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Gut-derived IL-13 contributes to growth via promoting hepatic IGF-1 production

Microbiome volume 12, Article number: 248 (2024) Cite this article

Abstract

Background

The gut microbiota has a profound effect on immunity and metabolic status of the host, which has increasingly attracted research communities. However, the intrinsic mechanism underlying the interplay among these three aspects remains unclear.

Results

Different immune states were established via shaping the population structure of gut microbiota with antibacterial agents. The gut microbiota population structures altered with the subtherapeutic level of antibacterial agents facilitated growth phenotype in both piglets and infant mice. Notably, increased colonization of Prevotella copri was observed in the intestinal microbiota, which shifted the immune balance from a CD4+ T cell-dominated population toward a T helper 2 cell (Th2) phenotype, accompanied by a significant elevation of interleukin-13 (IL-13) levels in the portal vein, which was found to display a strong positive correlation with hepatic insulin-like growth factor-1 (IGF-1) levels. Subsequent investigations unveiled that gut-derived IL-13 stimulated the production of hepatic IGF-1 by activating the IL-13R/Jak2/Stat6 pathway in vitro. The IGF-1 levels were increased in the muscles, leading to an upregulation of and resulted the increased genes associated with related to myofibrillar synthesis and differentiation, which ultimately improving the growth phenotype.

Conclusions

Our findings highlight the modification of gut immunity states as a central strategy for increasing anabolism of the host, which has significant implications for addressing human undernutrition/stunting, sarcopenia, obesity and related comorbidities.

Video Abstract

Introduction

The gut microbiota profoundly affects the metabolic process of the host, resulting in significant phenotypic differences, such as increased muscle mass, bone density, and adipose tissue accumulation [1, 2]. This influence is evidenced by observations that abnormal gut microbiota composition is associated with undernutrition/stunting [3], obesity [4], and sarcopenia [5], which can be partially alleviated through fecal microbiota transplantation from healthy individuals [3,4,5,6]. The phenomenon that promoting anabolism by the modification of gut microbiota and immunity has been well recognized and utilized in the farm industry since antibacterial agents were supplemented in feed in the early 1950s. Subtherapeutic levels of antibacterial agents (SLAs) have consistent and reproducible growth-promoting effects and are far more potent than any other alternatives tested thus far [7,8,9]. The degree of growth promotion may vary, but the percentage increase averages between 4 and 15%, and the feed utilization is improved by 2 to 5% [9, 10]. Although SLAs have been banned to be administered for growth promotion in animal feeds because they frequently create optimum selection pressures for the propagation of both pathogenic and commensal resistant bacteria in the gut of the animals by eliminating competition from non-resistant strains, the clarification of the influence of the SLA-induced intestinal microbiota shift on the host growth phenotype may contribute to further understanding the crosstalk between the intestinal microbiota and host’s immunity and metabolism.

Billions of microbes dwelling in the digestive tract can modulate the innate and adaptive immune responses of the reactive host cells. The immune activation, in turn, can inhibit the anabolism through the actions of proinflammatory cytokines on the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis with increasing energy consumption by the immune system [11,12,13]. Paradoxically, moderate immune activation can also promote anabolic metabolism of the host during the early stages of the juveniles. IGF-1, an important anabolic hormone, is reported to be affected by gut microbes. Germ-free (GF) mice exhibit lower levels of IGF-1 in the liver compared to conventionally raised mice [14]. Colonization of GF mice with conventional microbiota leads to elevated IGF-1 levels [15]. Similarly, in colonized Drosophila with conventional microbiota, the activity of IGF-like peptide activity is increased [16, 17]. Administration of therapeutic doses of antibacterial agents can deplete microbiota in conventional mice, resulting in decreased hepatic IGF-1 levels [15]. However, the precise mechanisms underlying the discrepancies in hepatic IGF-1 level caused by the gut microbiota shift remain unclear.

Piglets grow through exponential weight gain, which is conducive to displaying the effects of experiments on growth promotion. Additionally, pigs are optimal animals for human research because of their similar genetics, anatomy and physiology, especially in the gastrointestinal tract [7]. Here, 21-day-old piglets were administered orally with a SLA and compared with conventionally raised piglets or piglets fed an antibacterial agent cocktail (therapeutic doses of each component) in terms of gut microbiota, immunity states, and hepatic IGF-1 level. Next, the type conversion of SLA-mediated gut immunity was determined, and the resulted active immune mediators that facilitate the production of hepatocyte IGF-1 were screened out in the portal vein. Then, the microbiota taxa contributing to hepatic IGF-1 production were finalized. In addition, the effects mediated by the SLA in piglets were recapitulated in mice to confirm their general applicability across species. Our investigation into the mechanism underlying the SLA-promoted growth promotion not only extends the understanding of the interplay between gut microbiota and host’s immunity and metabolism, but also offers a novel approach to address human metabolism-related disorders and to maximize animal genetic potential for growth.

Methods

Ethics

The research performed in this study complies with all relevant ethical regulations. All animal procedures were approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University, China (Approval No. HZAUSW-2023–0019 for the swine experiments, and No. HZAUSW-2022–0020 for the mouse experiments), and all methods were performed in accordance with the relevant guidelines and regulations.

Animal experiments

Experimental animals and feeding conditions

SLAs promote the growth of both male and female juvenile animals [10, 18, 19]. To reduce variance caused by the sex difference, the male animals were chosen for experiments [14]. A total of 42 barrows (Landrace, weaned at 21 days old) with an initial body weight of 7.76 ± 0.81 kg were selected from 10 litters and fed in pens padded with rubber mats at 25 ± 2 °C. The piglets had free access to food and water. SPF BALB/c male mice (weaned at 21 days old) were housed in autoclaved cages with standard laboratory chow (GB14924.3–2010) and sterile water and with an ambient temperature of 23 ± 2 °C, humidity of 55 ± 10%, and a dark–light cycle of 12 h.

Piglets supplemented with subtherapeutic levels of antibacterial agents

Twenty-one-day-old barrows were randomly assigned to three dietary groups as follows: (1) a corn-soybean meal basal diet (CR, n = 16), (2) the basal diet containing 50 mg/kg quinocetone [20] (Qui, a classic antibacterial growth-promoting agent, n = 16), and (3) the basal diet containing an antibacterial agent cocktail (AC, a mixture of 10 mg/kg ampicillin, 60 mg/kg metronidazole and 22 mg/kg neomycin for 14 days, and 15 mg/kg vancomycin for another 14 days, n = 10) [18, 19, 21, 22]. The pigs were housed in pens with 5 to 6 piglets per pen and had free access to water and feed. The basal diets were formulated to meet National Research Council nutrient requirements (Table S1). The feeding trial lasted for 28 days. The individual body weight and feed consumption were measured. The weight gain and feed conversion ratio (the feed consumption: weight gain) were calculated at the end of the trial.

Mice supplemented with subtherapeutic levels of antibacterial agents

Twenty-one-day-old SPF BALB/c male mice were randomly divided into three treatments (n = 22). Subtherapeutic quinocetone (Qui) was added to drinking water at a dose of 1 μg/g body weight. An antibacterial agent cocktail [22] (AC, a mixture of 1 mg ampicillin per mL water, 1 mg metronidazole per mL water and 1 mg neomycin per mL water) was added to the drinking water for 14 days, which was followed by 0.5 mg vancomycin per mL water for another 14 days [18, 19, 21, 22]. Mice conventionally raised (CR) without any antibacterial agent in the drinking water were used as the control. The daily water intake in mice averaged about 15 mL water per 100 g body weight [10]. The water containers were changed daily.

Mice supplemented with Prevotella copri

Twenty-one day-old SPF BALB/c male mice pretreated with AC in drinking water for 2 weeks to deplete the gut microbiota were alternatively administered intragastric garage (1 × 109 CFUs) and rectal delivery (0.5 × 107 CFUs) of Prevotella copri (P. copri) for 14 days (n = 8). The P. copri suspension was orally administered with a sterile sodium bicarbonate solution at a final concentration of 1.2% to protect the microbes from gastric acid damage. The AC-pretreated mice were similarly administered sterile sodium bicarbonate solution as the control (n = 8). Gut immunity and growth parameters and hepatic IGF-1 were evaluated at day 28 of the trial.

Administration of recombinant IL-13 in mice

Twenty-one day-old SPF male mice pretreated with AC for 2 weeks were intraperitoneally injected with 3 different doses (1, 2 and 5 ng/g, n = 14) of RIL-13 for 14 days, and the AC-pretreated mice were intraperitoneally injected with the same volume of sterile sodium bicarbonate solution as the control (0 ng/g RIL-13, n = 12). Considering the bioavailability or degradation of cytokines after they were injected the peritoneal cavity [23,24,25], the dose of injected RIL-13 was higher than IL-13 level measured in the circulating blood.

The culture, supernatant preparation, and inactivation of Prevotella copri

The P. copri (ATCC) was cultured in Brain Heart Infusion Broth, Brain Heart Infusion, DSMZ medium (BHI/BHIB) with 10% sterile defibrinated sheep blood at 37 °C under anoxic conditions (10% H2, 10% CO2, and balance N2).

For extraction of the bacterial supernatant, BHI/BHIB medium was inoculated with the P. copri and incubated anaerobically until reaching exponential phase. Then, the cultures were centrifuged and the supernatant was freeze-dried, and the concentrate powder was immediately preserved at − 80 °C.

For the inactivation of the P. copri, the cultured bacterial suspension was centrifuged at 4000 rpm for 5 min, and the precipitate was washed with sterile PBS, centrifuged and resuspended in PBS (3 × 109 CFU/mL). Th2 cells were inoculated in 96-well plates and exposed to the inactivated P. copri (0, 3.2 × 103, 1.6 × 104, 8 × 104 or 4 × 105 CFU) for 12 or 24 h. Then, the cell culture was collected and centrifuged at 5000 rpm, 4 °C, for 10 min. The concentration of IL-13 in the supernatant was then evaluated.

Cell culture

The immortalized porcine hepatocyte cell line (Hep-Li) was provided by the State Key Laboratory for the Diagnosis and Treatment of Infectious Diseases of Zhejiang University (Hangzhou, China) and cultured in high-glucose DMEM containing 10% FBS and 1% penicillin–streptomycin. The mouse hepatocyte cell line (NCTC1469) was cultured in high-glucose DMEM containing 10% horse serum. The human hepatocellular carcinoma cell line (HepG2) was cultured in high-glucose DMEM containing 10% FBS. Different concentrations (1, 10 and 20 ng/mL) of recombinant IL-13 (RIL-13, PeproTech) or recombinant IL-10 (RIL-10, MCE) were supplemented when the hepatocyte confluence reached 70% (DMEM containing 1% FBS or HS). To evaluate the effects of Jak2 and Stat6 on the production of IGF-1 in hepatocytes, Hep-Li, NCTC1469, or HepG2 cells were treated with 0, 1, 3, or 5 μM of BMS-911543 (inhibitor of Jak2) and 0.1 μM, 0.15 μM, and 0.2 μM of AS1517499 (inhibitor of Stat6) and then exposed to 20 ng/mL of RIL-13 for 24 h.

Lymphocytes in the mesenteric lymph nodes (MLNs) are commonly used indicator for gut immunity [24,25,26]. The MLNs of BALB/c mice were isolated, and naive CD4+ T cells were sorted through flow cytometry. Mouse splenic lymphocytes were isolated and treated with mitomycin C (3:1), at 37 °C, for 30 min. The nonmitotic splenic lymphocytes were washed with PBS three times, centrifuged, and re-suspended in RIPM 1640 medium. Naive CD4+ T cells (1 × 106 cells/mL) were activated with nonmitotic splenic lymphocytes and cultured with phytohemagglutinin (10 μg/mL) and RIL-2 (10 ng/mL) in neutral or polarizing conditions: Th2, IL-4 (1000 U/mM), and anti-IL-12 (10 μg/mL). The polarized Th2 cells were incubated in the presence or absence of inactivated P. copri (0, 3.2 × 103, 1.6 × 104, 8 × 104, or 4 × 105 CFU) or the supernatant concentrate of the P. copri preparation (0, 0.05, 0.25 or 2.5 mmol/L) for 12 or 24 h.

Mouse primary hepatocytes were isolated from BALB/c mice. Briefly, the liver was fully digested via inferior vena cava perfusion using perfusion medium A and then digest medium A at a rate of 5 mL/min for each medium [27]. The livers were then excised and minced, which was followed by filtration through a 70-μm cell strainer and centrifuged at 50 × g for 3 min to obtain primary hepatocytes, which were further purified with a 50% Percoll solution. Hepatocytes were then seeded onto collagen-coated plates and cultured in DMEM supplemented with 10% FBS.

The swine primary hepatocytes were isolated from piglets using the method described in Bader et al. [27]. Briefly, the liver was removed from the abdominal cavity and blood vessels visible on the cut surface was cannulated and perfused at a flow rate of 20–40 ml/min with perfusion buffer I (8.3 g/L NaCl, 0.5 g/L KCl, 2.4 g/L HEPES and 0.19 g/L EGTA). The liver was further perfused with the perfusion buffer II (8.3 g/L NaCl, 0.5 g/L KCl, and 2.4 g/L HEPES), which was followed by collagenase type IV medium at 37 °C. The liver capsule was disrupted and its parenchyma was suspended in Hank’s buffer. The cell suspension was filtered and centrifuged at 50 × g for 2 min. The cells were resuspended in DMEM containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. All cells were incubated at 37 °C and 5% CO2 humidified incubators.

C2C12 myoblasts grown in incubators at 37 °C and 5% CO2, and proliferating cells were cultured in DMEM supplemented with 10% FBS. For myogenic differentiation, C2C12 cells were cultured in DMEM containing 1% HS. All cells were grown to 80% confluence before differentiation was induced.

Gut microbiome analysis

Ileal and colonic mucosal samples were freshly collected from mice under different treatments. At least three mucosal regions were collected for each gut segment of each mouse, snap frozen in liquid nitrogen, and stored at – 80 °C until ready for extraction. The DNA of the microbes on the mucosa of the ileum and colon were isolated using the M5 HiPer Bacteria Genomic DNA Kit (Mei5Bio).

16S rRNA gene sequencing

An aliquot of 0.5 g of ileum and colon contents in piglets was used for DNA extraction with a DNeasy PowerSoil kit (Qiagen) according to the manufacturer’s recommendations. The concentration and quality of DNA were measured using a Nanodrop 2000 spectrophotometer (Thermo Scientific) and visualized by 2% agarose gel electrophoresis. The hypervariable regions V3-V4 of the bacterial 16S rRNA gene were amplified with the forward primer 338F (5-ACTCCTACGGGAGGCAGCA-3) and the reverse primer 806R (5-GGACTACHVGGGTWTCTAAT-3) by PCR. The resulting amplicons were used for construction of sequencing libraries using a TruSeq DNA PCR-Free Sample Preparation Kit (Illumina). Then, the libraries were sequenced on the Illumina MiSeq platform with a paired-end 250-bp (PE250) at Shanghai Personal Biotechnology, Co., Ltd.

The quality control of raw sequencing data of the 16S rRNA gene was performed using fastp. The processed reads were subsequently filtered and analyzed by QIIME2 (Quantitative Insight Into Microbial Ecology; version 2019.7). The 16S rRNA sequencing reads were quality filtered with Q30, and chimeras were removed with the DADA2 plugin. Subsequently, representative amplicon sequence variant (ASV) reads and a feature table were generated. The feature taxonomy of the 16S rRNA gene was assigned against a published database containing the sequences of atmMOB. All samples were resampled to the same level of sequencing to avoid the impact of sequencing depth on identifying microbial communities. The diversity indices included alpha diversity and beta diversity, and phylogenic trees (unrooted and rooted trees) were constructed in QIIME2.

Flow cytometry

T lymphocytes were isolated from the MLNs. To detect the frequencies of Th1, Th2, and Th17 cells in T lymphocytes in mice, the cells were stimulated with 1 μL PMA/ionomycin Mixture and 1 μL BFA/monensin Mixture for 6 h in advance. The cells were first stained using Fixable Viability Stain 510 (FVS, 1:1000), which was followed by surface staining with anti-APC-CyTM7 CD45 BV510 antibodies (1:1000), anti-FITC-mouse CD3ε antibodies (1:50), anti-PerCP-Cy5.5-mouse CD4 antibodies (1:50), and intracellular cytokines were then stained with anti-PE-mouse IFNγ antibodies (1:50) and anti-APC-mouse IL-4 (1:50) or anti-PE-mouse IL-17A antibodies (1:50). To detect frequencies of Treg cells in T lymphocytes in mice, the cells were stained using Fixable Viability Stain 510 (1:1000, FVS), anti-APC-mouse CD25 (1:50) antibodies, anti-FITC-mouse CD4 antibodies (1:50), and anti-mouse CD16/CD32 antibodies (1:50) or anti-PE-Cy7-CD8a antibodies (1:100), and then the cells were fixed and permeabilized using the Foxp3/Transcription Factor Fixation/Permeabilization set. Intracellular cytokines were stained with anti-PE-mouse Foxp3 antibodies (1:50). The cells were mixed well with antibodies and incubated in a darkroom at room temperature for 15 min. Then, the cells were resuspended in Flow Cytometry Staining Buffer in each tube ready for determination. The operations were conducted according to the instructions in the mouse Th1/Th2, Th17, and Treg Staining Kit. The cytokine and transcription factor levels (IFNγ, IL-17, Foxp3, or IL-4) are shown as the percentage of parent cells or the absolute number of positive cells in total lymphocyte. The information of antibodies and kits were shown in Table S4.

Quantification of serum cytokines and a histological analysis of muscles, fat, and growth plates

Blood samples were collected from the portal and jugular veins of piglets. The concentrations IFNγ, IL-2, IL-4, IL-13, IL-17, IL-22, and IL-10 in the colon and serum were quantified using porcine ELISA kits according to the manufacturer’s instructions. Hematoxylin and eosin (H&E) staining to analyze the fatty, muscular, and skeletal morphology was conducted using the method described Graca et al. [28]. The information of kits is shown in Table S4.

Cell transfection

Small interfering RNAs (siRNAs) of IL-4Rα, IL-13Rα1, and Scramble were synthesized by Suzhou GenePharma Co., Ltd, and the sequences are shown in Table S3. The NCTC1469 cells, which reached 60% confluence, were transfected with 50 pmoL of siRNA using jetPRIME (Polyplus-transfection SA) in accordance with the manufacturer’s instructions. The total proteins were extracted, and western blotting analysis was performed to confirm the protein levels of NCTC1469 at 36 h after transfection.

Three pairs of short hairpin RNAs (shRNA) were designed according to the Stat6 sequence. Stat6 shRNAs were synthesized by Suzhou GenePharma Co., Ltd, and the sequences are shown in Table S3. The ligation products were transformed into E.coli for their selected clones, and the plasmid DNA was isolated. The plasmids were digested, and the clones with the shRNA insert were selected. The reconstructed plasmids for transfection were purified with plasmid purification mini kit (Tiangen Inc.). NCTC1469 cells were cultured in DMEM supplemented with 10% HS and 2.05 mmol/L L-glutamine at 37 °C and 5% CO2. For transfection, the cells were seeded in 6-well plates at 1 × 106 cells/well and allowed to grow overnight to 75% confluence. They were transfected with a mixture of 2 μg plasmid DNA and 10 μL lipofectamine 2000 (Invitrogen) in 2 mL of serum-free medium. The cells were added to 500 μL FBS/well 6 h after transfection. The medium was replaced by normal medium containing 10% FBS at 24 h after transfection.

Indirect immunofluorescence assay

NCTC1469 and Hep-Li cells were seeded on coverslips in 24-well plates. The cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with methanol for 15 min, and blocked in 10% bovine serum albumin for 30 min. Indirect immunofluorescence assay was conducted by our previously described methods [29]. Fluorescence images were acquired with a fluorescence microscope (Thermo Fisher) or a confocal laser scanning microscope (Fluoviewver.3.1, Olympus).

Immunofluorescence staining

Intestinal segment samples were collected from the middle of the colon of piglets. The obtained segments were fixed in 4% paraformaldehyde and then embedded in paraffin. The segments were cut into approximately 5-μm-thick sections and stained with 5 μg/mL wheat germ agglutinin (WGA, Thermo Fisher) for 10 min. Subsequently, the slides were permeabilized with PBS buffer containing 0.2% Triton X-100 for 15 min and blocked with PBS buffer containing 5% BSA for 1 h. The slides were incubated with primary anti-CD3 antibody, anti-CD4 antibody, and anti-CD8 antibody at 4 °C overnight, which was followed by incubation with goat anti-rabbit IgG Alexa Fluor 488 at room temperature for 1 h. The slides were incubated with PBS buffer containing a 1 μg/mL DAPI solution for nuclear staining. Immunofluorescence images were obtained using a fluorescence microscope with the DP2-BSW software (Version 2.2, Olympus). The fluorescence values of each experimental group were analyzed after subtracting basal fluorescence (t = 0 h). The information of the materials is shown in Table S4.

Coimmunoprecipitation (Co-IP) assay

NCTC1649 cells were lysed in lysis buffer containing protease and phosphatase inhibitors. The cell-lysed solution was centrifuged at 230 × g and 4 °C for 10 min. The supernatant was incubated with a specific primary antibody by rotating at 4 °C overnight. Then, the incubated supernatant was added to 20 μL of 50% protein A + G-agarose slurry and rotated at 4 °C for 2 h. Protein A + G-agaroses containing antigen–antibody complexes were collected, rinsed with PBS, and centrifuged at 264 × g and 4 °C for 10 min. The immunoprecipitant was analyzed by western blotting.

Western blot

The cells and tissues were lysed using lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 μg/ml pepstatin, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 mM PMSF). Western blot assay was conducted by our previously described methods [29]. The antibodies used in the western blot assays were shown in Table S4.

Quantitative real-time PCR

The total RNA was extracted from cells or tissue using TRIzol reagent following the manufacturer’s instructions and reverse-transcribed to cDNA using a ABScript III RT Master Mix for the qPCR Kit (ABclonal). To confirm the abundance of abundance of major taxa, ileal and colonic mucous samples were collected and frozen and stored at − 80 °C freezer immediately. Total bacterial DNA was extracted from the samples (approximately 220 mg) using a M5 HiPer Stool Genomic Plus DNA Kit following the manufacturer’s procedures. Quantitative real-time PCR was performed using SYBR Green Gene Expression Assays. The details regarding specific primers are provided in Table S2, and the information of the materials and kits is shown in Table S4.

Body parameters

The bone mineral content (BMC), fat mass, and lean mass of the mice were measured using a dual energy X-ray bone densitometer (MEDIKORS, Gyeonggi, South Korea). The test mode was set with a high energy parameter of 80 kVp/1.0 mA and a low energy parameter of 55 kVp/1.25 mA. The InAlyzer 1.0 image processing system was used to analyze and process captured pictures of the lean, fat, and BMC in mice.

Bone length, compact bone substance, spongy bone, tissue mineral density, and the thickness of the cortical bone were evaluated using the computed tomography (120 kV/100 mAs, 0.5 s/0.27 mm, HP53.0).

Backfat thickness and eye muscle areas were measured using a B-scan-based technique (F: 3.6 MHz, D: 9 cm, PRC:13/5/H, PST: 2/2). Backfat in the area 1 cm lateral to the intersection point of the dorsal midline and its perpendicular line between the last third and fourth caudal rib was measured as the thickness of the subcutaneous fat layer between the skin and the fascia trunci profunda. The eye muscle area is the cross-sectional area of the longissimus dorsalis between the last third and fourth caudal rib.

Statistical analysis

The statistical analysis was performed using the Prism software version 8.2.1 (GraphPad, USA). Data were assessed for normal distribution and plotted in the figures. The differences between the two groups were assessed using two-tailed, unpaired Student’s t test with Welch’s correction. The differences among > 2 groups with one variable were assessed using one-way ANOVA. A two-way ANOVA with a Sidak post hoc test was used for > 2 groups with two variables. Taxonomic comparisons from 16S rDNA gene sequencing were analyzed with the Kruskal–Wallis test and the Bonferroni post hoc test. Pearson correlation coefficient (r) analysis was used to compare IL-13, IL-10, IL-22, or IL-4 in the portal vein and IGF-1 in the liver. Significant differences emerging above the results are indicated in figures as ∗ p < 0.05 and ∗∗ p < 0.01. Notable nonsignificant differences are indicated in the figures by “ns.”

Results

SLAs improve animals’ growth in their early stages

To evaluate the effects of SLAs on growth performance, 21-day-old male piglets were supplemented with quinocetone (Qui, a classic antibacterial growth-promoting agent) or an antibacterial agent cocktail (AC, a mixture of 4 antibacterial agents at therapeutic doses of each component [18, 19, 21, 22]) in the diet or conventionally raised (CR) for 28 days (Fig. 1A). The weight gain of piglets in Qui group was greater than that in AC and CR groups, but AC less than that in CR group during days 21 to 28 of the experiment (Fig. 1B). Compared with AC or CR, Qui treatment increased the body size (length, daily length gain, height and bust) and the weight of the liver and spleen in piglets (Fig. 1C, D). Eye muscle areas and backfat thickness are the most important predictors for the growth performance of animals. Qui-treated piglets had larger eye muscle areas and greater backfat thickness than CR- or AC-treated piglets (Fig. 1E and Fig. S1A-B). The bone parameters of the three treatments were evaluated with computed tomography. Compared with CR or AC, Qui increased the compact bone substance (CBS), tissue mineral density (TMD) and thickness of the cortical bone in the femur (Fig. S1C), and length of the tibia and femur of piglets (Fig. S1D). Likewise, compared with CR- and AC-treated piglets, Qui-treated piglets had thicker growth plates of the femur and tibia as shown by dual energy X-ray bone densitometer scanning and histological measurement (Fig. S1E). In addition, feed conversion ratio (FCR) in Qui-treated piglets (1.52) were better than those in CR- and AC-treated piglets (1.79 and 2.21, respectively) (Fig. 1F).

Fig. 1
figure 1

SLAs improve the growth of piglets. A In the experimental timeline, 21-day-old barrows were randomly assigned to three dietary groups as follows: (1) a corn-soybean meal basal diet (CR, n = 16), (2) the basal diet containing 50 mg/kg quinocetone [20] (Qui, a classic antibacterial growth-promoting agent, n = 16), and (3) the basal diet containing an antibacterial agent cocktail (AC, a mixture of 10 mg/kg ampicillin, 60 mg/kg metronidazole and 22 mg/kg neomycin for 14 days, and 15 mg/kg vancomycin for another 14 days, n = 10). B Body weight gain (n = 16). C Body length, daily length gain, height and bust (n = 8). D The weights of visceral organs, including the kidney, liver, spleen, inguinal lymph nodes (ILNs), and mesenteric lymph nodes (MLNs) (n = 9). E Imaging of eye muscle areas and subcutaneous backfat thickness (n = 8). F Feed conversion ratio was calculated as average daily feed intake divided by average daily gain (n = 8). Data are expressed as the mean ± SD. Differences among > 2 groups with one variable were assessed using one-way ANOVA. The two-way ANOVA with the Sidak post hoc test was used for ≥ 2 groups with two variables. * indicates a significant difference between two groups, P < 0.05, **P < 0.01. At the same time point, data labeled with different letters (a, b, c) indicate significant differences (P < 0.05), and ns indicates no significant difference. These statistical analysis methods and significance standards are also applicable to the following figures except for the extra description

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The effects of the SLA on the growth phenotype were further confirmed in mice. Twenty-one-day-old male mice were treated with Qui, AC and CR for 28 days (Fig. S2A). Qui enhanced the weight gain and the weight of the liver, spleen, and epididymal fat (an indicator for body fat) in mice compared with CR and AC (Fig. S2B, C). The total lean mass and fat mass of mice in CR treatment were greater than those in AC treatment, but less than those in Qui treatment (Fig. S2D). The average area of a single quadriceps muscle cell in Qui-treated mice was larger than that in CR- or AC-treated mice (Fig. S2E).

SLAs elevate hepatic IGF-1 in piglets and mice

Postnatal growth is regulated by activities of the somatotropic axis [30, 31], where GH secreted by the pituitary gland predominantly incites the synthesis of hepatic IGF-1 as an endocrine determinant for somatic growth [32]. In this study, GH and IGF-1 were measured in Qui-, AC-, and CR-treated piglets at day 28. There was no difference in the levels of hepatic and serum GH among the three groups (Fig. 2A). Intriguingly, Qui-treated piglets showed an increased IGF-1 level in the liver and jugular veins compared with CR- and AC-treated piglets (Fig. 2B). There was no difference in IGF-1 in the portal vein between Qui- and CR-treated piglets (Fig. S3A). IGF-1 in the liver can be transferred by insulin-like growth factor binding proteins (IGFBPs) to muscles, where it binds its receptor on the surface of muscular cells, phosphorylates AKT and promotes muscular cell proliferation [14]. In our study, the levels of IGF-1 in porcine muscles (quadriceps and anterior tibialis) of Qui group were greater than those of AC and CR group (Fig. S3B). The increased IGF-1 in muscles of piglets and mice resulted in the alteration of its downstream substances, including phosphorylated/total AKT ratio and genes related to myofibrillar synthesis (MyHCI, MyHCIIa, MyHCIIx, and MyHCIIb) and differentiation (Myogenin and MyoD) (Fig. 2C–E and Fig. S3C-D). In mice, Qui-induced IGF-1 alterations in the tissues were similar to those in piglets (Fig. S3C). These results indicate that the SLA promoted the development of skeletal muscles via inducing production of IGF-1 in the liver. IGFBPs, mainly IGFBP1 and IGFBP3, regulate IGF-1 activity via releasing and sequestrating IGF-1) [33, 34]. In our study, Qui and AC decreased IGFBP3, but did not alter IGFBP1 in the circular blood compared with the CR (Fig. S3E). However, the roles of IGFBPs in SLAs-induced growth promotion need to be investigated.

Fig. 2
figure 2

SLAs elevate hepatic IGF-1 in piglets. A The protein levels of growth hormone (GH) were measured with ELISA in serum from the jugular vein and liver of piglets treated with Qui, AC, and CR (n = 9). B The protein levels of insulin-like growth factor 1 (IGF-1) in the serum of the jugular vein and the protein and mRNA levels of hepatic IGF-1 were assayed (n = 9). C The protein levels of phosphorylated AKT/total AKT in the quadriceps and anterior tibialis muscles (n = 9). D and E The mRNA levels of genes related to myofibrillar synthesis (MyHCI, MyHCIIa, MyHCIIx, and MyHCIIb) and differentiation (Myogenin and MyoD) in the quadriceps (D) and anterior tibialis muscles (E) (n = 6)

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SLAs alter the composition of gut microbiota

To investigate SLAs-enriched gut microbes, 21-day-old piglets were treated with Qui, AC and CR for 28 days. The results showed that 515 OTUs were between CR and Qui groups, and 1329 OTUs, 1531 OTUs and 154 OTUs were unique at the species level for CR-, Qui-, and AC-treated piglets, respectively (Fig. 3A). The microbial species richness in the colonic and ileal mucosa was lower in the AC group than in the Qui group. However, there was no difference in other alpha diversity metrics between Qui- and CR-treated piglets based on Chao and Shannon indices (Fig. 3B and Fig. S4A). Unanimous shifts existed within the microbial communities in the piglets exposed to Qui, AC, and CR (Fig. 3C and Fig. S4B). Function prediction analysis demonstrated that differences existed in the immunity-related system, digestive system, endocrine system, cell growth and death, transport, and catabolism (Fig. S4C). KEGG analysis indicated that these changes in colonic flora composition were mainly involved in β-alanine metabolism, alanine aspartate and glutamate metabolism, butanoate metabolism starch, and sucrose metabolism and pentose and glucuronate interconversions (Fig. 3D). Furthermore, Qui caused shifts in the taxonomic composition. At the genus level, Qui induced higher abundances of Prevotella, Faecalibacterium, and Oscillospira in both colonic and ileal mucosa compared to CR (Fig. 3F, G and Fig. S4D-F). Notably, the highly enriched Prevotella in piglets exposed to Qui is consistent with other reports in SLA-treated piglets (Fig. 3H) [33, 35,36,37,38], which indicates that Prevotella may play an important role in growth promotion by SLAs. At the species level, Prevotella copri (P. copri) was highly enriched on the colonic mucosa of Qui-administered piglets in this study (Fig. 3E, G). The increased abundance of P. copri was also confirmed on the colonic mucosa of Qui-treated mice compared with CR-treated mice (Fig. 3I). Considering the commensalism of P. copri in variety of mammals, it was chosen for further studying its role in growth promotion by the SLA.

Fig. 3
figure 3

SLAs affect the microbiota composition on the colonic mucosa of piglets. Weaned piglets were treated with Qui, AC, and CR for 28 days (n = 3 per group). A The unique and shared genera shown by the Venn diagram in the different groups (n = 3). B Microbial community diversity showed by Shannon index and microbial community abundance shown by the Chao index (n = 3). C Pan curve demonstrates the relation between total number of operational taxonomic unit (OTUs) and the number of samples (n = 3). D The top 20 most enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. E The differential abundance of microbiota taxa on the colonic mucosa in the genus (up) and species (down) levels using MetagenomeSeq analysis. Significant differences are marked with colored dots or rings, and non-significant differences are marked with gray rings. F, G Phylogenetic tree of piglets’ colon-associated 16S rRNA sequences reported in this study. Each row in y axis is a different phylotype. Microbial community composition on the colonic mucosa of CR-, Qui-, and AC- treated piglets at the genus (F) and species (G) levels. The genus (Lactobacillus, Shigella, Chlamydia, Prevotella, Megasphaera, Oscillospira, Faecalibacterium, SMB53, Dialister, Lachnospira, ordered top to bottom) are color coded F. The species (Lactobacillus_salicarius, Lactobacillus_helveticus, Faecalibacterium_prausnitzii, Lactobacillus_hamsteri, Bulleidia_p-1630-c5, Lactobacillus_agilis, Prevotella_copri, Lactobacillus_delbrueckii, Prevotella_stercorea, Pediococcus_acidilactici, ordered top to bottom) are color coded G. Each column is a different group. For each genus and species type, the clone abundance is indicated by a color scale value. H The Abundance of Prevotellaceae was analyzed at the genus level in the intestinal tract of piglets supplemented with SLAs [33, 35,36,37,38] (i: chlortetracycline; ii: chlortetracycline; iii: bacitracin zinc and colistin sulfate; iv: chlortetracycline, bacitracin zinc and colistin sulfate; iv: chlortetracycline and olaquindox; v: chlortetracycline, olaquindox and enramycin; vi: chlortetracycline, olaquindox and virginiamycin; vii: bacitracin methylene disalicylate; viii: ixaureomycin and kitasamycin; ix: chlortetracycline; x: amoxicillin). I The relative abundance of P. copri on the colonic mucosa of Qui-, CR-, and AC-treated mice (n = 9). The two-sided Wilcoxon rank-sum test P value is presented to compare the difference of bacterial abundance among different groups

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Gut microbiota shifted by SLAs skews CD4 + T cell population toward a Th2 phenotype

SLAs shift gut microbiota and thereby would modulate the innate and adaptive immune responses of the reactive host cells [9, 10, 39]. In our study, the gut immunity exposed to the SLA-insulted microbiota was investigated. The fluorescence intensities of CD4+, CD3+, and CD8+ T cells were measured at day 28 posttreatment. There were more CD4+ T cells in Qui-treated piglets than in CR-treated piglets, whereas there were fewer CD8+ T cells in the piglets exposed to Qui and AC than in the piglets treated with CR. There was no change in the fluorescence intensities of CD3+ T cells between Qui- and CR-treated piglets (Fig. 4A). The Qui treatment increased the ratio of CD4+/CD8+ in the colon of piglets (Fig. 4B). Based on characteristic cytokines, effector CD4+ T cells can be differentiated into different subtypes, such as Th1 (IFNγ and IL-2), Th2 (IL-13, IL-4), Th17 (IL-17 and IL-22), and regulatory T (Treg, IL-10) cells [40]. In the colon (Fig. S5A) and portal vein (Fig. 4C) of piglets, Qui increased IL-4, IL-13, IL-22, and IL-10 compared with CR and AC, but decreased IFNγ and IL-17 compared with CR. AC treatment decreased IL-4, IL-13, IL-22, IL-10, IFNγ, and IL-17 compared with CR. There was no difference in IL-2 among the three groups. The effects of the SLA on immune responses were further confirmed in the colon of mice (Fig. S5B-F). Compared with CR, Qui treatment increased CD4+ T cells and decreased CD8+ T cells in the mesenteric lymph nodes (MLNs (a commonly used indicator for gut immunity [26, 41, 42]) of mice (Fig. S5B). Furthermore, compared with CR-treated mice, Qui treatment increased Th2 cells, while AC and Qui treatments reduced Th1, Th17, and Treg cells in the MLN of mice, which were also reflected by their corresponding representative cytokines (Fig. S5C-D). Intriguingly, Qui decreased the ratio of IFNγ+Th1/IL-4+Th2 cells compared with CR and increased the ratio of CD25+Foxp3+Treg/IL-17+Th17 cells in the MLN of mice compared with AC and CR (Fig. S5E). In addition, Qui treatment increased the mRNA levels of IL-13, IL-22, and IL-10, but decreased the mRNA levels of IL-2 and IFNγ in the colon of mice (Fig. S5F). The gene expression levels of IL-2, IL-13, IL-10, and IL-22 in CR-treated mice were greater than those in AC-treated mice (Fig. S5F). Furthermore, there were no difference among groups in the splenic and circulating protein level of IL-13 in mice (Fig. S6I). Collectively, our study demonstrated that Qui skewed the Th1/Th2 paradigm toward Th2 phenotype in the intestinal tract.

Fig. 4
figure 4

The gut microbiota shift skews CD4+ T cell population toward a Th2 phenotype in piglets. A Representative merged fluorescence images and the analysis of CD3+, CD4+ and CD8+ cells in the colon of piglets (n = 6) treated with Qui, CR, and AC. Scale bar = 200 μm. B The ratio of CD4+/CD8.+ fluorescence intensity in the colon of piglets (n = 6). C The protein levels of IL-2, IFNγ, IL-4, IL-13, IL-17, IL-22, and IL-10 in the portal vein of piglets (n = 6). Swine hepatocytes (Hep-Li cells; D, E) were treated with different concentrations (0, 1, 10, and 20 ng/mL) of recombinant IL-13 (RIL-13) for 12 or 24 h. The levels of IGF-1 in Hep-Li cells were evaluated by immunocytochemistry (D) and western blotting (E) (n = 3)

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Th2 CD4+cells-derived IL-13 upregulates hepatic IGF-1 in favor of growth

Post-neonatal growth is mainly regulated by the GH/IGF-1 axis [39, 43]. Because SLAs modify gut immunity via shifting intestinal microbiota, and increasing IGF-1 in the liver rather than GH in the systemic blood or liver, we hypothesize that SLAs-shifted gut immunity may affect the production of hepatic IGF-1 through certain cytokines transferred from the intestinal tract to the liver. The portal vein which carries blood from the intestinal tract to the liver is the most important route for gut microbiota to communicate with the liver. Considering the aforementioned phenotypic conversion of gut immunity induced by Qui, the elevated cytokines derived from gut Th2 and Treg cells in the portal vein were analyzed to determine whether they were implicated in the production of hepatic IGF-1. The results demonstrate that IL-13, IL-10, IL-4, and IL-22 in Qui-treated piglets were positively correlated (r = 0.902, 0.896, 0.770, and 0.756, respectively) with hepatic IGF-1 gene expression (Fig. S6A). Next, the two highest correlated cytokines (IL-13 and IL-10) were chosen to stimulate hepatocytes. Western blotting and immunocytochemistry showed that recombinant IL-13 (RIL-13) rather than IL-10 (RIL-10) increased the production of IGF-1 in a dose- and time-dependent manner in swine hepatocytes (Hep-Li) and mouse hepatocytes (NCTC1469) at 12 and 24 h (Fig. 4D, E and Fig. S6B-D). Then, IL-13 was used for the subsequent experiment. RIL-13 also increased IGF-1 levels in human hepatocellular carcinoma (HepG2) and primary hepatocytes isolated from piglets and mice (Fig. S6E-G). The blood in the portal vein mainly converges from the intestinal tract and the spleen vein and is drained directly into the liver [44]. Compared with CR piglets, Qui treatment caused increased IL-13 in the colon and portal vein of piglets, and in the colon of mice (Fig. 4C and Fig. S5A, F), but not in the spleen and peripheral blood of piglets and mice (Fig. S6H, I). These results indicate that upregulated IL-13 in the gut was delivered to the liver, where it promoted the production of IGF-1.

To evaluate the role of IL-13 in mediating growth dynamics, the male mice were pretreated with AC for 14 days and followed by intraperitoneal injection of different doses (0, 1, 2, and 5 ng/g) of RIL-13 for 14 days (Fig. 5A). AC pre-treatment caused a decrease in bacterial abundance on the ileal and colonic mucosa and in weight gain (Fig. S7A-B). RIL-13 enhanced weight gain of the AC-pretreated mice in a dose-dependent manner during days 9 to 14 of the trial (Fig. 5B). RIL-13 increased total lean mass, BMC, average cross-sectional area of single quadriceps femoris muscle cell, epididymis fat weight, and spleen and liver weight at day 14 after its application (Fig. 5B–D and Fig. S7C-E). Importantly, the intraperitoneal injection of RIL-13 led to increased IGF-1 in the liver and quadriceps, but not IL-13 in the peripheral blood (Fig. 5E and Fig. S7F). RIL-13 by itself did not induce myoblast cells (mouse C2C12) to express IGF-1 (Fig. S7G, H). These results indicate that IL-13 promotes growth via facilitating the production of IGF-1 in the liver.

Fig. 5
figure 5

Gut-derived IL-13 upregulates hepatic IGF-1 in favor of the growth of infant mice. A The experimental timeline, to shift gut immunity via depleting gut microbiota, 21-day-old SPF BALB/c male were pretreated with AC (1 mg ampicillin per mL water, 1 mg metronidazole per mL water and 1 mg neomycin per mL water, or 0.5 mg vancomycin per mL water) for 2 weeks. To verify whether RIL-1 intraperitoneal injection could recover growth, the AC-treated mice received intraperitoneal administration (i.p.) of RIL-13 (0, 1, 2, or 5 ng/g, n = 14) for another 2 weeks. B The weight gain in RIL-13-injected mice (n = 12) and the weight of the liver in mice on day 15 after RIL-13 injection (n = 8). C The total lean mass and fat mass were assessed by dual energy X-ray densitometer scanning in mice on day 15 after RIL-13 injection (n = 8). D Photos and hematoxylin–eosin staining of quadriceps femoris muscles (n = 3). Scale bars = 100 μm. E The levels of IGF-1 in the liver and quadriceps of mice (n = 9)

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IL-13 induces production of IGF-1 by activating Jak/Stat signaling pathway in vitro

IL-13 can bind with a heterodimeric receptor composed of IL-4Rα and IL-13Rα1 or with IL-13Rα2 [45]. IL-13Rα2 is not believed to contribute to IL-13 signaling since it possesses a short cytoplasmic tail without signaling motifs [45]. The functional heterodimeric receptor sequentially associates with Janus tyrosine kinases (Jaks) and signal transducers and activators of transcription (Stats) upon activation [46, 47]. The components of this IL-13R complex have been shown to vary in different cell types [48], but have not been reported in hepatocytes. The ability of IL-13R to selectively activate Stat proteins contributes to signal specificity [48]. IL-13Rα1 by itself binds IL-13 with low affinity; however, in the presence of IL-4Rα, IL-13Rα1 binds IL-13 at a high affinity [49]. In this trial, the expressions of IL-13Rα1 or IL-4Rα were inhibited by transfection with siRNA-IL-4Rα, siRNA-IL-13Rα1, or both (Fig. S8A, B), and this inhibition decreased IGF-1 expression in NCTC1469 cells exposed to RIL-13 (Fig. 6A and Fig. S8C). These results, together with the aforementioned RIL-13-induced upregulation of IGF-1 (Fig. 4D, E and Fig. S6), showed that IL-13R is necessary and sufficient for IGF-1 production of hepatocytes. Next, the downstream components of the IL-13R-activated signaling pathway were investigated in various hepatocyte across species. Among members of Jak and Stat families, Stat6 and Jak2 were phosphorylated in response to IL-13 in NCTC-1469 cells (Fig. 6B). These results were further confirmed in primary hepatocytes of mice (Fig. S8D). In addition, activated Stat6 were observed to translocate to the nucleus in NCTC1469 cells (Fig. 6C). In contrast to RIL-13 as an agonist, BMS-911543 (a Jak2 inhibitor) decreased IGF-1 in a dose-dependent manner in hepatocytes (Hep-Li, NCTC1469, and HepG2) of pigs, mice, and humans (Fig. 6D and Fig. S8E). Likewise, the expression of IGF-1 in the hepatocytes exposed to RIL-13 was inhibited by AS 1517499 (a Stat6 inhibitor) (Fig. 6E and Fig. S8F), which was further confirmed by the knockdown of Stat6 in NCTC1469 cells (Fig. 6F). Together, our data indicate that Jak2 and Stat6 were involved in IL-13R signal transduction for the production of hepatocyte IGF-1 upon exposure to IL-13.

Fig. 6
figure 6

Jak2 and Stat6 mediate IL-13-induced production of IGF-1. A The level of IGF-1 in NCTC1469 cells upon exposure to 20 ng/mL RIL-13 after transfected with siRNAs against IL-4Rα and IL-13Rα1 (n = 9). B STAT1-6, Jak1-3, and Tyk2 were phosphorylated in NCTC1469 cells when exposed to 20 ng/mL of RIL-13 (n = 3). C Confocal fluorescence microscopy showed that p-Stat6 was transferred into the nucleus of NCTC1469 cells exposed to 0 or 20 ng/mL of IL-13 for 12 or 24 h, scale bar = 100 μm. p-Stat6: red, DAPI: bule. D Hep-Li and NCTC1469 cells produced IGF-1 upon exposure to 20 ng/mL of RIL-13 and after they were treated with 0, 1, 3, or 5 μM of BMS-911543 (n = 3). E Hep-Li and NCTC1469 cells produced IGF-1 upon exposure to 20 ng/mL of RIL-13 after treated with 0, 0.1, 0.15, or 0.2 μM of AS1517499 (n = 3). F NCTC1469 cells produced IGF-1 upon exposure to 20 ng/mL of RIL-13 after transfected with sh-Stat6-1, sh-Stat6-2 and sh-Stat6-3, or sh-scramble (sh-NC) (n = 3). G (I and II) The phosphorylation cascade initiated by 20 ng/mL of RIL-13 was examined in NCTC1469 cells at 24 h. The phosphorylation states were analyzed using anti-phosphotyrosine immunoprecipitation (PY-99), which was followed by detection with an anti-IL-4Rα or anti-IL-13Rα1 antibody (I left and II left). The cell lysate was immunoprecipitated with antibodies against IL-4Rα (I right) and IL-13Rα1 (II right), and immune complexes were detected with an antibody against phosphotyrosine. (III) Jak2 kinase was immunoprecipitated from IL-13-treated or untreated hepatocytic lysates, which was followed by detection of IL-13Rα1 (left). The cell lysate was immunoprecipitated with an antibody against IL-13Rα1, which was followed by detection with an antibody against Jak2 (right). (IV) NCTC1469 cells were treated with 20 ng/mL of RIL-13 for 24 h, and the lysate was immunoprecipitated with the PY-99. The immunoprecipitated proteins were immunoblotted with Stat6 antibody (left). The cells were lysed and immunoblotted with an anti-phosphorylated Stat6 (Tyr 641) antibody (right)

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The ligation of phosphorylated components provoked by IL-13 was examined in NCTC1469 cells. The phosphorylation status of RIL-13-treated or RIL-13-untreated hepatocytic lysates was assessed with anti-phosphotyrosine immunoprecipitation (PY-99), which was followed by the detection of the immunoprecipitate with an IL-4Rα or IL-13Rα antibody on a western blot (Fig. 6G I left and II left). This was confirmed by performing trials with antibodies against to IL-4-Ra or IL-13Rα1 for immunoprecipitation, which was followed by detection with an antibody against phosphotyrosine (Fig. 6G I right and II right). The phosphorylation of IL-4Rα and IL-13Rα1 was increased in the hepatocytes exposed to RIL-13 (Fig. 6GI and II). Kelly-Welch et al. reported that IL-13Rα1 rather than IL-4Rα interacts with Jak2 [45]. Then, whether activated IL-13Rα1 associated with Jak2 was investigated. Jak2 kinase was immunoprecipitated from RIL-13-treated or -untreated hepatocytic lysates, which was followed by detection of IL-13Rα1 with its antibody. Antibodies against Jak2 immunoprecipitated IL-13Rα1 indicated that the recruitment of Jak2 to IL-13Rα1 was IL-13 dependent (Fig. 6G III left). Similarly, western blotting with an antibody against IL-13Rα1 in the immunoprecipitate followed by detection with antibody to Jak2, (Fig. 6G III right). These results indicated the association of IL-13Rα1 with Jak2 despite the lack of direct evidence here. Jaks can trigger the phosphorylation of Stats. Stat6 was detected in PY-99 immunoprecipitates in the lysates of hepatocytes exposed to RIL-13, which was confirmed by executing trials with anti-phospho-Stat6 antibody for immunoprecipitation, and followed by detection with the antibody to phosphotyrosine (Fig. 6G IV). Together, IL-13 utilizes IL-13R/Jak2/Stat6 signal transduction to promote IGF-I expression in hepatocytes in vitro.

The Prevotella copri facilitates gut-derived IL-13 secretion in mice

To determine whether the P. copri enriched by Qui promoted the production of IL-13 via altering the phenotype conversion of gut immunity, SPF mice pretreated with AC in drinking water for 14 days to deplete the gut microbiota received an intragastric garage and rectal delivery alternatively of the P. copri for 14 days, and mice were treated with saline alone as the control (Fig. 7A). The colonized P. copri with a high abundance on the mucosa of the ileum and colon of mice was found at day 14 of the trial (Fig. S9A). The mice administered with the P. copri showed a pronounced growth (Fig. 7B) with lean mass, fat mass, liver, and spleen increased (Fig. 7C, D). However, the bone mineral content in the P. copri-administered mice was not different from that in the control (Fig. S9B). The P. copri administration elevated IGF-1 in the liver, fat, and quadriceps muscles of mice (Fig. 7E). Additionally, the colonization of the P. copri increased IL-13, IL-4, and IL-10 mRNA, but decreased IL-12 mRNA in the colonic mucosa (Fig. S9C). Interestingly, IL-4+ Th2 cells were increased in response to the P. copri administration (Fig. 7F), while IFNγ+Th1 cells were reduced compared with the control (Fig. 7G), which led to decreased Th1/Th2 (Fig. 7J). The P. copri did not alter Treg and Th17 cells (Fig. 7H, I), but increased Treg/Th17 (Fig. 7J). These results indicate that the P. copri tips the gut immunity state to facilitate Th2 and Treg cells.

Fig. 7
figure 7

Prevotella copri promotes growth and shifts CD4+ T cell population toward Th2 phenotype in mice. A In the experimental timeline, to shift gut immunity via depleting gut microbiota, 21-day-old SPF BALB/c mice were pretreated with AC (1 mg ampicillin per mL water, 1 mg metronidazole per mL water and 1 mg neomycin per mL water, or 0.5 mg vancomycin per mL water) in drinking water for 2 weeks. To verify whether P. copri could recover growth, the AC-treated mice were alternatively administered intragastric garage (1 × 109 CFUs) and rectal delivery (0.5 × 107 CFUs) of P. copri for 14 days. Mice were similarly treated with saline as the control (n = 8). The i.g. and r.d. represent intragastric garage and rectal delivery, respectively. B, C Weight gain (B) and the weight of the liver, spleen, or epididymal fat (C) (n = 8). D Total lean mass and fat mass were assessed by dual energy X-ray densitometer scanning (n = 8). E The protein level of IGF-1 in the liver, fat and quadriceps muscles (n = 9). F–J Lymphocytes were isolated from the mesenteric lymph nodes of P. copri-treated mice, and the frequencies of CD4+IL-4+ (F), CD4+IFNγ+ (G), CD4+IL-17+ (H), and CD4+Foxp3+ cells (I), and the proportions of Th1/Th2 and Th17/Treg cells (J) were measured with flow cytometry (n = 6). K The polarized Th2 cells of mice were incubated with a supernatant concentrate of the P. copri preparation (0, 0.25 mmol/L) for 12 or 24 h. IL-13 was determined by ELISA (n = 6). Data are expressed as the mean ± SD. The differences between the two groups were assessed using two-tailed, unpaired Student’s t test with Welch’s correction

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Although IL-13 can be secreted by various cell types, it is predominantly produced by Th2-polarized CD4+ T cells [38]. To determine whether the P. copri facilitates production of IL-13, primary CD4+ T cells from mouse MLNs were incubated under Th2 polarization conditions. The polarized Th2 cells were supplemented with the inactivated P. copri or supernatant concentrate of the P. copri preparation. The inactivated the P. copri did not affect IL-13 expression in Th2 cells of mice (Fig. S9D-F), but the supernatant concentrate induced IL-13 expression in Th2 cells at 12 and 24 h (Fig. 7K).

Discussion

The immune response may produce various metabolic modifications via pro-inflammatory cytokines (such as IL-1, TNF-α, IL-6, and IL-13) acting on the GH/IGF-1 axis [50,51,52]. This is supported by the fact that the administration of recombinant IGF-1 abrogates pro-inflammatory cytokine-induced net protein loss and enhances peripheral glucose clearance in lambs [53], and that muscular protein synthesis is correlated with IGF-1 in immunity-challenged animals [54]. Proinflammatory cytokines are also associated with altered nutrient uptake and utilization, interrupted anabolic processes, and amplified catabolic activities [55, 56]. It is assumed that during an immune challenge, proinflammatory cytokines elaborate a homeostatic response in which nutrients deviate from tissue growth to support immune function [55, 56].

Scientists have been attempting to dampen the immune challenge (inflammatory response) to improve profitability and animal welfare. SLAs in the diets of younger animals have shown to improve growth performance compared with conventionally raised animals, and the effects are more pronounced when animals are raised in unhygienic environments compared to sanitary ones [12]. However, SLAs lack growth-promoting effects under GF condition [9]. These results indicate that SLAs improve juvenile animal growth by targeting gut microbiota and altering immunity states. Therefore, we propose a hypothesis in which certain immune cytokines derived from specific gut immune states facilitate the growth of the juveniles. In this study, we screened out IL-13 and discovered its strong potential for promoting the production of hepatocyte IGF-1 and improving growth in SLA-treated piglets and mice. IL-13 is secreted by multiple types of cells, including T cells, mast cells, basophils, eosinophils, and airway smooth muscle cells, but it is predominantly produced by TH2-polarized CD4+ T cells [57]. The inflammatory response characteristics of IL-13 may be associated with its tissue origins and immune states. Studies have proven that it plays an important role in several allergic disorders in humans, such as asthma and allergic dermatitis [57, 58]. On the contrary, it can also function as an anti-inflammatory cytokine [59]. It may be implicated in the hepatic fibrosis of systemic sclerosis as an anti-inflammatory cytokine by activating IL-13Rα1/Tyk2/Stat1/Stat6 signaling pathways in macrophages and fibroblasts [55, 60]. Wynes and Riches reported that Th2 cytokines IL-4 and IL-13 induced macrophage insulin-like growth factor-I expression [61]. In our study, the intraperitoneal administration of recombinant RIL-13 promoted the production of hepatic IGF-1 via IL-13R/Jak2/Stat6 signal transduction in vitro. The pleiotropic properties of IL-13 may be attributed to the activation of its distinct downstream signaling pathways in different tissues and cells. Most cytokines demonstrate marked redundancy in that different cytokines exhibit the same biological function. Although IL-10, IL-22 and IL-4 were all correlated with porcine hepatic IGF-1, their correlation coefficients and effects were less pronounced than those for IL-13. Further investigation is needed to determine if these cytokines act synergistically and to what extent they contribute to the production of hepatocyte IGF-1.

SLAs with different types and activities were effective across juvenile species, including children [62] and juvenile mammalian animals [9], suggesting their potential targeting of conserved microbiota in the intestinal tract. Our study showed that the SLA significantly influenced the composition of intestinal microbiota in piglets, which is consistent with the findings reported by Cho et al. (2012) [10]. Specially, the P. copri was enriched on the colonic mucosa of SLA-treated piglets and mice, leading to tip the gut immunity axis from CD4+ T cell population toward a Th2 phenotype. The P. copri is a ubiquitous inhabitant in the gut of both humans and animals, and its high abundance is associated with a diet rich in indigestible plant polysaccharides. It plays a beneficial role by fermenting dietary fiber [63]. In adult pigs, the P. copri is associated with fat accumulation [64]. These discrepancies in effects of the P. copri likely depend on factors such as dietary composition, age, gut microbial ecosystem, or immune state. In addition to P. copri, other intestinal bacteria, such as Faecalibacterium prausnitzii, were also enriched by the SLA. The Faecalibacterium prausnitzii is recognized as a probiotic and possesses anti-inflammatory properties. This finding underscores the importance of considering multiple microbes in shaping gut immunity.

Conclusion

Gut-derived IL-13 facilitates the production of hepatocyte IGF-1 by activating the IL-13R/Jak2/Stat6 pathway and improves the growth phenotype. Gut immunity modification may be a novel strategy for correcting metabolic disorders and alleviating persistent stunting or undernutrition that still affects one fourth children worldwide, which remains a global health challenge.

Data availability

The raw data generated or analyzed during this study are included in this article (and its supplementary information files). Other data that support the findings of this study are available from the corresponding author upon reasonable request.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

The authors would like to thank Pro. Libao Ma for providing the formula of weanling piglet feed and Dr. Jinlong Zhang for his guidance on piglet breeding. We thank Yongwei Zhu for his comparative analysis of the data on different bacterial populations in studies processed by SLAs. We would like to thank Academician Lanjuan Li, for providing the Hep-Li cell line. We are also thanking to Personalbio company for help with statistical analysis providing the graphical abstract about 16s RNA data.

Funding

This work was supported by the National Key R & D Program of China (2023YFD1801400), the Natural Science Foundation of China (32072938;32172930) and the Fundamental Research Funds for the Central Universities of China (2662022DKDY006).

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  1. College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China

    Ning Ma, Haolong Wang, Qiuhua Li, Mengyu Chang, Jiandi Zhu, Sha Nan, Qiulin Zhang, Panpan Guo, Ruiling Yin, Jinrui Sun, Huikang Wang, Qianghui Lei, Zhenli Liu, Mingxing Ding & Yi Ding

  2. College of Veterinary Medicine, Nanjing Agricultural University, Jiangsu, 210095, People’s Republic of China

    Qiao Li

  3. School of Animal Science and Technology, Hainan University, Haikou, 570228, People’s Republic of China

    Diqi Yang

  4. School of Life Sciences, Hubei University, Wuhan, 430070, People’s Republic of China

    Ke Ming

  5. College of Animal Science and Technology, Northwest A&F University, Shanxi, 712100, People’s Republic of China

    Shen Zhuang

  6. College of Biomedicine and Health, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China

    Xiaoshu Zhou

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Contributions

NM performed all experiments and the data analysis. XSZ and YD designed the project. HLW and QHL helped with experimental work. NM, QHL, HLW and JDZ contributed to the piglets handling. MYC, NS, and QLZ helped to set up experimental mice models. DQY, SZ and KM helped with flow cytometry analysis. QL, JRS and HKW helped with siRNA experiments. SN, RLY and QHL helped with experimental work about lentiviral shRNA knockdown. PPG and ZLL helped with the experiments of cultured bacteria. MXD supervised the project and obtained the funding for the project. XSZ and YD edited the manuscript. The authors read and approved the final version of the manuscript.

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Correspondence to Xiaoshu Zhou or Yi Ding.

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Ma, N., Wang, H., Li, Q. et al. Gut-derived IL-13 contributes to growth via promoting hepatic IGF-1 production. Microbiome 12, 248 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40168-024-01929-3

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Microbiome

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