Effect of oral administration of Limosilactobacillus reuteri on intestinal barrier function and mucosal immunity of suckling piglets

Abstract The aim of present study was to evaluate the effect of Limosilactobacillus reuteri (L. reuteri) on the intestinal barrier function and mucosal immunity of piglets at different suckling phase. In this study, fifty-four neonatal piglets (Rongchang × Yorkshire) were orally administered with 1.0 × 1010 CFU L. reuteri I5007 (dissolved in 2 ml 0.1% peptone) or 2 ml 0.1% peptone by gavage every day. The experiment had lasted for 20 days from the birth. Six piglets from each group were slaughtered at 10- and 20-day-old, respectively. Intestinal samples were collected for the evaluation of target genes by real-time PCR and western blot. The results showed that L. reuteri I5007 significantly increased the average daily gain, decreased the diarrhoea incidence, and improved the intestinal morphology of piglets (p < .05). Oral administration of L. reuteri I5007 also elevated the mRNA and protein level of jejunal and ileal Zonula Occludens-1, Claudin-1, and Occludin to enhance the intestinal mucosal barrier function. Additionally, L. reuteri I5007 treatment modulated toll-like receptors (TLRs) and porcine host defense peptides (HDPs) expression, such as increased the mRNA levels of ileal TLR9 and PBD2 at 10-day-old, jejunal TLR9 and ileal PG1-5 at 20-day-old, the protein level of ileal TLR2 at 20-day-old, and influenced the protein and mRNA level of ileal TLR4. These findings indicated that oral administration of L. reuteri I5007 could improve the intestinal mucosal barrier and enhance the intestinal innate immunity of piglets during suckling period. Highlights L. reuteri increased the average daily gain and decreased the diarrhoea incidence of piglets. L. reuteri increased the expression of Zonula Occludens-1, Claudin-1, and Occludin in jejunal and ileal mucosal. L. reuteri influenced the expression of toll-like receptors and porcine host defense peptides.


Introduction
Probiotics were defined as 'live microorganisms that, when administered in adequate amounts, confer a health benefit on the host' in a guideline issued by the FAO/WHO group (Hill et al. 2014;Sanchez et al. 2017). Numerous studies have shown that probiotics exert manifold beneficial effects on the intestinal health of the host, such as maintaining gut mucosa homeostasis, strengthening the intestinal structure, increasing tight junction formation, boosting the immune function, attenuating the inflammatory diseases of gastrointestinal tracts (GIT), etc. (Arsene et al. 2021;Camilleri 2021). The GIT of neonatal animals is immature and susceptible to infection by pathogenic bacteria. Probiotics will help young animals to establish a healthy intestinal environment (Daniel 2017;Pluske et al. 2018;Yang et al. 2018). Oral administration of Lactobacillus delbrueckii in suckling piglets improved the antioxidant status and stimulated the immune response after the weaning. Wang Y et al also reported that administration of Lacticaseibacillus rhamnosus (L. rhamnosus) GG to piglets at the suckling period can improve the biological, physical and immunological barriers of intestinal mucosa of pre-weaning piglets . Another probiotic, Ligilactobacillus salivarius (L. salivarius) B1 can stimulate the maturation of the intestinal immune system and elevate the immunomodulatory activities for piglets in early life (Zhang et al. 2011). Co-administration of Bacillus subtilis RJGP16 and L. salivarius B1 enhances the intestinal mucosal immunity of newborn piglets by increasing the expression of interleukin (IL) 6, porcine beta-defensin (PBD) 2, and toll-like receptor (TLR) 2 and elevating the number of immunoglobulin (Ig) A producing cells (Deng et al. 2013).
Tight junctions (TJ) are the important components of the intestinal barrier and crucial for the maintenance of barrier integrity. The expression and distribution of TJs (such as ZO-1, Claudin-1, and Occludin) directly affect intestinal epithelium barrier function (Groschwitz and Hogan 2009;Ulluwishewa et al. 2011). Probiotics reinforce immune functions through cytokines and signalling pathways by regulating TLRs. TLR2 plays an important role in barrier defense and immune response, and recognises a variety of microbial components by interacting with the co-receptor TLR6 (Takeuchi et al. 2001;Melmed et al. 2003;Kang et al. 2009). TLR4 can recognise the bacterial lipopolysaccharide and it is involved in defense against pathogens. L. reuteri reduces intestinal inflammation via modulation of TLR4 and NF-jB signalling (Kawai and Akira 2011;Liu et al. 2012). As an innate immune receptor, TLR9 recognises bacterial DNA to trigger the activation of host immune defences. L. rhamnosus HN001 attenuates necrotising enterocolitis in newborn mice and piglets, because its DNA exerts the protective effects and the protection requires activation of TLR9 (Gribar et al. 2009;Good et al. 2014). Host defense peptides (HDPs) are produced in the epithelial cells and immune cells of the gut and are known for their antimicrobial properties and immunomodulatory activities. HDPs play essential roles in the innate immune system and can be activated under normal physiological conditions to maintain and enhance the defensive barriers (Becker et al. 2010;Zeng et al. 2013;Mansour et al. 2014;Robinson et al. 2015).
Limosilactobacillus reuteri (L. reuteri) is one of the dominant Lactobacillus species in the GIT of humans and animals, which plays an important role in intestinal mucosal barrier function of the host Wang et al. 2018). Nii et al. (2020) reported that administration of L. reuteri protects against pathogen infection and enhances mucosal barrier function in newly hatched chicks (Nii et al. 2020). L. reuteri ZJ617 alleviated LPS-induced intestinal tight junction protein destruction and intestinal inflammatory in the piglets (Zhu et al. 2021). L. reuteri D8 exerts a beneficial effect on piglets by promoting intestinal development and enhancing intestinal innate immunity Wu et al. 2020). Administration of L. reuteri KT260178 improves the growth and reduces morbidity of neonatal piglets by improving intestinal microbiota profile and regulating the antioxidant and immune status of the body (Yang et al. 2020). Oral administration of L. reuteri can promote intestinal development and maintain the mucosal barrier in newborn piglets, but the effect of continuous administration of L. reuteri on the intestinal mucosal barrier and immunological function of piglets at different suckling phases is unknown.
This work aimed to investigate the effects of L. reuteri I5007 on the performance, intestinal morphology, the expression of TJ proteins, TLRs and porcine HDPs, and systematically evaluate its beneficial effect on suckling piglets.

Ethics approval
In this study, all animal protocol was in accordance by the Guidelines for the Administration of Affairs Concerning Experimental Animals. The procedure of slaughter trial was approved by the Institute Ethics Committee of the Chongqing Academy of Animal Science (approval number: xky-20190715).
Bacterial strain and culture condition L. reuteri I5007 (provided by Dr. Shiyan Qiao) was grown in De Man, Rogosa and Sharpe medium at 37 C for 22 h in an anaerobic environment. The bacterial cells were harvested and washed with phosphate-buffered saline (PBS) twice, then re-suspended in skim milk for vacuum desiccation. The dried bacteria cells were stored at 4 C and subpackage in sterile and sealed packets. The colony forming units (CFU) of freeze-dried powder was calculated by plate count and dissolved to a certain concentration with 0.1% peptone before the trial.

Animal treatment
Fifty-four neonatal piglets (Rongchang Â Yorkshire) from 6 litters were selected and assigned into a control (CN) and a L. reuteri I5007 (LR) group on the basis of litter origin and body weight (nine piglets/litter, three litters/group). The trial was started at 1-day-old and lasted for 20 days, the piglets from LR group and CN group were orally administered with 1.0 Â 10 10 CFU L. reuteri I5007 (dissolved in 2 mL 0.1% peptone) or 2 mL, 0.1% peptone by gavage every day, respectively. All piglets had received colostrum from the sows and had free access to sow milk and drinking water. The sows were housed under similar conditions in separate buildings and fed a basal diet formulated to meet the NRC (2012) requirements of nutrients for lactating sows. The incidence of diarrhoea (%) was calculated as [(number of piglets with diarrhoea Â number of days of diarrhoea)/ (total number of piglets in the experiment Â number of days of the whole experiment)] Â 100%. The breeder evaluated the diarrhoea and the piglets were categorised as diarrhoeic if they had faeces in liquid form at least one day (NRC 2012).

Sample collection
All of the piglets were weighed individually at the age of 1, 10, and 20 days, respectively. Six piglets from each group (two piglets per litter) were randomly selected for euthanasia at 10-and 20-day-old, respectively. The intestinal tissues from the middle duodenum, jejunum and ileum were collected and fixed in 4% paraformaldehyde solution after being gently rinsed with saline, respectively, as previously described (Zhang et al. 2011). Mucosa from the jejunum and ileum were scraped with a glass slide and immediately frozen in liquid nitrogen, then, stored at -80 C for RNA extraction and western blotting analysis.

Intestinal morphology analysis
The fixed segments of the duodenum, jejunum, and ileum were removed from the 4% paraformaldehyde solution, then, washed and embedded in paraffin immediately. The segments were cut into 5 lm thick sections and stained with haematoxylin and eosin (HE) as previously described (Hu et al. 2017). All images were obtained using a BA200 digital microscope (Motic, Xiamen, China) and the villus height and crypt depth of each sample were measured with Image-Pro Plus 6.0 (Media Cybernetics, USA).

Quantification of gene expression by realtime PCR
Total RNA of intestinal mucosa was extracted using a Trizol reagent (Takara, Dalian, China) according to the manufacturer's protocol. The cDNA was reverse transcribed using the PrimeScript TM RT reagent kit (TaKaRa, Dalian, China) and real-time PCR (RT-PCR) was performed using the SYBRV R Premix Ex Taq TM II kit (TaKaRa, Dalian, China). The reactions were run in triplicate and analyses were conducted using the QuantStudio TM 6 Flex RT-PCR system (Life Technologies, CA, USA). Relative expression of mRNA was normalised by the endogenous control GAPDH and all primers are listed in Table 1. The mRNA expression of zonula occludens-1 (ZO-1), claudin-1, occludin, TLR2, TLR4, TLR6, TLR9, PBD2, PBD3, protegrins (PG) 1-5, epididymis protein 2 splicing variant C (PEP2C) was detected by RT-PCR. RT-PCR reactions were performed in 384-well plates at 95 C for 30 s, followed by 40 cycles of 95 C for 5 s and 60 C for 30 s. Relative expression was calculated using the 2 -᭝᭝CT method.

Statistical analysis
The data were analysed by unpaired Student's twotailed t-test with SPSS17.0 Software (SPSS, Inc., Chicago, IL, USA). The results were presented as means and standard error of means (SEM). The level of significance was set at p < .05, and .05< p < .1 was considered as a tendency.

Results
Effects of L. reuteri I5007 on the performance and diarrhoea of suckling piglets As shown in Table 2, the piglets treated with L. reuteri I5007 had a significantly higher average daily gain at 11-20 day period and a lower diarrhoea incidence than control piglets (p < .05). However, oral administration of L. reuteri I5007 had no significant effect on body weight and average daily gain at 1-10 day period.
Effects of L. reuteri I5007 on intestinal morphology of suckling piglets L. reuteri I5007 significantly increased the ratio of villus height to crypt depth (VCR) (p < .05) and also tended to increase the villus height of the ileum at 10-day-old (p < .1). Compared with a control group, no significant difference was observed in duodenal and jejunal morphology in the L. reuteri I5007 group (Table 3 and Figure 1).

Effect of L. reuteri I5007 on the mRNA and protein expression of TJ proteins in suckling piglets
In the jejunal mucosa, L. reuteri I5007 significantly increased the mRNA level of occludin and the protein  expression of ZO-1 and Claudin-1 (p < .05), and tended to increase the mRNA level of ZO-1 at 10-day-old (p < .1); additionally, at 20 days, the mRNA and protein expression level of ZO-1 and Claudin-1 were all significantly higher for the L. reuteri I5007 group than for the control group treatment (p < .05) (Figure 2). In the ileal mucosa, the mRNA and protein expression of Occludin were elevated by L. reuteri I5007 treatment at 20-day-old (p < .05); but, no significant difference was observed in TJ proteins between the two groups at 10-day-old (Figure 3).

Effect of L. reuteri I5007 on the mRNA and protein expression of TLRs in suckling piglets
The mRNA expression of TLR2, TLR4, TLR6, and TLR9 and protein levels of TLR2 and TLR4 in the jejunal and ileal mucosa were measured and presented in Figures   4 and 5, respectively. In the jejunal mucosa, the mRNA expression level of TLR-9 was significantly increased in the L. reuteri I5007 group at 20-day-old (p < .05), but no significant difference was observed in the expression levels of TLR2, TLR4, and TLR6 compared with the control group (Figure 4).
In the ileal mucosa, L. reuteri I5007 treatment significantly elevated the mRNA expression of TLR9 and the protein level of TLR2 at 10-day-old; additionally, the mRNA expression of TLR4 at 20-day-old and the protein level of TLR4 at 10-day-old were significantly reduced by oral administration of L. reuteri I5007 (p < .05) ( Figure 5).

Effect of L. reuteri I5007 on the mRNA expression of porcine HDPs in suckling piglets
The relative levels of mRNA transcription of porcine HDPs (including PBD2, PBD3, PG1-5, and PEP2C) in the jejunal and ileal mucosa were determined by RT-PCR ( Figure 6). The relative abundances of mRNA for PBD2 at 10-day-old and PG1-5 at 20-day-old were significantly increased in the ileal mucosa with L. reuteri I5007 treatment compared with the control (p < .05). But, L. reuteri I5007 did not change the mRNA expressions of PBD3 and PEP2C in the jejunal and ileal mucosa.

Discussion
In this study, we investigated the effects of oral administration of L. reuteri I5007 on the performance, intestinal morphology and intestinal mucosal barrier function in newborn piglets. We found that administration of L. reuteri I5007 significantly improved performance in suckling piglets, consistently with a result by Liu et al., which showed that L. reuteri I5007 increased average daily gain at early life (Liu et al. 2017). In the present study, L. reuteri I5007 improved intestinal morphology by increasing the ratios of villi heights to crypts depths and the villi heights of the ileum in suckling piglets. Villi and crypts are important components of the digestive tract, which are associated with the structure and function of the small intestine (Pluske et al. 1997;Judkins et al. 2020). The increased villi height improves the ability of small intestine to absorb nutrients from food, this may explain why the bodyweight was also increased in the LR group.
Many studies have shown that intestinal bacteria regulate intestinal barrier function by changing the expression of TJ proteins (Buckley and Turner 2018). In our present study, administrated with L. reuteri I5007 increased the mRNA expression of ZO-1, claudin-1, occludin and the protein expression of ZO-1 and Claudin-1 in the jejunal mucosa, and increased the expression of Occludin in the ileal mucosa of piglets at 20-day-old. A previous study showed that dietary supplementation with L. reuteri LR1 increased the expression of ZO-1 and Occludin in the jejunum and ileum of weaned pigs (Yi et al. 2018). L. reuteri I5007 significantly increased the expression of ZO-1, Claudin-1, and Occludin in vivo and in vitro . These data indicated that L. reuteri enhanced the intestinal barrier function by evaluating the expression of TJ proteins. In our study, L. reuteri affected the TJs expression differently between jejunal and ileal mucosa. The jejunum takes almost 80% of the small intestine in piglets, where digests and absorbs mostly nutrients of food (Kim and Duarte 2021). Recent research indicated that effective probiotic bacteria associated with jejunal mucosa could maintain or improve the intestinal health of suckling piglets (Adhikari et al. 2019). Thus, probiotic L. reuteri only increased some TJs in the jejunum but not in the ileum.
The intestinal tract is a complicated system that can defense against pathogens. TLRs are one of the mechanisms in immune defense against fungal, bacterial and viral pathogens in the intestine (Kamdar et al. 2013;McDermott and Huffnagle 2014;Groeger and Meyle 2019). Our study detected the expression of TLR2, TLR4, TLR6, and TLR9 in the jejunal and ileal mucosa, showing that L. reuteri I5007 increased the expression of TLR9 at the gene level, and induced TLR2 expression and reduced TLR4 expression of piglets. The results are consistent with the results obtained in the previous study, which showed that L. reuteri LR1 increased TLR2 expression and decreased TLR4 expression in the ileum of weaned piglets (Yi et al. 2018). Another report showed that Lactiplantibacillus plantarum modulated the intestinal innate immunity of young piglets by preventing the upregulated expression of TLR4 in the jejunal mucosa induced by ETEC K88 (Yang et al. 2021). Bacillus amyloliquefaciens SC06 significantly reduced the expression of TLR6, but had no significant difference for TLR2, TLR4, and TLR9 in the jejunum of piglets (Du et al. 2018). L. salivarius administration significantly lowered the expression levels of TLR2 and TLR4 in mesenteric lymph nodes (Sun et al. 2020). These researches indicated that different probiotics have distinct influences on the expression of TLRs.
Several studies have indicated that porcine HDPs (such as PBD2, PBD3, PG1-5, and PEP2C) are involved in intestinal innate immune defense (Veldhuizen et al. 2008;). Among them, PBD2 was confirmed to improve the integrity of intestinal mucosal barrier and attenuate gut inflammation in dextran sodium sulphate (DSS)-treated mice (Han et al. 2015). In this study, we found that the mRNA expression of PBD2 and PG1-5 was increased by L. reuteri I5007 treatment, indicating that L. reuteri I5007 may help to protect from pathogen infection and elevate innate immunity in the intestine of piglets. The beneficial effect of L. reuteri I5007 had mostly observed thanks to the decrease in the diarrhoea incidence.
This research indicated that L. reuteri could improve the intestinal barrier function and the mucosal immunity of suckling piglets. However, a limitation of the study is the absence of the positive control group with infected piglets.

Conclusion
In summary, this study showed that L. reuteri I5007 could improve the intestinal barrier function and mucosal immunity of suckling piglets by elevating the expression of TJ proteins (ZO-1, Claudin-1, and Occludin), TLRs (TLR2, TLR4, and TLR9) and porcine HDPs (PBD2 and PG1-5), suggests that L. reuteri I5007 can exert a beneficial effect on piglets in early life.

Disclosure statement
No potential conflict of interest was reported by the authors.

Data availability statement
The data that support the findings of this study are openly available from the corresponding author upon reasonable request.