Modulatory Impacts of Probiotic on the Synthesis of Claudins and Mucins in the Gastrointestinal Tract of Broiler ChicksABSTRACT
This study investigated the effect of 3 strains of probiotic bacteria on mRNA synthesis of some claudins (claudins1(CLD1), CLD3, and CLD5), and mucins (MUC5AC and MUC2) in broilers’ digestive systems. Chicks were assigned to one of three groups: a control group receiving no probiotics, and two experimental groups receiving starter rations with 0.2% and 0.4% probiotics (mixture of Streptococcus faecalis, Clostridium buthricum, and Bacillus mesentericus) over a 14-days period. Proventriculi, ilea, ceca, and cola samples were collected at days 0, 7, and 14 (D0, D7 and D14) for gene expression analysis. Results revealed that CLD1 expression was lower in probiotic groups at D0 and D14 in the proventriculus and colon, respectively, yet higher at D7 in the ileum and colon. In probiotic-fed chicks, CLD5 mRNA synthesis was downregulated at D14 in all segments, except for the proventriculus; and it was upregulated at D7 in colon, and at D14 in the proventriculus. MUC5AC expression was elevated at D7 in the proventriculus and D14 in the ileum with probiotic feeding, whereas MUC2 was significantly downregulated at D14 in the ileum. These findings suggest a protective role of probiotic against pathogenic stimuli affecting the synthesis of claudins and mucins in the gastrointestinal tract of broilers, potentially enhancing intestinal barrier function and resilience against pathogenic challenges.
Keywords:
Claudins; tight junctions; mucins; gut health; mucosal barrier; probiotics
INTRODUCTION
Chicken’s intestinal mucosal surface is a primary site for potential interaction with various pathogens. In broiler chickens, the intestinal mucosa serves as an efficacious barrier between the pathogenic contents in the lumen and the host’s underlying tissues, suggesting that the intestinal mucosal tissue is a key factor in determining gut health and overall activity in chickens (Rinttilä & Apajalahti, 2013).
In newly hatched chicks, the immune system in the small intestine is initially underdeveloped and undergoes substantial morphological, biochemical, and molecular transformations within the first two weeks after hatching (Sato et al., 2009). Supporting the mucosal barrier function in the digestive system of chicks is crucial, as it establishes a dynamic equilibrium among the microbiota, mucus layer, lining epithelium, and underlying immunocytes (Schenk & Mueller, 2008).
Goto & Kiyono (2012) reported that maintaining the integrity of the intestinal epithelium is crucial for nutrient absorption and protecting the host from pathogens. The mucosal barrier system, which includes the lateral modifications of the lining epithelium known as cell junctions, the secreted mucin layer, and active leukocytes, is vital in preventing infections. Tight junctions (TJs) are lateral modifications in the lining epithelium that form an extracellular barrier that shields the underlying tissues from most toxins and pathogens. TJs function to tightly bind adjacent cells, reducing solute diffusion via the intercellular space, and establishing a distinct boundary between the apical and basolateral regions of cellular barriers. One of these TJs proteins is the claudin (CLD) family, which form the primary structure of tight junctions. Members of the CLD family engage in lateral interactions with other CLD proteins in neighboring epithelial cells (Ariyadi et al., 2012). Along with TJs proteins such as occludin and tricellulin, CLDs create the tight junction complex within the plasma membrane (Forster, 2008).
In chickens, CLD1, 3 and 5 have been identified in the oviduct (Ariyadi et al., 2012). The expression patterns of CLD1 and CLD3 have already been studied in chicken embryos (Simard et al., 2006).
CLD1 is found in high-resistance stratified epithelia such as the epidermal tissue, while CLD3 is located in the most constricted part of the nephron, and CLD5 forms tight junction fibers in the endothelium, implying that CLD1, CLD3, and CLD5 are likely involved in forming tight junctions in various epithelial tissues. In birds, CLD1 expression is higher in immature testes compared to adult testes in pheasants (Phasianus colchicus) (Park et al., 2011). Additionally, the expression of CLD3, CLD5, and CLD16 in the intestinal epithelium is higher at two days posthatching than at twenty days prehatching (Ozden et al., 2010). Furthermore, CLD1, CLD3, and CLD5 expression in the oviductal mucosa of hens is lower at the molting than the laying stage, indicating that CLDs may be influenced by physiological conditions (Ariyadi et al., 2012).
The entire surface lining of the chicken gastrointestinal tract is coated with a mucus layer that acts as a diffusion barrier separating its lumen from the absorptive cells. This mucus layer consists primarily of mucins, which are produced and secreted by mucus-secreting cells and support innate immune defense mechanisms by shielding against pathogenic invasions, preventing enteric diseases, and improving digestion and nutrient uptake (McCool et al., 1995).
Research indicates that two days before hatching, the chicken’s small intestine already exhibits villus structures and has the ability to digest and absorb carbohydrates. This early development is essential for preparing the chick’s gastrointestinal tract for efficient post-hatch feeding and nutrient absorption. The intestinal mucus layer serves as its primary defense mechanism, protecting epithelial cells from luminal threats such as mechanical forces during digestion, enzymes, and gut bacteria. It is crucial for supporting the colonization of beneficial bacteria, maintaining a favorable environment for digestion, and aiding in nutrient transport from the lumen to the underlying epithelium. The production and maintenance of this mucus layer are performed by mucin-secreting cells (Duangnumsawang et al., 2021).
Mycotoxins in feed reduce MUC2, MUC5AC, and MUC5B mRNA and protein levels, while also decreasing total mucin production. This reduction may indicate increased intestinal permeability and a heightened susceptibility to toxin exposure (Wan et al., 2014; Huang et al., 2019; Wu et al., 2019). Additionally, MUC2 gene expression decreases in quails fed diets with graded levels of crude protein, but increases with graded levels of threonine (Emadinia et al., 2020). Furthermore, adding turmeric, thyme, and cinnamon to broiler feed can increase MUC2 gene expression in the small intestine (Sangani et al., 2014). The innate immune response, involving genes associated with mucin synthesis (MUC2, MUC5AC, MUC13), is modulated in the jejunum of broilers by including fishmeal in the diet in response to challenges from Eimeria and Clostridium perfringens (Kitessa et al., 2014).
Administering probiotics can influence both systemic and mucosal immune functions, improve intestinal barrier integrity, and modify the microbial environment in the gut (Shanahan, 2010). Probiotic bacteria have been observed to enhance intestinal barrier functions by promoting the production of mucus and antimicrobial peptides (Dykstra et al., 2011), stimulate mucosal IgA responses, improve the synthesis and distribution of TJs like zonula occludens (ZO1) (Zhou et al., 2010), prevent apoptosis of epithelial cells, and induce protective molecules (Khailova et al., 2010).
Probiotics are believed to work through various mechanisms, such as maintaining a balanced gut microbiota; supporting gut development and integrity; regulating the immune system to prevent inflammation; enhancing metabolism; improving feed intake and digestion; and counteracting enterotoxins while stimulating the immune response (Brisbin et al., 2010).
This study explored how a combination of three probiotic bacteria (namely Streptococcus faecalis, Clostridium butyricum, and Bacillus mesentericus) may affect tight junction proteins (claudins CLD1, CLD3, CLD5) and mucin genes (MUC5AC, MUC2) in the digestive tract of broiler chicks.
MATERIALS AND METHODS
Birds
Acquisition and Handling of Broiler Chicks
Male one-day-old Chunky broilers were sourced from Fukuda poultry hatchery, Okayama, Japan. The chicks were handled following the protocols established by the Hiroshima University Animal Research Committee.
Bird Treatment and Tissue Collection
The chicks were categorized into three groups and fed a starter ration with or without probiotic that was comprised of corn starch powder enriched with Streptococcus faecalis (>1×10^8/g), Clostridium butyricum (>1×10^7/g), and Bacillus mesentericus (>1×10^7/g) (Provided by TOA pharmaceutical company, Tokyo, Japan). The control group was fed with a starter ration containing 0% probiotic (corn starch was added as a control), 0.2% probiotic and 0.4% probiotic (control group, probiotic group I and probiotic group II). The chicks were raised for 14 days and tissue samples were collected from the proventriculus, ileum, cecum, and colon on days 0 (D0; one day old), day7 (D7), and day14 (D14).
RNA isolation, cDNA synthesis and gene expression analysis
RNA isolation was carried out from the mucosa of the proventriculus, ileum, cecum, and colon, followed by cDNA synthesis that was used for quantitative and qualitative PCR analysis. Reverse transcription PCR was used to assess the expression of CLD1, CLD3, CLD5, MUC5AC, and MUC2 in each segment. The PCR cycles consisted of 35 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C (CLD3, CLD5, MUC5AC, and MUC2) or 58°C (RPS17 and CLD1) for 30 seconds, and extension at 72°C for 1 minute, followed by a final extension at 72°C for 6 minutes. The primers used in this study are listed in Table 1. Real-time PCR data were analyzed using the 2-△△ct method to calculate the relative gene expression of the target genes, normalized against the RPS17 housekeeping gene (Livak and Schmittgen, 2001). The thermal protocol included 50 cycles at 95°C for 10 seconds and at 58°C (for CLD1, CLD3, and MUC2), 60°C (for CLD5, MUC5AC, and MUC2), and 62°C (for RPS17) for 30 seconds. The complete protocol of RNA extraction, cDNA, and PCR analysis and the related reagents and equipments has been outlined in our earlier studies (Mohammed et al., 2016; Mohammed and Radey, 2021).
Statistical Analysis
Differences in gene expression levels among the groups at each time point were statistically analyzed using Tukey’s test. A p-value of less than 0.05 was considered statistically significant.
RESULTS
Feeding probiotics did not have a significant impact on the body weight of chicks at D7 or D14 (Fig. 1).
At the gene expression analysis level, the mucosal tissue collected from all the segments of control group expressed the three examined CLDs, MUC5AC and MUC2 (Fig. 2).
Reverse transcription-PCR analysis for the expression of mucins and claudins. The expression of MUC5AC, MUC2, CLD1, CLD3 and CLD5 were identified in the proventriculus, ileum, cecum, and colon of broiler chicks of control groups.
The influence of feeding probiotics on CLDs mRNA synthesis in the digestive tract of broilers were investigated by administrating two different concentrations of probiotics for 14 days (Fig. 3 and 4). CLD1 mRNA expression was downregulated at D7 in the proventriculus (Fig. 3A) and at D14 in the colon (Fig. 3D) in the probiotic groups. In contrast, CLD1 expression of probiotics group II and probiotic group I was upregulated at D7 in the ileum (Fig. 3B) and colon (Fig. 3D), respectively. Within the control groups, the expression of CLD1 was lower at D14 than D0 and D7 in the proventriculus (Fig. 3A) and was higher at D14 than D0 and D7 in the ileum (Fig. 3B). There were no significant differences in CLD1 levels in the cecum across all treatment and control groups (Fig. 3C). A similar level was observed for CLD3 expression, which did not show any significant differences among all treatment and control groups.
Effects of probiotics-feeding on the expression of CLD1 in the proventriculus (A), ileum (B), cecum (C) and colon (D) of broiler chicks. Values are mean ± SE of fold changes in expression. Control groups, and probiotics groups I and II were fed 0, 0.2 or 0.4% probiotics, respectively. D0, D7 and D14 refer to the probiotic’s treatment days (D0 = one-day-old chicks). *, **, a, b Values are significantly different among treatments (p<0.05 or p<0.01, Tukey’s test).
Effects of probiotics-feeding on the expression of CLD5 in the proventriculus (A), ileum (B), cecum (C) and colon (D) of broiler chicks. Values are mean ± SE of fold changes in expression. Control groups, and probiotics groups I and II were fed 0, 0.2 or 0.4% probiotics, respectively. D0, D7 and D14 refer to the probiotic’s treatment days (D0 = one-day-old chicks). *, **, a, b Values are significantly different among treatments (p < 0.05 or p < 0.01, Tukey’s test).
The expression of CLD5 in the proventriculus was lower at D14 than D0 and D7 in control groups, while it was significantly upregulated in probiotic group I and II in comparison to the control at D14 (Fig. 4A). In the ileum, CLD5 expression was higher at D14 than D0 and D7 in control groups, while it was lower in both probiotic groups at D7, and in probiotic group II at D14 than in the control (Fig. 4B). In the cecum, no significant differences were observed among the control groups across all time points or between the control and probiotic groups at D7. However, probiotic supplementation led to a significant downregulation of CLD5 expression at D7 (Fig. 4C). In the colon, CLD5 transcript level was higher at D7 compared to D0 and D14 within the control groups. At D7, CLD5 expression was upregulated in probiotic group I compared to the control group, while it was significantly downregulated in both probiotic groups at D14 (Fig. 4D).
MUC5AC mRNA synthesis in the proventriculus was significantly downregulated at D7 and D14 in control groups, while it was upregulated in probiotic groups compared to the control group at D7 (Fig. 5A). In the ileum, the expression of MUC5AC within control groups was higher at D7 than D0, with no significant difference from D14. At D14, the expression of MUC5AC was significantly higher in probiotic group II than in the control group (Fig. 5B). No significant differences of MUC5AC expression among control and probiotic groups in the cecum and colon were found (Data not shown).
Effects of probiotics-feeding on the expression of MUC5AC in the proventriculus (A) and ileum (B) of broiler chicks. Values are mean ± SE of fold changes in expression. Control groups, and probiotics groups I and II were fed 0, 0.2 or 0.4% probiotics, respectively. D0, D7 and D14 refer to the probiotic’s treatment days (D0 = one-day-old chicks). *, **, a, b, Values are significantly different among treatments (p < 0.05 or p < 0.01, Tukey’s test).
In the proventriculus, MUC2 expression remained consistent across both treatment groups and controls throughout the experiment (data not shown). The expression of MUC2 in the ileum was higher at D14 than D0 and D7 in control groups, while in probiotic groups the expression was significantly downregulated in comparison to the control group at D14 (Fig. 6A). Feeding probiotics did not affect the expression of MUC2 in the cecum and colon, while it was higher only at D7 within control groups (Fig. 6B and 6C).
Effects of probiotics-feeding on the expression of CLD5 in the proventriculus (A), ileum (B), cecum (C) and colon (D) of broiler chicks. Values are mean ± SE of fold changes in expression. Control groups, and probiotics groups I and II were fed 0, 0.2 or 0.4% probiotics, respectively. D0, D7 and D14 refer to the probiotic’s treatment days (D0 = one-day-old chicks). *, **, a, b Values are significantly different among treatments (p < 0.05 or p < 0.01, Tukey’s test).
DISCUSSION
This study reports the profound effect of probiotic supplementation on CLD1, 3, 5, MUC5AC and MUC2 in broiler chick proventriculus, ileum, cecum, and colon. The major findings of this study are: (1) CLD1 expression was lower in probiotic groups than in the control at D7 in the proventriculus, while it was higher in the colon and ileum; (2) CLD5 expression was higher in probiotic groups at D14 in the proventriculus while it was lower in the ileum, cecum and colon; (3) MUC5AC expression was higher in probiotic groups at D7 and D14 in the proventriculus and the ileum, respectively; (4) probiotic downregulated MUC2 mRNA synthesis only in the ileum at D14.
Previous studies have indicated that probiotics can facilitate the maintenance and repair of the intestinal mucosal barrier following damage. For instance, Escherichia coli Nissle 1917 prevents the mucosal barrier damage by enteropathogenic E. coli and restores its integrity. This protection is carried out by elevated expression and redistribution of TJ proteins, specifically zonula occludens 2 (ZO2), leading to the reconstruction of the tight junction complex (Stetinova et al., 2010). Similarly, Lactobacillus casei DN-114001 (Parassol et al., 2005) and VSL#3, a mixture of prebiotics and probiotics (Dai et al., 2012), maintain intestinal barrier function through similar mechanisms.
Tight junction proteins play a critical role in maintaining intestinal integrity by forming a barrier within the cellular space (Zhang & Kim, 2014). CLD1 is pivotal as a central component of TJs, crucial for barrier establishment. Occludin contributes significantly to the formation and maintenance of TJs, while ZO1 is a TJ-specific protein with multiple domains that provide intracellular scaffolding to support and regulate TJ structure. Previous studies have demonstrated that probiotics can mitigate TJ damage by enhancing ZO1 protein synthesis (Emami et al., 2020). Furthermore, in chicks fed probiotics, there was increased expression of occludin and ZO1 in the ileum (Rahimi et al., 2009). Mountzouris et al. (2019) also found that diets supplemented with complex probiotics boosted mRNA transcript levels of ZO1, CLD1, and occludin in the jejunum of broiler chickens. In agreement with these reports, our results revealed that CLD1 transcript level was upregulated by probiotic in the ileum and colon at D7, while CLD5 was upregulated at D0 and D7 in the proventriculus and colon, respectively.
Although we did not detect significant differences in CLD3 expression, previous studies have reported that supplementation with probiotics, prebiotics, or essential oils led to higher CLD3 mRNA expression on days 8 and 14 compared to a negative control, with probiotic-fed birds showing higher CLD3 expression only on day 8. These differences in expression may be attributed to the structural role of CLD family proteins in TJ complexes, particularly their extracellular domains, which can be attachment sites for pathogens like Clostridium perfringens enterotoxins, influencing gut integrity (Saitoh et al., 2015; Eichner et al., 2017).
Higher CLD3 expression in birds receiving probiotics or probiotic/prebiotic/essential oil supplements might be due to these products mitigating the negative effects of necrotic enteritis. Conversely, lower CLD3 expression in the negative control birds could be a defense mechanism to a reduction in attachment sites for Clostridium perfringens enterotoxins, thereby diminishing their impact (Emami et al., 2020). Curcumin as a feed additive can also increase mRNA abundance of ZO1 and CLD1 in Caco2 cells and alleviate IL1β-induced disorganization of ZO1, CLD1, CLD5, and CLD7in intestinal epithelial cells, improving barrier function and reducing paracellular permeability (Ozden et al., 2010; Ghosh et al., 2018).
Supporting our findings, Mountzouris et al. (2019) reported that lower expression of CLD proteins could be a normal physiological response for conserving nutrients and energy in the absence of pathological inflammation or pathogenic challenge. Similar observations were made regarding the expression of antimicrobial peptides (avian beta-defensins and cathelicidins) in the gastrointestinal tract of broiler chicks, where probiotics alone did not enhance their expression. However, when challenged with Salmonella Minnesota lipopolysaccharides, probiotic-fed chicks showed increased expression of these molecules compared to control groups (Mohammed et al., 2016; Mohammed & Radey, 2021).
In line with our findings on MUC5AC expression, previous research has indicated that mRNA expression of mucins in chick intestines can increase under conditions such as starvation, probiotic supplementation, and pre-hatching administration of carbohydrates (Smirnov et al., 2006). In the oviduct, MUC5AC was also affected by the laying cycle in hens (Ariyadi et al., 2012) and was stimulated by both estrogen and LPS in the chicken oviduct (Ariyadi et al., 2013).
Probiotic use can stimulate mucin production, enhancing protection against numerous pathogens in broilers where MUC2 gene expression was higher in the probiotic group compared to the Salmonella entertidis or Salmonella typhimurium challenged broiler chicks (Zhen et al., 2018). The chemical barrier formed by MUC2 proteins plays a crucial defense role in the protection against enteric pathogens and their toxins.
Supporting our results, Mountzouris et al. (2019) reported that supplementation with a viable probiotic form reduced cecal MUC2 expression more than an inactivated probiotic form or no probiotic. Intestinal mucin, like IgA, provides protection on gut mucosal surfaces against luminal threats (Capaldo et al., 2017). Therefore, the decrease in MUC2 expression observed with viable probiotic supplementation could be attributed to less challenging conditions or a lower state of inflammation, as supported by reduced TLR and NF-κB expressions (Mountzouris et al., 2019).
In conclusion, consistent with the findings of Wu et al. (2022), our study indicates that probiotic supplementation can decrease oxidative stress, modulate TJs and mucins related genes, and promote growth performance in broilers. This enhancement of the mucosal barrier functions in the digestive tract may reduce the risk of enteric pathogenic invasion in broiler chicks.
ACKNOWLEDGEMENTS
The authors thank Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Al Ahsa, Saudi Arabia for financial support (Grant NO: KFU242621) and Toa Pharmaceutical Co., Ltd. (Tokyo, Japan) for providing probiotics.
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FUNDING
This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Al Ahsa, Saudi Arabia [Grant No. KFU242621].
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DATA AVAILABILITY STATEMENT
Data will be available upon request.
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DISCLAIMER/PUBLISHER’S NOTE
The published papers’ statements, opinions, and data are those of the individual author(s) and contributor(s). The editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions, or products referred to in the content.
Data will be available upon request.
