The rumen is the most important site for digestion and absorption in ruminant animals. Rumen digestion is performed by symbiotic microbes, and the major fermentation productions, short-chain fatty acids (SCFAs), absorbed by the rumen epithelium, meet 50–70% energy requirement of the animals. However, persistent contact of the rumen epithelium with trillions of bacteria brings serious threatens to the host healthy. To maintain a harmonious symbiosis with these exogenous antigens, the host has to be tolerant of commensals. Tolerance is defined as a state in which the immune system of the host rendered non-reactive toward commensals (van Baarlen et al., 2009). The commensals in the tolerance state contribute to the energy absorption of the host by modulating the expression of host genes that participate in breakdown and absorption of energy substrates/diverse and fundamental physiological functions (Hooper and Gordon, 2001). The acquiring of tolerance needs the activation of innate and adaptive responses in the prevention of an inappropriate inflammatory response (Wood et al., 2012; Belkaid and Hand, 2014; Swiatczak and Cohen, 2015). In animals, the gastrointestinal (GI) microbiota has a crucial function in building and maintaining gut tolerance. A dysbiosis of GI microbiota predisposes the host to various kinds of immune and metabolic diseases (Ohland and Jobin, 2015).

Previous studies have shown that diet contribute to the immune homeostasis by promoting the SCFAs productions of GI microbiota, which modulate the immune responses via G protein-coupled receptors (GPRs) and histone deacetylases (HDACs) pathways (Tan et al., 2014). A study on mice revealed that diet enhanced immune tolerance also by affecting the interactions of pattern-recognition receptors (PRPs) and commensals in GI tract (Zelante et al., 2013). Our previous studies have shown that the increased intake of non-fiber carbohydrate (NFC), which can be rapidly fermented by GI microbes, causes the significant increases in the concentrations of butyrate, acetate and propionate in goat rumen (Yan et al., 2014; Lu et al., 2015). However, yet little is known about the diet-induced specification of ruminal microbiota and their effects on the immune tolerance of rumen epithelium.

Toll-like receptors (TLRs) have a critical role in suppressing inflammation of the GI epithelium. Mice exhibits an increased susceptibility to intestinal inflammation when they are deficient in myeloid differentiation primary response 88 (MyD88), an important component of the TLR signaling cascade (Frantz et al., 2012; Chu and Mazmanian, 2013). TLRs are also reported to suppress the inflammatory responses by reducing inflammatory cytokine productions (Oosting et al., 2014), by conditioning tolerance CD103⁺ dendritic cells (DCs) (DePaolo et al., 2012), and by modulating the development of regulatory T cells (Tregs) (Kramer et al., 1996). Although, no similar report concerning the functions of TLRs in maintaining the rumen homeostasis is presently available, the study of Malmuthuge et al. (2012) has revealed that TLR 1–10 is constantly expressed in ruminal epithelium. We therefore speculate that the interactions between ruminal microbiota and epithelial TLRs play important roles in promotion of immune tolerance during dietary modulation.

In this study, a combination of molecular microbiology and immunology methods has been applied to investigate the synergetic responses of the rumen microbiota and the expressions of TLRs at the apical surface to the switch of dietary NFC from 15 to 31%. The expressions of SCFA-absorption-related genes were investigated to understand the effects of altered microbiota on the epithelium functions. These results provide a better understanding of the nature of the host–microbe interactions.

The study was approved by the Animal Care and Use Committee of Nanjing Agricultural University, in compliance with the Regulations for the Administration of Affairs Concerning Experimental Animals (The State Science and Technology Commission of P. R. China, 1988).

Our results demonstrated that the diversity and richness of the microbial community was increased in the MNFC group. Phylogenetic analysis of the detectable OTUs revealed the expansion occurred in all major lineages. Ecological and evolutionary theory suggests that the more diverse communities make a greater contribution to ecosystem functioning. Bell et al. (2005) has proposed that, if each species uses a slightly different nutrient resource and occupies a highly specific niche in the community, then the more diverse microbiota should be able to capture energy, and resistance to invading pathogens more efficient. According to this principle, the MNFC diet should benefit rumen immunity and metabolism, since a more highly diversified ruminal microbiota was observed in the MNFC group. In the present study, the Verrucomicrobia was most significantly expanded after a 4-week period of MNFC feeding. However, this was not detected in the rumen microbiota of goats suffered with diet-induced subacute ruminal acidosis (SARA) (Mao et al., 2016). Previous studies have shown that the bacteria belonging to the Verrucomicrobia play important roles in the acquiring of immune tolerance in mice gut (Derrien et al., 2011). A decrease or absence of these species in the mammalian gut was associated with a decline of host immunity (Png et al., 2010; Swidsinski et al., 2011). Notably, our LEfSe analysis revealed that opportunistic pathogens, namely Rikenella (Alkadhi et al., 2014), Anaeroplasma (Kalshingi et al., 2015), and Olsenella (Vieira Colombo et al., 2016) were significantly shrunk in MNFC group, which might reduce the probability of inflammation in the rumen epithelium as well as ruminant animals. In addition, the concentration of ruminal butyrate was significantly increased in MNFC group. The study of Gantois et al. (2006) has shown that butyrate is able to suppress the expression of virulence genes in bacteria. Thus, the results of our study taken together indicate that an MNFC diet makes commercials bolster their resistance to pathogens. This is, obviously, beneficial to the immunity and health of the host.

Our study has also demonstrated that, in the rumen epithelium, the upregulated expressions of TLR 1, 6, 10, and MyD88 expressions were associated with the downregulated expressions of IL-1β, 6, and 10 after a 4-week period of MNFC feeding. Previous studies have shown that, upregulated expressions of pro- and anti- inflammatory cytokines were associated with the unhealthy changes of animals, such as pathogen/virus infection and SARA (Chang et al., 2015; Zhang et al., 2015). In such unhealthy state, pro-inflammatory cytokines enhanced the attack of the immune cells to the pathogens by activating inflammatory responses, and the anti-inflammatory cytokines inhibited immune hurt to the tissue by suppressing inflammatory responses. Accordingly, the synergic decreases of pro-inflammatory cytokines IL-1β and 6 and anti-inflammatory cytokine IL-10 in MNFC group indicated the enhanced tolerance of epithelium to the rumen microbiota.

The study of Oosting et al. (2014) has revealed that antagonistic antibody blocking or siRNA silencing of TLR10 results in the enhanced production of IL-1β and 6 in cultures of human peripheral blood mononuclear cells (PBMCs). Our in vivo findings agree with the study of Oosting et al. (2014) by indicating an inhibitory property of TLR10 signaling on the expression of pre-inflammatory cytokines in the rumen epithelium. In humans, TLR10 is suggested to exert its function through the formation of heterodimers with TLR2 (Guan et al., 2010; Oosting et al., 2014; Stappers et al., 2015; Ammerdorffer et al., 2016). However, the upregulation of TLR10 expression was not associated with any significant change of TLR2 in our study. By searching for the locations of TLRs in the goat genome annotation release 101 in NCBI (Dong et al., 2013), we noted that TLR10 is located on chromosome 6, together with TLR1 and TLR6, whereas TLR2 is located on chromosome 17, independently. Moreover, Opsal et al. (2006) have observed the synergy in the expression of TLR1 and TLR6 in cattle rumen. We therefore speculate that the inhibitory effect of TLR10 signaling needs both ligations of the TLR1/TLR10 complex and of the TLR6/TLR10 complex in ruminal epithelium. However, more evidence is required from the further studies.

Our CCA revealed that the abundance of the commensal bacterium in Verrucomicrobia subdivision 5 was highly related to the expression of TLR10. Previous studies have found that Akkermansia muciniphila, the cultured species of Verrucomicrobia, plays an important role in maintaining gut homeostasis (Png et al., 2010; Swidsinski et al., 2011; Hansen et al., 2012; Rajilic-Stojanovic, 2013). This species has also been suggested to be a co-evolutionary bacterium with the mammalian gut (Belzer and de Vos, 2012). An in vivo study in gnotobiotic mice has shown that A. muciniphila modulates the expression of pathways related to the immune tolerance of the gut epithelium (Derrien et al., 2011). Thus, we speculated that, in the present study, the suppression of the expressions of pro-inflammatory cytokines might have been caused by the species in Verrucomicrobia subdivision 5 through the activation of TLR10 signaling.

Higher ruminal concentrations of SCFAs were observed in the MNFC group. This indicated that commensals, induced by the NFC-rich diet, contributed to the increase of SCFAs in the rumen fluid. Previous studies have shown that the upregulated expressions of SCFA^-/HCO₃^- exchangers PAT-1, AE-2, and DRA were associated with the enhanced uptake of luminal SCFA into the rumen epithelium (Gabel et al., 1991; Kramer et al., 1996; Aschenbach et al., 2009). The upregulated expressions of SCFA- absorption -related transporters NHE1 and 3, Na⁺/K⁺ ATPase, and vH⁺ ATPase contributed to maintain the homeostasis of intracellular pH in a relatively low ruminal pH (Yang et al., 2012). The upregulated expressions of lactate transporters MCT1 and 4 were associated with the increased transportation of SCFA metabolic products lactate and ketone bodies from the rumen epithelium into the blood for energy supplement (Aschenbach et al., 2011). Accordingly, these data showed that commensals, induced by the NFC-rich diet, were beneficial to the energy absorption of rumen epithelium

The simultaneous increase of the diversity and richness in the microbiota community, coupled with the decreases in the expressions of pro-inflammatory and anti-inflammatory cytokines, indicates that the NFC-rich diet promoted the tolerance of epithelium to the rumen microbiota. The increased SCFAs productions and upregulated expressions of genes related to the SCFA absorption indicates that the commensals are promoted by the NFC-rich diet. Altogether, our study indicates that a new balanced state is established following 31% NFC intake, and that it is characterized by the facilitated transport of ruminal SCFA into the blood for animal growth. These results provide novel sights into the sustainable development of livestock production through dietary intervention and the development of the next generation of probiotics with the ability to maintain immune homeostasis. They also give hints concerning the recognized ligands of TLR10.

HS analyzed data and wrote the paper; ZL designed the research; ZL and ZC performed the experiments; YW and ZS approved the version to be published.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.