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Exhaustive Exercise Induces Gastrointestinal Syndrome through Reduced ILC3 and IL-22 in Mouse Model

Hou, Pengfei; Zhou, Xi; Yu, Li; Yao, Yu; Zhang, Yong; Huang, Yujie; Chen, Mengtin; Yi, Long; Mi, Mantian

Author Information
Medicine & Science in Sports & Exercise: August 2020 - Volume 52 - Issue 8 - p 1710-1718
doi: 10.1249/MSS.0000000000002298

Abstract

Erratum

The authors of “Exhaustive Exercise Induces Gastrointestinal Syndrome through Reduced ILC3 and IL-22 in Mouse Model” () report an error Figure 3. The amended figure is presented hereinafter.

FIGURE 3
5+ images

Medicine & Science in Sports & Exercise. 53(3):671-672, March 2021.

Exercise has beneficial effects for human health and is helpful for the protection against several metabolic diseases such as obesity, type 2 diabetes, and cardiovascular diseases (1). Moderate exercise (3.0–5.9 metabolic equivalents) is helpful for the promotion of musculoskeletal and cardiovascular adaptations associated with improved health. However, substantial exposure to exercise stress might be detrimental to human health, leading to the disorder, dysfunction, or even injury of certain tissues and organs, especially of the gastrointestinal tract (2). Evidence has shown that the prolonged and/or exhaustive exercise (e.g., marathon) would cause “gastrointestinal syndrome (GIS),” manifested with diarrhea, cramping, nausea, and gastric pain, resulting in the reduction of the exercise performance. Recent reports demonstrated that 86% elite athletes suffered from severe or moderate severe GIS (3).

There have been a substantial amount of studies investigating the causal mechanisms involved in the exercise-induced GIS. Up to present, the exercise-associated GIS was associated with several factors, including splanchnic blood flow redistribution and subsequent intestinal ischemia, cytokine responses, sympathetic activation, gastrointestinal motility, and malabsorption (4–6). Particularly, a hypothesis regarding the circulatory–gastrointestinal pathway is widely accepted (2), which involves the redistribution of blood flow to working muscle and peripheral circulation, subsequently reducing total splanchnic perfusion, finally leading to the perturbation of gastrointestinal integrity and function. Such gastrointestinal ischemia and intestinal energy exhaustion lead to the dysfunction of several intestinal cell types, subsequently a consequence of increased intestinal barrier permeability (7). However, the mechanism remains unknown.

The intestinal mucosal barrier is composed of four parts including the physical, chemical, biological, and immunologic barriers, respectively. The integrated intestine barrier can prevent the translocation of harmful substances such as lipopolysaccharide (LPS) from the lumen to the bloodstream. The physical barrier is maintained by the intestinal epithelial cells, which are connected to each other by tight junction (TJ) proteins such as claudins, occludin, and zona occludens (ZO-1, ZO-2, and ZO-3) (8). Because of the special construction, intestinal villi are more sensitive to splanchnic hypoperfusion, and hypoxia and acidosis are the critical immune suppressors (9). Meanwhile, the survival of lymphocyte is only dependent on blood glucose and glutamine (10). The glutamine supplementation can combat with exercise-induced GIS possibly through the maintaining of the intestinal barrier function (11). Furthermore, recent studies identified innate lymphoid cells (ILC), non-B non-T lymphocytes, play a key role in the maintenance of the intestinal barrier function. ILCs are divided into three major groups, T-bet+ ILC1, GATA3+ ILC2, and RORγt+ ILC3. Among them, RORγt+ ILC3 is mainly distributed in the intestinal lamina propria and an important source of interleukin (IL)-22, a critical cytokine responsible for the modulation of the crosstalk between epithelial cells, certain immune cells, and the commensal microflora (12,13). Mice deficiency in IL-22 suffers from severe intestinal inflammation and destruction of the epithelial barrier, with a higher susceptibility to Citrobacter rodentium infection (14). Upon activation by IL-22, intestinal epithelial cells express antimicrobial proteins and mucus-associated molecules. IL-22 could also upregulate the expressions of certain TJ proteins in the model of LPS-induced intestinal epithelial injuries (15). Thus, we hypothesized that ILC3 and IL-22 might be highly involved in the occurrence of exercise-associated GIS.

MATERIALS AND METHODS

Animals and experimental protocol

Male C57BL/6 mice weighing 20–22 g at 8 wk of age were housed at a controlled temperature (22°C–25°C) and humidity (50%–55%). The mice were maintained on a 12-h light/12-h dark cycle in the Experimental Animal Center of Third Military Medical University (Chongqing, China). They were administrated with the chow diet (10% fat, 70% carbohydrate, 20% protein; D12450B, Research Diets), whereas food and water were changed every 3 d and were provided ad libitum. Sixteen mice were randomly divided into two groups (eight mice/group), nonexercise (NE) and exhaustive exercise (EE) groups. Mice in the NE group were kept in cages as normal. For acclimatized training, mice in the EE group were placed in a motorized treadmill (SANS Biological Technology, Jiangsu, China) in the morning, running at a speed of 15 m·min−1 once a day for 7 d, including 2 d of resting (weekend) and 5 d in the treadmill for 10 min. On day 8, mice in the EE group ran at the speed of 20 m·min−1 until exhaustion. The exhaustion was defined as the inability to run for 20 s, although the mice were continuously prodded by the electrode slice (16). The mice in the two groups were weighed. Five mice from each group were sacrificed immediately, and the other three mice were used for gut permeability experiment. The liver, small intestine, and colon tissues were removed and stored at −80°C. All animal experiments were approved by the Institutional Animal Care and Use Committees of Third Military Medical University (Chongqing, China; Approval SYXC-2015-00169) and followed the National Research Council Guidelines.

Gut permeability assay

Mice were orally administered with a fluorescein isothiocyanate (FITC)–dextran (40,000 kDa) (Sigma-Aldrich, catalog no. 46944, 125 mg·mL−1) at a dose of 600 mg·kg−1 body weight (17). The blood samples were collected 2 h later and then were centrifuged at 6000× rpm for 10 min at 4°C. The serum was diluted with the equal volume of phosphate-buffered saline (PBS) and analyzed using a SpectraMax M2 microplate reader (Molecular Devices, San Jose, CA) with an excitation wavelength at 485 nm and an emission wavelength at 535 nm as previously described (17). The standard curve was applied for calculating the concentration of FITC–dextran through a serial dilutions of FITC–dextran in PBS.

Histological analysis

The small intestines and colons were dissected and fixed in 4% formaldehyde for at least 24 h. Then, the samples were embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin–eosin. The microscopic examination was performed, and photographs were taken using a light microscope (Nikon, Tokyo, Japan).

Ultrastructure observation by transmission electron microscopy

The small intestine and colon tissue samples were fixed in 2.5% glutaraldehyde for 18–20 h, followed by postfixed in buffered 1% osmium tetroxide for 1 h, dehydrating using ascending grades of ethanol and dry acetone, and embedding in epoxy. Ultrathin sections of 70 nm were cut, mounting on 200-mesh hexagonal copper grids, staining with 2% uranyl acetate and lead citrate, and observed using a JEM-1400 microscope (JEOL, Tokyo, Japan). A minimum of 10 photomicrographs per mouse were taken randomly from each sample at 30,000× magnification, and at least three mice in each group were used for the quantitative analysis.

Immunofluorescence staining

Segments of small intestines and colons were harvested and fixed in 4% paraformaldehyde. After that, 5-μm-thick, paraffin-embedded sections of the colon were prepared and incubated in 0.5% of Triton X-100 for 1 h at room temperature. The sections were then blocked with 5% bovine serum albumin at room temperature for 30 min and were subsequently incubated with the primary antibodies against proliferating cell nuclear antigen (PCNA; 1:200; Proteintech, Rosemont, IL) and E-cadherin (1:100; Proteintech) overnight at 4°C, respectively. Furthermore, the sections were incubated with the goat antimouse Cy3 (1:300; GB21301; ServiceBio, Wuhan, China) or FITC (1:300, GB22301; ServiceBio) antibodies at room temperature for 2 h. After washing with PBS, the sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China). Subsequently, they were washed and mounted with Fluoromount-G (SouthernBiotech, Birmingham, AL). The fluorescence was visualized by a laser scanning fluorescence microscopy (Leica TCS SP5; Leica). The mean fluorescence intensity was measured using ImageJ software.

Quantitative real-time polymerase chain reaction

Total RNA was extracted from intestine tissues of mice using TRIzol reagent (Invitrogen Life Technologies, Grand Island, NY). Reverse transcription of mRNA into cDNA was carried out with PrimeScript RT master mix (Takara Bio, Japan). Polymerase chain reaction (PCR) assay was performed using a SYBR Premix Ex Taq (Takara) with qTOWER 2.2 (Analytik Jena, Jena, Germany). Each sample was processed in triplicate and normalized to β-actin with the 2−△△CT method. The prime sequences are listed in Table 1.

T1
TABLE 1:
Primers used for real-time reverse transcription PCR.

Western blot analysis

Proteins were extracted from the intestine by using tissue lysis buffer (Thermo Scientific, Waltham, MA) with a protease inhibitor cocktail (Roche Diagnostic, Mannheim, Germany). Samples were separated in sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked by 5% dried skimmed milk for 1 h and were incubated with the corresponding primary antibodies under rotation overnight at 4°C. The membranes were incubated for 1 h with the peroxidase-conjugated secondary antibody at room temperature, and the proteins were visualized by Fusion FX (Vilber Lourmat, Paris, France) with Millipore Immobilon ECL substrate (Millipore, Inc.). The primary antibodies used were ZO-1 (1:1000 dilution; Invitrogen), Occludin (1:1000 dilution; Abcam), Claudin-1 (1:1000 dilution; Invitrogen), sirtuins-3 (SIRT3, 1:1000 dilution; Cell Signaling Technology), hypoxia-inducible factor-1a (HIF-1a; 1:500 dilution; Cell Signaling Technology), and β-actin (1:1000 dilution; Santa Cruz), respectively.

Isolation of lymphocytes from intestinal lamina propria lymphocytes

As for the isolation of lamina propria lymphocytes (LPL) from the intestine, the intestines were cut open longitudinally followed by the removal of associated fat tissues and Peyer’s patches. Then, the intestines were washed with iced PBS and shaken in D-Hank’s (Beyotime) medium containing 5 mM of ethylenediaminetetraacetic acid, 1 mM of dl-dithiothreitol, and 10 mM of HEPES for 15 min at 37°C three times to remove intraepithelial lymphocytes and intestinal epithelial cell (IEC). The tissues were then digested in Hank’s medium containing DNase I (0.5 mg·mL−1) and collagenase II (Gibco, Waltham, MA) for 30 min at 37°C three times and were homogenized manually through 70-μm cell strainers for collecting the LPL. Mononuclear cells were then harvested from the bottom of a 30% Percoll (Sigma, St. Louis, MO) gradient after a spin at 2000× rpm for 10 min at room temperature.

Flow cytometry analysis

The isolated LPL were stained with antibodies against CD45 (30-F11), CD3ε, NKp46, CD127, fixable viability dye-510, and CD4 (GK1.5; BD Biosciences, Franklin Lakes, NJ) for 20–30 min at 4°C. The intracellular staining was performed with an anti-RORγt (B2D; BD Biosciences) antibody with a FoxP3 transcription factor buffer Kit (eBioscience, San Diego, CA). For IL-22 staining, the LPL was stimulated with 50 ng·mL−1 of recombinant IL-23 (R&D Systems, Minneapolis, MN), 100 ng·mL−1 phorbol-12-myristate 13-acetate, and 1 mg·mL−1 of ionomycin, with 10 ug·mL−1 brefeldin A (BD Biosciences) for 4 h at 37°C, 5% CO2. After stimulation, cells were first surface-stained a combination of the antibodies listed previously and subsequently incubated with fixation/permeabilization buffer (BD Biosciences) at 4°C for 20 min. As for intracellular transcription factor and cytokine staining after fixation, cells were incubated for 30 min at room temperature with an anti–IL-22 antibody (eBioscience). Cells were acquired on a BD FCSverse flow cytometer (BD Biosciences) and analyzed with FlowJoV10 software.

Statistical analysis

All analyses were conducted in SPSS 19.0 (Chicago, IL). All experimental data are expressed as mean ± S.E.M. Statistical differences among groups were determined with Student’s t-test (for two groups). P values less than 0.05 were considered statistically significant. Each experiment was performed a minimum of three times.

Results

Exhaustive exercise induced intestinal injury and inflammation in mice

There were no significant differences in body weight and average daily food intake between the NE and EE groups (Supplemental Digital Content 1, Body weight and daily food consumption between the NE and EE groups, https://links.lww.com/MSS/B932). After the exhaustive exercise, histological staining was conducted in the small intestine and colon of mice. Compared with the NE group, the integrated structure of mucosal in the small intestine and colon of mice from the EE group was changed with notable congestion and inflammatory infiltration (Fig. 1A). Besides, the expression of PCNA in the colon tissues of mice was detected by the immunofluorescence staining. As shown in Figures 1B and C, the expression of PCNA in the EE group was decreased compared with that in the NE group, suggesting a decreased cell proliferative activity in colon tissues followed by the exhaustive exercise. Moreover, the mRNA levels of tumor necrosis factor α (TNF-α), IL-6, and interferon-γ (IFN-γ) in the intestine were examined by quantitative real-time PCR (qRT-PCR) assay. The mRNA expressions of the indicated genes were significantly increased in the intestine of mice from the EE group (Figs. 1D–F). Before the exhaustive exercise, the mRNA levels of IFN-γ and TNF-α were not changed (Supplemental Digital Content 2A–B, Acclimatized training dose not induce inflammation and intestinal barrier dysfunction in mice, https://links.lww.com/MSS/B933). Overall, the results demonstrates that exhaustive exercise results in an increased intestinal injury and inflammation in the intestine.

F1
FIGURE 1:
Exhaustive exercise induced intestinal injury and inflammation in mice. A, Representative photographs of small intestine and colon sections with hematoxylin–eosin staining (top row, 200× magnification; bottom row, 400× magnification). B, Representative images of PCNA immunofluorescence staining in NE and EE group colons. Red fluorescence represents the expression of PCNA protein, and blue fluorescence with DAPI represents cell nucleus. C, The bar charts show the quantification of fluorescence intensity. The mRNA expression levels of certain inflammatory markers TNF-α (D), IFN-γ (E), and IL-6 (F) in intestine tissues were assessed by qRT-PCR assay. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the NE group by independent-samples Student’s t-test for all statistical analyses.

Exhaustive exercise induced a disrupted intestinal barrier integrity

Next, the effect of exhaustive exercise on intestinal barrier integrity and permeability, as well as the expression of the associated genes, was analyzed. Exhaustive exercise led to an obviously increase of intestinal permeability in the EE group compared with that in the NE group (Fig. 2A). Moreover, the mRNA levels of ZO-1 and Occludin were decreased remarkably in the EE group (Figs. 2B, C). The acclimatized training had no significant effect in the mRNA levels of ZO-1 and Occludin (Supplemental Digital Content 2C–D, Acclimatized training dose not induce inflammation and intestinal barrier dysfunction in mice, https://links.lww.com/MSS/B933). The protein levels of ZO-1, Occludin, and Claudin-1 were also reduced in the EE group (Figs. 2D–G), implying a disruption of the intestinal barrier integrity induced by exhaustive exercise. Observed by the immunofluorescence staining, the expression of E-cadherin was reduced in the intestine and colon of mice from the EE group, revealing a disrupted intestinal epithelial adherence junctions in the intestine and colon of mice followed by exhaustive exercise (Figs. 2H–J). Besides, the morphological ultrastructure of intestinal villi was determined by transmission electron microscopy assay. Mice from the EE group showed less microvillus in the small intestine and colon, accompanied by the damaged IEC with mitochondrial swelling and vacuolation (Fig. 2K). Taken together, exhaustive exercise results in a disrupted intestinal barrier integrity, manifested with increased paracellular permeability, decreased the expressions of TJ proteins and damaged IEC in the intestine and colon of mice.

F2
FIGURE 2:
Exhaustive exercise induced a disrupted intestinal barrier integrity. A, Intestinal permeability was measured by oral administration of 4000-Da FITC–dextran. Blood samples were taken, and the serum levels of FITC–dextran were determined using a microplate reader. The relative mRNA expression levels of ZO-1 (B) and occludin (C) in intestine tissues were assessed by qRT-PCR. D, The protein expression levels of ZO-1, Occludin, and Claudin-1 were evaluated by Western blotting. E–G, Relative protein levels were quantified by densitometry. H, Representative images of E-cadherin immunofluorescence staining in the small intestine and colon between the NE and EE groups. Green fluorescence represents the expression of E-cadherin protein aggregate form, and blue fluorescence with DAPI represents cell nucleus. I–J, The bar charts show the quantification of fluorescence intensity. K, Intestinal epithelial cell from the NE and EE group mice detected using transmission electron microscopy (magnification, 30,000x; scale bar, 500 nm). *P < 0.05, **P < 0.01, ***P < 0.001. Two groups were compared by independent-samples Student’s t-test for all statistical analyses.

Exhaustive exercise results in a reduced IL-22 level and ILC3 in the LPL

We further detected whether the alterations of intestinal inflammation, barrier integrity, and permeability induced by the exhaustive exercise were associated with the disorder of certain intestinal immune cells and the associated cytokines. IL-22 is mainly secreted by ILC3, Th17, and Th22 cells, and played a key role in maintaining the intestinal barrier and antimicrobial function. A notably decreased mRNA level of IL-22 was found in the intestine of mice in the EE group (Fig. 3A), but not in the acclimatized training mice (Supplemental Digital Content 2E, Acclimatized training dose not induce inflammation and intestinal barrier dysfunction in mice, https://links.lww.com/MSS/B933). Furthermore, the protein expression of IL-22 in the LPL was detected by an intracellular staining with the corresponding antibodies. The percentage of IL-22–produced CD45+ cells in the intestine of mice from the EE group was dominantly less than that from the NE group (Figs. 3B, C). As previous studies showed, IL-22 produced in CD3 non–T cells (particulary ILC3) in the LPL of mice was more than that in CD3+ T cells (such as Th17 and Th22 cells; Supplemental Digital Content 3A, The expression of IL-22 in CD3 or CD3+ cells of LPL, https://links.lww.com/MSS/B934). However, there was no difference in the percentage of IL-22+CD3+CD4+ T cells in the LPL between the NE and EE groups (Figs. 3D–F). Moreover, several relative genes including IL23 and IL1β, which were involved with the production of IL-22 in the intestine of mice, were measured by qRT-PCR assay. The mRNA expressions of the indicated genes were decreased significantly in the EE group compared with the NE group (Figs. 3G–H). Furthermore, the intestinal LPL was gated on CD45+CD3CD127+RORγt+ ILC3, and the two subpopulations of ILC3, Nkp46+ILC3 (NCR+ILC3), and CD4+ILC3 (LTi) were also analyzed. Compared with the NE group, there was a remarkable decrease of intestinal ILC3 in the EE group, in particular the NCR+ILC3 rather than LTi (Supplemental Digital Content 3B, The expression of IL-22 in CD3 or CD3+ cells of LPL, https://links.lww.com/MSS/B934), suggesting that the reduced percentage IL-22 in the intestine of mice might be resulted from less ILC3 followed by the exhaustive exercise (Figs. 3I–K). To conclude, the results indicate that exhaustive exercise results in a decreased expression of IL-22 in the LPL of mice, which is closely associated with the reduced percentage of ILC3 rather than Th17 and Th22 in the intestine of mice.

F3
FIGURE 3:
Exhaustive exercise results in a reduced IL-22 level and ILC3 in the LPL. A, The relative mRNA expression levels of IL-22 in intestine tissues were assessed by qRT-PCR assay. B, Intestinal LPL was stimulated by phorbol-12-myristate 13-acetate, IL23 (50 ng·mL−1), and ionomycin with Golgi Stop for 4 h. IL-22 expression in CD45+ cells was analyzed by flow cytometry. Experiments were independently repeated at least three times. C, Frequencies of IL-22–expressing cells in the CD45+-gated cell populations of the intestines were shown. D, IL-22 expression in CD3+CD4+ cells was analyzed by flow cytometry (cells were gated by CD45). Experiments were independently repeated at least three times. E–F, Frequencies of IL-22–expressing cells in the CD3+CD4+-gated cell populations of the intestines were shown. G–H, The relative mRNA expression levels of IL1β and IL23 in intestine tissues were assessed by qRT-PCR. I, ILC3 (CD45+CD3CD127+ RORγt+), NCR+ ILC3 (CD45+CD3CD127+RORγt+ Nkp46+CD4), and LTi (CD45+CD3CD127+RORγt+Nkp46CD4+) in intestine LPL were analyzed by flow cytometry. Experiments were independently repeated at least three times. J–K, Percentages of all cell populations of the intestine LPL from the NE and EE group mice were shown.*P < 0.05, **P < 0.01, ***P < 0.001. Two groups were compared by independent-samples Student’s t-test for all statistical analyses.

Exhaustive exercise led to the glucose exhaustion and the altered metabolic homeostasis

Then, we examined the energetic metabolism alteration of mice after the exhaustive exercise. There was a significant decrease of hepatic glycogen in the liver of mice from the EE group by the periodic acid–Schiff reaction assay (Figs. 4A, B). The blood glucose level in the EE group was obviously lower than that in the NE group after exhaustive exercise (Fig. 4C). However, compared with the NE group, the blood lactic acid level was notably higher in the EE group (Fig. 4D). These data indicated that exhaustive exercise might induce energy exhaustion in mice. To further confirm whether the exhaustive exercise could induce certain changes of metabolic phenotype in the intestine of mice, the expressions of SIRT3 and HIF-1a were measured by Western blot. SIRT3 is a key mitochondrial gene that regulates the oxidative phosphorlation, whereas HIF-1a contributes to the glycolysis process. As expected, a decreased expression of SIRT3 and an increased expression of HIF-1a were observed in the EE group, implying that a reduced IL-22 level and ILC3 in the LPL of mice might be resulted from the alteration of the metabolic phenotype induced by the exhaustive exercise (Figs. 4E–G). The results indicate that exhaustive exercise might result in an energy substance exhaustion as well as a reduced oxidative phosphorlation and increased ischemia–hypoxia in the intestine of mice, leading to an immunosuppressive effect of certain immune cells such as ILC3 in the intestine.

F4
FIGURE 4:
Exhaustive exercise led to glucose exhaustion and altered metabolic homeostasis. A, Representative photographs of liver sections with periodic acid–Schiff staining (200× and 400× magnification). B, The bar charts showed the quantification of mean density. The blood glucose (C) and lactic acid (D) levels in each group were measured. E, The protein expression levels of HIF-1a and SIRT3 were evaluated by Western blotting. F–G, Relative protein levels were quantified by densitometry. *P < 0.05, **P < 0.01, ***P < 0.001. Two groups were compared by independent-samples Student’s t-test for all statistical analyses.

DISCUSSION

As expected, we found, for the first time, that the acute high-intensity running-induced GIS in a mice model was closely associated with a reduced percentage of ILC3 and IL-22 level in the intestinal lamina propria. This work might provide a novel preventive and therapeutic target toward the populations suffering from the exercise-induced GIS. Previous studies have demonstrated that substantial exposure to exercise induces considerable physiological changes in the gastrointestinal tract. The term, “exercise-induced gastrointestinal syndrome (GIS),” has recently applied to describe physiological responses to exercise that perturbs and compromises gastrointestinal integrity and function (18). Many athletes suffer from GIS when they participate in the training or competitions, accompanied by the decreased exercise performance. Almost 30%–90% GIS have been reported in endurance sports, especially running (3). One reasonable explanation for exercise-induced GIS might be splanchnic hypoperfusion, which results in the disrupted intestinal barrier integrity and function as well as increased intestinal permeability (2). However, the mechanisms remain elucidated. The primary aim of this study was to investigate the role of intestinal physical and immune barriers in the occurrence of acute running-induced intestinal permeability and examine their possible association with running-induced GIS. Our results show that the acute high-intensity running could induce a disrupted intestinal barrier integrity and an aggravated inflammation in a running-to-exhaustive mice model, possibly due to the intestinal mucosa hypoperfusion. The impaired intestinal integrity and function might be associated with impaired epithelial barrier as well as reduced percentage of ILC3 and IL-22 level in the LPL (Fig. 5). To our knowledge, this is the first study that directly links an intestinal immune barrier, particularly ILC3 and IL-22 in the LPL, to exercise-induced GIS. This finding sheds light on the mechanism involved in the intestinal responses followed by the acute exercise.

F5
FIGURE 5:
Exhaustive exercise–induced gastrointestinal symptoms were involved with reduced ILC3 and IL-22 in the intestinal lamina propria. The acute high-intensity running induces glycogen exhaustion and intestinal mucosa hypoperfusion, followed by an increased intestinal permeability, which is associated with impaired epithelial barrier as well as reduced percentage of the ILC3 and IL-22 levels in the LPL, finally leading to the occurrence of GIS.

A large variety of studies showed that prolonged and strenuous physical exercise increases intestinal permeability, allowing luminal endotoxins to translocate through the intestinal barrier and reach the bloodstream (19). Meanwhile, high-intensity exercise also increased the intestinal permeability through the downregulation of TJ proteins. Dokladny and colleagues (20) also found that high-intensity exercise (e.g., 1 h of running at 70% V˙O2max) could induce the dysfunction of almost all the epithelial cell types and disordered TJ proteins, finally leading to the increased intestinal permeability. However, in our study, we also found that the adherence junction maintained by E-cadherins was decreased significantly after exhaustive exercise. Moreover, there were severe mitochondrial injuries and reduced microvillus of IEC after acute exercise. To our knowledge, the injured mitochondria could increase oxidative stress and trigger cell apoptosis through the mitochondria-dependent pathway (21). The reduced microvillus may increase the susceptibility of bacterial infection (22). Overall, the decreased intercellular junctions coupled with the injury IEC contributed to the higher intestinal permeability and intestinal inflammation, which is the reason why occurrence of exhaustive exercises led to gastrointestinal symptoms. This is the first report on exhaustive exercise–induced disruption of intestinal barrier integrity, but the mechanisms were still not elucidated.

The immune system and its responses to any specific stimulus are coordinated to protect the body’s tissues against pathogenic agents (23). More and more studies have shown that high-intensity exercise would hurt the athletes’ immune system and damage their resistance to illness (24). However, most of the literature available just focused on the peripheral blood and spleen instead of the regional immunity of intestinal mucosa (25). In this study, we investigated the alterations of intestinal mucosal immune changes between the NE and EE groups. T-bet+ ILC1, GATA3+ ILC2, and RORγt+ ILC3 are three major subgroups of ILCs. RORγt+ ILC3 can produce IL22 and IL-17, and it is mainly distributed in the lamina propria of the intestinal mucosa. In recent years, several studies focused on the protective role of IL-22 in the maintenance of barrier integrity and anti-infection capability through promoting the IEC to secrete antimicrobial proteins, such as S100A8/A9 and RegIIIβ/γ (15). The IL-22+CD4+ T cells, which have been termed as“Th22 cells” and ILC3, produce IL-22 in the intestine. To our knowledge, there is no report regarding the association of ILC3 and IL22 with exhaustive exercise–induced GIS. According to current study, we found that the level of IL-22 in the intestine was significantly reduced after exhaustive exercise. However, we did not find any change of IL-22 secreted by Th22 cells in mice that suffered from exhaustive exercise. Interestingly, our study has demonstrated that the proportion of ILC3 was obviously reduced in the EE group mice. Moreover, we found that the expressions of IL-1β and IL-23 in the intestine, which could promote ILC3 to secret IL-22, were significantly reduced after exhaustive exercise. Moreover, there was a study showing that treatment with IL-22 induced intestinal epithelial cell expression of TJ proteins (e.g., Claudin-1 and ZO-1) and facilitated transepithelial resistance to LPS-induced damage, indicating that IL-22 protects intestinal mucosa from inflammation via maintenance of epithelial barrier integrity (15). Therefore, our data indicate that exhaustive exercise might induce the immunosuppressive effect with a decreased proportion of the ILC3 and IL-22 level, leading to the increased intestinal permeability and decreased anti-infection effect.

High-intensity exercise also changes the neuroendocrine factors, metabolic factors, and blood flow, whereas ischemic stress and oxidant damage can affect the intestinal barrier function and immune system (10). According to our study, decreased hepatic glycogen and blood glucose after exhaustive exercise cannot be neglected, and glucose is the primary substance of energy source for the immune cells (26,27). Carbohydrate beverage ingestion before, during, and after 2.5 h of exercise was associated with higher plasma glucose levels, fewer perturbations in blood immune cell counts, and a diminished proinflammatory and anti-inflammatory cytokine response (28). Carbohydrate availability could alter mucosal immune responses to exercise (29). On the other hand, the alteration of HIF-1a and more lactic acid in the EE group demonstrated that hypoxia–ischemia occurred in the intestinal microenvironment. According to the previous studies, HIF-1 shaped various immune responses and immune metabolism (30). Hypoxia–ischemia could occur as early as 10 min of high-intensity exercise, and splanchnic hypoperfusion could occur 20 min into a 60-min bout of cycling at 70% V˙O2max (31). After then, reperfusion in the period of recovery induces xanthine oxidase reaction releasing hydrogen peroxide, a potent free radical, which causes tissue breakdown (32). The lower level of SIRT3 may contribute to the mitochondrial dysfunction and reactive oxygen species–related apoptosis pathway (33). These results all together indicated that EE would induce the alteration of substance and energy metabolism. The disruption of metabolic homeostasis in the EE mice may conduct the significant mechanism to the reduced intestinal barrier function after exhaustive exercise. Different metabolic pathways determined by SIRT3 or HIF-1a could lead to the changed levels of certain metabolites such as coenzyme A, which may subsequently affect the immunophenotype of certain immune cells such as ILC3. However, the more precise metabolic mechanism will be explained by some new techniques such as metabolomics.

In summary, exhaustive exercise would affect the intestinal permeability with reduced ILC3 and IL-22 in the LPL, which might be associated with the altered energy metabolism in the intestine. This work will help establish essential strategies to maintain the intestinal permeability and mucosal immunity to prevent the GIS. Certain dietary supplements might contribute to the prevention against perturbed intestinal function followed by substantial exercise (34). Carbohydrate, glutamine, and probiotics supplementation have been proposed to improve the exercise-induced perturbation of immune and intestinal permeability (35). Thus, it is an urgent demand to find new targets in intestinal mucosal and to provide new preventive strategies for athletes.

This work was supported by the Major Project of Military Logistics Scientific Research (No. AWS17J014) and the Military Pre-research Foundation of Army Medical University (No. 2016XYY03). The results of the study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation. The results of the present study do not constitute endorsement by the American College of Sports Medicine. The authors thank Dr. Youcai Deng (Third Military Medical University), Dr. Yufeng Wang (The First Hospital of Jilin University), and Dr. Luxi Chen (Ohio State University) for their assistance with the isolation of intestinal lamina propria lymphocytes from mice and the analysis of ILC3 by flow cytometry. We also thank Dr. Xiaohong Yao and Jie Zhou (Third Military Medical University) for the pathological assessment of the HE stained sections.

All authors declare no conflict of interest.

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Keywords:

GASTROINTESTINAL SYNDROME; ILC3; IL-22; EXHAUSTIVE EXERCISE; LAMINA PROPRIA

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