The Gut Immune System: A New Frontier in Gut Health Research

Coccidiosis is an ubiquitous intestinal protozoan infection of poultry which seriously impairs the growth and feed utilization of infected animals (Shirley and Lillehoj, 2012; Lillehoj and Lillehoj, 2000). Conventional disease control strategies rely heavily on chemoprophylaxis costing the industry large amounts of money. The existing vaccines comprise live virulent or attenuated Eimeria strains with limited scope of protection against an ever evolving and widespread pathogen. The continual emergence of drug resistant strains of Eimeria, coupled with the increasing regulations and bans on the use of anticoccidial drugs in commercial poultry production, urges the need for novel approaches and alternative control strategies. Due to the complexity of the host immunity and the parasite life cycle, a comprehensive understanding of the host-parasite interactions and protective immune mechanisms becomes necessary for successful prevention and control practices. Recent progress in functional genomics technology has facilitated the identification and characterization of host genes involved in immune responses as well as parasite genes and proteins that eliciting protective host response (Kim et al., 2008; Kim et al., 2010; Kim et al., 2011).

While natural infection with Eimeria spp. induces immunity, vaccination procedures on a commercial scale have shown limited effectiveness and disease control remains largely dependent on routine use of anti-coccidial drugs. Available live vaccines are composed of either virulent or attenuated strains with the major disadvantage which consists of the large number of live parasites making them laborious and costly to produce. Although live oocyst vaccines represent a limited but useful alternative to anticoccidial drugs, a vaccine composed of parasite antigens/antigen-encoding genes that elicit specific immunity is eminently preferable. While it would be cost-effective to produce recombinant vaccines (proteins or DNA), the difficulty remains to identify which antigens or genes are responsible for eliciting protective immunity and how these recombinant vaccines should be delivered and presented to the bird’s immune system. Also, such subunit vaccines would eliminate the danger of emerging resistant strains which encounter the live vaccines but until efficient vaccines become commercially available, the poultry industry is forced to rely upon prophylactic chemotherapy to control the disease. Further, the introduction of alternative prevention/treatment measures, such as non-chemical feed supplements that effectively enhance productivity and non-specific immunity, may help limit the use of anticoccidials. However, the lack of efficient vaccines, the increasing incidence of drug resistant strains, and the escalating public anxiety over chemical residues in meat and eggs mandate the development of alternative control methods. 

A comprehensive understanding of protective immunity to coccidiosis is required before we can develop alternative strategies to control coccidiosis. Although both circulating and secretory antibodies, specific for coccidia parasites, have been detected in serum, bile and intestine (Lillehoj and Ruff, 1987; Yun et al., 2000), the antibody titers in serum and intestine do not correlate with the level of protection after oral infection with coccidia (Lillehoj and Ruff, 1987). In general, antibodies are the hallmark of host immune response to Eimeria parasites, but do not seem to be involved in protection against coccidiosis (Lillehoj and Lillehoj, 2000). Extensive experimental evidence supports the notion that immunity mediated by lymphocytes and their secreted products, such as cytokines, mediates antigen-specific protection against challenge infection with Eimeria (Lillehoj and Lillehoj, 2000; Lillehoj et al., 2004; Lillehoj et al., 2012). In contrast to the plethora of mammalian cytokines, only a few chicken homologs have been described; the major ones including IFN-γ, IL-1, 2, 6 (Schneider et al., 2001), 8, and 15 (Lillehoj et al., 2004; Staeheli et al., 2001). More recently, a series of new chicken cytokines and their receptors (Min et al., 2002; Jeong et al., 2011; Jeong et al., 2012) have been described, including IL-17 (Min and Lillehoj, 2002; Yoo et al., 2009), 18 (Schneider et al., 2000), 16 (Min and Lillehoj, 2004), 12 (Degen et al., 2004), and Th2-type cytokines, such as IL-4, 5 (Koskela et al., 2004), IL-10 (Rothwell et al., 2004), 13 and the granulocyte-macrophage colony-stimulatory factor (GM-CSF) (Avery et al., 2004). The IL-17 family cytokines are the newest cytokines described recently and have been associated with Th17 CD4+ T cell population which is distinct from the classical Th1 and Th2 lymphocyte lineages (Iwakura, 2011). Although traditionally thought of as a component of adaptive immunity, Th17-related cytokines are now recognized as part of the rapid response that develops during the initial phases of immune system activation. Once secreted, these cytokines, and others, regulate the interactions between mucosal epithelia and their associated lymphocytes to eradicate invading pathogens, and to restore immune homeostasis. In the case of avian coccidiosis, the immunoregulatory roles of the newly described proinflammatory IL-17 cytokine family in the host response to parasite infection deserves continued study (Min et al., 2013). Involvement of multiple cytokines in different stages of coccidia infection supports a notion that host immune response to coccidiosis is cell-mediated and complex (Hong et al., 2006). The complexity of cytokine response associated with different species of Eimeria infections was investigated using a genome-wide transcriptional microarray (Kim et al., 2011). 

Recent studies documented that the dietary immunomodulation of gut immunity in broiler chickens using natural dietary supplements, such as TLR ligands, DFMs and plant-derived phytochemicals that interact with innate sensing molecules to stimulate innate immunity, is a promising alternative strategy that can be applied to many infectious diseases besides coccidiosis where traditional prevention methods show limitations (Lillehoj and Lee, 2012). Furthermore, the underlying immune mechanisms involved in various dietary strategies using TLR ligand-, DFM- and plant phytochemical-mediated immune enhancement of innate immunity should be investigated in order to maximize its effect and to develop a rational synergistic approach for disease control. Some examples of the immune modulation strategies which we have been developing to increase host protective immunity to coccidiosis and to mitigate the use of antibiotics in poultry production include molecular vaccine (Jang et al., 2010; Jang et al., 2011a, 2011b, 2011c), probiotics (Lee, et al., 2010a, 2010b), passive immunization using hyperimmune IgY antibodies (Lee et al., 2009a, 2009b) and dietary immune modulation using plant-derived phytonutrients (Lillehoj et al., 2011; Lee et al., 2007, 2008, 2009, 2010a, 2010b, 2011a, 2011b).

One of the initial steps triggering the innate immune response involves germ-line encoded, highly conserved innate immune sensing molecules of PRRs which include TLRs, nucleotide-binding oligomerization domain proteins (NODs), retinoid-inducible gene 1 (RIG-1) and C-lectin binding receptors. Eimeria parasites, the causative pathogens of coccidiosis, contain several components which are stimulatory for immune cells, and they activate innate immunity and inflammatory response. In 2005, our laboratory (Lillehoj, 2004) showed that a conserved antigen of sporozoites of Eimeria, profilin, is a parasite PAMP which stimulates T-lymphocytes and induces IFN-γ production. Recently, a significant effect of the oil-based ISA 71 VG, or aqueous nanoparticle-based Montanide IMS 1313 N VG (IMS 1313) adjuvant on recombinant vaccination against coccidiosis was demonstrated (Jang et al., 2011a). These latest studies have opened other doors for the development of recombinant vaccines against coccidiosis and illustrate the importance of elucidating the underlying molecular mechanism of vaccination.

Commensal bacteria on the intestinal mucosa contain many probiotics ligands (such as long surface appendages, polysaccharides and lipoteichoic acids) which can communicate with PRRs inducing downstream signaling pathways that lead eventually to probiotic (health-promoting) effects. Direct-fed microbials (DFM) and their associated ligands can modulate host innate immune response. We have recently evaluated several field isolates of B. subtilis strains by the continuous feeding of young broiler chickens with the spore-supplemented standard poultry diet to investigate the probiotic effects of Bacillus strains. Depending on the B. subtilis strain, feeding diets supplemented with B. subtilis spores increased the various intestinal intraepithelial T cell subpopulations, cytokine mRNA levels, and macrophage function. Following an E. maxima challenge infection, DFM-fed chickens showed an enhanced disease resistance with higher body weight gain and decreased intestinal lesions as compared with the uninfected control birds (Lee et al., 2010a, 2010b). Detailed immune pathways that were affected by Bacillus treatment were further examined using a high-throughput gene expression analysis. Various immune-related genes, especially ones associated with the inflammatory response, were up-regulated in the gut of probiotic-treated chickens.

One promising new avenue to achieve this goal is the use of natural foods and herbal products to enhance host defense against microbial infections and tumors. A growing body of scientific evidence demonstrates the health-promoting effects of plant-derived phytochemicals, chemical compounds derived from plants or fruits. "Phytonutrients" refer to phytochemicals or compounds from edible plants that have been used as health-promoting agents by many cultures for several millennia. In 400 B.C.E., Hippocrates prescribed willow tree leaves (containing salicylic acid) to abate fever. There is abundant evidence from epidemiological studies that phytochemicals can significantly reduce the risk of cancer and may reduce high blood pressure, pain, and asthma. The most popularly used drug for cancer chemotherapy worldwide is Taxol (paclitaxel), a phytochemical initially extracted and purified from the Pacific Yew tree. Taxol possesses anti-viral, anti-bacterial, and anti-cancer properties. Many other phytochemicals with potent medicinal properties are currently in clinical trials for treatment of a variety of diseases. Lycopene, for example, from tomatoes is in clinical trials for cardiovascular diseases and prostate cancer. Its beneficial effects may be due to its anti-oxidant and anti-inflammatory effects. While numerous studies have showed disease prevention or immune enhancing effects resulting from oral feeding of plants, only a few reports have examined the specific effects of plant-derived phytochemicals on gut defenses. The intestinal mucosal system plays a central role in the exclusion and elimination of harmful dietary substances in humans and animals. Part of the intrinsic gut defense mechanisms are mediated by the lymphoid system and the intestine contains a relatively large component of lymphatic tissues.

Recent studies from our laboratory (Lillehoj et al., 2011) provided clear evidence that the dietary supplements of natural phytochemicals activate innate immunity in poultry and, in particular, enhanced protective immune responses against avian coccidiosis. Phytochemicals are plant- or fruit-derived chemical compounds which possess the health benefits including promotion of tumor killing and the increased resistance to infectious diseases caused by bacteria, virus and parasites. However, very limited information is available on the mode of action of most of health-promoting plant phytochemicals. Therefore, in order to obtain a basic understanding of how dietary supplements, such as plant and fruit extracts, exert immunostimulatory effects in poultry, we carried out in vitro and in vivo feeding trials using an intestinal protozoan disease model, avian coccidiosis. In various in vitro studies, culture of chicken spleen lymphocytes with crude extracts from milk thistle, turmeric, shiitake and reishi mushrooms, persimmon, tomato, safflower leaf, plum fruit, and cinnamaldehyde induced significantly higher cell proliferation compared with the untreated control cells. Stimulation of chicken macrophages with crude extracts of milk thistle, shiitake and reishi mushrooms, persimmon, raspberry, safflower leaf, plum fruit, and cinnamaldehyde resulted in a robust nitric oxide production to the levels that were similar with those induced by recombinant chicken IFN-γ. Most of the phytochemical extracts and cinnamaldehyde inhibited the growth of chicken tumor cells in vitro. The levels of mRNAs encoding IL-1β, IL-6, IL-12, IL-18, and tumor necrosis factor superfamily member 15 (TNFSF15) were enhanced in macrophages that were treated with extracts of turmeric or shiitake mushroom as compared with the untreated control (Lee et al., 2009, 2010b, 2011a).

Cinnamaldehyde also directly reduced the viability of Eimeria tenella parasites at 10 and 100 μg/ml (P<0.05 and P<0.001, respectively), as compared with the media controls (Lee et al., 2011a). The effects of plant extracts on enhancing the various in vitro parameters of protective immunity have been positively correlated with their ability to protect against microbial infections. In several in vivo trials, the feeding of broiler chickens with diets supplemented with extracts of mushroom, safflower, plum, and cinnamaldehyde consistently enhanced innate immunity and provided enhanced protection against live, oral parasite challenge infections. For example, mushroom extracts and cinnamaldehyde significantly protected chickens against weight loss characteristically seen during coccidiosis and promoted parasite killing as indicated by reduced fecal oocyst shedding. Dietary supplementation of young broiler chickens infected with Eimeria acervulina using plum, safflower leaf extracts, curcuma, capsicum, and cinnamaldehyde increased body weight gain, reduced fecal oocyst shedding, and increased cell-mediated immunity as measured by the transcriptional changes in key cytokines such IL-1β, IL-6, IL-15, and IFN-γ (Lee et al., 2006, 2008, 2009, 2010a, 2011a).

Furthermore, combination of two phytonutrient mixtures, VAC (carvacrol, cinnamaldehyde, and capsicum oleoresin), and MC (capsicum oleoresin and turmeric oleoresin), were evaluated for their effects on chicken immune responses following immunization with a recombinant Eimeria profilin protein. Following immunization and infection, chickens fed the VAC- or MC-supplemented diets showed increased body weights, greater profilin antibody levels, and/or greater lymphocyte proliferation as compared with non-supplemented controls. Immunized chickens fed the MC-supplemented diet exhibited increased MHC class II⁺, CD4⁺, CD8⁺, TCR1⁺, or TCR2⁺ T cells as compared with nonsupplemented controls while chickens on the VAC-containing diet displayed an increase in K1+ macrophages. Finally, the dietary supplementation with VAC or MC alters immune parameters following recombinant protein vaccination and shows vaccine-stimulated immunity against avian coccidiosis (Lee et al., 2011 b). 

In view of increasing consumers’ concerns about drug residues in the food chain, the poultry industry will eventually discover alternative methods to control economically important avian diseases. Chickens will continue to provide a major and increasing supply of the world’s animal protein. It is hard to imagine disease control in the field without the use of anticoccidial drugs, but it is probable that the current methods of control will continue unabated and supplemented with drug-free alternatives. There are, however, increasingly negative political views towards in-feed medication of livestock (especially within Europe) and an overall increasing negative view on the use of prophylactic chemotherapy provides a significant spur for work on the immunological control of avian coccidiosis. Application of the recently described innovative technology in immunomodulation and vaccination may lead to the development of alternatives to prophylactic medication. With rapidly developing technologies in functional genomics and computational biology, it is anticipated that new paradigms for coccidiosis control will be formulated. It is possible that the use of genetic tools could become one way of combating parasites, in synergy with other strategies of coccidiosis control such as vaccination, nutrition, and management. 

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