Innate Immune Training in Chickens for Improved Defense against Pathogens: A Review

ABSTRACT

The avian immune system plays a vital role in poultry production to obtain good productibility and products that are safe and of high quality. Historically, adaptive immunity has been the main target of vaccination. However, over the past decade, innate immunity has been reported to be enhanced in different animals through vaccination and feed additives. This enhancement is due to innate immune memory termed “trained immunity,” in which epigenetic and metabolic reprogramming play significant roles. Although reports on trained immunity in poultry are limited, several studies have suggested that vaccinations and feed additives affect the innate immunity. This review discusses the possible effects of vaccination and β-glucan on innate immunity for potential incorporation in advanced strategies to enhance the defense function in poultry while considering the information on trained immunity in mammals.

INTRODUCTION

The well-being of chickens is an important concern in poultry production as it is essential for good productivity, product safety, and animal welfare management. In particular, the immune system plays a key role in the prevention of pathogenic infections. The immune system is comprised of innate immunity, the first line of defense, and adaptive immunity, which provides protection against specific pathogens via the actions of lymphoid cells and antibodies. The enhancement of adaptive immunity by vaccination, in part through the immune memory of lymphocytes, has been well documented[1]. Moreover, whereas it was previously believed that innate immunity did not form immune memory, reports over the past decade suggest that vaccination and certain substances such as β-glucan induce innate immune memory in different animals. This phenomenon, termed “trained immunity,” produces hyperresponsiveness in the innate immune system[2,3,4]. Establishing a procedure that leads to trained immunity is thus beneficial for enhancing the defense system against infections. Although reports on trained immunity in poultry are limited, several studies have suggested that vaccinations and feed additives affect the innate immunity. This review discusses the possibility of inducing trained innate immunity through vaccination and β-glucan supplementation to enhance the defense functions of innate immunity in chickens.

ADAPTIVE IMMUNITY IN CHICKENS

During the adaptive immune response, major histocompatibility complex (MHC) molecules present short antigen peptides on the cell surface for inspection by immune cells, particularly T cells, which allows the occurrence of appropriate immune responses[5]. Among CD8+ and CD4+ T cell subsets[6], CD8+ T cells comprise cytotoxic lymphocytes that kill infected cells and inhibit viral replication. They recognize antigen peptides derived from proteins located in the cytoplasm or nucleus that are bound to classical MHC class I molecules. Alternatively, CD4+ helper T cells recognize antigen peptides bound to classical MHC class II molecules derived from proteins originating in intracellular vesicles of antigen presenting cells including dendritic cells, macrophages, and B cells. B cells receive signals from those activated helper T cells that induce antigen-specific B cell proliferation and affinity maturation of B cells, resulting in the generation of both plasma cells synthesizing antigen specific antibodies and memory B cells[7]. Following the elimination of antigens through this immune response, effector cells die or enter a resting state, resulting in surviving memory lymphoid cells[1]. These memory cells are responsible for immune memory, which responds quickly and strongly when individuals are re-infected with the same antigens.

Vaccination also contributes to the immune memory effected by T and B cells, albeit to varying degrees. Marek’s disease (MD) vaccines can become latent with concomitant life-long immunity[8], whereas turkey rhinotracheitis (TRT), infectious bronchitis (IB), and Newcastle disease (ND) vaccines may replicate in the respiratory tract for a maximum of 2 weeks and provide immunity for 12–50 weeks[9,10]. Gough and Alexander[11] compared the duration of immunity in chicks vaccinated with a live IB vaccine via aerosol, intraocular, and drinking water routes, revealing different immunity durations. Moreover, Schijns et al.[1] reported that the level of memory cell formation and working duration of immunity caused by vaccines are associated with the type of vaccine (live or killed), delivery route, age, and immune status of the birds.

INNATE IMMUNITY IN CHICKENS

Following infection, pathogen-associated molecular patterns (PAMPs) are recognized by pattern recognition receptors, such as toll-like receptors (TLRs), nod-like receptors (NLRs), and dectin, in the innate immune system. Such stimulus then activates transcription factors such as nuclear factor-κB (NF-κB), leading to the expression of innate immune molecules including proinflammatory cytokines, cytokines, chemokines, and antimicrobial peptides[12,13] (Fig. 1). TLR3 and TLR7 recognize double-stranded (dsRNA) and single-stranded ribonucleic acid (ssRNA), respectively, from viruses. TLR21 recognizes unmethylated CpG deoxynucleic acids (CpG-DNA) in microbes, whereas TLR2, 4, 5, and 15 recognize bacterial peptidoglycans, lipopolysaccharides (LPS), flagellin, and virulence-associated bacterial proteinases, respectively[12]. Cytokines such as interleukin (IL)-1β and IL-6, synthesized as a result of PAMP recognition by TLRs, play key roles in regulating leukocyte activities and the synthesis of antimicrobial peptides and interferons (IFNs) that remove pathogens. Antimicrobial peptides including avian β-defensins (AvBDs) and cathelicidin kill the invading pathogens, such as enveloped viruses, bacteria, and fungi, by breaking down their membranes[14,15]. AvBDs are primarily synthesized by epithelial cells and leukocytes, including heterophils[16,17,18,19], in response to direct TLR stimulation or indirectly through stimulation by the pro-inflammatory cytokines synthesized upon TLR stimulation[12,20]. Cathelicidin synthesis is also increased by the IL-1β and IL6 synthesized in response to infection by microbes in the oviduct mucosa[21].

Fig. 1.

  Toll-like receptors (TLRs), their corresponding ligands, and signal pathways to induce innate immune factor expression. Interaction of TLRs with ligands sends signals to activate the transcription factor, NF-κB, which induces innate immune molecules such as proinflammatory cytokines, cytokines, chemokines, and antimicrobial peptides. TLR2, 4, 5, and 15 are expressed on the surface of the cell membrane (shown in brown), whereas TLR3, 7, and 21 are in endosome in the cytoplasm (shown in tan).

The innate immune system in chickens, including the expression of pattern recognition receptors, cytokines, and antimicrobial peptides, first appears during the embryonic phase and is maintained even in chicks and mature birds, although antimicrobial peptide synthesis is reduced at four days post-hatching[19,22,23]. Maternal immunoglobulin Y (IgY) in the yolk is absorbed by the embryo and catabolized by the chick during the first 14 days post-hatching[24,25]. However, although the chick may begin to independently synthesize IgY approximately five days post-hatching[24], the expression of IgY and IgM in the small intestine remains minimal on post-hatching days 4 to 10[26]. Therefore, enhancement of the innate immunity in chicks at the early post-hatching stage would be beneficial to prevent infection by pathogenic agents.

TRAINED IMMUNITY

Adaptive immunity can form an immune memory by reserving memory lymphocytes that respond to the original stimulating antigens. Vaccination can enhance adaptive immunity by inducing the production of specific antibodies against the antigens contained in the vaccine and, in turn, the formation of memory lymphocytes. Previously, innate immunity was not considered to form immune memory. However, recent studies have suggested that the innate immune system previously exposed to certain stimuli demonstrates an enhanced immunological response to secondary triggers, indicating memory functions[27]. This trained immunity[28] is mediated by prototypical innate immune cells such as natural killer (NK) cells and monocytes/macrophages[4].

Epidemiological data suggest that vaccination with live attenuated vaccines, such as Bacillus Calmette–Guérin (BCG), measles, and oral polio vaccines, results in increased overall childhood survival[29,30]. As vaccination reduces infection by different pathogenic agents, not only specific adaptive immunity but also nonspecific innate immunity were considered to be enhanced. In particular, BCG vaccination leads to increased IFN-γ production and the release of monocyte-derived cytokines, such as tumor necrosis factor (TNF) and IL-1β, in response to unrelated bacterial and fungal pathogens[31,32]. The enhanced function of circulating monocytes is accompanied by increased TLR4 expression[31], suggesting that vaccination may enhance the response to unrelated antigens. Thus, innate immunity is currently accepted as being enhanced by vaccination, with trained immunity being involved in that process[33].

The concept of trained immunity originated in the field of infectious diseases; namely, the training of innate immune cells, such as monocytes, macrophages, and/or NK cells, by infection or vaccination enhances immune responses against microbial pathogens following restimulation[34]. Trained immunity has been documented in plants, invertebrates, and mammals, including humans[4,35,36], although studies on poultry are limited. Moreover, a recent report indicates that trained immunity can also be triggered via the inclusion of certain feed additives such as β-glucan.

EPIGENETIC AND METABOLIC REPROGRAMMING FOR TRAINED IMMUNITY IN MAMMALS

Epigenetic reprogramming

Trained immunity is based on two key pillars—the epigenetic and metabolic reprogramming of cells[28,33]. The key epigenetic markers for trained immunity in mammals are the acquisition of histone 3 lysine 27 acetylation (H3K27ac) at distal enhancers and the consolidation of histone 3 lysine 4 trimethylation (H3K4me3) marks at the promoters of stimulated genes[4,28,31,32]. These histone modifications may lead to more rapid and enhanced recruitment of transcription factors and associated gene expression of innate immune factors after a secondary challenge, even with an unrelated stimulus. Therefore, BCG and some other vaccinations have been suggested to induce trained immunity protecting nonspecifically against infections through the epigenetic reprogramming of innate immune cells[31].

Metabolic reprogramming

Trained immune activation requires rapid access to a supply of substrates to initiate the numerous metabolic processes associated with the immune response. Metabolic reprogramming, one of the mechanisms involved in trained immunity, is therefore also necessary to induce the long-term functional upregulation of innate immune cells is[29]. Trained immunity via metabolic reprogramming involves changes in cellular metabolic pathways such as glycolysis, oxidative phosphorylation, and fatty acid and amino acid metabolism in innate immune cells, including monocytes, macrophages, and NK cells[2,28,33,37]. β-glucan is one of the substances that can induce metabolic reprogramming in innate immune cells[37,38]. In a recent review, Kalafati et al.[39] described that changes in these cellular metabolic pathways, as well as cholesterol biosynthesis, can dictate innate immune cell plasticity. Moreover, such metabolic reprogramming has been suggested to increase the capacity of innate immune cells to respond to secondary stimulation[37].

DO VACCINATION AND Β-GLUCAN SUPPLEMENTATION ENHANCE INNATE IMMUNITY IN CHICKENS?

Effects of vaccination

Enhanced expression of innate immune molecules, including pattern recognition receptors (i.e., TLRs, NLRs, and dectin), cytokines, and antimicrobial substances, leads to improved resistance to infectious diseases. Kang et al.[40] examined the effects of multiple routine vaccinations (i.e., IB, MD, ND, and infectious bursal disease) during the first 14 days post hatching on the expression of innate immune molecules and histone modifications in the ovaries of 21-day-old chicks. The expression of TLR2 and 21 was upregulated in the ovaries of vaccinated chicks, whereas that of tumor necrosis factor superfamily (TNFSF) 15 and some AvBDs (i.e., AvBD1, 2, 4, and 7) decreased. Moreover, the densities of histone 3 lysine 9 dimethylation (H3K9me2) and histone 3 lysine 9 acetylation (H3K9ac) were significantly higher in the vaccinated group than in the control group; conversely, H3K4me2/3 and H3K27ac did not markedly differ between the two groups (Fig. 2). The authors therefore suggested that vaccination positively or negatively affected the expression of innate immune molecules, including TLRs, TNFSF15, and AvBDs, in chick ovaries and may be associated with epigenetic reprogramming via histone modifications in ovarian cells[40].

Fig. 2.

  Effects of multiple routine vaccinations on histone modification in the ovaries of chicks. (a-d) Fold change in histone modifications between vaccinated and unvaccinated chicks are shown: (a) di-methyl histone H3 (Lys9), H3K9me2; (b) di-methyl and tri-methyl histone H3 (Lys4), H3K4me2/3; (c) acetyl histone H3 (Lys9), H3K9ac; and (d) acetyl histone H3 (Lys27), H3K27ac. Chicks in the vaccine group (■) received the infectious bronchitis vaccine and Marek’s disease vaccine on day 1, mixed vaccines of Newcastle disease and infectious bronchitis on day 7, and infectious bursal disease vaccines on day 14. Control chicks (□) were administered water or dilution buffer in lieu of vaccines. Samples were collected on day 21, and the densities were examined using western blot analysis. Values represent the mean ± SEM of the densities relative to histone H3 (n = 8). Asterisks indicate significant differences between the control and vaccine groups (*P < 0.05, **P < 0.01). Data from Kang et al.[40] reproduced with permission of the publisher.

Shimizu et al.[41] examined the effects of vaccination using the live attenuated Newcastle disease virus and avian infectious bronchitis virus (ND/IB) vaccine, in addition to those of attenuated MD vaccination on the expression of TLRs and AvBDs in the kidneys of broiler chicks. The results demonstrated that IB/ND vaccination upregulated the expression of TLR7 and TLR21 (Fig. 3), whereas MD vaccination upregulated the expression of the four AvBDs in the kidneys of 3-day-old but not of 10-day-old chicks. These results suggest that IB/ND vaccination modulates the recognition of viral ssRNA and microbial unmethylated CpG-DNA by TLR7 and TLR21, whereas MD vaccination enhances AvBD synthesis in the chick kidney for several days post-vaccination although such enhancement likely declines or disappears by the 10^th day post-inoculation.

Fig. 3.

  Effects of Marek’s disease (MD) and avian infectious bronchitis/Newcastle disease (IB/ND) vaccinations on the expression of toll-like receptors (TLRs) 7 and 21 in the chick kidneys. Day-old chicks were vaccinated with IB/ND or MD vaccines, and TLR gene expression was examined three days post-vaccination using real-time PCR. Chicks in the control group (Con) received no vaccine. Solid bars represent the median values within each group. Asterisks indicate significant differences between the two groups (*P < 0.05, **P < 0.01). Data from Shimizu et al.[41] reproduced with permission from the publisher.

Recent studies have examined the effects of the live mixed ND/IB vaccine on the expression of innate immune molecules in the chick gastrointestinal tract[17,42]. In the proventriculus of chicks that received ND/IB vaccination on day old, the expression levels of TLR7 and TLR21 were higher than those in non-vaccinated chicks at seven days post-hatching[17,42], whereas the expression level of only TLR21 was higher than that in non-vaccinated chicks at 11 days post-hatching (Fig. 4)[17]. The expression of TLRs in the ileum was, however, not affected by vaccination[17,42]. These results suggest that the effects of ND/IB vaccination on TLR expression differ among the gut segments. Furthermore, liquid chromatography-mass spectrometry analysis revealed that the relative amounts of AvBD2, 6, and 7 proteins in the proventriculus were higher in LPS-challenged chicks than in non-challenged chicks. However, vaccination did not affect AvBD levels in LPS-challenged or non-challenged chicks[17]. Therefore, ND/IB vaccination is likely to induce TLR7 and TLR21 in the proventriculus of chicks, whereas it may not affect the sensitivity to LPS-mediated induction of AvBD synthesis via TLR4.

Fig. 4.

  Effects of Newcastle disease and infectious bronchitis (ND/IB) vaccination on the expression of toll-like receptors (TLRs) in the chick proventriculus. Day-old chicks were administered phosphate-buffered saline (control; C) or the ND/IB vaccine (V), and proventricular tissues were collected on the 11^th day post-hatching. Dots indicate the values for each individual. Bars represent mean ± SEM (n = 10). Asterisks indicate significant differences between the control and vaccinated groups (**P < 0.01, n = 10). Data from Yoshimura et al.[17] reproduced with permission from the publisher.

Evaluation of the effects of vaccinations in different organs has revealed that TLR21 expression is upregulated via multiple vaccinations containing ND/IB or through vaccination with ND/IB in the ovary, kidney, and proventriculus of chicks[17,40,41,42]. TLR7 expression is also upregulated by the ND/IB vaccine in the kidney and proventriculus[41,42] for at least 7 to 11 days post-vaccination. The increase in TLR7 expression suggests an enhanced ability to recognize ssRNA viruses, including ND and IB viruses. The upregulation of TLR21 expression by ND/IB vaccination may enhance the ability to recognize microbial CpG-DNA, which is a PAMP unrelated to the viral antigen (i.e., genomic ssRNA) in the vaccine. Conversely, ND/IB vaccination may not cause long-term upregulation of AvBD expression, as routine multiple vaccinations rather downregulate AvBD expression in chick ovaries[40]. MD vaccination has also been shown to upregulate their expression in the kidney at three days, but not at 10 days, post-inoculation[41]. The duration of elevated TLR expression following ND/IB vaccination in chickens remains to be examined. However, trained immunity in mammals is reportedly maintained for three months to one year after vaccination[28].

Several mammalian studies on trained immunity have described the effects of live-attenuated vaccines, such as BCG, measles, and polio vaccines[29,39]. In comparison, the ND/IB vaccines used to study the effects of vaccines on innate immunity in the chick kidney and proventriculus were live vaccines. Kang et al.[43] used an inactivated Salmonella enteritidis (SE) vaccine to examine the effects on innate immunity and histone modifications in the ovarian follicles of laying hens. They observed increased expression of some TLRs (i.e., TLR2-1, 4, and 15) recognizing bacterial molecular patterns and some AvBDs (i.e., AvBD1, 2, 4, and 7), together with increased density of histone dimethylation (H3K9me2) in the follicular theca one week after vaccination as compared to the values in non-vaccinated birds. These results suggest that vaccination with inactivated SE upregulates innate immune molecules, including TLR and AvBD, in the ovaries of laying hens, in association with histone modifications. In turn, Gu et al.[44] reported that intranasal immunization with inactivated whole cells of Acinetobacter baumannii induced rapid, efficient, and broad protection against certain gram-negative bacterial pneumonia in mice, dependent on trained immunity mediated by alveolar macrophages. Thus, numerous reports have described the enhancement of innate immunity by live-attenuated vaccines; nevertheless, this phenomenon may also be caused by part of the inactivated vaccine. Future studies should therefore consider the use of live and killed vaccines as well as DNA and RNA vaccines for the training of innate immunity.

Effects of β-glucan

β-glucans are glucose polymers found in fungal cell walls, plants, and some bacteria[45]. They are recognized by dectin-1, a pattern recognition receptor, in innate immune cells including macrophages, neutrophils, and NK cells[45,46]. β-glucans prime leukocytes to enhance the responsiveness to secondary pathogen challenges in a nonspecific manner[45,47,48]. A study in mice demonstrated that the administration of β-glucans protected against infection with Staphylococcus aureus, a pathogenic gram-positive bacteria[49], suggesting that mouse immunity was enhanced by β-glucans. Moreover, studies in mammals have revealed that immunity trained by metabolic reprogramming may be induced by β-glucan[3]. The profound changes in cellular metabolic pathways, such as glycolysis, oxidative phosphorylation, and fatty acid and amino acid metabolism, that occur in innate immune cells after priming with β-glucan increase the capacity of the cells to respond to secondary stimulation[37]. β-glucan-induced trained immunity is also reported to improve phagocytosis, nitric oxide production, myeloperoxidase activity, and TNF-α gene expression in calf monocytes through the upregulation of genes in the TLR2/NF-κB pathway[38].

Several studies have examined the effectiveness of β-glucan supplementation on the growth performance and immunity of broiler chicks. The effects of dietary β-glucan on body weight remain controversial, as both no change in weight gain (with β-glucan alone)[50] and improved growth performance (with a combination of mannan-oligosaccharide and β-glucan)[51] have been reported. Alternatively, several reports have suggested that dietary β-glucan modulates immune function. For example, β-glucan increased the proliferative and phagocytic activity of macrophages and the percentage of CD4+ and CD8+ T cells in intestinal intraepithelial leukocytes[52]. Lowry et al.[53] reported that β-glucan as a feed additive upregulated the functional abilities of heterophils in chicks and significantly reduced organ invasion by SE. Moreover, dietary β-glucan altered the intestinal cytokine–chemokine balance[50] and intestinal immune-related gene expression profiles, favoring an enhanced Th1 cell response during coccidiosis[54]. In studies by Verwoolde et al.[48,55], chicken monocytes primarily stimulated with β-glucan plus IL-4, followed by secondary stimulation with LPS, demonstrated elevated expression of colony-stimulating factor 1 receptor (CSF-1R), the activation markers CD40 and MHC class-II, and enhanced nitric oxide (NO) production. The results suggested that trained innate immunity may be induced in primary chicken monocytes by β-glucan. The authors also compared the effects of β-glucan treatment between the monocytes of laying and broiler hens and demonstrated that IL-1β mRNA expression, inducible nitric oxide synthase (iNOS), and hypoxia-inducible factor 1α expression was increased in both breeds. However, the enhancement of CD40 expression and NO production was only observed in layers, suggesting differential effects of β-glucan between the two breeds[55].

Thus, studies on the effects of β-glucan supplementation suggest the possibility of enhancing innate immunity in chickens. Metabolic reprogramming may participate in this mechanism, as suggested by the reported effects of β-glucan in mammals.

Do vaccinations and β-glucan supplementation cause trained immunity?

The modulated TLR7 and TLR21 expression following ND/IB vaccination, and altered intestinal innate immune-related gene expression upon β-glucan supplementation support that these treatments enhanced the innate immunity in chickens. This enhancement may fall under the category of trained immunity, considering existing reports on mammals. Further examination is needed to determine the contribution of other factors including the response to secondary stimuli, confirm the existence of epigenetic and metabolic reprogramming for innate immune molecule synthesis and cellular functions, and establish the duration of innate immunity elevation. Understanding these issues will support the development of procedures for inducing trained immunity in chickens.

FUTURE PROSPECTS AND STRATEGIES

Prevention of pathogenic infections is vital for the production of healthy chickens. Immunodefense plays an important role in prevention, with vaccines used toward this end to enhance immunity. However, drug-resistant pathogens, variant viruses, and pathogens for which vaccines have not yet been developed may appear on farms. Enhancing immunity in response to different pathogens is crucial to address these issues. Unlike adaptive immunity, innate immunity responds to various pathogens with less specificity via pattern recognition receptors and cellular and humoral immune factors. Therefore, the enhancement of both adaptive and innate immunity will provide birds with better defense mechanisms. BCG and several other vaccinations, in addition to β-glucan supplementation, can induce trained immunity in mammals[31,37,45]; however, studies on trained immunity in chickens remain limited. Nevertheless, some vaccinations such as the ND/IB vaccine upregulate TLRs, and β-glucan stimulates innate immune cells, suggesting the possibility of innate immunity enhancement. The results suggest that these effects likely reflect trained immunity induced by vaccination and β-glucan. The determination of markers of successfully trained immunity, such as specific histone modifications and intracellular metabolic events, will support the exploration of more effective vaccines and feed additives in the future.

Furthermore, the enhancement of innate immunity by trained immunity is recommended as a strategy for preventing infections that have not been considered in poultry. Therefore, more effective vaccines and feed additives are needed to induce trained immunity.

In summary, the enhancement of innate immunity is beneficial for the non-specific prevention of pathogenic infections. Recent reports have suggested that trained immunity may be induced in chickens through vaccination and feed additives, similar to the effects in mammals. Development of vaccines and feed additives for this purpose is an important strategy for the enhancement of poultry defense functions by innate immunity.