Dietary supplementation with ovine serum immunoglobulin modulates correlations between mucin, microbiota and immunity proteins in the growing rat

1 INTRODUCTION

Human and animal plasma-derived oral immunoglobulin (Ig) fractions are used as supplements in animal diets, food, nutraceuticals and other pharmaceutical formulations (Balan, 2011; Balan, Han, Dukkipati, & Moughan, 2014; Pierce, Cromwell, Lindemann, Russell, & Weaver, 2005). Several peer review publications are found in the literature for Ig preparation, among which human, bovine and porcine bloods are well studied (Pierce et al., 2005; Wilson, Evans, Weaver, Shaw, & Klein, 2013). In the past few years, remarkable progress has been achieved in the usage of functional immune proteins such as immunoglobulins (Ig) in both humans and in animals (Balan & Moughan, 2013; Balan, Sik-Han, & Moughan, 2019; Rahman, Van Nguyen, Icatlo, Umeda, & Kodama, 2013; Wilson et al., 2013). Recent studies have shown the various biological effects of an oral Ig fraction in different animal models, including its positive effects on immunity (Balan, 2011; Balan, Han, Rutherfurd, Singh, & Moughan, 2009; Balan et al., 2019), intestinal growth (Pierce et al., 2005) and gut barrier function (Garriga et al., 2005).

Gastrointestinal tract (GIT) mucosal surface is made up of a complex organization of epithelium, immune cells and resident microflora (McCracken & Lorenz, 2001). Mucus plays an important role in GIT surface integrity, and malfunction of mucus secretion and/or mucin expression may result in numerous pathologies such as inflammatory intestinal diseases and cancer (Forstner, 1995). Mucins are the high molecular weight glycoproteins secreted by intestinal goblet cells that are located in the columnar epithelium (Balan, 2011). As mucins play a vital part in safeguarding the underlying epithelium, any quantitative modification in mucus secretion may change this defensive barrier and have important physiological implications (Balan, 2011; Balan et al., 2019; Moughan, Rutherfurd, & Balan, 2013).

Furthermore, both qualitative and quantitative aspects of the GIT bacterial community can be modified by diet (David et al., 2014). Thereby, the physical efficiency of the protective barrier within the GIT might eventually be dependent at least in part upon an interaction between the gut microflora and the mucus layer, which itself can be modified by oral administration of various bioactive components (Han et al., 2014; Han, Balan, Molist Gasa, & Boland, 2011).

Ig prepared from ovine serum (from New Zealand lamb) was tested for apparent antimicrobial effects using an in vitro study (Han, Boland, Singh, & Moughan, 2009). Han et al. (2009) reported that ovine serum Ig exerted inhibitory and binding activity to Gram-positive and Gram-negative microbial pathogens, as well as their lipopolysaccharide and enterotoxin. Following which, a series of peer-reviewed publications are found in the literature suggesting the various positive biological effects of ovine Ig preparation in different challenged and unchallenged animal models. When compared to casein-based diet, ovine serum Ig supplemented diet was found to positively modulate growth, immunity, gut microbiota and gut mucins in both challenged (Balan, Han, Rutherfurd, Singh, & Moughan, 2011; Balan, Han, Rutherfurd-Markwick, Singh, & Moughan, 2011; Balan & Moughan, 2013; Balan et al., 2019) and unchallenged (Balan, 2011; Balan, Han, Lawley, & Moughan, 2013; Balan et al., 2009; Balan, Han, Rutherfurd-Markwick, Singh, & Moughan, 2010; Balan, Han, Singh, & Moughan, 2011) rodent models. Furthermore, recently a report was found showing that ovine Ig significantly reduces plaque formation and significantly modulates the oral and peripheral immunity in the cats (Balan et al., 2019).

Probiotic bacteria such as lactic acid bacteria have been shown to stimulate mucus production in the GIT (Hecht, 1999). Lactobacillus species contained in a probiotic formula influenced the mucin secretion in vitro (Caballero-Franco, Keller, De Simone, & Chadee, 2007). Recently, Han et al., (2014) reported that consumption of ginseng led to increased number of total bacteria, Lactobacillus strains and mucin gene expression and their correlation analysis suggested that the expression of the mucin gene was significantly correlated with the number of Lactobacillus strains and total bacteria. Moreover, we have shown in rats that by feeding ovine serum Ig resulted in an increase in the gut IgA IgG, mucin levels and changes in the composition and levels of gut microbiota (Balan, 2011; Balan et al., 2013, 2019; Balan, Han, Singh, et al., 2011). Based on above findings, it could be hypothesized that there could be strong connection among gut immunoglobulins, mucin protein, gene expression, goblet cells count and number of gut bacteria. Therefore, the objective of this study was to evaluate the relationship among the number of bacteria, number of goblet cells, gut mucin gene expression, mucin protein and immunity protein levels of rats fed a diet containing freeze-dried ovine Ig (FD).

2 MATERIALS AND METHODS

3 RESULTS AND DISCUSSION

In the stomach content, a significant correlation was found between the Muc5Ac gene expression and mucosal mucin protein (r = .92, p < .001) (Table 1). There is also evidence of a significant positive relationship between chyme mucin protein and mucosal mucin protein (r = .84, p-value = .002). A significant correlation was also evident between the Muc5Ac gene expression and chyme mucin protein (r = .68, p = .03). Correlation was slightly lower as there was large variation between two values from treatment (ovine serum Ig) group (Table 1).

The mucus forms a continuous gel which covers the epithelial surface (Balan, 2011). The GIT mucosal layer is an important barrier between the enterocytes and the GIT lumen. The gut barrier aids to preserve a close mutual benefit between gut microbes and the host animal. In addition, the functionality of this barrier is conserved by a complex network of physical, physiological and immune factors, which are comprised of dietary influences, the host's environment and the indigenous microbial flora of the gut (Balan, 2011; Balan et al., 2019). In humans, there are at least nineteen different mucin genes that have been documented (Han et al., 2014). Among them, thirteen mucin genes are expressed in the GIT. Muc2, the key secretory mucin, is restricted to the goblet cells, whereas Muc3 and Muc4, the transmembrane mucins, are present in both goblet cells and enterocytes (Han, Deglaire, Sengupta, & Moughan, 2008; Trompette et al., 2004). The major GIT mucin in rats is found to be Muc2, which is produced by goblet cells. Mucin constitutes the first line of defence against pathogenic bacteria and is the key constituent of the GIT barrier (Han et al., 2014).

In the ileum (Table 2), a significant (p < .004) correlation was observed among the mRNA levels of mucin (Muc2, Muc3 and Muc4) genes. There was also evidence of a strong relationship between digesta mucin and mucosal mucin protein concentrations. There was a significant (p < .004) positive relationship between villi goblet cells and crypt goblet cells. Furthermore, villi and crypt goblet cell numbers were significantly (p < .05) correlated with Muc2 gene expression. The number of total Lactobacillus in the ileal digesta was significantly (p = .006) correlated with digestal total bacteria number. However, there was no (p > .05) relationship between number of enterobacteria with either total bacteria or total Lactobacillus in the digesta samples (Table 2). The number of enterobacteria was positively correlated with mucosal IgG protein (p = .028), while negatively correlated with each digesta IgA protein (p = .003) and mucosal mucin protein concentrations (p = .007). There was a significant (p = .04) relationship between digesta IgG concentrations and Muc2 gene expression. Similarly, digesta IgA concentrations significantly (p < .05) correlated with the digesta IgG. Mucosal IgG protein concentrations significantly (p < .05) correlated with mucin (mucosal and digestal) protein concentrations and goblet cells (villi and crypt) numbers. Furthermore, the mucosal IgA protein was positively correlated with mucosal IgG (p = .005), mucin (p < .008) (mucosal and digestal) protein concentrations and number of goblet cells (p < .013) (villi and crypt), while negatively correlated with total Lactobacillus (p = .041) and total bacteria (p = .048).

There were significant changes evident in ileal correlation results from the unchallenged (uninfected) growing rats fed the ovine serum Ig (FD) when compared to the results obtained using both BD and FD fed rats (Table 4).

Correlation analysis revealed that for FD the expression of Muc2 was significantly (p < .05) correlated with mucosal mucin protein concentrations. The expression of Muc3 and Muc4 genes was each significantly (p < .05) correlated with villi goblet cell numbers. The number of total bacteria in the ileal digesta was significantly (p < .05) correlated with mucosal mucin protein concentration and Muc2 gene expression. Also, digesta IgA (p = .03) and mucosal IgA protein concentrations (p = .001) were each negatively correlated with number of enterobacteria. Similarly, negative correlation of mucosal IgA protein concentration with each total Lactobacillus and total bacteria was not evident when only FD data were used in the correlation analysis.

The immunological defence against various potential pathogens in the GIT is facilitated by intestinal IgA in a process known as immune exclusion where intestinal IgA binds to the bacteria, pathogen or its antigen, thereby inhibiting their translocation (Amin, Diebel, & Liberati, 2007). Puri, Rattan, Bijlani, Mahapatra, and Nath (1996) reported that diets supplemented with Enterococcus faecium (SF68) stimulated immune function in dogs by increasing the concentrations of intestinal and plasma IgA. Reports also found in the literature showed that feeding dietary fructooligosaccharides to healthy men improved bifidobacteria, lactobacilli and lactic acid content in the gut (Bruggencate, Bovee-Oudenhoven, Lettink-Wissink, Katan, & Van Der Meer, 2006). Lactobacillus sp. fed to mice up-regulated the levels of intestinal IgA, serum IgA and splenocyte proliferation (Kaburagi et al., 2007).

The indigenous microbiota provides protection against infection from certain pathogens. “Colonization resistance” or “bacterial antagonism” demonstrates the capability of the host to prevent the colonization of the GIT by exogenous pathogens. It is essential to understand that interplay between microbes, within the GIT, results in both direct and indirect modes of protection from pathogens. Modification of this interplay may not only disturb the integrity of the mucosal barrier, but it may also assist certain pathogens to overcome or evade mucosal immune responses (Balan, 2011).

One of the most important constituents of the mucosa is mucin, which is synthesized, stored and secreted by goblet cells (Han et al., 2014). The greater presence of goblet cells results in an improved synthesis of mucus, which has important immune and non-immune functions that would help to protect the epithelial surface (Balan, 2011; Balan et al., 2019). Some of our results are similar to the recent findings of Han et al., (2014) where they found the expression of Muc2 gene significantly correlated with the number of total bacteria (Table 3, 4, & 5). Similarly, they suggested that the number of Lactobacillus strains were significantly correlated with the number of total bacteria (Tables 2 and 4). Furthermore, the amount of digesta mucin protein was correlated with Muc4 gene expression (Tables 2, 3, and 5). Paturi, Butts, Stoklosinski, and Ansell (2012) found a significant correlation between the expression of Muc3 gene and number of goblet cells (Paturi et al., 2012).

In the colon (Table 3), our correlation analysis showed the expression of Muc2 gene was significantly (p < .05) correlated with the Muc4 gene expression, digesta mucin protein concentration, goblet cell number and digesta total bacteria. Mucin protein concentration in digesta was significantly (p < .05) correlated with the mucosal mucin protein concentrations, goblet cell number and digestal total Lactobacillus respectively. Similarly, mucosal mucin protein concentrations significantly (p < .05) correlated with crypt goblet cells, number of enterobacteria and Lactobacillus. However, in the colon, unlike ileal results, there was not a significant (p > .05) relationship between digesta total bacteria and digesta mucin protein concentrations, crypt goblet cell numbers and the number of Lactobacillus (Table 3). There was a significant (p = .047) negative relationship between digesta IgA concentrations and number of enterobacteria in colonic digesta. Furthermore, mucin (mucosal and digestal) protein concentrations, number of crypt goblet cells, number of Lactobacillus and digesta IgG concentrations were each significantly (p < .05) negatively correlated with mucosal IgA protein concentrations.

Similar to ileal findings, there were significant changes evident in colonic correlation results from the unchallenged growing rats fed the ovine serum Ig (FD) when compared to the results obtained using both BD and FD fed rats (Table 5). Muc2 and Muc4 gene expressions were each significantly (p < .05) correlated with mucosal mucin protein concentrations for FD. The number of total bacteria in the colonic digesta was significantly (p < .05) correlated with mucosal mucin protein, digesta mucin protein, crypt goblet cell numbers and mucosal IgA protein concentration. The mucosal IgA protein concentration was positively correlated with digesta (secretory) IgA (p = .05) protein concentrations, while negatively correlated with number of enterobacteria (p = .002). Similarly, digesta IgA (p = .006) protein concentrations were negatively correlated with number of enterobacteria.

Reports have been found in the literature suggesting an increase in goblet cells by dietary intervention, such as probiotic bacteria and keratinocyte growth factor (Fernández-Estívariz et al., 2003; Vinderola, Matar, & Perdigón, 2007). Other investigators have found that chronic protein depletion, or protein-energy under-nutrition, reduced goblet cell number or mucin synthesis in rodent and pig intestinal models respectively (Balan, 2011; Balan et al., 2019; Sherman, Forstner, & Roomi, 1985). Reports have also shown that feeding pigs with either plasma or colostrum resulted in a significantly higher villi and crypt goblet cell numbers (King, Morel, Pluske, & Hendriks, 2008). Peptides, from a casein hydrolysate, were also found to stimulate the expression of mucin genes in the rat jejunum (Claustre et al., 2002). Han et al. (2008) reported that feeding rats with hydrolysed casein influenced mucin gene expression in the rat intestine, in vivo. The greater ileal endogenous nitrogen loss, found in that study, might have resulted from a higher secretion of mucins into the intestine, with diet hydrolysed casein. Also, they reported that hydrolysed casein promoted a significant increase of Muc3 mRNA in the small intestinal tissue and Muc4 mRNA in the colon. Secretory activity of intestinal goblet cells is found to be increased by the dietary fibre (alginate and ulvan) and short-chain fatty acids (Barcelo et al., 2000). Caballero-Franco et al. (2007) reported that among the three bacterial groups (Lactobacilli, Bifidobacteria and Streptococci) present in a probiotic formula, Lactobacillus species strongly influenced the mucin secretion in vitro. Similarly, Hecht (1999) suggested that the probiotic bacteria such as lactic acid bacteria have been shown to stimulate mucin production in the GIT (Hecht, 1999).

Some of our results are similar to the recent findings of Paturi et al., (2012) where they found the significant correlation between the expression of Muc3 gene and number of goblet cells in the colon tissue (Table 4). Moreover, reports are also found in the literature showing strong relationship between goblet cells numbers and Muc2 gene expression in Eimeria papillata infection. Authors have found that infection of mice with E. papillata resulted in significant reduction of the goblet cell numbers and Muc2 gene expression (Dkhil, Delic, & Al-Quraishy, 2013).

An understanding of the detailed mechanistic basis of the effects of ovine serum Ig can be drawn based on the correlation study. Passive inhibition of the adhesion of the pathogenic bacteria to the host GIT could be mediated by antibodies present, especially in the FD (Han et al., 2009). It is likely that animals are exposed to pathogens to a greater degree than humans from a Westernised society. As a result, it is possible that an animal's immune system would be stronger and better adjusted to counter-attack the full spectrum of microbial pathogens by producing antibodies against them. It is well documented that animal serum contains antibodies that are effective against a variety of microbial pathogens such as E. coli, Salmonella, Listeria, C. difficile, rotavirus and others (Balan, 2011; Balan, Han, Rutherfurd, et al., 2011; Balan, Han, Rutherfurd-Markwick, et al., 2011; Balan et al., 2019; Han et al., 2009; Pierce et al., 2005). Therefore, serum Ig would provide passive antimicrobial protection (and from subclinical infection) by exclusion of opportunistic microbial pathogens. Stronger intestinal function could be obtained through supporting elevated numbers of total bacteria and lactic acid bacteria (Lactobacillus sp, L. johnsonii, Leuconostoc citreum, Weissella cibaria) which in turn/or vice versa enhance the gut mucin content and immunity such as digesta (secretory) IgA (Balan, 2011; Balan et al., 2013, 2019; Balan, Han, Rutherfurd-Markwick, et al., 2011; Balan & Moughan, 2013; Maijó et al., 2012). The increased colonization of total bacteria and lactobacilli into the GIT may impact bacterial pathogenesis by enhancing the immune system by immunostimulation, such that this could have conferred greater immune-mediated protection against pathogens (Jain, Yadav, & Sinha, 2008), and by increasing mucin gene expression in goblet cells which result in increase in mucin thickness and secretion. Ovine Ig has also been found to function like a prebiotic (Balan et al., 2014, 2019). Ovine Ig resisted digestion by the digestive enzymes and reached the large intestine, where it was utilized by the resident microbiota, such as the growth of lactobacilli and other beneficial bacteria in the gut (Balan et al., 2014, 2019).

In conclusion, this study suggests that feeding freeze-dried ovine Ig results in a strong positive correlation between number of bacteria (i.e. Lactobacillus and total bacteria), mucin (gene expression, goblet cells and protein) and immunity (IgA and IgG) proteins in growing rats.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

ANIMAL WELFARE STATEMENT

The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. This work was approved by the Massey University Animal Ethics Committee (MUAEC 06/132) and procedures complied with the New Zealand Code of Recommendations and Minimum Standards for the Care and Use of Animals for Scientific purposes (New Zealand Animal Welfare Advisory Committee, 1995).