Effects of substituting soybean meal with corn on immune function and gene expression of gut TLR4 pathway of growing goats

Introduction

Adequate protein in the diet is a vital source of essential amino acids that play an important role in the physiological process (Wu, 2009; de Vries-ten Have et al., 2020). Soybean meal is among the most common sources of protein in the diet of ruminants. Forage resources in arid or semi-arid regions are limited and feeding an adequate and biologically available protein to livestock might be difficult or even impossible (Firmenich et al., 2020). In ruminants, the excessive dietary nitrogen converted to ammonia in rumen and produced urea in the liver, which was excreted in the urine and resulted in water pollution, soil acidification, and other environmental problems (Hristov et al., 2011). Moreover, protein is the most expensive component of animal rations. A low protein diet is desirable for economic and environmental reasons in ruminant feeding. Due to efficient rumino-hepatic circulation of urea (Muscher et al., 2010), ruminants can recycle N more effectively than monogastric species to cope with a reduced protein and therefore diminished N intake as long as the energy supply is adequate (Firmenich et al., 2020). Williams, Verstegen & Tamminga (2001) reported that about 68% of the energy value of fermented carbohydrates was provided to the host as the form of volatile fatty acids. As the least expensive and an excellent carbon and energy source in diets (Jeong et al., 2021), carbohydrate has a protein-sparing effect in ruminants.

Several human and animal studies have shown that protein malnutrition triggers immune dysfunction, including altered innate and adaptive immune responses, impairment of immune system and increased the risk of infectious disease (Rytter et al., 2014; Vlasova et al., 2017; Miyazaki et al., 2018). The innate immune system consists of phagocytic cells (e.g., neutrophils, monocytes, macrophages, and dendritic cells), natural killer (NK) cells, and cells that release inflammatory mediators (basophils, mast cells, and eosinophils) (Carroll & Forsberg, 2007). Cellular indicators such as white blood cells, lymphocytes, neutrophils, monocytes, basophils and eosinophils were used to reflect the change of immune function in growing goats feed a substitution of soybean meal with corn diet in this study. The innate immune system is the first line of defense against invading pathogens by either direct mechanisms such as phagocytosis and cytokine production or indirectly by activating adaptive immunity (Mian, Paradis & Schiffrin, 2014). Adaptive immune system, which consists of cell mediated (T cell--mediated) and humoral immunity (B cell--mediated), is characterized by the production of inflammation-related cytokines and antibodies respectively (Wolf & Ley, 2019).

The cytokines secreted by T-helper 1 (Th1) cells, including tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-1 beta (IL-1β), and IL-2, are involved in cell-mediated immunity while Th2 cytokines (IL-4, IL-5, and IL-13) have shown anti-inflammatory effect and promote humoral immunity (Ewer et al., 2021; Schulze-Koops & Kalden, 2001). The studies in dam and mice showed that interferon-beta (IFN-β) could inhibit the release of IFN-γ and also has anti-inflammatory and pro-inflammatory effects (Boivin et al., 2015; Bolivar et al., 2018). It has been reported that the cells in neonatal goats displayed a more robust Th-1 response to various TLR agonists than adult cells (Tourais-Esteves et al., 2008). In a general statement, there may be a specific immune response bias modulation in a susceptible environment. B cells can mediate different biological effector functions by switching from immunoglobin M (IgM) to IgG, IgA or IgE expression ( Brightbill et al., 2010). It has been reported that IFN-γ can promote the secretion of IgG2a and IgG3 antibodies, while Th2 cytokines, such as IL-4, IL-5, and IL-13, arouse IgG1 antibody production by B cells ( Kovacs-Nolan et al., 2009). The release of anti-inflammatory cytokines and specific antibodies by the adaptive immune system, are important in protecting from infections (Nakayama et al., 2017). Thus, cytokines and antibody levels were used as the targets to evaluate the immune activities of protein restricted goats.

The intestine also serves as the largest compartment of the immune system and contains the largest number of immune cells of any tissue in the body (Vighi et al., 2008; Mowat & Agace, 2014). Toll-like receptors (TLRs) are a family of at least 10 proteins that functions as pattern recognition receptors (PRRs) and play an essential role in animal immunity (Medzhitov, 2001; Vandana et al., 2019). TLR4 is an important member of the TLR family in the gut. It has been recognized as a receptor for lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria that can activate TLR4/ myeloid differentiation factor 88 (MyD88)-dependent and TLR4/MyD88-independent signaling pathways ( Brown et al., 2011), resulting in production of pro-inflammatory cytokines and strong inflammatory response (Akira, Takeda & Kaisho, 2001; Ren et al., 2019). Since malnutrition, in particular the protein–energy malnutrition is closely related to inflammation (Sauerwein et al., 1997). We hypothesized that protein deficiency can cause inflammatory disorder in the gut and there may be a Th1-type response bias in goats exposed to a highly infectious environment due to protein malnutrition. The present study may help shed new light on applied nutrition approaches to improve malnourished goat performance and health during the critical life-stage transition (growth stage).

In addition, previous studies have suggested malnutrition presents a greater risk in female goats during growth, pregnancy and lactation periods (Heersink et al., 2010; Castel, Shahar & Harman-Boehm, 2006). In the present study, we focus on reducing dietary protein under isoenergetic conditions, and an extremely low protein diet was formulated by replacing soybean meal with corn. The objective of this study was to evaluate the effect of substituting soybean meal with corn on activation of the TLR pathway and potential impact on immune response bias towards Type 1 or Type 2 (immune cellular indicators, anti-inflammatory cytokines and antibody levels) using growing female goats as experimental animals. Through the model for ruminant nutrition, gaining a deeper understanding between protein nutrition and immune function.

Materials and Methods

All procedures were approved by the Institutional Animal Care and the Use Committee of the Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, China.

Results

Effects of a CRS diet on the expression of genes related to TLR4 pathway in the gut of growing goats

Agarose gel electrophoresis of total RNA extracted from gut samples (n = 8/per group) were showed in Fig. S1. The results showed that the integrity of RNA met the experimental requirements. The efficiency of primers calculated from the slope (between −3.19 and −3.45) was between 94.92% and 105.69%. The above results indicated that the amplification efficiency of primers was good. The stability of reference gene in the experiment has been tested across all tissue samples. The results showed that GAPDH was a classic internal gene (Fig. S2).

The mRNA expression of inflammatory cytokines in duodenum, jejunum, ileum and colon were shown in Fig. 1. Expression of TLR4 (P = 0.001), TNF-α (P < 0.001), IFN-β (P < 0.001) and IL-1β (P = 0.041) in colon were significantly up-regulated in growing goats fed a CRS diet. As shown in Fig. 2, the mRNA expression of the key nodes (TIRAP, IRAK1, TRAF6, NF-κB, MAPK1 and AP-1) in the TLR4/MyD88-dependent pathway were not altered by a CRS diet even though the expression of MyD88 was significantly up-regulated (P = 0.019) in the colon. For the TLR4/MyD88-independent pathway, the expression of TBK1 was significantly up-regulated in the duodenum (P = 0.001) and colon (P = 0.005), and the expression of IRF3 (P = 0.004) was significantly up-regulated only in the colon (Fig. 3).

Discussion

In the present study, there was no difference in final body weight of goats between the CON (19.81 ± 0.78 kg) and CRS groups (19.97 ± 0.62 kg), while the crude protein intake in the CON group (59.51 ± 1.37 g/d) was significantly higher than that in the CRS group (28.95 ± 0.78 g/d) and the percentage of protein restriction is approaching to 49% (unpublished data). It has been reported that protein-malnourished mice were leukopenic, with reduced numbers of neutrophils, lymphocytes and monocytes (Mello et al., 2014). In our study, the concentration of Bas was increased in CRS, which was unexpected. Similarly, a previous study has shown that an increase in the count of circulating neutrophils was observed in cattle fed a high starch diet (Francesio et al., 2020). It is possible that the high level of dietary starch may be a factor resulting in the increased concentration of Bas. Previous studies have shown that carbohydrates could regulate the production of pro-inflammatory cytokines by inducing expression of genes of inflammatory molecules and regulating innate and adaptive immunity (Braun & Duvillard, 2004; Xu, Liu & Komai-Koma, 2004). Since bacterial endotoxins derived from gram-negative bacteria accumulate in the digestive tract especially in the hindgut of ruminants fed a high concentrate diet (Khafipour, Krause & Plaizier, 2009; Tao et al., 2014), the high starch in CRS may result in more endotoxins and therefore inducing increased Bas.

In our study, the CRS failed to reduce specific immunoglobulin (IgA, IgM and IgG) production, which was consistent with the results reported by Kefyalew et al. (2021) who elucidated that reducing dietary protein levels from 14.5% to 12.5% of DM did not affect the formation of circulating total immunoglobin concentration and specific antibodies (IgA and IgM) in growing lambs. In addition, our results showed that the protein-restricted diet had no effect on IL-4, IL-5 or IL-13 level in serum. In contrast, a study using goats as experimental animals has shown that maternal protein restriction during late gestation decreased plasma IgG and IgM concentrations of neonatal goat kids even though there were no differences in any plasma immune parameters at 6 weeks of age (He et al., 2014). Another experimental studies in both animals and humans have shown that sufficiently severe protein deficiency would interfere with antibody formation and increase the susceptibility to disease (Scrimshaw, 2007; Tang et al., 2018). The degree of severity of malnutrition might be another factor that results in the variable immunoglobulin concentrations in malnourished subjects (Gross & Newberne, 1980).

Li et al., 2014 have noticed that a TLR4 signaling pathway was required for recombinant Brucella abortus BCSP31-induced Th1 immune response in vivo and in vitro. Pro-inflammatory genes (TNF-α, IFN-β and IL-1β) are the downstream signals of TLR-4, and the expression of TLR4, TNF-α (Th1 cytokine) and IFN-β in the colon of CRS group were significantly up-regulated while the expression of IL-1β (Th1 cytokine) in the colon tends to be increased in the CRS group compared with the CON group. It suggests that CRS diet might activate the TLR4 pathway and promote the secretion of intestinal inflammatory cytokines. Based on the results that a low protein diet may increase the mRNA expression of Th1 cytokine and it appears that immune modulation is at a local level opposed to systemic. Interestingly, few changes in the genes related to TLR4/MyD88-dependent pathway was observed induced by the CRS diet, whereas the expression of the key nodes in the TLR4/MyD88-independent pathway, such as IRF3 and TBK1, were up-regulated. In agreement with our results, a previous study has shown that the occurrence of inflammation in sheep epithelial cells following capsular polysaccharide and M. ovipneumoniae treatment was linked to a MyD88-independent signaling pathway (Jiang et al., 2017). Furthermore, it has been reported that MyD88-independent signaling accounts for most of the LPS response (Vaure & Liu, 2014). In agreement with this notion, the CRS diet induced the release of a pro-inflammatory cytokine such as IL-1β, TNF-α and IFN-β mainly through a MyD88-independent pathway, and colon may be the easiest targeted section in the intestinal tract. A limitation of this study was that the higher starch content in the low protein diets which might influence the results. The intestinal immune system was sensitive to these challenges. It has to be considered that the low protein content and the high starch content might be factors, which influenced the present results. Therefore, further studies should focus on the relationships between low-protein, high-starch diet and intestinal immune mechanisms to facilitate the animal health. From a broader point of view, the findings of the present study are of importance for humans as well, especially for children. People and children in underdeveloped countries of the world might not be able to consume a sufficient amount of dietary protein whereas low-priced carbohydrates are available. As of 2019, there were 149 million stunted under-four children and 50 million emaciated children worldwide (https://www.unicef.org/reports/state-of-worlds-children-2019). According to a recent study children die of severe deficits of dietary protein and energy account for 11.4% of deaths under the age of 5 years (Black, Victora & Walker, 2013). Although the potential mechanism of CRS diet in regulating the production of proinflammatory cytokines remains largely unknown, our findings using goats as experimental animals may provide a better understanding on how malnutrition affecting the immune system for humans especially for children.

Conclusion

In this study, the immune system in growing goats was not profoundly changed by the CRS diet. Nevertheless, the expression of pro-inflammatory cytokines TNF-α (Th1 cytokine) and IFN-β were significantly increased and IL-1β (Th1 cytokine) was tended to be increased in colon of goats fed the CRS diet. Moreover, the CRS diet upregulated the expression of TBK1 and IRF3 in the TLR4/MyD88-independent pathway in the colon. Thus, the CRS diet may induce a proinflammatory response through a TLR4-MyD88-independent pathway and colon may be the easiest targeted section in the intestinal tract. The immune modulation induced by a CRS diet was at a local level opposed to systemic.

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