity cut-off of 97%. The Good's coverage index is 0.998 ± 0.0001, indicating an adequate sequencing depth, which is further proved by rarefaction analysis (Supplementary Fig. S3).
Estimators of alpha-diversity were calculated after rarefying the sequence depth to 19345 reads per sample, in order to avoid bias ( Supplementary Fig. S3). According to Chao1 and Shannon index, both low-dose penicillin and metronidazole decreased bacterial richness in 2 weeks, while penicillin on its own increased bacterial diversity (Fig. 3b), which was similar to a previous study 12 . The impact of both antibiotics on alpha-diversity was transient, as the difference became insignificant one week later (Fig. 3c).
In order to preserve data from rare species, which may play important roles in gut homeostasis 18 , we used unrarefied sequences for other analysis in our study. Principle component analysis (PCA) revealed low-dose penicillin caused unique structural changes of fecal microbiota during and after treatment, as was shown by clustering of samples in plots (Fig. 3d,e). However, Venn diagrams showed the majority of OUTs were preserved and shared among groups under low-dose antibiotic treatment (Fig. 3f-h). The fecal microbiota were mainly composed of Bacteroidetes and Firmicutes in all groups, followed by far less abundant Proteobacteria, Candidate division TM7 and Actinobacteria (Fig. 3i). On genus level, low-dose treatment only caused small shifts of microbiota composition (Fig. 3j), compared with obvious dysbiosis induced by large-dose antibiotics in previous reports 8,9 .
Scientific RePoRtS | 7:43662 | DOI: 10.1038/srep43662 Key bacterial alterations under low-dose penicillin treatment. In order to clarify which bacteria might be responsible for low-dose penicillin's unique impact on intestinal inflammation, we conducted the LEfSe analysis for microbial biomarker discovery. By testing both the consistency and effect size of difference in bacteria abundance between groups, LEfSe suggests key bacteria at different taxonomy levels 19 . According to our experimental results, we combined control with metronidazole group into a super-group (each as a subgroup), as was opposed to penicillin group. According to the results, the key alteration caused by 2-week low-dose penicillin treatment was a decrease of Enterohadus, Lactobacillus, streptococcus, Candidatus Arthromitus, Allobaculum, and Turicibacter; and an increase in Bacteroides, staphylococcus, Marvinbryantia, Roseburia, Peptococcus, Oscillibacter, Escherichia-Shigella and Stenotrophomonas. At phylum level, low-dose penicillin treated mice featured a decrease of Firmicutes and an increase of Bacteroidetes and Gammaproteobacteria (Fig. 4a-c). One week after penicillin cessation, the disparity between two groups became less prominent, as was indicated by a smaller number of biomarkers at all levels. The key alterations in penicillin treated mice included a decrease in Prevotella, Gemella, Ruminococcaceae, Candidatus Arthromitus, Allobaculum, Turicibacter and Erysipelotrichaceae; and an increase in Gordonibacter, Alistipes, Rikenella, Streptococcus and Marvinbryantia ( Supplementary Fig. S4).
Some of the key bacteria are potentially associated with IBD, such as Prevotella [20][21][22] , Gemella 20 and Erysipelotrichaceae 23 . Among them, Candidatus Arthromitus, or segmented filamentous bacterium (SFB), has been known for its special immune-stimulating capacity for years 24 . Due to the limitation of culturing technique, we conducted PCR to investigate the bacteria's distribution among mice. Most mice upon purchase were SFB-positive, while some were SFB-free. After one day's treatment of penicillin, the load of SFB dropped dramatically (Fig. 4d). We also sought to define the minimum dose of penicillin for SFB eradication and found as little as 0.1 μ g/d · g bodyweight of penicillin (equals to 0.6 mg/L penicillin in drinking water) had the same effect with higher dosages (Fig. 4e). Penicillin's suppression of Il17 expression and Th17 differentiation is SFB dependent. According to previous studies, adhesive mouse-specific SFB plays a crucial role in Th17 differentiation 16,17,25,26 . So we sought to clarify if eradication of SFB is responsible for penicillin's effect. SFB's colonization occurs at weaning, and declines in an age-dependent manner 27 (see also Supplementary Fig. S4). At week 9, all mice became free of SFB 27 . We then treated 9-week-old mice with low-dose penicillin, and found no difference in SI-LP Th17 count between penicillin and control groups (Fig. 5a,b). In another group of SFB-free weanling mice, penicillin caused a much less and insignificant drop of Il17a expression (Fig. 5c). To sum up, the decrease of Th17 only occurred in SFB-positive mice. Moreover, ileal Serum amyloid A (SAA), which is responsible for SFB's immune-stimulating capacity 28,29 , was also suppressed by penicillin ( Supplementary Fig. S5).
However, there is still a possibility that penicillin's effect partly relies on the enriched taxa after treatment, apart from the suppressed ones. To test this, we transplanted bacteria from caecal content of antibiotic treated mice, to 3-week SFB-positive weanling mice through gavage. The recipient mice failed to show a trend of Il17a suppression (Fig. 5d). This further strengthened the idea that it was not the enriched genera, but the decreased genera, especially SFB, which were responsible for the penicillin-induced IL-17 inhibition. This is in consistency with a recent theory 10 .
We also induced DSS colitis in a group of SFB-free mice. This time, the weight variance and histology showed similar degrees of inflammation among different groups ( Fig. 5e-g).

Discussion
In the present study, we examined the impact of low-dose antibiotic pre-treatment on intestinal inflammation with a mouse model. Among the tested antibiotics, penicillin yielded an unexpected protective effect against DSS colitis. A 2-week penicillin treatment led to a prominent drop of Il17a gene expression in intestinal immune tissues and a decrease of Th17 cells in SI-LP. We also found the effect of low-dose penicillin was associated with the eradication of commensal bacteria SFB, which is highly sensitive to this antibiotic.
Penicillin was the first antibiotic isolated and used against bacterial infection. The bacteriocidal effect depends on the inhibition of DD-transpeptidases by β -lactam rings, which causes a disbalance between building and breaking down of bacterial cell walls. Penicillin and other β -lactams are among the top antibiotics consumed worldwide [30][31][32] . A recent meta-analysis pooling risk-factor studies of IBD in western countries concluded that penicillin is the only antibiotic that wasn't associated with IBD onset, while fluoroquinolones and metronidazole were most strongly related with IBD 3 . In our study, a 2-week pre-treatment of enrofloxacin or metronidazole failed to aggravate later DSS colitis, while penicillin turned out to be protective. This further indicates the complexity of the association between antibiotics and IBD pathogenesis. Some basic science researches have revealed penicillin's extra-anti-infective effects on hosts through the modulation of gut microbiota. Zhang et al. reported that penicillin therapy could inhibit neutrophil aging and further alleviate inflammation-related tissue damage of sickle cell disease or endotoxin-induced septic shock 33   risk factors of IBD can also be triggers of gut microbiota alterations 35 . There are several specific microorganisms considered as possible causative agents of the disease, such as Mycobacterium avium subspecies paratuberculosis (MAP), and Escherichia coli 36 . With the help of low antibiotic dose, which didn't cause severe dysbiosis and suppressed only a few genera, we were able to define a group of key bacteria and highlight the importance of SFB. SFB, or Candidatus Arthromitus as the genus name, is a commensal bacterium mainly inhabits the small intestine, forming attachment sites on epithelia cells 37 . SFB genome sequencing revealed a lack of ability of biosynthesis, indicating that its life style is highly dependent on host cells 38 . SFB had been unculturable, until recently, a team developed an SFB-host cell co-culturing system 26 . Previous studies with electron microscopes found prominent morphology changes of SFB within 3-5 hours after the hosts were given a large dose of penicillin, inferring its sensitivity to penicillin 39 . We think the high demand for cell wall construction for numerous intracellular septa during a rapid life cycle is the reason why SFB could be eradicated with such a low dose of penicillin in our experiments 26 . Low-dose metronidazole, while decreasing SFB load, was not able to achieve a complete wipe-out. This makes penicillin a specific and ideal tool for SFB manipulation.
The importance of SFB lies on its unique ability to elicit physiological inflammation. When adhering to epithelial cells, SFB is able to promote Th17 differentiation, IgA secretion and antimicrobial peptide production 26 . Though commonly detected in various young animals as a commensal 27 , SFB is reported to exacerbate autoimmune encephalitis and arthritis in mouse model 40,41 . In the case of intestinal inflammation, SFB could induce colitis in SCID mice transferred with CD4 + CD45RB high T cells, when co-colonizing with a cocktail of other bacteria 42 . SFB was also found highly coated with IgA, which turned out as a marker of colitogenic microbes 23 . Moreover, though not usually found in grown-up animals or adult human, there are case reports providing evidence that SFB could be detected at certain inflammatory sites both in UC and CD patients 43,44 . Our finding reinforces the notion that SFB, though not able to cause intestinal inflammation on its own, could be part of the pathogenesis 42 .
SFB's pro-inflammatory potential was mainly attributed to its ability to inducing IL-17-producing cells, including Th17. Similar to a previous report 12 , we found down-regulation of Th17 cells in SI-LP by low-dose penicillin. Th17 cells are an important part of the immune system. Researchers have discovered an enrichment of balancing selection in genes related to IL-17 production 45 . This implies a need for balance between microbial defense and inflammation during evolution 45 . Under normal condition, Th17 cell can promote neutrophil function and antibacterial peptide production, but they may also trigger autoimmune diseases including IBD, rheumatoid arthritis, psoriasis and multiple sclerosis 46 . Several genes in IL-17/IL-23 pathway have been observed as IBD risk loci, including IL-23R, IL-22, RORC, IL-21, Stat3 and AHR 34,47 . SFB is recently reported to induce Th17 differentiation through activation of an IL-23R/IL-22 circuit 28 . Thus, SFB and IL-17/Th17 are closely associated with IBD pathogenesis. However, whether the alleviation of DSS colitis was caused by Th17 inhibition is still to be elucidated. IL-17, though usually viewed as a pro-inflammatory cytokine, plays a rather complicated role in human IBD as well as experimental colitis. While the dual blockade of IL-17A and IL-17F attenuated intestinal inflammation 48 . Th17-deficient mice presented even more severe DSS colitis than wild type ones 49 . While antibodies targeting IL-17A proved ineffective in treating CD patients 50 , some small-molecule inhibitors have shown efficacy in clinical trials 51,52 . Overall, there is a complex and delicate balance between the protective and pathogenic roles of IL-17 and Th17 cells. Targeting IL-17 during a more acute phase of intestinal inflammation, or even as a prophylactic therapy ahead of disease flare, could be a more rational approach 53 . Here in the present study, why did the inhibition of IL-17 yield anti-inflammatory results? We put forward several explanations. First of all, penicillin achieved a dual suppression of Il17a and Il17f expression, the latter as a more pro-inflammatory cytokine. Secondly, penicillin-treated mice were not deprived of all Th17 cells, which may preserve more protective capacity in compare with the case of IL-17 knock-out mice. Thirdly, the pre-treatment had put mice in a low-Th17 state before DSS colitis, mimicking a prophylactic therapy. DSS colitis is featured with massive neutrophil infiltration in intestinal tissue. Th17 cells and neutrophils can reciprocally recruit each other in inflamed sites, forming a vicious cycle 54 . When Th17 cell differentiation is inhibited by penicillin, the vicious cycle can be less strong. Overall, we suggest that the down regulation of IL-17 and Th17 could be responsible for penicillin's protective effects through eradication of SFB.
There are several limitations in our research. We failed to conduct further experiments with other bacteria that also had biological importance according to LEfSe analysis, especially Turicibacter and Allobaculum, of which behavior was highly similar to SFB under penicillin pressure. Secondly, we used conventional mice to achieve a more real-life results with a more diverse microbiome, yet at the possible risk of inter-study variations. Further researches with germ-free or gnotobiotic mice together with new culturing methods for key bacteria are needed for better control of the composition of gut microbiota. Thirdly, we applied DSS colitis model in the study, while chemically induced inflammation cannot fully recapitulate human IBD. However, there is another report in which the authors directly examined the colitogenic effect of SFB-containing bacterial cocktails with a T-cell transferred colitis model 42 . Interestingly, they found that SFB together with a cocktail of bacteria were effective to trigger intestinal inflammation, which is very supportive of our conclusion. Genetically modified mice should be used in future to elucidate the compound effects of SFB and IBD risk genes especially in IL-17/IL-23 pathway.
In conclusion, through a highly specific manipulation of sensitive bacteria, a short-term treatment of low-dose penicillin has shown its potential to interfere with development of intestinal immune system in early life. It may partly answer the epidemiologic question why different antibiotic exposure imposed distinct risk on IBD onset. Our study may further indicate the possibility of achieving a favorable immune state among a certain group of patients with IBD, or other autoimmune diseases, by fine-tuning the gut microbiota.

Methods
Animals and antibiotic treatment. Three-week-old C57BL/6 mice were purchased from SLAC laboratory (Shanghai, China) and maintained under conventional barrier conditions with a 12-hour light/dark cycle in Shanghai Jiao-Tong University, School of agriculture and biology, or the experimental animal centre of Tongji University. All cages and water were autoclaved prior to use, with bedding and chow irradiated. Low dose antibiotics were supplied in drinking water ad libitum at the concentration of 6.67 mg/L, which equals to 1 μ g/g bodyweight according to previous reports 12,55 . Antibiotic intervention began on the next day of animal receipt and lasted for 2 weeks. In order to maintain the concentration of antibiotics against degradation, penicillin and metronidazole were renewed every day, with enrofloxacin every three days [56][57][58] . All animal experimentations were approved by the scientific research ethical committee of Renji Hospital affiliated to Shanghai Jiao-Tong University. Procedures involving animals and their care were conducted following NIH guide for the care and use of laboratory animals (eighth edition).
Induction of colitis and determination of clinical scores. Acute colitis was induced with 2.5% (w/v) DSS (molecular weight 36000-50000, MP Biomedicals, US) dissolved in drinking water for 7 days. As described in a protocol, newly-made DSS solution was given on day 0, day 2 and day 4, followed by regular water on day 7 until termination of the experiment on day 8-10 59 . DSS solution was always given 1 week after antibiotic cessation, in order to exclude the direct effect of antibiotic residue in animals. Body weight was determined every day, as well as stool consistency and rectal bleeding state. Occult blood was assessed with a urine-fecal occult blood test kit. An established scoring system for intestinal bleeding was used as an index of disease activity 59 . Hemotoxylin and eoxin (HE) staining and histologic analysis. Following euthanasia on day 9, 0.5 cm of distal colons were dissected, washed in saline and fixed in 4% paraformalin for 24 h at 4 °C. The tissues were then embedded in paraffin and sectioned to 4 μ m thickness. HE staining was performed by an autostaining machine following standard protocols, while images acquired by an Olympus BX43 microscope. Based on a previously described scoring system, each tissue was assigned with four scores for epithelial damage, inflammatory infiltration of mucosa, submucosa and muscularis/serosa. The scores were multiplied with an index according to the extent of tissue damage 60 .

Microbial community analysis by 16S rRNA gene sequencing using Illumina technology.
Mouse feces were collected into autoclaved eppendorf tubes and stored at − 80 °C after snap frozen in liquid nitrogen. Fecal genomic DNA was extracted with OMGA-soil DNA kit following the manufacturer's instruction. Hypervariant region V3-V4 of bacterial 16S rRNA gene was amplified with the primer 338F Processing of sequencing data. Raw fastq files were quality-filtered using QIIME 61 (version 1.17).
Reads which could not be assembled were rejected. Operational taxonomic units (OTUs) were clustered by UPARSE 62 (version 7.1 http://drive5.com/uparse/) with 97% similarity and chimeric sequences were identified and removed using UCHIME. The taxonomy of each sequence was analyzed by RDP Classifier against Silva (SSU115) 16S rRNA database with 70% confidence threshold 63,64 . Rarefaction analysis was performed by Mothur and alpha-diversity indexes were compared using rarefied data. Principle component analysis (PCA) plot and Venn diagram were implemented by R programming language. Significant changes in relative abundance of microbial taxa were detected by linear descriminant analysis effect size (LEfSe) method 19 . Segmented Filamentous Bacteria (SFB) quantification by real-time PCR. Fecal genomic DNA was extracted by Tiangen Fecal Genomic DNA Extraction Kit under the manufacturer's instruction. Quantitative PCR was carried out by Applied Biosystems 7500 with SYBR Premix Ex Taq quantitative PCR Kit using the universal bacteria primers (5′ -ACTCCTACGGGAGGCAGCAGT-3′ and 5′ -ATTACCGCGGCTGCTGCG-3′ ) 17 , mouse-specific SFB primers (5′ -TGAGCGGAGATATATGGAGC-3′ and 5′ -CATGCAACTATATAGCTATATGCGG-3′ ) and rat-specific SFB primers (5′ -TGAAGCGGAGGTAGATGGA-3′ and 5′ -GCAACTATATAGCTGTATGCGG-3′ ) 29 . PCR reactions were performed as indicated by Takara protocol. The results were expressed as fold changes of relative abundance variation of SFB. Each mouse was tested for SFB upon arrival.
Tissue RNA extraction and quantification of gene expression with real-time PCR. Tissues were stored in RNAlater (Qiagen) at − 20 °C upon harvesting. Total RNA was isolated using TRIzol (Invitrogen, CA, USA) according to the manufacturer's instruction. Quantification real-time PCR was performed with SYBR Premix Ex Taq quantitative PCR Kit in a StepOnePlus apparatus. Results were analyzed with Δ Δ CT method and normalized to the gene Gapdh. Primers used in the experiment are included in Supplementary Table S1.

Small intestinal lamina propria lymphocyte isolation and flow cytometry.
Mouse ilea were collected at sacrifice. Peyer's patches were carefully excised. Ilea were then cut longitudinally and washed with saline to fully remove intestinal content. Before further processing, tissues were stored at 4 °C in complete 1640 containing 5% fetal bovine serum (FBS; Gemini bio, US), 200 U/ml penicillin, 200 μ g/ml streptomycin, 10 μ g/ml amphotericin B and 100 μ g/ml gentamycin. Mucus and epithelium were removed by two 20 mins washes at 37 °C in calcium-free PBS containing 5 mM dithiothreitol (DTT), 5 mM EDTA, 5% FBS, 200 U/ml penicillin, 200 μ g/ml streptomycin, 10 μ g/ml amphotericin B and 100 μ g/ml gentamycin. The remaining tissues were excised into fine pieces and washed in PBS, and then digested in complete 1640 containing 1% collagenase (Worthington Biochemical, US), 1% DNase (Sangon, Shanghai, China) and 2% FBS, at 37 °C and 150 rpm, for 20 mins. After a 7 s vortex for adequate isolation, cells were eluted by passing through a 70-micron strainer, washed with PBS and resuspended in 40% Percoll (GE Healthcare, US) in complete 1640. Cell suspension were laid onto 80% Percoll and centrifuged at 750 g for 15 mins at 4 °C without braking. Lamina propria mononuclear cells (LPMCs) were collected at the interface and used for further experiment. For flow cytometry analysis, LPMCs were stimulated with 50 ng/ml phorbol-12-myristate 13-acetate (PMA; Sigma, US) and 750 ng/ml ionomycin (Sigma) for 4 hrs at 37 °C in the presence of monensin (BD biosciences, US). Following stimulation, cells were permeablized by Perm/ Wash buffer set (BD biosciences) under the manufacturer's instruction and stained with anti-CD4-FITC (BD biosciences), anti-TCRβ -PE (BD biosciences), anti-IL-17A-PE (BD biosciences) and anti-IFNγ -APC (ebioscience). Cells were acquired on a Fortessa flow cytometry and analyzed with Flowjo 7.6.1 software.
Manipulation of gut microbiome by gavage. Cecal content from donor mice were taken after sacrifice, suspended in 10 volumes (w/v) 20% glycerol/PBS solution and stored at − 80 °C. Upon use, frozen suspension was thawed and centrifuged at 4 °C for 8 mins (8000 g). The pellet was resuspended with the same volume of PBS. 100 μ L of the suspension was given to each recipient by oral gavage using 22 ga * 25 mm plastic feeding tubes. Gavage was performed every week for 3 weeks, in order to maintain a stable microbial composition. No adverse effect was observed during experiments.
Statistical analysis. Data are presented as mean ± SEM. Shapiro-wilk test and Levene's test were used for determination of normality and homogeneity of variance, respectively. According to distribution of data, comparisons between different groups were carried out with one-tailed unpaired t-test (between 2 groups), one-way ANOVA corrected for multiple comparison with SNK tests (among more than 2 groups), or Wilcoxon rank sum test (for data unsuitable for t-test or ANOVA). Differences were considered statistically significant at P < 0.05. Analysis was performed with SPSS 19 software.