Dietary alpha-ketoglutarate enhances intestinal immunity by Th17/Treg immune response in piglets after lipopolysaccharide challenge

Abstract

Alpha-ketoglutarate (AKG) is important for improving intestinal and systemic immune function. This study aimed to explore whether AKG enhances gut immunity in lipopolysaccharide (LPS)-challenged piglets by modulating the immune-related helper T cells 17 (Th17)/regulatory T cells (Treg) balance pathway. A 2 × 2 factor design was used on 24 pigs, with the major factors being diet (basal diet or 1% AKG diet) and immunological challenge (saline or LPS). Piglets were fed with a basal or AKG diet for 21 d and then received intraperitoneal injection of LPS or saline. The results demonstrated that AKG supplementation enhanced growth performance compared with the control group (P < 0.05). AKG improved the ileal morphological structure (P < 0.01). Finally, AKG supplementation increased interleukin (IL)-10, transforming growth factor beta-1, forkhead box P3, and signal transducer and activator of transcription 5 genes expression whereas decreasing IL-6, IL-8, IL-1β, tumor necrosis factor-α, IL-17, IL-21, signal transducer and activator of transcription 3 and rar-related orphan receptor c genes expression (P < 0.05). These findings suggested that dietary AKG can improve the growth performance of piglets. Meanwhile, dietary AKG can alleviate LPS-induced intestinal inflammation through Th17/Treg immune response signaling pathway.

Dietary alpha-ketoglutarate can improve the growth performance of piglets. It can alleviate intestinal inflammation induced by lipopolysaccharide through the Th17/Treg immune response signaling pathway and thereby improve intestinal immunity.

Introduction

The intestine is the digestive, immune, and endocrine organ of weaned piglets. The immature digestive and immune systems cause a variety of problems, including gastrointestinal dysfunction (Pluske et al., 2018) and low feed intake (Humphrey et al., 2019). Immune stress is known as a critical issue in the modern pig industry. Piglets are frequently harmed by the stimulation of substances, such as pathogens and non-pathogens (endotoxin, heterologous protein). This stimulation activates the intestinal immune system, resulting in the release of a cascade of cellular inflammatory factors [interleukin (IL)-6, tumor necrosis factor-α (TNF-α)] (Waldner and Neurath, 2015).

The balance of helper T cells 17 (Th17) and regulatory T cells (Treg) is critical in maintaining normal immune function in animals. Many factors can affect the Th17/Treg balance, including T cell receptor signaling, costimulatory and cytokine signaling, and forkhead box P3 (FOXP3) stability (Lee et al., 2018). Th17 cells produce pro-inflammatory cytokines (IL-17, IL-22) that induce autoimmune and inflammatory responses, whereas Treg cells produce anti-inflammatory cytokines [IL-10, transforming growth factor beta1 (TGF-β1)] to suppress immune responses and maintain immune homeostasis (Yan et al., 2020). Previous studies have linked abnormal Th17/Treg ratios to chronic inflammation and autoimmune diseases (Fan et al., 2020). Consequently, many researchers are working on nutrition strategies to regulate the intestinal immunity of piglets.

Piglets’ growth performance (Chen et al., 2017), protein metabolism (Chen et al., 2018), and immune function are all influenced by alpha-ketoglutarate (AKG) (Wu et al., 2022). AKG supplementation also has immune and regulatory effects via downregulating mRNA expression of the cytokines IL-22 and TNF-α and enhancing gut homeostasis (Li et al., 2019). AKG is essential for maintaining gut health. AKG supplementation can improve intestinal morphology in early-weaned piglets by lowering intestinal inflammatory cytokine concentrations and mRNA expression (IL-1β, IL-6, and IL-12) (Tian et al., 2021). Whether the effect of AKG on intestinal immunity in piglets via modulation of Th17/Treg biology-related immune signaling has not been reported. The injection of LPS was used in this study to establish an intestinal inflammation model. This study aimed to test whether AKG could enhance growth performance. And whether AKG could reduce intestinal inflammation caused by lipopolysaccharide (LPS) by altering Th17/Treg immune response.

Materials and Methods

Pig care and experimental design

In this study, all animal experimental procedures were confirmed by Sichuan Agricultural University Animal Care and Use Committee (SICAU-2022-05). Twenty-four weaning piglets (castrated; Duroc × Yorkshire × Landrace; 7.36 ± 0.98 kg initial body weight) were distributed to four treatment groups in a 2 × 2 factorial arrangement including diet (with or without AKG) and immune challenge (saline or LPS). After 21 d of feeding with 0% or 1% AKG, the piglets received the intraperitoneal injection of Escherichia coli LPS (100 μg/kg body weight; E. coli serotype 055: B5; Sigma Chemical Inc., St. Louis, MO, United States) or the same dose of 0.9% saline. Each treatment has six replicates, and one replicate had one pig. Room temperature and humidity were approximately 30 °C and 50%, respectively. In the whole experiment, all piglets were provided ad libitum with clean water. The basal diets (Table 1) were performed for all nutrients in accordance with the National Research Council (2012) requirements. The concentration of AKG (Shanghai Yuanye Bio-Technology Co., Ltd, China) was selected in agreement with a previous study (He et al., 2016). LPS dose was selected according to the criteria presented by Liu et al (2022). To prevent the effects on intestinal feed intake, all piglets were fasted for 4 h before slaughter on day 21 of the experiment.

Table 1.

Ingredient composition of experimental diets (%)

Ingredient	Content of basal diet (%)	Ingredient	Content of AKG diet (%)
	7 to 25 kg		7 to 25 kg
Corn	32.15	Corn	32.15
Extruded corn	27.54	Extruded corn	27.54
Soybean oil	1.72	Soybean oil	2.35
Glucose	1.70	Glucose	0.07
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
		α-Ketoglutarate	1.00
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.

CP, crude protein; SID, standardized ileal digestible.

Determination of growth performance indicators

At the end of day 21, all piglets were weighed at days 1 and 21 of the formal trial, and the average daily weight gain (ADG), average daily feed intake (ADFI), and F:G (ADFI/ADG) for the whole period were calculated.

Intestinal sample collections

Then, 4 h after all pigs received intraperitoneal injections (E. coli LPS solution or saline), the pigs were electrocuted and sacrificed, and ileal samples were separated and immediately harvested. The samples were washed with 0.9% NaCl (4 °C) and quickly stored at −80 °C (liquid nitrogen) for further analysis.

Intestinal morphology analysis

The ileal samples were fixed in 4% paraformaldehyde solution, collected, and embedded in paraffin. Villus height, crypt depth, and crypt width were analyzed by Image Pro Plus (Media Cybernetics, Bethesda, MD, United States).

Enzyme-linked immunosorbent assay analysis

The method of enzyme-linked immunosorbent assay (ELISA) analysis was performed on the basis of our previous study (Liu et al., 2021a). Briefly, ileum samples were dissolved in radioimmunoprecipitation assay buffer, sonicated, and centrifuged (4 °C). The protein concentrations of IL-6 and TGF-β1 were determined with an ELISA kit.

Real-time polymerase chain reaction analysis

Total RNA isolation, reverse transcription, and quantitative real-time polymerase chain reaction (RT-PCR) were performed according to previously described method (Gu et al., 2021). Briefly, total RNA of the ileum was extracted using Trizol (Takara, Dalian, China). The specific primers of the genes were synthesized by Takara Biotechnology Company (Takara, Dalian, China; Supplementary Table S1). Quantification by RT PCR was carried out on real-time PCR system (ABI 7900HT, Applied Biosystems). The 2^−ΔΔct method was used to calculate the relative genes expression.

Western blotting

Western blotting was performed according to the previous study (Liu et al., 2021b). Briefly, protein concentration was detected by bicinchoninic acid (BCA) kit. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Millipore, Eschborn, Germany) was used to isolate and transfer the samples. The membrane was washed and blocked. Then, the membrane was incubated with the primary antibody overnight (4 °C). The primary antibodies were as follows: β-actin (1:1,000; 4970S) and rar-related orphan receptor γt (RORγt; 1:500; 13205-1-AP) and forkhead box P3 (FOXP3; 1:500; bs-10211R). All the primary antibodies were supplied by Proteintech Group, Inc. (Wuhan, China). After overnight, the membrane was washed and incubated with a secondary antibody (90 min). The protein bands were visualized with an enhanced chemiluminescence kit with the imaging system of high-sensitivity multifunction (ChemiDocTM, Bio-Rad). Afterward, the Image Lab Software was used to determine the value.

Statistical analysis

Growth performance data were analyzed with independent-samples t-test and other data were evaluated with two-way statistical analysis (general linear model) of variance and significance tests using the SPSS 26.0 software (IBM, Chicago, IL, United States). The main effects of the model included LPS, AKG, and the interactive effects of the two. Duncan’s multiple range test was used to analyze significant differences in groups. P < 0.05 was deemed statistically significant.

Results

Effects of AKG on the growth performance of weaning piglets

The growth performance of weaning piglets is shown in Figure 1. Relative to the control group, AKG supplementation significantly enhanced ADG (Figure 1A) and ADFI (Figure 1B).

Figure 1.

Effects of alpha-ketoglutarate supplementation on the growth performance of piglets. The experimental period lasted 21 d and the growth performance of the piglets was measured and calculated before LPS challenge. CON = the control group; AKG = the alpha-ketoglutarate group; ADFI = average daily feed intake; ADG = average daily weight gain; FCR = feed conversion rate. ^a,b,c,dLabeled means in a row without a common letter differ at P < 0.05. Values are expressed as the mean ± standard error (n = 6).

Effects of AKG on the ileal morphology of weaning piglets after LPS challenge

Ileal morphology is shown in Supplementary Figure S1 and Table 2. Relative to the control group, LPS challenge enhanced the crypt depth and reduced the villus height and villus height/crypt depth ratio (VCR) (P < 0.01), whereas dietary AKG enhanced the villus height and VCR, and decreased crypt depth (P < 0.01). AKG and LPS have an interactive effect on villus height, villus area, and VCR (P < 0.01).

Table 2.

Effects of AKG supplementation on the ileal morphology of piglets after 4 h LPS challenge

Item	Saline		LPS		SEM	P-value
	CON	AKG	CON	AKG		Diet	LPS	Interaction
Villus height, μm	225.24b	417.95a	171.24c	223.14b	8.681	<0.01	<0.01	<0.01
Villus area, μm	91486.34c	277997.12a	47143.60d	107962.11b	9985.774	<0.01	<0.01	<0.01
Crypt depth, μm	236.19b	116.99c	351.15a	207.83b	12.295	<0.01	<0.01	0.339
VCR	0.98b	3.79a	0.53c	1.10b	0.132	<0.01	<0.01	<0.01

^a,b,c,dLabeled means in a row without a common letter differ at P < 0.05.

Values are expressed as the mean ± standard error (n = 6).

CON, control; LPS, lipopolysaccharide; AKG, alpha-ketoglutarate. VCR, villus height/crypt depth ratio.

Effects of AKG on protein concentration and the relative mRNA levels of inflammatory cytokine of ileal tissue in weaning piglets after LPS challenge

The ileal protein concentrations of IL-6 and TGF-β1 are shown in Table 3. Compared with the control group, LPS challenge enhanced IL-6 protein concentration (P < 0.01) and reduced TGF-β1 protein concentration (P < 0.01). AKG supplementation reduced IL-6 protein concentration (P < 0.01) and enhanced TGF-β1 protein concentration (P < 0.01). AKG and LPS had an interactive effect on IL-6 and TGF-β1 protein concentrations (P < 0.01).

Table 3.

Effects of AKG supplementation on IL-6/TGF-β1 protein concentration in the ileal tissue of piglets after 4 h LPS challenge

Item	Saline		LPS		SEM	P-value
	CON	AKG	CON	AKG		Diet	LPS	Interaction
IL-6, pg/mg prot	387.58b	211.45c	478.07a	254.17d	6.367	<0.01	<0.01	<0.01
TGF-β1, pg/mg prot	31.09b	45.38a	24.82c	30.50b	0.967	<0.01	<0.01	<0.01

^a,b,c,dLabeled means in a row without a common letter differ at P < 0.05.

Values are expressed as the mean ± standard error (n = 6).

IL-6, interleukin-6; TGF-β1, transforming growth factor-beta 1; CON, control; LPS, lipopolysaccharide; AKG, alpha-ketoglutarate.

The ileal relative mRNA levels of inflammatory cytokine are shown in Table 4. Compared with the control group, LPS challenge up-regulated the gene expression levels of IL-6, IL-8, IL-1β, and TNF-α (P < 0.01). AKG supplementation down-regulated the gene expression levels of IL-6, IL-8, IL-1β, and TNF-α. AKG and LPS had an interactive effect on the expression of IL-6, and TNF-α mRNA (P < 0.05).

Table 4.

Effects of AKG supplementation on the relative mRNA levels of inflammatory cytokine in the ileal mucosa of piglets after 4 h LPS challenge

Item	Saline		LPS		SEM	P-value
	CON	AKG	CON	AKG		Diet	LPS	Interaction
IL-6	1.0000b	0.6337c	1.5510a	0.8357b	0.068	<0.01	<0.01	0.019
IL-1β	1.0000b	0.6761c	1.3178a	0.8938b	0.065	<0.01	0.001	0.452
IL-8	1.0000b	0.6507c	1.5083a	1.0225b	0.067	<0.01	<0.01	0.320
TNF-α	1.0000b	0.6715c	1.5525a	0.7280c	0.075	<0.01	<0.01	0.003
IL-10	1.0000b	1.4603a	0.3553c	0.9552b	0.088	<0.01	<0.01	0.436

^a,b,c,dLabeled means in a row without a common letter differ at P < 0.05.

Values are expressed as the mean ± standard error (n = 6).

IL, interleukin. CON, control; LPS, lipopolysaccharide; AKG, alpha-ketoglutarate.

Effects of AKG on the mRNA expression of Th17/Treg-related signaling pathways of the ileum in weaning piglets after LPS challenge

The effects of AKG on the mRNA levels of Th17/Treg response-related signaling pathways are shown in Table 5. Compared with the control group, LPS challenge up-regulated the gene expression levels of IL-17, IL-21, STAT3, and RORγt (P < 0.01), and down-regulated the gene expression levels of IL-10, TGF-β1, FOXP3, and STAT5 (P < 0.01). AKG supplementation down-regulated the gene expression levels of IL-17, IL-21, STAT3 and RORc (P < 0.01), and up-regulated the gene expression levels of IL-10, TGF-β1, FOXP3, and STAT5 (P < 0.01). AKG and LPS had an interactive effect on the expression of TGF-β1 and STAT5 mRNA (P < 0.05).

Table 5.

Effects of AKG supplementation on the relative mRNA levels of cytokines related to Th17/Treg-related signaling pathways in the ileal mucosa of piglets after 4 h LPS challenge

Item	Saline		LPS		SEM	P-value
	CON	AKG	CON	AKG		Diet	LPS	Interaction
IL-17	1.0000c	0.6967d	1.7447a	1.2825b	0.082	<0.01	<0.01	0.342
IL-21	1.0000b	0.6651c	1.4050a	0.8792b	0.060	<0.01	<0.01	0.126
STAT3	1.0000b	0.6682c	1.6317a	1.1448b	0.065	<0.01	<0.01	0.246
RORγt	1.0000b	0.5414c	1.3061a	0.8424b	0.082	<0.01	0.001	0.976
TGF-β1	1.0000b	1.2764a	0.4705c	0.9977b	0.040	<0.01	<0.01	0.005
FOXP3	1.0000b	1.4766a	0.4843c	1.1223b	0.051	<0.01	<0.01	0.132
STAT5	1.0000b	1.2133a	0.5016c	1.1853a	0.049	<0.01	<0.01	<0.01

^a,b,c,dLabeled means in a row without a common letter differ at P < 0.05.

Values are expressed as the mean ± standard error (n = 6).

STAT, signal transducer and activator of transcription; RORγt, rar-related orphan receptor γt; TGF-β1, transforming growth factor beta1; FOXP3, forkhead box P3. CON, control; LPS, lipopolysaccharide; AKG, alpha-ketoglutarate.

Correlation analysis of Th17/Treg signaling pathway and intestinal immunity

The correlation analysis results between Th17/Treg response signaling pathway and intestinal immunity in weaned piglets are shown in Supplementary Table S2. The mRNA expression levels of STAT3, RORγt, IL-17, and IL-21 were positively correlated with the mRNA expression levels of IL-6, IL-1β, TNF-α, and IL-8 and were negatively correlated with the mRNA expression levels of TGF-β1 and IL-10 (P < 0.05). The mRNA expression levels of FOXP3 and STAT5 were negatively correlated with the mRNA expression levels of IL-6, IL-1β, TNF-α, and IL-8, and were positively correlated with those of TGF-β1 and IL-10 (P < 0.05).

Effects of AKG on the protein expression levels of RORγt and FOXP3 in the ileum of weaning piglets after LPS challenge

The effects of AKG on the protein expression levels of RORγt and FOXP3 of weaning piglets are shown in Figure 2. ­Compared with the control group, LPS decreased FOXP3 protein expression and increased RORγt protein expression (P < 0.01). AKG supplementation increased FOXP3 protein expression and decreased RORγt protein expression (P < 0.01).

Figure 2.

The effects of alpha-ketoglutarate (AKG) supplementation on the ratios of RORγt/β-actin and FOXP3/β-actin in the ileal mucosa in piglets after 4 h LPS challenge. RORγt = rar-related orphan receptor γt; FOXP3 = forkhead box P3. CON = piglets were fed with basal diet and challenged with saline; LPS = piglets were fed with basal diet and challenged with lipopolysaccharide; AKG = piglets were fed with AKG diet and challenged with saline. AKG_LPS = piglets were fed with AKG diet and challenged with LPS. Values are expressed as the mean ± standard error (n = 3).

Discussion

We demonstrated that AKG supplementation improved ADG and ADFI under normal and nonchallenged conditions, indicating that AKG supplementation can improve the growth performance of weaned piglets. This result is consistent with that of a previous report (Wang et al., 2016). The main reason that dietary addition of AKG can improve the growth performance of piglets may be that it is rapidly absorbed in the front part of small intestine to supply energy to intestinal cells, synthesize essential amino acids such as glutamate and proline to promote protein synthesis and early bone growth, and improve the level of blood growth hormone (Chin et al., 2014). LPS is a component of the outer wall of gram-negative bacteria and induces barrier dysfunction, inflammation, and disease by activating pro-inflammatory mediators such as IN-6 and TNF-α (Mercer et al., 1996). AKG can improve the intestinal health and reduce immune stress (Hou et al., 2010). Intestinal morphology can partly reflect the health of intestinal tract (Song et al., 2022). AKG protected intestinal morphology by increasing villus height and decreasing crypt depth. This result was consistent with previous research findings. He et al. (2017) reported that AKG enhanced villus height and crypt depth in the duodenum, jejunum, and ileum. This may be because AKG provides energy for intestinal epithelial cell metabolism and promotes the growth and development of intestinal cells (He et al., 2015). These findings suggested that AKG can enhance the intestinal morphology of piglets.

To further explore the mechanism of AKG in intestinal health, we demonstrated gene expression of immune factors associated with Th17/Treg balance. Th17 cells can protect the intestinal mucosa and induce intestinal inflammation by releasing pro-inflammatory cytokines (Omenetti et al., 2019). Th17 cell differentiation occurs in three stages. Firstly, initiation of Th17 cell differentiation (IL-6 and TGF-β); followed by expansion of Th17 cell differentiation (IL-21); and lastly, differentiation of mature Th17 cells at later stages (IL-23) (Guo and Zhang, 2021). In our study, AKG reduced the relative mRNA expression of inflammatory factors. AKG has consistently been shown to have immunomodulatory effects on the intestine of weaned piglets (Wu et al., 2021). AKG also reduced the expression of related Th17 (IL-17, IL-21, STAT3, and RORγt), suggesting that AKG may have an immunoregulatory effect on intestinal inflammation by inhibition of pro-inflammatory factors release and the differentiation of Th17 cells.

Treg cells not only maintain immune homeostasis but also prevent autoimmune diseases (Horwitz et al., 2019). Previous research has shown that Treg cells play an important role in inflammatory response control, primarily through the secretion of TGF-β1, IL-10, and other anti-inflammatory cytokines in various immune diseases (Fang and Zhu, 2020). AKG supplementation increased the relative expression of IL-10, consistent with previous research that AKG has anti-inflammatory properties (Hua et al., 2022). Furthermore, AKG significantly affects the decrease of the TGF-β1, STAT5, and FOXP3 mRNA relative expression caused by the LPS challenge, indicating that the immune regulation of AKG on piglets with intestinal inflammation is mediated through the TGF-β1/STAT5/FOXP3 signaling pathway.

In conclusion, dietary AKG can improve the growth performance of piglets, and dietary AKG can alleviate intestinal inflammation induced by LPS through the Th17/Treg immune response signaling pathway.

Glossary

Abbreviations:

ADFI

average daily feed intake

ADG

average daily weight gain

AKG

, alpha-ketoglutarate

CP

crude protein

ELISA

enzyme linked immunosorbent assay

FOXP3

forkhead box P3

IL

interleukin

LPS

lipopolysaccharide

NaCl

sodium chloride

RORγt

rar-related orphan receptor γt

RT-PCR

reverse transcription-polymerase chain reaction

SID

standardized ileal digestible

STAT

signal transducer and activator of transcription

TGF-β1

transforming growth factor beta1

Th17

helper T cells 17

TNF-α

tumor necrosis factor-α

Treg

regulatory T cells

VCR

villus height/crypt depth ratio

Funding

This work was supported by the Specific Research Supporting Program for Discipline Construction in Sichuan Agricultural University (number 03570126).

Conflict of Interest Statement

There are no conflicts to declare.