Xylo-oligosaccharides enhance intestinal and thymic immunity by modulating pyroptosis, gut microbiota, and Th17/Treg immune response in lipopolysaccharide-challenged piglets

Abstract

Xylo-oligosaccharides (XOS) have been shown to improve the immune system of weaned piglets, but the molecular mechanism of their action remains unclear. Therefore, this study aimed to investigate the impact of XOS on intestinal and thymic immune function in weaned piglets challenged with lipopolysaccharide (LPS) and elucidate the underlying mechanism. In a 2 × 2 factorial arrangement, consisting of diet treatment (basal diet vs 0.02% XOS diet) and immunological challenge [saline vs LPS], 24 piglets were randomly divided into 4 groups (n = 6): CON group, basal diet + saline; LPS group, basal diet + LPS; XOS group, 0.02% XOS diet + saline; XOS_LPS group, 0.02% XOS diet + LPS. Piglets were fed either the basal or XOS diet for 21 d, followed by intraperitoneal injections of normal saline or LPS on the 22nd day. Ileum, thymus, and colon samples were collected 4 h after the intraperitoneal saline or LPS injection. The piglets fed the XOS diet had higher average daily feed intake and average daily weight gain (P < 0.05). The XOS diet increased ileal villus height and decreased crypt depth. XOS also enhanced ileal and thymic antioxidant enzyme activities, anti-inflammatory cytokine expression, and decreased malondialdehyde levels and mRNA abundance of pro-inflammatory cytokines in piglets (P < 0.05). The XOS diet also downregulated the ileal and thymic NOD-like receptor family pyrin domain containing 3 and gasdermin-D gene and protein expression associated with pyroptosis (P < 0.05). Moreover, The XOS diet increased the mRNA abundance of forkhead box P3, signal transducer and activator of transcription 5, and transforming growth factor beta 1 while decreasing signal transducer and activator of transcription 3 and retinoid-related orphan receptor-gammat mRNA abundance (P < 0.05). The XOS diet enhanced forkhead box P3 protein expression and reduced retinoid-related orphan receptor-gammat protein expression following the LPS challenge (P < 0.05). At the same time, The XOS diet affected the gut microbiota and increased levels of short-chain fatty acids (P < 0.05). In conclusion, XOS may modulate ileal and thymic immune function in weaned piglets following a 4-h LPS challenge by affecting gut microbiota, pyroptosis, and Th17/Treg immune responses.

Dietary xylo-oligosaccharides can enhance ileal and thymic immunity in weaned piglets by modulating pyroptosis, Th17/Treg immune response, and intestinal microorganisms.

Introduction

The intestine is the largest immune organ in the animal body and plays an important role in regulating immune function, promoting digestion and absorption of nutrients, and defending against the invasion of viruses and antigens. The immune system of piglets is not fully developed, and they are vulnerable to attacks by pathogens, which affects the immune homeostasis of the intestine, further causes intestinal villus atrophy, induces intestinal oxidative stress and inflammatory response, and destroys the integrity of the intestinal barrier (Campbell et al., 2013). The thymus is an essential component of the immune system in piglets, performing critical functions in the proliferation, differentiation, and maturation of T cells, specifically CD4⁺ helper T cells and CD8⁺ cytotoxic T cells. In the thymus, immature thymocytes undergo a highly regulated development process, progressing from double-negative to double-positive stages before becoming mature single-positive CD4⁺ or CD8⁺ T cells, which are then released into peripheral tissues. Through these processes, the thymus plays a crucial role in establishing and regulating cell-mediated immune responses (Wang et al., 2020). When exposed to unfavorable stimuli, the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome assembles and activates gasdermin-D (GSDMD), resulting in the release of substantial pro-inflammatory cytokines. This cascade ultimately leads to cellular death, known as pyroptosis (Bergsbaken et al., 2009). Pyroptosis is a form of programmed cell death that exhibits inflammatory properties and holds significant importance in the immune response (Yu et al., 2021b). Our previous study demonstrated that pyroptosis induced by lipopolysaccharide (LPS) not only exacerbates the disorder of thymic immune function in piglets but also triggers a thymic inflammatory response (Liu et al., 2023b). Other studies have demonstrated that inhibiting pyroptosis can alleviate inflammation, restore immune function, and reduce oxidative stress (Liu et al., 2022b; Wang et al., 2023; Xiao et al., 2023). Therefore, targeting the pyroptosis-related signaling pathway may be a potential alternative for improving intestinal and thymic immune function in piglets.

The regulation of the immune system is significantly influenced by the Th17/Treg immune response. The Th17 and Treg cells are both subsets of CD4⁺ T cells (Zhang et al., 2021). Th17 cells have a pro-inflammatory function primarily through the secretion of cytokines such as interleukin-17 (IL-17) and IL-21 (Korn et al., 2009). The differentiation of Th17 cells is induced by transforming growth factor beta (TGFB), which collaborates with the signal transducer and activator of transcription 3 (STAT3) to activate the retinoid-related orphan receptor-gammat (RORγt; Chaudhry et al., 2009). The promotion of CD4⁺ T cells to the Th17 cell lineage is facilitated by the upregulation of RORγt, a crucial protein involved in Th17 cell differentiation (Veldhoen et al., 2006). Conversely, Treg cells, which are regulated by the transcription factor forkhead box P3 (FOXP3), suppress the function of Th17 cells and secrete anti-inflammatory cytokines (IL-10 and TGFB) to attenuate the inflammatory response (Raffin et al., 2020). Furthermore, the impact of TGFB on Treg cell differentiation requires the initial activation of STAT5, which subsequently triggers the expression of FOXP3, ultimately leading to Treg cell differentiation (Ohkura et al., 2012). There are few reports on the study of Th17/Treg immune response in piglet thymus. Previous research on Th17/Treg in piglets mainly focuses on intestinal immunity. For example, adding chitosan oligosaccharides, α-ketoglutaric acid and glutamate to piglet diets can effectively regulate Th17/Treg immune responses and improve intestinal immune function (Yu et al., 2021a; Liu et al., 2023a, 2023c). Therefore, it is reasonable to infer that Th17/Treg immune response may be a potential target for restoring thymic immune function.

The impact of intestinal microorganisms on immune function is widely recognized (Peng et al., 2021). Previous studies have confirmed a close relationship between the Th17/Treg immune response and intestinal microflora (Omenetti and Pizarro, 2015). The differentiation of Th17 cells and Treg cells requires the participation of specific microorganisms (Atarashi et al., 2011; Omenetti and Pizarro, 2015). In mice that were colonized by 17 species of Clostridium, the level of Treg cells significantly increased. This resulted in the synthesis and release of IL-10, which alleviated 2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice (Atarashi et al., 2013). In addition, metabolites of gut microbes, such as short-chain fatty acids (SCFA), are translocated to the thymus, affecting the development of T cells in the thymus (Hebbandi Nanjundappa et al., 2022). For pyroptosis, existing studies support the potential effects of intestinal microorganisms on it (Zhao et al., 2023). Intestinal microorganisms may inhibit the activation of NLRP3 through SCFA to inhibit the occurrence of cell pyroptosis (Bao et al., 2024). Conversely, pathogenic bacteria in the intestine can activate cell pyroptosis by upregulating the expression of NRLP3 by producing LPS (Bao et al., 2024; Liu et al., 2024). Therefore, alterations in the intestinal microflora may impact the intestinal and thymic immune function of the piglets.

Xylo-oligosaccharides (XOS) are primarily found in agricultural byproducts, such as corn cobs, sugarcane bagasse, cottonseed hulls, wheat bran, wheat straw, rice straw, rice husks, and bark. As a functional oligosaccharide, XOS plays a crucial role in immune regulation, antioxidation, and antibacterial activity (Chen et al., 2021a). An in vitro study reported that XOS promotes macrophage proliferation, reduces levels of malondialdehyde (MDA), tumor necrosis factor-alpha, IL-1β, and IL-6 induced by LPS, and enhances superoxide dismutase activity (Liu et al., 2023e). In mice, XOS reduced systemic inflammation, modulated gut microbiota, and increased SCFA content (Hansen et al., 2013). Broiler chickens fed XOS diets showed improved growth performance by enhancing intestinal barrier function, modulating microbiota, and exerting positive effects on immunity (Yuan et al., 2018; Chen et al., 2021b; Wang et al., 2022). Studies on piglets have shown that XOS can reduce the expression of intestinal pro-inflammatory cytokines, promote the secretion of IL-10 and sIgA, inhibit the activation of pro-inflammatory signaling pathways, regulate intestinal microorganisms, and increase the content of SCFA (Yin et al., 2019; Pang et al., 2021; Wang et al., 2021). However, the impact of XOS on thymic immune function in piglets is not well understood. At present, there is no report on the effect of XOS on Th17/Treg immune response. Therefore, the aim of this study is to investigate the effect of XOS on the intestinal and thymic immunity of piglets after LPS challenge and to explore its possible mechanisms.

Material and Methods

Animal and experimental design

Sichuan Agricultural University Animal Care and Use Committee approved the use of experimental animals and animal-related experimental procedures (SICAU-2022-05). The study is a complete randomized design with a 2 × 2 factorial arrangement: diet treatment (basal diet vs 0.02% XOS diet) and immunological challenge (saline vs LPS). Twenty-four piglets (Duroc × Landrace × Yorkshire), weaned at 21 ± 1 d of age, were randomly assigned to 4 treatment groups (n = 6). The 4 treatment groups were as follows: the basal diet + saline (CON group); 0.02% XOS diet + saline (XOS group); the basal diet + LPS (LPS group); 0.02% XOS diet + LPS (XOS_LPS group). First, 24 piglets were randomly divided into 2 groups, one group was fed with basal diet and the other group was fed with XOS diet for 21 d, during which the daily feed intake of each piglet was recorded. On the 22nd d, all piglets were weighed after fasting for 12 h, and then half of the piglets fed with basal diet or XOS diet were intraperitoneally injected with 100 µg/kg body weight E. coli LPS (055: B5; Sigma, USA) or the same volume of saline, thereby generating the above 4 treatment groups. The basal diet (Table 1) was formulated to meet the nutritional requirement standard of Southern and Adeola (2012) for piglets. XOS (containing 35% XOS with 65% maltodextrin as the carrier) was made from birch wood and obtained from Yibin Yatai Biotechnology Co., Ltd. (Sichuan, China). The 0.02% XOS addition was selected based on a previous study and the supplier’s recommendation (Wang et al., 2021). During the experimental period, piglets were raised in cages (1.1 × 2.0 m) and had access to the diet and water ad libitum. The room temperature was maintained at 26 ± 3 °C. The injection dose of LPS was based on our previous study (Liu et al., 2023b). Four hours after LPS and saline treatment, all piglets were euthanized in a humane manner (electric shock), and thymus, ileum, and colonic digesta samples were collected and stored in liquid nitrogen for further analysis.

Table 1.

Composition and nutrient levels of basal diet

Ingredient	Content of basal diet, %	Ingredient	Content of XOS diet, %
Corn	32.15	Corn	32.15
Extruded Corn	27.54	Extruded Corn	27.54
Soybean oil	1.72	Soybean oil	1.80
Glucose	1.70	Glucose	1.60
Whey powder	5.00	Whey powder	5.00
Dehulled Soybean meal (46% CP)	13.24	Dehulled Soybean meal (46% CP)	13.24
Soybean protein concentrate	4.00	Soybean protein concentrate	4.00
Extruded soybean	8.00	Extruded soybean	8.00
Fish meal (67% CP)	3.00	Fish meal (67% CP)	3.00
L-Lysine-HCl (78.8%)	0.57	L-Lysine-HCl (78.8%)	0.57
DL-Methionine (99%)	0.11	DL-Methionine (99%)	0.11
L-Threonine (98.5%)	0.21	L-Threonine (98.5%)	0.21
L-Tryptophan (98%)	0.02	L-Tryptophan (98%)	0.02
		Xylo-oligosaccharides	0.02
Choline chloride (50%)	0.15	Choline chloride (50%)	0.15
Limestone	0.64	Limestone	0.64
Monocalcium phosphate	1.35	Monocalcium phosphate	1.35
NaCl	0.30	NaCl	0.30
Vitamin premix1	0.05	Vitamin premix1	0.05
Mineral premix2	0.25	Mineral premix2	0.25
Total	100.00	Total	100.00

Nutrient level3	Content, %
Digestible energy, Mcal/kg	3.54
Crude protein, %	19.47
Calcium, %	1.16
Total phosphorus, %	0.66
SID-Lysine, %	1.38
SID-Methionine, %	0.41
SID-Threonine, %	0.81
SID-Tryptophan, %	0.21

¹The vitamin premix provides the following per kilogram of diet: VA 18,000 IU; VD3 6,000 IU; VE 48 IU; VK3 6 mg; VB1 6 mg; VB2 15 mg; VB6 7.2 mg; VB12 720 μg; D-pantothenic acid 30 mg; nicotinic acid 60 mg; folic acid 3 mg; biotin 3 mg.

²The mineral premix provides the following per kilogram of diet: copper (CuSO4 •5H2O) 6 mg; iron (FeSO4•H2O) 100 mg; zinc (ZnSO4 •H2O) 100 mg; manganese (MnSO4•H2O) 4 mg; iodine (KI) 0.14 mg; selenium (Na2SeO3) 0.3 mg.

³Nutrient levels are calculated values.

Intestinal morphology analysis

Ileal tissue sections were prepared by Wuhan Servicebio Company (Wuhan, Hubei, China). Briefly, ileum samples were fixed with 4% paraformaldehyde for 24 h and embedded in paraffin, and then cut into 5-μm-thick sections. The sections were then stained with hematoxylin-eosin. Villus height and crypt depth in intestinal sections were observed under a light microscope. Villus height and crypt depth were measured in 30 intact villi in each section sample.

Antioxidant status

Similar to our previous study (Liu et al., 2023b), the ileum and thymus samples were homogenized with phosphate-buffered saline at the ratio of 1:9 (wt:vol). The supernatant was separated by centrifugation to determine the concentration of protein for analysis. Following the manufacturer’s advice (Nanjing Jiancheng, China), the concentration of MDA and the activities of catalase (CAT), glutathione (GSH), glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD) were detected in the ileum and thymus of piglets by commercial assay kits.

ELISA assay

The protein contents (IL-6, ml025981; TGF-β1, ml002363) in the ileum of piglets were determined by the commercial ELISA kits of Shanghai Enzyme Union Biotechnology Co., Ltd (Shanghai, China). The ELISA assay was consistent with our previous experiment (Gu et al., 2021). The absorbance of the sample was measured at a wavelength of 450 nm following the manufacturer’s instructions. Subsequently, the protein concentration of IL-6 and TGF-β1 was calculated.

Real-time qPCR analyses

According to our previous study (Liu et al., 2022b), total RNA from the ileum and thymus was extracted with the TRIzol reagent. The cDNA was synthesized through the extraction of total RNA using the Prime Script RT kit (TaKaRa, China). Real-time qPCR assays were run with SYBR® Green I PCR Reagent Kit (TaKaRa, China) by the Prism Sequence Detection System (7900HT ABI, Applied Biosystems, USA). The relative mRNA abundance was calculated by 2^−ΔΔCt method (normalized to β-actin). The primer sequence is shown in Table S1.

Western blotting analysis

Western blot analysis was based on a previous study (Liu et al., 2023a). The ileum or thymus samples (0.1 g) were homogenized in 900 µL of RIPA lysis buffer containing 1% phenylmethanesulfonyl fluoride and 2% phosphatase inhibitor cocktail A at 4 °C. The ileum and thymus homogenates were detected for total protein and separated on 10% polyacrylamide gels. Electrophoresis was performed at 80 V for 30 min and then at 120 V for 90 min until bromophenol blue reached approximately 1 cm from the bottom of the gel, and the proteins were transferred to polyvinylidene fluoride membranes (Millipore; Billerica, MA, USA). The membranes were then incubated with specific primary and secondary antibodies (SA00001-2; 1:2500; RRID: AB_2722564; Proteintech, Wuhan, China) for immunoblotting. The primary antibody was diluted in primary antibody diluent (P0023A-100ml; Beyotime, Shanghai, China). The specific primary antibodies include FOXP3 (bs-10211R; 1:1000; Bioss, Beijing, China), RORγt (13205-1-AP; 1:1000; RRID: AB_2180306; Proteintech, Wuhan, China), GSDMD (DF12275; 1:1000; RRID: AB_2845080; Affinity Biosciences, Jiangsu, China) and NLRP3 (19771-1-AP; 1:1000; RRID: AB_10646484; Proteintech, Wuhan, China). The membrane was finally immersed in an ECL chemiluminescence kit (Beyotime, Shanghai, China) for visualization.

Colonic chyme SCFA determination

According to our previous study, SCFA were isolated and determined from colonic digesta samples in a gas chromatogram with a membrane-thickness capillary column (Liu et al., 2022a). Take 0.7 g of sample and mix with 1.5 mL of ultrapure water, collect the supernatant after centrifugation; take 1 mL of supernatant, add 0.2 mL of 25% (w/v) metaphosphoric acid solution and 23.3 μL of 210 mmol/L crotonic acid solution, mix, and centrifuge; take 0.3 mL of supernatant and add 0.9 mL of chromatographic methanol (1:3 dilution), centrifuge again, and take the supernatant and filter it with a 0.22 μm filter membrane. Then take 1 μl of the filtrate for capillary gas chromatography analysis (Capillary column: 30 m × 0.53 mm × 1 μm).

16S rRNA analysis

The colonic digesta 16S rRNA analysis method in this study was identical to that used in our previous study (Liu et al., 2022a). Genomic DNA was extracted using the FastDNA® Spin Kit (MP Biomedicals, Irvine, CA, United States). The extracted genomic DNA was assessed for both concentration and purity using 1% agarose gel electrophoresis. Amplification of the 16S rRNA gene was performed using an ABI Gene Amp 9700 PCR Thermocycle Instrument (Applied Biosystems, Inc., Carlsbad, CA, United States). Subsequently, PCR products from each sample were visually identified through electrophoresis on a 2% agarose gel. To recover the PCR products, the AxyPrepDNA Gel Recovery Kit (Axygen, Union City, CA, United States) was utilized. The concentration-normalized PCR products were then subjected to sequencing using the Miseq platform (Illumina Inc., San Diego, CA, United States).

Statistical analysis

16S rRNA analysis

The pair-end reads obtained from Miseq sequencing are merged together, and the quality of the sequence is carefully controlled and screened. FLASH software (version 1.2.11) was used to splice paired-end reads: 1) the tail bases with quality values less than 20 were filtered, low-quality regions were truncated, and reads shorter than 50 bp and containing N were removed; 2) reads were spliced based on a minimum overlap of 10 bp, with an allowable mismatch rate of 0.2; 3) samples were distinguished and the direction was adjusted based on barcode and primers, with no barcode mismatch and a maximum of 2 base mismatches in primers. Usearch (version 7.0.10901) is utilized, with a similarity threshold of 97%, to complete the clustering of operational taxonomic units (OTU). Subsequently, the 16S rRNA gene sequence is compared with the Silva 16S rRNA database (version 1382). The OTU clustering data (version 1.30.24) is employed for the analysis of the alpha diversity index. To assess the presence of significant differences between samples, principal coordinate analysis (PCoA) is carried out using Qiime (version 1.9.15), followed by ANOSIM analysis based on the Bray-Curtis distance.

Growth performance analyses

Growth performance data were analyzed by T-test between the basal diet and the 0.02% XOS diet.

General statistical analyses

Additional data were analyzed using univariate in the general linear model of SPSS 27 (Chicago, USA). The sources of variation were diet (basal diet vs 0.02% XOS diet), LPS (saline vs LPS), and their interaction. When the difference was significant or trend, the data were further analyzed by one-way ANOVA with Duncan’s multiple range tests. The results were expressed as mean ± SEM. P < 0.05 is set as a significant difference, 0.05 ≤ P < 0.10 is set as a trend.

Correlation analysis

The Pearson correlation test was used to assess the correlation between the different indicators. Correlation analysis of a certain intestinal microorganism is only performed when its interactions are significant or have a trend.

Results

Effects of dietary XOS on piglet growth performance

The growth performance of piglets is shown in Figure 1. There was no significant effect on the feed conversion rate (FCR) of piglets fed XOS diets (P > 0.05). However, piglets fed the XOS diet had significantly higher average daily feed intake (ADFI; P < 0.05) and average daily weight gain (ADG; P < 0.05).

Figure 1.

Effects of dietary xylo-oligosaccharides on growth performance of piglets. (A) Dietary xylo-oligosaccharides increases average daily weight gain in piglets. (B) Dietary xylo-oligosaccharides increases average daily feed intake in piglets. (C) Dietary xylo-oligosaccharides had no effect on feed conversion rate. Values are mean ± stand error (n = 12). Different superscript letters indicate P < 0.05 by T-test. CON, the basal diet; XOS, 0.02% XOS diet.

Effects of XOS on ileal morphology of weaned piglets after LPS challenge

The roles of XOS on the ileal morphology of piglets are shown in Table 2 and Figure S1. Significant interactions were observed in villus height and the ratio of villus height to crypt depth (V:C) between XOS and LPS (P < 0.01). Compared with the CON group, XOS increased villus height and the V:C, and decreased crypt depth (P < 0.01); LPS reduced villus height and V:C (P < 0.01).

Table 2.

Effects of xylooligosaccharides on the morphology of the ileum in weaned piglets after LPS challenge

Item	Saline		LPS		SEM	P-value
	CON	XOS	CON	XOS		Diet	LPS	Interaction
Villus height, μm	242.86b	382.97a	162.84d	205.56c	2.208	<0.010	<0.010	<0.010
Crypt depth, μm	250.48b	149.57c	416.38a	283.69b	12.676	<0.010	<0.010	0.224
V:C	0.98b	2.57a	0.40d	0.73c	0.013	<0.010	<0.010	<0.010

The data are presented as the mean ± pooled standard error (n = 6). Different superscript letters within the same row indicate statistical significance at P < 0.05 based on one-way ANOVA. CON, the basal diet; XOS, 0.02% xylooligosaccharide diet; LPS, lipopolysaccharide; V:C, the ratio of villus height to crypt depth.

Effects of dietary XOS on antioxidant status of the ileum and thymus in piglets after LPS challenge

The indexes related to the antioxidant status of piglets are shown in Table 3. In the ileum, compared with the CON group, LPS increased MDA levels, but decreased the activities of T-SOD, CAT, GSH-Px, and the levels of GSH (P < 0.01). Compared with CON group, XOS increased the activities of T-SOD, CAT, GSH-Px, and the levels of GSH, but decreased the content of MDA (P < 0.01). Significant interactions between XOS and LPS were found in CAT and T-SOD (P < 0.01).

Table 3.

Effect of xylo-oligosaccharides on antioxidant index of the ileum and thymus of weaned piglets challenged by lipopolysaccharide

Item	Saline		LPS		SEM	P-value
	CON	XOS	CON	XOS		Diet	LPS	Interaction
Ileum
MDA, nmol/mg prot	2.84c	2.23d	4.15a	3.41b	0.079	<0.010	<0.010	0.443
T-SOD, U/mg prot	100.60c	133.44a	51.29d	110.77b	2.232	<0.010	<0.010	<0.010
CAT, U/mg prot	28.27c	47.83a	23.04d	34.38b	1.005	<0.010	<0.010	<0.010
GSH, μmol/g prot	10.84b	16.15a	6.12c	13.66ab	1.307	<0.010	0.012	0.403
GSH-Px, U/mg prot	942.29b	1184.57a	675.63c	968.64ab	49.789	<0.010	<0.010	0.616
Thymus
MDA, nmol/mg prot	13.92b	8.94c	20.41a	11.92b	0.742	<0.010	<0.010	0.028
T-SOD, U/mg prot	221.32c	259.30a	159.55d	238.06b	6.153	<0.010	<0.010	0.022
CAT, U/mg prot	43.42b	90.43a	13.95c	52.01b	4.501	<0.010	<0.010	0.332
GSH, μmol/g port	75.74b	129.40a	16.36c	62.97b	4.047	<0.010	<0.010	0.394
GSH-Px, U/mg prot	1761.20b	2277.18a	754.06d	1343.95c	129.633	<0.010	<0.010	0.779

^a,b,cDifferent superscript letters in the same row indicate P < 0.05 by one-way ANOVA.

CAT, catalase; CON, the basal diet; GSH, glutathione; GSH-Px, glutathione peroxidase; LPS, Intraperitoneal injection of lipopolysaccharide; MDA, malondialdehyde; T-SOD, total superoxide dismutase; XOS, 0.02% xylo-oligosaccharide diet. Values are mean ± pooled standard error (n = 6).

In the thymus, the piglets treated with LPS had higher levels of MDA and lower activities of T-SOD, CAT, GSH, and GSH-Px (P < 0.05). In contrast, the piglets fed the XOS diet had lower levels of MDA and higher activities of T-SOD, CAT, GSH, and GSH-Px (P < 0.05). In the thymus, the interaction of diet × LPS was observed in MDA content and T-SOD activity (P < 0.05), but not in CAT, GSH, and GSH-Px (P ≥ 0.05).

Effects of dietary XOS on inflammatory cytokines gene levels of the ileum and thymus in piglets after LPS challenge

The gene expression of inflammatory cytokines is shown in Table 4. In the ileum, in comparison to the CON group, the consumption of XOS in the diet led to a decrease in the abundance of pro-inflammatory cytokine mRNA (tumor necrosis factor-α [TNF-α], IL-1β, IL-6, IL-8, IL-17A, IL-21]; P < 0.01). Conversely, it increased the abundance of IL-10 mRNA (P < 0.01). On the other hand, the LPS resulted in an increase in mRNA abundance for pro-inflammatory cytokine (TNF-α, IL-1β, IL-6, IL-8, IL-17A, IL-21) in the ileum of weaned piglets (P < 0.01). However, it led to a decrease in IL-10 mRNA abundance (P < 0.01). Additionally, a significant interaction between XOS and LPS was observed for IL-21 mRNA levels (P < 0.01).

Table 4.

Effects of xylo-oligosaccharide on the ileal and thymic gene expression after LPS challenge

Item	Saline		LPS		SEM	P-value
	CON	XOS	CON	XOS		Diet	LPS	Interaction
Ileum
TNF-α	1.00b	0.50c	1.30a	0.93b	0.057	<0.010	<0.010	0.314
IL-1β	1.00b	0.51c	1.44a	0.84b	0.087	<0.010	<0.010	0.541
IL-6	1.00b	0.41d	1.47a	0.76c	0.050	<0.010	<0.010	0.259
IL-8	1.00b	0.50c	1.42a	0.83b	0.062	<0.010	<0.010	0.463
IL-10	1.00b	1.60a	0.44c	1.15b	0.053	<0.010	<0.010	0.343
IL-17A	1.00b	0.46c	1.35a	0.89b	0.064	<0.010	<0.010	0.528
IL-21	1.00b	0.41c	1.14a	0.93b	0.044	<0.010	<0.010	<0.010
TGF-β1	1.00c	1.27a	0.43d	1.12c	0.036	<0.010	<0.010	<0.010
STAT3	1.00b	0.39c	1.37a	0.91b	0.045	<0.010	<0.010	0.117
STAT5	1.00b	1.28a	0.51c	1.21a	0.056	<0.010	<0.010	<0.010
RORγt	1.00b	0.29c	1.26a	0.86b	0.050	<0.010	<0.010	<0.010
FOXP3	1.00b	1.31a	0.47c	0.90b	0.057	<0.010	<0.010	0.307
NLRP3	1.00b	0.48c	1.41a	0.89b	0.078	<0.010	<0.010	0.950
GSDMD	1.00b	0.46c	1.13a	0.91b	0.056	<0.010	<0.010	<0.010
Thymus
IL-18	1.00c	0.26d	1.42a	1.16b	0.039	<0.010	<0.010	<0.010
IL-10	1.00b	1.47a	0.63c	1.11b	0.069	<0.010	<0.010	0.974
IL-17A	1.00b	0.55c	1.75a	1.08b	0.094	<0.010	<0.010	0.284
IL-21	1.00b	0.64c	1.71a	1.11b	0.096	<0.010	<0.010	0.227
TGFB1	1.00b	1.51a	0.57c	1.14b	0.068	<0.010	<0.010	0.665
STAT3	1.00b	0.43c	1.48a	1.01b	0.067	<0.010	<0.010	0.503
STAT5	1.00c	2.67a	0.48d	1.29b	0.115	<0.010	<0.010	<0.010
RORγt	1.00b	0.44c	1.72a	1.18b	0.085	<0.010	<0.010	0.921
FOXP3	1.00b	2.43a	0.46c	1.30ab	0.184	<0.010	<0.010	0.126
NLRP3	1.00b	0.47c	1.88a	1.02b	0.114	<0.010	<0.010	0.174
GSDMD	1.00c	0.47d	1.39a	1.16b	0.053	<0.010	<0.010	0.012

^a,b,cDifferent superscript letters in the same row indicate P < 0.05 by one-way ANOVA.

CON, the basal diet; IL, interleukin; LPS, Intraperitoneal injection of lipopolysaccharide; XOS, 0.02% xylo-oligosaccharide diet. Values are mean ± pooled standard error (n = 6).

In the thymus, the piglets treated with LPS had lower gene expression of IL-10 and higher gene expression of IL-17A, IL-18, and IL-21 (P < 0.05). Conversely, the piglets fed the XOS diet had higher IL-10 mRNA levels as well as lower mRNA abundance of IL-17A, IL-18, and IL-21 (P < 0.05). There was an interaction of diet × LPS at the mRNA level of IL-18 from the thymus (P < 0.05).

Effects of dietary XOS on the ileum and thymus pyroptosis in piglets after LPS challenge

In the ileum and thymus, the mRNA and protein expression of GSDMD and NLRP3 were higher in the piglets treated with LPS (P < 0.05), but the expression of GSDMD and NLRP3 genes and proteins in piglets fed the XOS diet was downregulated (P < 0.05). The interaction of diet × LPS only affected the mRNA expression of GSDMD (P < 0.05; Table 4; Figures 2 and 3).

Figure 2.

Effects of xylooligosaccharides on pyroptosis and Th17/Treg immune response related protein expression in the ileum of weaned piglets challenged with lipopolysaccharide. (A) Relative protein expression of NLRP3, GSDMD, RORγt and FOXP3 (n = 3). (B) Protein concentrations of IL-6 and TGF-β1. The data are presented as the mean ± standard error (n = 6). Different superscript letters within the same row indicate statistical significance at P < 0.05 based on one-way ANOVA. CON, control group; XOS, the diet was supplemented with xylooligosaccharide group and treated with saline; LPS, basal diet group with Escherichia coli lipopolysaccharide treatment; LPS_XOS, the diet was supplemented with xylooligosaccharide group with Escherichia coli lipopolysaccharide treatment; RORγt, RAR-related orphan receptor c; FOXP3, forkhead box P3; IL-6, interleukin-6; TGF-β, transforming growth factor beta.

Figure 3.

Effects of xylo-oligosaccharides on pyroptosis and Th17/Treg immune response related protein expression in the thymus of weaned piglets challenged with lipopolysaccharide. Values are mean ± standard error (n = 3). Different superscript letters indicate P < 0.05 by one-way ANOVA. CON, the basal diet + saline group; LPS, the basal diet + LPS group; XOS, 0.02% XOS diet + saline group; XOS_LPS, 0.02% XOS diet + LPS group; FOXP3, forkhead box P3; GSDMD, gasdermin-D; NLRP3, NOD-like receptor family pyrin domain containing 3; RORγt, retinoid-related orphan receptor-gammat.

Effects of dietary XOS on Th17/Treg immune response in the ileum and thymus of piglets after LPS challenge

The expression of Th17/Treg immune response-related genes and proteins in the ileum and thymus of piglets is shown in Table 4, Figures 2 and 3. In the ileum, a significant interaction between diet and LPS was observed in the mRNA abundance of TGF-β1, STAT5, and FOXP3 (Table 4; P < 0.01). Compared to the CON group, the inclusion of dietary XOS significantly increased the mRNA abundance of TGF-β1, STAT5, and FOXP3, while it significantly decreased the mRNA abundance of STAT3 and RORγt (P < 0.01). On the other hand, LPS significantly reduced the mRNA abundance of TGF-β1, STAT5, and FOXP3, and significantly increased the mRNA abundance of STAT3 and RORγt (P < 0.01). A significant interaction between diet and LPS was observed in the relative protein expression of FOXP3 (Figure 2; P < 0.01). Compared with the CON group, XOS increased the relative protein expression of FOXP3 and decreased the relative protein expression of RORγt (P < 0.01). However, LPS decreased the relative protein expression of FOXP3 and increased the relative protein expression of RORγt (P < 0.01). XOS significantly enhanced the protein concentration of TGF-β1 and decreased the protein concentration of IL-6 (P < 0.01), which was contrary to LPS (Figure 2; P < 0.01).

In the thymus, the piglets treated with LPS had higher mRNA abundance of STAT3, RORγt, and the protein expression of RORγt; had lower mRNA abundance of TGFB1, STAT5, FOXP3, and the protein expression of FOXP3 (P < 0.05). The piglets fed XOS diet had higher mRNA expression of TGFB1, STAT5, and FOXP3 and the protein expression of FOXP3; lower mRNA expression of STAT3 and RORγt and the protein expression of RORγt (P < 0.05). In the thymus, the mRNA expression of STAT5 showed the interaction of diet × LPS (P < 0.05; Table 4; Figure 3).

Effect of XOS on the composition of gut microbiota in piglets after LPS challenge

All samples of the 4 groups generated 1,289 OTUs, including 1,044 special OTUs in CON group, 935 special OTUs in XOS group, 809 OTUs in LPS group, and 975 OTUs in XOS_LPS group (Figure 4A). Subsequently, the community richness (sobs, chao, ace index) and coverage (coverage index) of each group at the OTU level were analyzed. Significant interaction effects were observed between the diet and LPS administration on the sobs, chao, and ace indices (P < 0.05). In comparison to the CON group, the community richness was lower in the LPS group, as evidenced by lower sobs, chao, and ace indices (P < 0.05). When compared to the CON group, the XOS group also showed a significant reduction in the chao and ace indices (P < 0.05; Figure 4B). The PCoA analysis on the OTU level displayed minimal variation between the CON group and XOS_LPS group, while a considerable dissimilarity was observed between the XOS group and LPS group (Figure 4C).

Figure 4.

Effects of dietary xylo-oligosaccharides treatment on intestinal microbiota of piglets. (A) Bacterial OTUs (operational taxonomic units) level Venn diagrams. (B) sobs, chao, ace and coverage indexes. (C) weighted UniFrac Principal coordinate analysis (PCoA) plot. (D) Phylum level composition of intestinal microbiota in piglets. (E) Genus level composition of intestinal microbiota in piglets. Values are mean ± standard error (n = 5). Different superscript letters indicate P < 0.05 by one-way ANOVA. CON, the basal diet + saline group; LPS, the basal diet + LPS group; XOS, 0.02% XOS diet + saline group; XOS_LPS, 0.02% XOS diet + LPS group.

The relative abundance of gut bacterial communities at the phylum level is depicted in Figure 4D and Supplementary Table S2. At the phylum level, 8 bacteria (relative abundance > 0.1%) were screened, including Firmicutes, Bacteroidota, Actinobacteriota, Spirochaetota, Proteobacteria, Cyanobacteria, Desulfobacterota, and Synergistota. Significant interaction effects between diet and LPS were found in Firmicutes, Bacteroidota, and Synergistota (P < 0.05). The relative abundance of Firmicutes were higher in the piglets treated with LPS (P < 0.05). Additionally, the piglets treated with LPS had lower relative abundance of Synergistota and Bacteroidetes (P < 0.05). In contrast, the piglets fed the XOS diet had lower relative abundance of Firmicutes and a higher relative abundance of Bacteroidetes and Synergistota (P < 0.05).

As shown in Figure 4E and Supplementary Table S2, bacteria with relative abundance > 1% at the genus level were screened. The abundance of Prevotellaceae_NK3B31_group, norank_f__norank_o__RF39, Prevotellaceae_UCG_003 was lower in the piglets treated with LPS, but the abundance of Blautia, Coprococcus, Ruminococcus_gauvreauii_group, Holdemanella, Marvinbryantia was higher (P < 0.05). The piglets treated with LPS showed a trend toward lower abundance of Parabacteroides (P = 0.076), norank_f__Eubacterium_coprostanoligenes_group (P = 0.059), UCG_002 (P = 0.058), and Treponema (P = 0.063). Piglets fed the XOS diet had significantly higher abundances of Terrisporobacter, Turicibacter, UCG_005, Prevotellaceae_NK3B31_group, norank_f__norank_o__RF39, norank_f__Eubacterium_coprostanoligenes_group, Prevotellaceae_UCG_003, and lower the abundance of Lactobacillus, Blautia, Dorea, Holdemanella, Marvinbryantia (P < 0.05). Dietary XOS tended to increase the abundance of Clostridium_sensu_stricto_1 (P = 0.091), Parabacteroides (P = 0.070) and decreased the abundance of Coprococcus (P = 0.066), and Ruminococcus_gauvreauii_group (P = 0.056). Significant interactions between diet and LPS was observed in Clostridium_sensu_stricto_1, Terrisporobacter, Prevotella, Turicibacter, UCG_005, Faecalibacterium, unclassified_f__Lachnospiraceae, Prevotellaceae_NK3B31_group, norank_f__Muribaculaceae, norank_f__Eubacterium_coprostanoligenes_group, Prevotellaceae_UCG_003 (P < 0.05).

Effects of XOS on SCFA composition of piglet colonic digesta after LPS challenge

Table 5 shows the effect of XOS on the composition of SCFA in piglet colonic digesta. The levels of acetate (AA), propionate (PA), isobutyrate (IBA), butyrate (BA), isovalerate (IVA), and valerate (VA) in colonic digesta when piglets were challenged with LPS were lower (P < 0.05). However, the piglets fed the XOS diet had higher levels of AA, PA, IBA, BA, IVA, and VA in the colonic digesta (P < 0.05). Moreover, the interaction between diet and LPS affected the levels of AA, PA, BA, IVA, and VA (P < 0.05).

Table 5.

Effects of xylo-oligosaccharides on SCFA composition in colonic digesta of weaned piglets challenged with lipopolysaccharide

Item (mg/g)	Saline		LPS		SEM	P-value
	CON	XOS	CON	XOS		Diet	LPS	Interaction
AA	3.05b	6.29a	1.34d	2.62c	0.139	<0.010	<0.010	<0.010
PA	1.73b	3.06a	0.64d	1.00c	0.068	<0.010	<0.010	<0.010
IBA	0.03c	0.05a	0.02d	0.04b	0.002	<0.010	<0.010	0.684
BA	1.15b	2.01a	0.41d	0.56c	0.047	<0.010	<0.010	<0.010
IVA	0.09c	0.20a	0.07d	0.13b	0.005	<0.010	<0.010	<0.010
VA	0.21b	0.45a	0.12d	0.16c	0.010	<0.010	<0.010	<0.010

^a,b,cDifferent superscript letters in the same row indicate P < 0.05 by one-way ANOVA.

CON, the basal diet; AA, acetate; BA, butyrate; IBA, isobutyrate; IVA, isovalerate; LPS, Intraperitoneal injection of lipopolysaccharide; PA, propionate; VA, valerate; XOS, 0.02% xylo-oligosaccharide diet. Values are mean ± pooled standard error (n = 6).

Correlation analysis

The correlation analysis in the ileum is shown in Figure S2A. Parabacteroides was positively correlated with FOXP3 and IL-10 and negatively correlated with STAT3, RORγt, NLRP3, GSDMD, TNF-α, IL-1β, IL-6, IL-17A, and IL-21 (P < 0.05). Prevotellaceae_NK3B31_group was found to be positively correlated with IL-10 and FOXP3 (P < 0.05). On the other hand, it showed a negative correlation with STAT3, RORγt, GSDMD, NLRP3, TNF-α, IL-1β, IL-6, and IL-8 (P < 0.05). The content of AA, PA, IBA, BA, IVA, and VA were positively correlated with IL-10, TGFB1, FOXP3, and STAT5, and negatively correlated with STAT3, RORγt, GSDMD, NLRP3, IL-8, IL-17A, TNF-α, IL-1β, IL-6, and IL-21 (P < 0.05). The gene abundance of NLRP3 and GSDMD was significantly positively correlated with the gene abundance of STAT3 and RORγt, but negatively correlated with the gene abundance of STAT5, FOXP3, TGFB1, and IL-10 (P < 0.05).

The correlation analysis in the thymus is shown in Fig. S2B. Parabacteroides was positively correlated with FOXP3, STAT5, and TGFB1 and negatively correlated with STAT3, RORγt, GSDMD and IL-18 (P < 0.05). Prevotellaceae_NK3B31_group was found to be positively correlated with IL-10, FOXP3, STAT5, and TGFB1 (P < 0.05). On the other hand, it showed a negative correlation with STAT3, RORγt, GSDMD, NLRP3, IL-18, IL-17A, and IL-21 (P < 0.05). The content of AA, PA, IBA, BA, IVA, and VA were positively correlated with IL-10, TGFB1, FOXP3, and STAT5, and negatively correlated with STAT3, RORγt, GSDMD, NLRP3, IL-18, IL-17A, and IL-21 (P < 0.05). The gene abundance of NLRP3 and GSDMD was significantly positively correlated with the gene abundance of STAT3 and RORγt, but negatively correlated with the gene abundance of STAT5, FOXP3, and TGFB1 (P < 0.05).

Discussion

Our prior studies showed that dietary XOS can relieve LPS-induced jejunal injury through the endoplasmic reticulum-mitochondrial system pathway in piglets (Liu et al., 2024a). We expanded our current study to the ileum and thymus to examine the role of XOS in immunity with the same animals. XOS possesses a range of biological activities, such as anti-inflammatory, antioxidative, and antibacterial effects (Liu et al., 2023d). After a 21-d feeding experiment, it was observed that XOS supplementation significantly increased the ADG and ADFI of piglets. However, it had no significant effect on FCR. This is consistent with previous studies that have reported positive effects of dietary XOS on piglet growth (Chen et al., 2021b; Pang et al., 2021). Lipopolysaccharide (LPS), the main component of Gram-negative bacteria cell walls (many intestinal pathogens such as Escherichia coli being such bacteria), enables the LPS challenge model to simulate the host’s immune response to bacterial cell wall components during infection. Once in the body, LPS can elicit immune stress (Escribano et al., 2014). In order to evaluate the immunomodulatory properties of XOS, we implemented a porcine model of immune stress by administering intraperitoneal LPS injection. This approach stimulates the secretion of pro-inflammatory cytokines and induces the development of oxidative stress. In the present study, the LPS challenge resulted in lower antioxidant enzyme activity, higher MDA levels, and higher pro-inflammatory cytokine levels. This was accompanied by a decrease in villus height and V:C, as well as an increase in crypt depth. These outcomes validate the effective establishment of the piglet stress model, which is consistent with the findings of our previous research (Liu et al., 2023b). XOS significantly elevates serum activities of GSH-Px, CAT, and SOD, while concurrently reducing MDA levels. This leads to an enhancement of the body’s antioxidant capacity (Pang et al., 2021). Our data demonstrates that XOS supplementation counteracts the adverse effects induced by LPS, underscoring the pivotal role of XOS in preventing oxidative stress and restoring normal villi morphology in piglets. This is similar to the results of another study (Chen et al., 2021b).

LPS prompts an excessive release of pro-inflammatory cytokines, culminating in severe inflammation (Escribano et al., 2014). Our results indicate that dietary XOS alleviates the LPS-induced inflammatory response, characterized by reduced levels of IL-17A and IL-21 in the ileum and thymus, alongside an increase in IL-10 levels. Additionally, XOS reduces the mRNA expression of IL-1β and IL-8 in the ileum, while increasing the mRNA expression of IL-10 (Chen et al., 2021b). Other studies have also observed that XOS decreases pro-inflammatory cytokine expression and elevate IL-10 levels in the blood of piglets (Escribano et al., 2014; Wang et al., 2021). Nevertheless, Yin et al reported that dietary XOS solely decreased serum levels of interferon-gamma, with no effect on IL-1β, IL-6, and IL-10 levels, which may be attributed to XOS dosage variations (Yin et al., 2019). Therefore, considering these findings and the current study’s results, XOS can alleviate the LPS-induced inflammatory response and exert an anti-inflammatory effect.

LPS initiates the NLRP3 inflammasome through mitochondrial dysfunction and the release of reactive oxygen species, which directly triggers pyroptosis. This process culminates in the release of pro-inflammatory cytokines (Zhou et al., 2011). This mechanism was previously confirmed in our research (Liu et al., 2023b). To elucidate the anti-inflammatory mechanisms of XOS, we investigated its role in pyroptosis-related signaling pathways. NLRP3 serves as a pivotal initiator of pyroptosis and participates in NLRP3 inflammasome formation (He et al., 2016). The NLRP3 inflammasome cleaves downstream GSDMD, leading to the formation of pores in the lipid membrane, facilitating the release of pro-inflammatory cytokines, and further promoting pyroptosis (Shi et al., 2015). In this context, XOS inhibited the upregulation of NLRP3 and GSDMD expression induced by LPS, consequently reducing pro-inflammatory cytokine levels. These findings suggest that XOS impedes pyroptosis by modulating the expression of pyroptosis-related mRNA and proteins in the ileum and thymus. At present, the role of XOS in cell pyroptosis is very limited and further research is still needed.

The anti-inflammatory effects of XOS are closely linked to the Th17/Treg immune response. Th17/Treg immune response relies on the involvement of TGFB, STAT3, STAT5, RORγt, and FOXP3 in immune function regulation (Mangan et al., 2006; Veldhoen et al., 2006; Heltemes-Harris and Farrar, 2012). Th17 cells secrete IL-17 and promote inflammation (Korn et al., 2009). In contrast, Treg cells inhibit Th17 cell activity, leading to the secretion of TGFB and IL-10, mitigating inflammation (Raffin et al., 2020). In this study, XOS decreased the expression of IL-17A, RORγt, and STAT3, while increasing the expression of IL-10, TGFB1, FOXP3, and STAT5. These findings contrast with the effects of LPS, highlighting that XOS hinder Th17 development, bolster the anti-inflammatory role of Treg cells, and regulates the ileal and thymic immune function. The effect of XOS on the immune function of the thymus in piglets has not been reported. However, a similar study suggested that the Th17/Treg balance plays a crucial role in regulating the impact of dietary chitooligosaccharides on intestinal immune function in piglets (Yu et al., 2021a). Additionally, glutamate and alpha-ketoglutarate employ the Th17/Treg immune response to regulate immune function in the piglet intestine (Liu et al., 2023a, 2023c). Therefore, based on these results, it is reasonable to infer that XOS modulates the ileal and thymic immune function via the Th17/Treg immune response.

Gut microbiota plays a key role in regulating host immune function (Peng et al., 2021). Therefore, establishing a healthy intestinal microbiota is critical to maintaining normal immune function and promoting healthy growth in piglets. At the phylum level, Firmicutes and Bacteroidetes are the major microbial phyla in the gut (Wang et al., 2019). By utilizing 16S rRNA gene pyrosequencing, it was observed in this study that the dietary addition of XOS resulted in a decrease in the abundance of Firmicutes and an increase in the abundance of Bacteroidetes. Consistent with previous findings in piglets fed XOS, a decrease in Firmicutes and an increase in Bacteroidetes were also reported (Wang et al., 2021). Similarly, a study in rats showed that XOS supplementation reduced the ratio of Firmicutes to Bacteroidetes in the intestines of rats fed a high-fat diet (Thiennimitr et al., 2018). Bacteroidetes are widely recognized for their contributions to the degradation of plant polysaccharides, the production of SCFA, and the modulation of intestinal immune function (Wang et al., 2021). These findings suggest that XOS can modulate immune function in piglets by increasing the abundance of Bacteroidetes while decreasing the abundance of Firmicutes. At the genus level, our study found an increase in the abundance of beneficial bacteria, such as Parabacterium and Prevotellaceae_NK3B31_group, with dietary XOS supplementation. The increase in Parabacteroides favors butyrate production and reduces the inflammatory response (Lei et al., 2021b). Mice fed XOS had higher Parabacteroides abundance and lower colon inflammation (Li et al., 2020). Prevotellaceae_NK3B31_group can degrade complex carbohydrates such as xylan and play an active role in the production of SCFA (Jiang et al., 2020). The abundance of intestinal Prevotellaceae_NK3B31_group is increased in growing pigs fed XOS diets (Sutton et al., 2021). Correlation analysis showed that the abundance of Parabacteroides and Prevotellaceae_NK3B31_group was significantly negatively correlated with gene expression of pro-inflammatory cytokines in the ileum and thymus, but significantly positively correlated with gene expression of IL-10. Parabacteroides were reported to be significantly negatively correlated with the expression of IL-6, IL-1β and TNF-α (Li et al., 2021). Prevotellaceae_NK3B31_group may inhibit pro-inflammatory cytokine expression (Liu et al., 2019). The above results demonstrated that the modulation of gut microbiota by XOS improved the ileal and thymic immune function of piglets. Correlation analysis shows that pyroptosis and Th17/Treg immune response are related to gut microbiota. Some studies have reported that intestinal microbial dysbiosis may activate pyroptosis, but this still requires further research (Huang et al., 2022; Chen et al., 2023). Gut microbiota is closely related to the balance between Treg and Th17 cells. Parabacteroides’ metabolite, bile acids, inhibits Th17 differentiation and reduces IL-17 levels (Sun et al., 2023). It has been reported that the increased abundance of Prevotellaceae_NK3B31_group in the intestine of piglets was positively correlated with the expression of STAT3, RORc, and FOXP3, and negatively correlated with the expression of IL17 and TGFB1 (Yu et al., 2021a). Thus, based on the above results, it can be shown that gut microbiota mediates the regulation of the Th17/Treg immune response by XOS. SCFA are one of the intestinal microbial metabolites that can exert immunomodulatory effects (Hebbandi Nanjundappa et al., 2022). In this study, dietary supplementation with XOS significantly increased intestinal levels of SCFA such as AA, PA, IBA, BA, IVA, and VA. SCFA plays a crucial role in regulating the gut microbiota and immune function of piglets. SCFA have been shown to enhance host immune function and reduce the release of pro-inflammatory cytokines by activating the expression of the FOXP3 gene and promoting the differentiation of naive T cells into Treg cells (Smith et al., 2013). In addition, this study also found a strong correlation between SCFA, Th17/Treg immune response and pyroptosis. The impact of SCFA on Th17/Treg immune response has been widely reported, but its impact on pyroptosis is still unclear and further research is needed. In summary, the dietary addition of XOS has the potential to improve ileal and thymic immune function in piglets by modulating the composition of the intestinal microbiota and promoting the production of beneficial SCFA.

Remarkably, this study unveiled a correlation between pyroptosis and the Th17/Treg immune response. Previous studies have established an inherent connection between pyroptosis and the Th17/Treg immune response. The NLRP3 inflammasome contributes to an increase in Th17 cells while inhibiting NLRP3 promotes Treg cell differentiation (de Castro et al., 2018). Similarly, NLRP3 impedes FOXP3 expression, leading to increased Treg cell production in various organs of mice lacking NLRP3 (Park et al., 2019). IL-17, secreted by Th17 cells, activates NLRP3 and GSDMD expression, thereby promoting pyroptosis in osteoblasts (Lei et al., 2021a). In macrophages, RORγ is indispensable for NLRP3 inflammasome activation (Billon et al., 2019).

Conclusion

In conclusion, the results of this study indicate that XOS modulates the ileal and thymic immune function in piglets 4 h after LPS challenge may be related to gut microbes, pyroptosis, and Th17/Treg immune responses.

Glossary

Abbreviations

AA

acetate

ADFI

average daily feed intake

ADG

average daily weight gain

BA

butyrate

CAT

catalase

CON

the basal diet

FCR

feed conversion rate

FOXP3

forkhead box P3

GSDMD

gasdermin-D

GSH

glutathione

GSH-Px

glutathione peroxidase

IBA

isobutyrate

IL

interleukin

IVA

isovalerate

LPS

lipopolysaccharide

MDA

malondialdehyde

NLRP3

NOD-like receptor family pyrin domain containing 3

OTU

operational taxonomic units

PA

propionate

PCoA

principal coordinate analysis

RORγt

retinoid-related orphan receptor-gammat

SCFA

short-chain fatty acids

STAT

signal transducer and activator of transcription

TGFB

transforming growth factor beta

T-SOD

total superoxide dismutase

VA

valerate

XOS

xylo-oligosaccharide

Conflict of interest statement

The authors declare no conflicts of interest.

Author Contributions

Weixiao Sun (Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing—original draft, Writing—review & editing), Guangmang Liu (Data curation, Funding acquisition, Methodology, Validation, Writing—review & editing), Fei Shen (Formal analysis, Methodology), De Wu (Conceptualization, Formal analysis, Methodology), Yan Lin (Formal analysis, Investigation, Methodology), Ruinan Zhang (Formal analysis, Validation), Lianqiang Che (Conceptualization, Formal analysis, Methodology), Bin Feng (Formal analysis, Investigation, Methodology), Yong Zhuo (Formal analysis, Investigation, Methodology), Shengyu Xu (Formal analysis, Investigation, Methodology), Hua Lun (Formal analysis, Methodology), Zhengfeng Fang (Formal analysis, Investigation, Methodology), Xuemei Jiang (Investigation, Methodology), Jian Li (Investigation, Methodology), and Jing Wang (Formal analysis, Methodology)

Data Availability

Data are available on request.

Ethics approval and consent to participate

Sichuan Agricultural University Animal Care and Use Committee approved the use of experimental animals and animal-related experimental procedures (SICAU-2022-05).

Consent for publication

Not applicable.