Alterations in intestinal microbiota composition coincide with impaired intestinal morphology and dysfunctional ileal immune response in growing-finishing pigs under constant chronic heat stress

Background Previous studies had shown that short-term acute heat stress (HS) affected the host’s metabolism and intestinal microbiota independent of feed intake (FI) reduction, and long-term calorie restriction caused intestinal morphological injuries and gut microbial alterations. However, research on the effects of constant chronic HS on intestinal microbial composition and the roles of FI reduction played in is limited. This study aimed to investigate the effects of 7-day constant chronic HS on the composition of intestinal microbes in growing-finishing pigs, and its relationship with pigs’ performance, intestinal morphology, and ileal immune response. Twenty-four growing-finishing pigs (Duroc × Large White × Landrace, 30 ± 1 kg body weight) were randomly assigned to three treatments (n = 8), 1) thermal neutral (TN) conditions (25 ± 1 °C) with ad libitum FI, 2) HS conditions (35 ± 1 °C) with ad libitum FI, 3) pair-fed (PF) with HS under TN conditions to discriminate the confounding effects of dissimilar FI, and the FI was the previous day’s average FI of HS. The small intestinal segments (duodenum, jejunum, and ileum) and feces were collected on d 8. Results Results indicated that HS drastically declined (P < 0.05) average daily gain (ADG) and average daily feed intake (ADFI) (about 61%) in comparison with TN, and caused hyperpyrexia, meanwhile PF caused hypothermia. Morphological observation by light and electron microscopes showed that both HS and PF treatment decreased (P < 0.05) the villus and microvillus height compared with TN. Additionally, HS increased (P < 0.05) protein expression of heat shock protein 70 in the duodenum, jejunum, and ileum. Furthermore, the expression of tight junction protein zonula occluden-1 (ZO-1) in the duodenum and ileum, and Occludin in the ileum were enhanced (P < 0.05) compared with TN and PF. Moreover, HS significantly enhanced (P < 0.05) the mRNA relative expression of inflammatory cytokines (TLR-2, TLR-4, and tumor necrosis factor-α (TNF-α), IL-6, IL-8, PG1–5, β-defensin 2 (pBD-2)), mucins (mucin-1 and mucin-2) and P65 protein level in the ileal mucosa tissue. Intestinal microbiota analysis by 16S rRNA sequencing showed lower (P < 0.10) α diversity in both HS and PF, and a separated cluster of β diversity among groups. Compared with TN, HS but not PF mainly reduced (FDR < 0.05) Bacteroidetes (phylum), Bacteroidia (class) and elevated the proportions of Proteobacteria (phylum, FDR < 0.05), Bacillales (order, FDR < 0.05), Planococcaceae (family, FDR < 0.05), Kurthia (genus, FDR < 0.05), Streptococcaceae (family, FDR < 0.10) and Streptococcus (genus, FDR < 0.10). Notably, Lactobacillales (order) was decreased (FDR < 0.05) by PF alone. Furthermore, the Spearman correlation analysis indicated that the microbes prevalent in HS were positively (P < 0.05) associated with intestinal morphological injuries indicators and ileal immune response parameters, and the microbes reduced in HS were negatively (P < 0.05) with the performance data. Conclusions Intestinal morphological injuries and ileal immune response caused by constant chronic HS independent of FI showed close connections with alterations in intestinal microbiota in growing-finishing pigs.

Conclusions: Intestinal morphological injuries and ileal immune response caused by constant chronic HS independent of FI showed close connections with alterations in intestinal microbiota in growing-finishing pigs.
Keywords: Constant chronic heat stress, Growing-finishing pigs, Ileal immune response, Intestinal microbiota, Performance Background Due to the global warming and intensively reared, heat stress (HS) has been considered as a common challenge to livestock industries for detrimental to animal performance, health, and welfare, mainly manifest in physical disorders, feed intake (FI) reduction, growth retardation, and if severe and prolonged enough even death [1][2][3][4][5]. Growing-finishing pigs are particularly susceptible to heat stress because of few functional sweat glands, thick layer of fat, and high metabolic heat production [6][7][8][9][10][11][12][13][14]. The intestine is susceptible to the high ambient environment, and HS can cause the increase of intestinal permeability and together with bacterial translocation, which will provoke the immune response via toll-like receptors pathway [15][16][17]. Especially, ileum was reported more sensitive to HS than other intestinal sections [10]. Gut microbiota changes induced by HS were speculated to activate TLR4/NF-κB signaling pathway contributing to inflammatory response in the colon of pigs [18]. However, the research about the ileal immune response and the associations with gut microbiota alterations is limited.
Intestine is colonized with commensal and pathogenic bacteria, and gut microbial composition can be affected by psychological, environmental, and physical stressors, and associated with metabolic, inflammatory, and infectious diseases [19,20]. HS was found to alter gut microbial composition in broilers [21,22], laying hens [23,24], ducks [25], goats [26], and pigs [27][28][29]. Our previous research indicated that changes in gut microbiota caused by 24-h acute HS showed interactions with the host's metabolism [28]. The negative consequences of HS during short term are usually presumed to be related to heat load and duration [4,10,27]. However, the alterations in the intestinal microbiota of growingfinishing pigs under constant chronic HS are still unclear.
To less heat production, FI was usually declined by HS, and FI reduction caused by HS was usually thought to be an indirect reason for morphology injuries, and alterations in gut microbiota. Numbers of studies have shown that long-term calorie restriction alone can also affect the intestinal epithelium integrity and gut microbiota [10,30,31]. Our previous study revealed that 24-h acute HS affects the gut microbiota independent of FI which may be because of too short duration [28]. Are the alterations in gut microbiota under longer time constant chronic HS partly associated with long-term FI reduction? Whereas there are few studies conducted to assess the confounding effects of constant HS via reduced FI on gut microbiota. In this study, we set a pairfed (PF) group, which aims to discriminate the confounding effects of dissimilar FI.
This study was conducted to assess the effects of 7-day constant chronic HS on the performance, small intestinal morphology, ileal immune response, and gut microbiota, and aimed to reveal the possible link between alterations in gut microbiota and FI, intestinal morphological injuries, and ileal immune response using a pair-feeding technique.

Animal management and experimental design
The treatments were similar as described before [28]. Briefly, a total of 24 ear-tagged pigs (Duroc × Large White × Landrace, 30 ± 1 kg body weight) housed in constant climate-controlled individual pens were randomly assigned to three treatments (n = 8), 1) thermal neutral (TN) conditions (25 ± 1°C) with ad libitum FI, 2) HS conditions (35 ± 1°C) with ad libitum FI, 3) pair-fed (PF) with HS under TN conditions to discriminate the confounding effects of dissimilar FI, and the FI was the previous day's average FI of HS. The relative humidity was maintained at 75~85% and the treatments lasted for 7 d. Before the trial, all pigs were adapted to TN conditions for one week. Body weight was recorded at the beginning of the experiment and immediately preceding the sacrifice. The pigs were individually fed third a day, and the FI of each pig was recorded. At the end of the experiment, the performance parameters (average daily gain, average daily feed intake, feed: gain ratio) were calculated. Fresh water was added into a graduated water bucket in every morning during the whole experiment, and daily water intake was calculated based on the amount of water added and residue. The rectal temperature was determined by a mercury thermometer. The body surface temperature was measured by a handheld infrared digital thermometer. All pigs received the same basal diet (Table 1) according to the NRC (2012) standard, and provided water ad libitum, and were routinely immunized and health-care according to the requirements. The pig house and metabolic cage were thoroughly disinfected before the test. The test was carried out in the metabolic room of the Institute of Animal Science of Guangdong Academy of Agricultural Sciences.

Sample collection
As the experiment finished, after fasting overnight, all pigs were slaughtered. The intestinal tissue was separated and divided into the duodenum, jejunum, and ileum, and placed on the surface of icebox for later use. The tissue of the proximal duodenum, middle jejunum, and distal ileum were annulus sampled. Dissected intestinal sections were opened and washed thoroughly with cold sterile phosphate-buffered saline. Mucosa sample was scraped from the intestinal inner side with a sterile slide. Fresh fecal samples from the rectum were collected into sterilized tubes. All samples were collected and immediately transferred into liquid nitrogen and stored at − 80°C until subsequent analysis. Annulus small intestine sections samples of about 1.5 cm thickness were placed directly in 4% paraformaldehyde for overnight fixation at room temperature for subsequent H&E staining, and samples about 1 mm thickness were fixed for 1 h in cold 1% osmium tetroxide incacodylate buffer for electron microscopy examination.

Digesta pH measurement
After each intestinal segment was separated, both ends were tied with sterile cotton thread, then an incision was cut in the middle part, after that a hand-held portable pH digital acidity meter (testo 205, Testo, Lenzkirch, Germany) was inserted into the digestive tract to determine the pH value of content.

Morphological examination
After dehydration, paraffin embedding, sectioning, dewaxing, hematoxylin-eosin (H&E) staining, dehydration, and sealing, the sample pieces were obtained. Nikon Eclipse E100 microscope equipped with Nikon DS-U3 imaging system (Nikon, Tokyo, Japan) was used to collect images, and Case Viewer software (3DHISTRCH Ltd., Budapest, Hungary) was used to measure intestinal villus height and crypt depth. At least five well-oriented villi were calculated and reported as one number per pig.

Electron microscopy examination
Tissue sections were fixed for 1 h in 1% cold osmium tetroxide incacodylate buffer. Preparations were fixed by 2.5% glutaral-paraformaldehyde mixture. After dehydration,

DNA extraction and 16S rRNA amplification
As previously reported [28], after total genome DNA of feces extracted and assessed, the V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified via PCR using the forward primer 341F (5′-CCTA YGGGRBGCASCAG-3′) and the reverse primer 806R(5′-GGACTACHVGGGTWTCTAAT-3′) with a sample-unique barcode. The amplification was carried out in 30 μL reaction system with 15 μL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs, MA, USA), 0.2 μmol/L of forward and reverse primers, and about 10 ng template DNA. Thermal cycling consisted of initial denaturation at 98°C for 1 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and elongation at 72°C for 30 s, and then a final extension at 72°C for 5 min. After quantified, PCR products were mixed at equal density ratios and then purified with Gene JETTM Gel Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing libraries were generated using Ion Plus Fragment Library Kit 48 rxns (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's recommendations. The library quality was assessed on the Qubit @ 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). At last, the library was sequenced on an Ion S5™ XL platform (Thermo Fisher Scientific, Waltham, MA, USA) and 400 bp/600 bp single-end reads were generated.

Bioinformatics analysis
As described before [28], after assigned to samples based on unique barcode, raw reads were obtained by cutting off the barcode and primer sequence. Quality control of the raw reads was performed using the software Cutadapt (V1. 9 com/muscle/) [32]. OTUs abundance information were normalized using a standard of sequence number corresponding to the sample with the least sequences. Subsequent analysis was based on the normalized data of OTUs abundance information. Bioinformatics and statistical analyses were performed using the QIIME (Ver-sion1.7.0) and R (Version 3.0.3, http://www.r-project. org/). A Venn diagram was generated using the R package 'Venn Diagram' to visualize the shared and unique OTUs among groups. Alpha diversity indices (Chao1, Shannon) were applied to analyze the richness and diversity of the sequences, and difference analysis between groups was performed with Wilcox rank-sum test. Beta diversity analysis was used to evaluate the structure and distribution of the microbial genetic communities among the samples, and beta diversity based on both weighted and unweighted Unifrac distance was calculated. ANOSIM test (non-parametric test) was used to detect differences in community structure between groups based on OTUs relative abundance, and generated a test statistic R to assess the difference between and within groups. The R value close to 1 suggests dissimilarity between groups, while the R value close to 0 suggests an even distribution of high and low ranks within and between groups. The reliability of ANOSIM test is represented by P-value, and P < 0.05 indicates significance. OTUs-based principal component analysis (PCA), unweighted Unifrac distance-based and weighted Unifrac distance-based Principal Coordinate Analysis (PCoA), and Binary-Jaccard based nonmetric Abbreviations: F Forward, R Reverse; TLR-2, toll-like receptor 2; TLR-4, toll-like receptor 4; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; IL-8, interleukin-8; PG1-5, protegrin 1-5; pBD-2, porcine beta-defensin 2 multidimensional scaling (NMDS) plot were performed to show the distribution of samples. NMDS plot was conducted with a conventional cut-off of < 0.20 for the stress value. Unweighted Pair-group Method with Arithmetic Means (UPGMA) Clustering was performed as a type of hierarchical clustering method to interpret the distance matrix using average linkage. Linear discriminant analysis effect size (LEfSe) analysis was used test to determine the biomarkers in different groups, and perform LDA scores to estimate the effect size (threshold ≥ 4). The significant differential bacteria of top 10 at each level were determined by Kruskal-Wallis test followed by Dunn test, and false discovery rate (FDR) values were estimated using the Bonferroni method to control for multiple testing. The Spearman correlation analysis was applied to analyze the associations of the significantly differential microbes with the performance, intestinal morphological injuries indicators, and ileal immune response parameters. The significance was tested by corr.test in the psych package from R, after which the visualization work was done by the pheatmap function in pheatmap package.

Statistical analysis
Data of the performance and physical responses variables, intestinal morphological injuries indicators, and ileal immune response parameters are presented as mean ± standard error (SEM). Analysis was performed using SPSS18.0 Software (IBM Corp, Armonk, NY, USA) and GraphPad Prism Version 5 (GraphPad Software Inc., San Diego, CA, USA). Statistical analysis of the difference was performed by one-way analysis of variance (ANOVA) with Fisher's least significant difference (LSD) post hoc test. Differences were considered to be significant at P < 0.05, and 0.05 ≤ P < 0.10 mean a trend.

Constant chronic HS decreased the performance and affected normal physiological responses
Because one head of pig in the HS group dead on the 3rd day of experiment, the subsequent analysis of HS group was based on the data of the remaining 7 pigs. Effects of constant chronic HS on the performance and physiological responses of growing-finishing pigs were listed in Table 3. After 7-day constant chronic HS or feed limited, final body weight was declined (P < 0.05) compared with TN, and even caused body weight loss (0.70-0.96 kg). HS or PF treatment significantly decreased (P < 0.05) ADFI, about 61% reduction compared to TN, as well as water intake, but the ratio of water to feed was increased (P < 0.05). Compared with TN, HS significantly elevated (P < 0.05) the rectal temperature and body surface temperature (jugular, back, hind leg), while PF significantly decreased (P < 0.05) body temperature.

Constant chronic HS decreased pH value in the small intestinal segments
As presented in Table 4, pH value of the digesta in the different small intestine sections (duodenum, jejunum, ileum) was measured. HS significantly decreased (P < 0.05) pH value of digesta in the jejunum and ileum compared with PF.

Constant chronic HS caused morphological injuries in the small intestine
As illustrated in Fig.1 and Fig.2, HS led to marked morphological injuries in the small intestine, such as villi tips desquamation and the lamina propria exposing. To quantify the extent of damage, villus height, microvillus height, and crypt depth were measured. As depicted in Tables 5 and 6, both HS and PF decreased villus and microvillus height (P < 0.05) in the duodenum, jejunum, and ileum compared with TN. In addition, HS also significantly decreased (P < 0.05) the ratio of villus height to crypt depth in different small intestine sections (duodenum, jejunum, and ileum).

Constant chronic HS elevated the expression of heat shock protein 70 and tight junction proteins
As shown in Fig.3, HS increased (P < 0.05) expression of HSP 70 in the duodenum, jejunum, and ileum, and induced up-regulation (P < 0.05) of the expression of tight junction protein ZO-1 in the duodenum and ileum, and Occludin in the ileum compared with TN and PF.

Associations of significantly differential intestinal microbes with the performance, intestinal morphological injuries indicators, and ileal immune response parameters under constant chronic HS
The Spearman correlation analysis between significantly differential intestinal microbes and the performance, morphological injuries indicators, and ileal immune response parameters was shown in Fig. 7.

Discussion
Previous studies showed that FI reduction is one of the main adaptations employed to reduce heat production in response to HS [4,7,12,28]. In line with previous results, Fig. 5 Effects of constant chronic heat stress on intestinal microbial communities. Venn diagram showed the proportion of common and special OTUs among groups (a). Alpha diversity index such as Chao1 (b) and Shannon (c) index indicated the diversity and evenness. Binary-Jaccard distance-based NMDS plot (d), OTUs-based PCA plot (e), unweighted unifrac distance-based PCoA plot (i), weighted unifrac distance-based PCoA plot (k) were used to display the distribution of the samples among groups. ANOSIM test was performed to detect differences in community structure between groups based on OTUs relative abundance (f, g, h). UPGMA clustering was conducted based on unweighted unifrac distance and weighted unifrac distance (j, i). Differences of α-diversity indices were determined by Wilcox rank-sum test, and differences of β-diversity indices were determined by ANOSIM test. * presented P < 0.05, *** presented P < 0.001, ns mean no significant difference. TN, thermal neutral conditions (25 ± 1°C); HS, heat stress conditions (35 ± 1°C); PF, pair-fed with HS under TN conditions (25 ± 1°C). n = 8 for TN and PF, n = 7 for HS we found that 7-day constant chronic HS caused hyperpyrexia, drastically ADFI declined (61%), and weight loss. Meanwhile, hypothermia in FI can be explained by calorie restriction which can lower core body temperature in endothermic animals [33].
It is well documented that intestinal epithelial cells under HS experiencing ischemia, hypoxia, inflammation, and oxidative stress can result in morphological injuries such as villus tips desquamation, villus height shorter, and lamina propria exposed [7,34]. Coincided, morphological injuries in the small intestine were also observed in heat-stressed pigs in the present study. Several studies found that HS can lead to intestinal barrier dysfunction and hyperpermeability by reducing and redistributing tight junction (TJ) proteins [34,35]. However, in this study, the results indicated a compensatory increase of TJ proteins (ZO-1, Occludin) in the duodenum and ileum along with HSP 70 elevated. HSPs, a class stressinducible protein, aids in protein folding, ubiquitination, renaturing, and repairing, were found to protect heatinduced intestinal tight junction barrier disruption by up-regulation and reorganization of TJ proteins [36]. It was reported that HS-induced up-regulation of Occludin expression in Caco-2 cells is mediated by activation of heat shock factor-1(HSF-1), which can promote the expression of HSPs [37]. In addition, HS exposure results in the overexpression of HSPs in organs of different species [7,[38][39][40][41][42]. Moreover, HSPs expression in the intestine were considered to be more sensitive to long-term stress than conventional stress markers such as serum concentrations of catecholamines, cortisol, lactate [38], and showed significant associations with intestinal immunity [42].
Increased intestinal permeability caused by HS may result in bacterial translocation and pathogen loads elevated, which can activate numerous bacterial endotoxinspecific recognition receptors in intestinal epithelial cells. Toll-like receptors are a class of classical recognition receptors, such as TLR-2 and TLR-4, which can recognize the damage-associated molecular patterns to produce several pro-inflammatory cytokines to evoke the host immune response during heat stress [15,17]. In this study, we found that HS up-regulated TLR2/4 mRNA expression meanwhile elevated the expression of proinflammatory cytokines TNF-α, IL-6, IL-8 as well as P65, indicated the activated intestinal immune response Brackets indicated the affiliation between species. Differences were determined by Kruskal-Wallis test followed by Dunn test, and false discovery rate (FDR) values were estimated using the Bonferroni method to control for multiple testing (* presented FDR < 0.05, ns mean no significant difference). n = 8 for TN and PF, n = 7 for HS under HS, which is consistent with previous report [18]. Epithelial cells can produce mucins and anti-microbial peptides, which can enhance the intestinal epithelial barrier function by selective microbial permeation and directly influencing microbial populations [43,44]. The increased gene expression of mucins (mucin-1, mucin-2) and anti-microbial peptides (PG1-5, pBD-2) may be speculated as another compensatory mechanism to endogenously protect the intestinal epithelial barrier. In addition, the results of a feed restriction test suggested Fig. 7 The Spearman correlation analysis between significantly differential microbes and the performance (A), small intestinal morphological injuries indicators (B), and ileal immune response parameters (C) under constant chronic HS. The heatmap of the correlation coefficient, the red represents positive correlation and the blue represents negative correlation, respectively (* presented P < 0.05, ** presented P < 0.01). Abbreviations: ADG, average daily gain; ADFI, average daily feed intake; V/C, the ratio of villus height to crypt depth; PG1-5, protegrin 1-5; pBD2, porcine beta-defensin 2; TLR-2, toll-like receptor 2; TLR-4, toll-like receptor 4; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; IL-8, interleukin-8 that 8-day 80% restricted intake caused 172 genes in immune response pathways upregulated in adipose tissue of weaning pigs [45]. However, the ileal immune response was not affected by PF in the current study. Too much FI reduction (61%) in the present study may cause malnutrition, which would explain the differences.
Firmicutes (phylum) and Bacteroidetes (phylum) have been reported as predominant microbial communities in pig gut microbiota [46,47]. This agrees with our data, Firmicutes (phylum) and Bacteroidetes (phylum) total comprise 67.33-86.80% in pigs' gut microbiota. Based on the results of our previous study [28], we prolonged HS duration to 7 days in the current study, and we found that 7-day constant HS also decreased the relative abundance of Bacteroidetes (phylum), and at the same time increased Proteobacteria (phylum) as the second abundant microbe instead of Bacteroidetes (phylum). Unfortunately, opportunistic pathogen Acinetobacter (genus), Kurthia (genus), and pathogen Streptococcus (genus) were observed prevalent in HS. The intensity of host response to HS is presumably decided by heat loads and duration [27], how the gut microbiota is changed by heat loads and duration need further investigations.
Increasing evidences have suggested the crosstalk between gut and host through microbiota [43,48,49]. Reduction of FI by HS appears to be affected by appetite-related signals, post-absorptive metabolism, and intestinal function and integrity [3,50]. A previous study has suggested that microbiota can participate in the regulation of host appetite-related pathways by its metabolites, for example, Escherichia coli can activate satiety via its by-product caseinolytic protease (Clp) B, which is an antigen-mimetic of anorexigenic factor αmelanocyte-stimulating hormone(α-MSH) [51]. Associations between FI and alterations in gut microbiota have been analyzed in pigs in our previous study, and we found that FI is positively associated with Bacteroidetes (phylum), Bacteroidia (class), Bacteroidales (order) and Prevotellaceae (family) and negatively associated with Proteobacteria (phylum), γ-Proteobacteria (class), Pseudomonadales (order), Moraxellaceae (family) and Acinetobacter (genus) in 24-h acute heat-stressed pigs [28]. Similarly, in the current work, FI was observed positively associated with Bacteroidales (order), and negatively associated with Proteobacteria (phylum), γ-Proteobacteria (class), Pseudomonadales (order), Moraxellaceae (family) and Acinetobacter (genus). In heat-stressed ducks, ADG was reported to be negative correlated with Proteobacteria (phylum), and positive associated with Firmicutes (phylum) in the jejunum [25]. Differently, in the present study, ADG showed no significant association with Firmicutes (phylum). We found that sampling location in the gastro-intestine, species, and statistical method would affect above correlation analysis results, therefor bacteria transplantation technique would be a better choice to explore the relationship between the microbiota and phenotypic characteristics.
In this paper, the intestinal morphological injuries indicators showed positive associations with Proteobacteria (phylum) and negatively with Bacteroidetes (phylum). Gut microbiota can influence the host through their by-products, such as short-chain fatty acids (SCFAs), which are not only energy sources but also beneficial to intestinal epithelial integrity [52]. Nevertheless, as one of the main of SCFAs producing bacteria [53], Bacteroidetes (phylum), Bacteroides (class), and Bacteroides (order) are lower in HS in present study. Moreover, butyrate-producing bacteria Ruminococcaceae (family) [54] was also reduced by HS in the current work. Declined of SCFAs producing bacteria means fewer SCFAs, and lead to intestinal epithelial integrity injuries. The results of jejunum microbiota in heat stressed-ducks were partly similar to our results that the morphological injuries were observed positively with the relative abundance of Proteobacteria (phylum) and negatively with Firmicutes (phylum), and this is a possible reason for different species [25]. Moreover, the prevalence of opportunistic pathogen Acinetobacter (genus), Kurthia (genus), and pathogen Streptococcus (genus) in HS caused intestinal inflammation and apoptosis, which may be another reason for morphological injuries.
We found the mucosal immune activation via TLR4/ nuclear factor kappa-B (TLR4/NF-κB) signaling pathway in the ileum by HS. The results of the fecal transplantation experiment also indicated that changes in gut microbial composition promoted by HS could activate the TLR4/NF-κB signaling pathway and cause inflammation in the colon of pigs [18]. Moreover, stress induces increased permeability of the gut allowing bacteria translocation and mucosal immune activation, which were reported to alter gut microbial composition in turn [48]. Crosstalk between the microbiota and intestinal immune system is critical in the maintenance of mucosal homeostasis. Accumulation evidences indicated that commensal and pathogenic bacteria and their metabolites participate in the metabolic process [43,48,49]. In this work, the results revealed that ileal immune response by HS positively with Proteobacteria (phylum), Streptococcaceae (family), Bacillales (order) and negatively with Bacteroidetes (phylum).
FI reduction caused by HS was thought to a main reason for morphology injuries and alterations in gut microbiota [6,10,31]. It is often difficult to discriminate the indirect influence of FI reduction under HS. Therefore, a pair-fed model was utilized in the current study, and we found a separate cluster of PF from TN and HS indicated the difference between PF and HS. Probiotics such as Lactobacillus (genus) were reported significantly favored by life-long feed restriction in the intestine of rodents [30]. Conversely, we found that PF decreased Lactobacillales (order) in the current study. Differences in species, growth stage, duration of feed restriction, and amount of FI reduction would explain the discrepancy.
Modifying feed behavior such as feed distribution frequency and size has been reported to affect the performance and metabolic of pigs reared in TN conditions [55]. The limitation of the present study is that the experimental design of PF model just considered the meal size but did not consider the change of feed frequency affected by HS. Fortunately, we found a study by Serviento and colleagues which reported that increased feed provision frequency to 8 times a day did not improve the HS response of pigs [56]. Therefore, further work is urgently needed in the exploration of how to improve the HS response of pigs via feeding management.

Conclusions
Intestinal morphological injuries and ileal immune response caused by constant chronic HS independent of FI showed close connections with alterations in intestinal microbiota in growing-finishing pigs. This study provided a better understanding of the mechanisms involved in heat stress response, in particular regarding the involved immune response and the epithelial barrier dysfunction. How the gut microbiota alterations under HS affect the host's gut immunity and the underly mechanism need to be further explored.