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. 2021 Nov 20;101(2):101615. doi: 10.1016/j.psj.2021.101615

Dietary hydrolyzed wheat gluten supplementation ameliorated intestinal barrier dysfunctions of broilers challenged with Escherichia coli O78

Yujun Wu *, Wenyu Wang †, In Ho Kim ‡, Ying Yang *,1
PMCID: PMC8715217  PMID: 34952261

Abstract

This study aimed to investigate a protective effect of hydrolyzed wheat gluten (HWG) on Escherichia coli (E. coli)-induced intestinal barrier dysfunctions in broilers. Broilers fed a basal diet unsupplemented or supplemented with HWG (0.5% or 1%) were intraperitoneally injected with either E. coli O78 suspension (108 CFU/mL) or equal volume of vehicle on d 18 of age. Blood and tissue samples were collected 3rd d post infection. The results showed that E. coli-infection increased immune-organ indexes of spleen and thymus, enhanced serum diamine oxidase (DAO) level, impaired ileal villus structure and reduced tight junction mRNA levels (Occludin, Claudin-1, ZO-1, P < 0.05), while increased mRNA levels of inflammatory cytokines (TNF-α, IFN-γ, IL-1β, IL-6, IL-8) and TLR4 in the ileum of broilers (P < 0.05). The effects of E. coli O78 challenge on organ indexes of spleen and thymus, serum DAO level, mRNA levels of tight junctions were alleviated by 1% HWG supplementation, the upregulations of IL-1β and TLR4 were prevented by 0.5% HWG supplementation (P < 0.05). In addition, increased IFN-γ of E. coli-infected broilers was abrogated by 0.5% or 1% HWG supplementation (P < 0.05). In summary, dietary HWG supplementation ameliorated intestinal barrier dysfunctions triggered by E. coli-infection in the ileum of broilers. HWG supplementation might be a nutritional strategy to improve the intestinal mucosal barrier function of broilers.

Key words: hydrolyzed wheat gluten, intestinal permeability, inflammatory cytokines, tight junction

Abbreviations: DAO, diamine oxidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Gln, glutamine; Glu, glutamate; Gly, glycine; HWG, hydrolyzed wheat gluten; IFN-γ, interferon-γ; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-8, interleukin-8; NRC, National Research Council; TLR4, Toll like receptor-4; TNF-α, tumor necrosis factor-α; ZO-1, zonula occluden-1

INTRODUCTION

The gastrointestinal tract is critical for nutrients absorption and intestinal homeostasis (Okumura and Takeda, 2017). The monolayer of enterocytes allows digestion and absorption of nutrients, while prevents the invasion of pathogens and toxins from penetrating the intestinal mucosa, otherwise potentially causing cellular injury (Citi, 2018). Dysfunction of the intestinal barrier has been reported to be associated with increased epithelial permeability and bacteria translocation, therefore resulting in the gastrointestinal disorders and even systemic inflammation (Camilleri et al., 2012).

Escherichia coli (E. coli) are a group of symbionts colonized in the gastrointestinal tracts of humans and animals (Sarowska et al., 2019). However, replication and colonization of pathogenic E. coli are associated with intestinal disorders and decrease digestion and absorption of nutrients in animals (Croxen et al., 2013). Avian pathogenic E. coli (APEC), a gram-negative flora, has been reported to adhere to epithelial cells and disrupt the intestinal integrity of birds, including both the meat type broilers, and laying hens (Wang et al., 2016). E. coli infection in birds leads to severe diarrhea, decreased feed intake, and reduced growth performance, therefore causing serious economic losses in poultry industries (Mellata, 2013). Several nutritional interventions have been attempted to ameliorate E. coli-induced intestinal barrier dysfunctions (Wang et al., 2016; Zhang et al., 2020), eventually improving the intestinal development and health outcomes of broilers.

Hydrolyzed wheat gluten (HWG), obtained by enzymatic hydrolysis of wheat gluten, has been widely used as the functional food to improve the health outcomes of humans and animals due to its high proportions of glutamine (Gln), glutamate (Glu), glycine (Gly) and bioactive peptides (Asrarkulova and Bulushova, 2019). Previous study has demonstrated that dietary supplementation with HWG can enhance the immune response and decrease the incidence of diarrhea in weaning piglets (Wang et al., 2011). Due to the high digestibility of HWG, it has been regarded as an ideal protein source for milk replacer for calves (Castro et al., 2016). Additionally, HWG administration alleviates deoxynivalenol-induced intestinal injury by promoting intestinal stem cell proliferation and differentiation in mice (Zhou et al., 2019). Dietary HWG inclusion improves the morphology of small intestinal in broilers (Van Leeuwen et al., 2004). However, it remains unknown whether dietary HWG supplementation can reduce E. coli challenge-induced intestinal barrier dysfunctions in broilers. Therefore, this study was conducted to investigate the protective effects of HWG supplementation on intestinal barrier functions and inflammatory response of E. coli-challenged broilers.

MATERIALS AND METHODS

E. coli Suspension Preparation

The E. coli O78 strain (CVCC1490; China Veterinary Culture Collection Center, Beijing, China) was cultured and used as previously described (Zhang et al., 2020). In brief, E. coli was aerobically cultured in Luria Borth liquid medium at 37°C for 16 h. After 10-fold serial dilution, the diluted suspensions were plated on MacConkey agar at 37°C for 24 h for bacteria enumeration. The final bacteria concentration was adjusted to 108 CFU/mL with sterile sodium chloride at pH 7.0.

Animals and Experimental Design

The experimental protocol was approved by the Animal Care and Use Committee of China Agricultural University (Beijing, China). A total of 360 1-day-old male Arbor Acre broilers were randomly allocated into 3 dietary treatments (0%, 0.5% or 1% HWG supplemented diets) with 8 replicates of 15 birds per replicate. Broilers in 0% HWG group were fed the basal diet (Table 1), which met the nutritional requirements of broilers (National Research Council, 1994), 0.5% or 1% HWG group were respectively fed 0.5% or 1% HWG supplemented diets. The HWG used in the study was provided by Zhengzhou Xinwei Nutritional Technology Co., Ltd. (Zhengzhou, China). The composition of hydrolyzed wheat gluten has been provided in the Table S1. The inclusion amount was based on the manufacturer suggestions, and nutrient contents were maintained across all diets. On d 18 of the age, 4 broilers per replicate from each treatment (3 dietary treatments) were randomly selected and isolated, then divided into 2 groups (E. coli infection or not) according to 3 × 2 factorial design. Broilers in infected and uninfected groups were intraperitoneally injected with 1 mL of either E. coli suspension (108 CFU/mL) or equal volume of vehicle. All birds had free access to individual feed and water throughout the whole study period.

Table 1.

Composition and nutritional levels of basal diet (air-dry basis).

Ingredients Contents (%) Nutritional levels1 Contents (%)
Corn 59.63 ME (calculated, MJ/kg) 12.35
Soybean meal 30.05 Crude protein (%) 21.05
Soybean oil 1.46 Lysine (%) 1.18
Corn gluten meal 4.69 Methionine (%) 0.55
Calcium hydrophosphate 1.90 Threonine (%) 0.81
Limestone 0.91 Tryptophan (%) 0.20
DL-Methionine 0.16 Calcium (%) 1.02
L-Lysine (78%) 0.20 Nonphytate Phosphorus (%) 0.46
NaCl 0.35
Choline chloride (50%) 0.30
Multivitamin2 0.02
Multimineral3 0.30
Santoquin 0.03
Total 100.00
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1

Nutritional levels were measured, except for ME.

2

The multivitamin provided the following per kg of diets: VA 12500 IU, VD3 2500 IU, VE 18.75 mg, VK3 2.65 mg, VB1 2 mg, VB2 6 mg, VB6 6 mg, VB12 0.025 mg, biotin 0.0325 mg, folic acid 1.25 mg, pantothenic acid 1.25 mg, and nicotinic acid 50 mg.

3

The multimineral provided the following per kg of diets: Cu 8 mg, Zn 75 mg, Fe 80 mg, Mn 100 mg, Se 0.15 mg, and I 0.35 mg.

The broilers were reared in the battery cages with a plastic wire-floor (0.06 m2/bird), placed in an environmentally controlled room. The room temperature was maintained at 33°C to 35°C during the first 3 d and then gradually decreased to 22°C by d 21. The lighting program was performed with 23 h lighting and 1 h darkness for the first 7 d, followed by 20 h light and 4 h darkness from d 8 until the end of the experiment.

Sample Collection

On the third day post challenge, one bird per replicate was randomly selected and weighed for sampling. Blood samples were aseptically collected from the wing vein. After centrifugation at 3,000 rpm for 10 min at 4°C, serum samples were obtained and stored at -80°C until further analysis. After euthanized by sodium pentobarbital (30 mg/kg BW), immune-related organs (spleen, thymus, and bursal) were separated and weighed to calculate the organ indexes (organ weight/body weight, %). Then, ileal segments were fixed in 4% paraformaldehyde solution for morphological analysis, and ileal mucosa was collected and stored at -80°C after snap-frozen for further genes quantification.

Serum Diamine Oxidase (DAO) Analysis

All serum samples were thawed and completely mixed before analysis. The serum DAO was measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (HY-60106, Beijing Sino-UK Institute of Biological Technology, Beijing, China).

Intestinal Morphology Analysis

Intestinal villus height and crypt depth were measured according to previous report (Qiu et al., 2021). Fixed intestinal tissues were dehydrated through a graded ethanol series (70–100%), then embedded in paraffin wax. Serial sections (5 μm of thickness) were cut by rotary microtome (LEICA RM2135, Leica Microsystems, CA), and stained with hematoxylin and eosin. Morphological structures of intestine were observed and evaluated under the bright field (100 ×) on a microscope (Carl Zeiss Microscopy LLC, NY). Five vertically crosscutting sections were selected for each sample. The vertical distances from the villous tip to villous–crypt junction was regarded as villus height, and from the villous–crypt junction to the lower limit of the crypt was seen as crypt depth. Ten well-orientated villi and their associated crypt per section were measured using an image analyzer (Lucia Software, Lucia, Za Drahou, Czechoslovakia). The mean value of 50 measurements per sample was submitted for statistical analysis.

RNA Extraction and Quantitative Real-Time PCR

Total RNA of ileal mucosa was extracted by Trizol reagent (Invitrogen, CA) following the RNA extraction instruction, while cDNA was obtained by using PrimeScript RT Kit (Takara, Shiga, Japan). Quantitative real-time PCR was performed according to the SYBR Premix Ex Taq II instructions (Takara, Shiga, Japan) and the reaction was conducted on an ABI-Prism 7500 Real-Time PCR System (Applied Biosystems, CA). Primers for RT-qPCR were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) (Table S2). Amplifications were performed in triplicate for each sample. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was regarded as the housekeeping gene and the relative expressions of target genes to that of the reference gene (GAPDH) was calculated according to the 2−ΔΔCt method.

Statistical Analysis

The data were evaluated using SPSS 19.0 software by two-way ANOVA, and the differences were considered significant at P < 0.05. When a significant dietary effect or an interaction between diet and challenge was observed, the data were further analyzed by using one-way ANOVA with Duncan's post hoc test.

RESULTS

Effects of Dietary Supplementation With HWG on Immune-Related Organs Indexes of Broilers

To evaluate the impacts of the dietary HWG supplementation or E. coli O78 challenge on the immune-related organs (spleen, thymus and bursal) of broilers, the organs were collected and the organ indexes were calculated (Figure 1). The results showed that dietary supplementation with 0.5% or 1% HWG did not alter the immune-related organs indexes of unchallenged broilers (P > 0.05), the spleen index and thymus index of broilers were significantly increased after E. coli O78 challenge (P < 0.05). For dietary HWG effects, 1% HWG supplementation significantly decreased thymus index of E. coli-challenged broilers (P < 0.05), while dietary supplementation with 0.5% HWG had no effects on immune-related organ indexes of E. coli-challenged broilers (P > 0.05).

Figure 1.

Figure 1

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Effects of dietary HWG supplementation on immune-related organs indexes of broilers on d 21 of age.

Spleen index (A), thymus index (B) and bursal index (C). Values are means ± SEM, n = 8. Means without a common letter differ, P < 0.05.

Effects of Dietary Supplementation With HWG on Serum DAO Level of Broilers

As the biomarker of the intracellular permeability, serum DAO level of broilers was tested (Figure 2). For the unchallenged broilers, dietary 0.5% or 1% HWG supplementation did not change the serum DAO activity (P > 0.05). E. coli O78 challenge significantly increased serum DAO activity of broilers (P < 0.05). Dietary 1% HWG supplementation significantly reduced serum DAO activity of E. coli-challenge broilers (P < 0.05), while 0.5% HWG group showed no significant difference (P > 0.05).

Figure 2.

Figure 2

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Effects of dietary HWG supplementation on serum DAO level of broilers on d 21 of age. Values are means ± SEM, n = 8. Means without a common letter differ, P < 0.05. Abbreviation: DAO, diamine oxidase.

Effects of Dietary Supplementation With HWG on Ileal Morphological Structure of Broilers

To identify intestinal morphological integrity after dietary HWG supplementation or E. coli challenge, ileum segments of broilers were fixed and evaluated. As shown in Figure 3, dietary supplementation with 0.5% or 1% HWG exhibited few significant modifications on the ileal morphological structure of unchallenged broilers. E. coli O78 challenge induced mucosal structure damage, which was alleviated by dietary 0.5% or 1% HWG supplementation for broilers, including the villi height and villi height/crypt depth ratio.

Figure 3.

Figure 3

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Effects of dietary HWG supplementation on ileal morphology of broilers on d 21 of age.

Ileum of 0% HWG group (A), 0.5% HWG group (B), 1% HWG group (C), 0% HWG + E. coli group (D), 0.5% HWG + E. coli group (E) and 1% HWG + E. coli group (F). Villi height (G), crypt depth (H) and villi height/crypt depth ratio (I) of ileal intestine. HWG, hydrolyzed wheat gluten; E. coli, E. coli O78. Values are means ± SEM, n = 6. Means without a common letter differ, P < 0.05.

Effects of Dietary Supplementation With HWG on mRNA Levels of Tight Junctions in the Ileum of Broilers

As the important components of intestinal barrier functions, the relative mRNA levels of tight junctions in the ileum of broilers were quantified (Figure 4). Dietary 0.5% or 1% HWG supplementation significantly upregulated the relative mRNA level of ZO-1 (P < 0.05), while not caused any changes in relative mRNA levels of Occludin and Claudin-1 in the ileum of unchallenged broilers (P > 0.05). In addition, the relative mRNA levels of Occludin, Claudin-1, and ZO-1 were reduced in the ileum of broilers after E. coli O78 challenge (P < 0.05). Although dietary supplementation with 0.5% HWG showed no differences in relative mRNA levels of tight junctions in E. coli-infected broilers (P > 0.05), dietary supplementation with 1% HWG significantly upregulated the relative mRNA levels of Claudin-1 and ZO-1 in the E. coli-challenged broilers (P < 0.05).

Figure 4.

Figure 4

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Effects of dietary HWG supplementation on mRNA levels of ileal tight junctions of broilers on d 21 of age.

Relative mRNA expressions of Occludin (A), Claudin-1 (B) and ZO-1(C). ZO-1, zonula occluden-1. Values are means ± SEM, n = 8. Means without a common letter differ, P < 0.05.

Effects of Dietary Supplementation With HWG on Relative mRNA Levels ofInflammatory Cytokines in Ileum of Broilers

To investigate the immune response to the dietary HWG supplementation or E. coli challenge, relative mRNA levels of inflammatory cytokines in the ileum were measured (Figure 5). As shown, dietary supplementation with 0.5% or 1% HWG did not alter the relative mRNA levels of inflammatory cytokines (TNF-α, IFN-γ, IL-1β, IL-6, IL-8, and TLR4) of the unchallenged broilers (P > 0.05), while E. coli O78 challenge significantly increased the relative mRNA levels of these inflammatory cytokines (P < 0.05). In addition, dietary supplementation with 0.5% HWG significantly reduced the relative mRNA levels of IFN-γ, IL-1β, and TLR4 in E. coli-challenged broilers (P < 0.05), while no significant effects were found of the TNF-α, IL-6, and IL-8 (P > 0.05). Dietary supplementation with 1% HWG significantly decreased the relative mRNA level of IFN-γ in E. coli-challenged broilers (P < 0.05), while no significant effects were found on the relative mRNA levels of TNF-α, IL-1β, IL-6, IL-8, and TLR4 (P > 0.05).

Figure 5.

Figure 5

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Effects of dietary HWG supplementation on ileal mRNA levels of inflammatory cytokines of broilers on d 21 of age.

Relative mRNA expressions of TNF-α (A), IFN-γ (B), IL-1β (C), IL-6 (D), IL-8 (E) and TLR4 (F). Values are means ± SEM, n = 8. Means without a common letter differ, P < 0.05. Abbreviations: IFN-γ, interferon-γ; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-8, interleukin-8; TLR4, Toll like receptor-4; TNF-α, tumor necrosis factor-α.

DISCUSSION

The intestinal structural and functional development of broilers in the early stage are vulnerable to pathogenic bacteria invasion, which can induce the intestinal mucosal damage and barrier dysfunction (Shao et al., 2013). Pathogenic E. coli infection is one of the most prominent problems with high morbidity and mortality in broiler industries, severely suppressing the growth of broilers and lowering the economic benefits (Kim et al., 2020). Previous studies indicate that early nutritional interventions can effectively promote immunological maturation, enhance intestinal barrier functions, and inhibit pathogens colonization, which consequently improve the broilers growth performance (Zhang et al., 2017; Lee et al., 2018). Hydrolyzed wheat gluten (HWG) is a protein mixture with high digestibility, comprising plentiful peptides and various functional amino acids, such as Gln, Glu and Gly (Wang et al., 2006). Although HWG has been widely used in piglets, calves, aquaculture and pets, few studies have been conducted in poultry industries. In this study, 2 doses of HWG were supplemented into the diets to explore the protective effects on intestinal barrier functions and immune response of broilers based on the E. coli-challenge model.

In general, pathogenic E. coli-infection is associated with reduced feed intake and intestinal mucosal damage (Mellata, 2013). In the present study, we found that E. coli O78 infection resulted in increased immune-related organs indexes (spleen index and thymus index) and impaired gut mucosal morphology of broilers, indicating that animal model of E. coli infection has been successfully established (Fadl et al., 2020). Previous studies have demonstrated that functional amino acids, like Gln and tryptophan (Trp), exerted positive role in anti-inflammation and cell proliferation, which might account for the amelioration of immune organic compensatory hypertrophy and the intestinal mucosal impairment of E. coli O78-induced broilers (Maiorka et al., 2016; Wang et al., 2020).

The intestinal permeability largely depends on the intestinal barrier integrity (Kelly et al., 2015). Intracellular DAO, serving as a biomarker of intestinal permeability, can be released into the circulation in large quantities, resulting in high level DAO in the blood when the intestinal barrier is damaged (Cai et al., 2019). In our study, elevation of serum DAO level after E. coli-challenge suggested that the intestinal mucosal barrier was damaged (Ding et al., 2019). The reduced serum DAO level in E. coli-challenged broilers after dietary 1% HWG supplementation can be inferred that Gln and bioactive small peptides in HWG were utilized by intestinal epithelial cells to maintain intestinal mucosal integrity and barrier functions (Zhou et al., 2019).

Tight junctions, including claudins, occludin, junctional adhesion molecule (JAM) and tricellulin, have been identified as main transmembrane proteins, interacting with cytosolic scaffold proteins, such as zonula occludens (ZO), corporately responsible for intestinal barrier functions and permeability (Suzuki, 2013). In this study, the mRNA upregulation of ZO-1 in the ileum of unchallenged broilers indicated that HWG could promote intestinal physical barrier under the general physiological conditions (Zihni et al., 2016). Previous studies have reported that pathogenic E. coli infection reduced the tight junctions and increased the intestinal permeability (Bhat et al., 2020; Bucker et al., 2020). In the current study, downregulated mRNA levels of Claudin-1 and ZO-1 by E. coli-challenge were prevented by dietary 1% HWG supplementation, suggesting that HWG could elevate the gene expressions of tight junctions to protect intestinal barrier integrity from the E. coli O78 invasion (Apper et al., 2016). In addition, the protective effects of Gln and Gly on epithelial tight junctions have been observed in our previous studies, in which dietary HWG may exert the main roles in the barrier defense (Wang et al., 2015; Li et al., 2016).

Accumulating evidence highlights that intestinal epithelial cells not only serve as a barrier, but also integrate the external and internal signals to coordinate the immune responses (McGuckin et al., 2009; Rescigno, 2011). The intestinal barriers impairment induced by pathogens or toxins are commonly companied with the inflammation (Camilleri et al., 2012). Generally, pathogenic E. coli recognizes the membrane TLR4 with its unique lipopolysaccharide (LPS) and activates downstream signaling pathway to produce large number of inflammatory cytokines, therefore triggering the inflammation (Keestra et al., 2013). In the present study, mRNA levels of ileal TLR4 and inflammatory cytokines (TNF-α, IFN-γ, IL-1β, IL-6, and IL-8) were elevated after E. coli O78 infection, indicating that E. coli infection triggered the inflammatory response of broilers (Wang et al., 2016). Interestingly, the upregulated mRNA levels of ileal IFN-γ, IL-1β, and TLR4 were abolished in E. coli-infected broilers by dietary HWG supplementation, suggesting that HWG might inhibit TLR4 signaling pathway to suppress the production of inflammatory cytokines, consequently alleviated the E. coli-induced inflammation (Liu et al., 2017). Previous studies have demonstrated that functional amino acids, such as Gln and Gly, could relieve the LPS-induced intestinal injury (Xu et al., 2018; Zhang et al., 2019). In the present study, high proportion of Gln, Gly and proline in the HWG may be responsible for the intestinal inflammatory alleviation of E. coli-infected broilers. Moreover, bioactive peptides (i.e., Gly-Glu), abundant in HWG, have been proved to mitigate the LPS-induced inflammation via depressing the production of proinflammatory cytokines (Jiang et al., 2009).

Taken together, dietary HWG supplementation depressed inflammatory response of E. coli-infected broilers by reducing the mRNA levels of TLR4 and inflammatory cytokines (TNF-α, IFN-γ, IL-1β, IL-6, and IL-8), alleviated intestinal mucosal damage and barrier dysfunctions of E. coli-challenged broilers through upregulating the mRNA levels of tight junctions (Claudin-1 and ZO-1). Our findings will provide the guidelines for the HWG application in poultry industries. However, more insightful mechanisms of the protective effects of HWG on the E. coli infection need further investigation.

ACKNOWLEDGMENTS

Funding Sources: This work was funded by the National Natural Science Foundation of China (No. 31572412, 31572410, 31625025, 31272450, and 31272451) and the “111” Project (B16044).

Ethical Approval: This research involves only animals (broilers), without any studies involving human participants. The animal use protocols in this study were reviewed and approved by the China Agricultural University Animal Care and Use Committee (Beijing, China).

DISCLOSURES

The authors declared no conflicts of interests.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2021.101615.

Appendix. Supplementary materials

mmc1.docx (17.5KB, docx)

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