Broilers and native chickens analyzed in this study represent distinct genotypes, that are characterized by different set of traits. Broilers are four-way crosses between heavily selected genetic lines. The extreme selection pressure implemented in contemporary broiler modulated not only growth and feed efficiency, but also mechanisms of the immune responses. Cheema, Qureshi (24) determined that broilers from 1957 had greater relative weight of bursa of Fabricius, caecal tonsils and spleen, and mounted higher humoral immune responses against SRBC in comparison to broiler from 2001. On the other hand, contemporary broiler scored higher in tests evaluating cellular and inflammatory immune responses (24). Genetics underlying such a shift in the immune responses due to selection towards growth rate has been attributed to variation accumulated in the genes TLR3 and PLIN3 (25). Native chickens analyzed in contrast to heavily selected broiler was Green-legged Partridgelike (GP). GP is a Polish heritage chicken line, unselected since 1960s and currently kept in conservative flocks (26). GP is a light-weight chicken, traditionally kept as a dual-purpose backyard chicken. It’s an excellent model for immunological studies (8, 27) due to conserved genetic status (28), as well as their resilience and sturdiness (26).
The intraperitoneal challenge with LTA and LPS antigens was performed to evaluate the level of the mucosal and systemic immune responses in caecal tonsils (Figure 2) and spleen (Figure 3) of the chickens stimulated in ovo. The intraperitoneal immunization is often referred to as mucosal vaccination, because it aims to stimulate local mucosal immune responses (i.e., secretory IgA production at the mucosal site). Mucosal vaccination has been used to develop local immunity against Salmonella typhimurium in chickens (29). In the current study, the chickens were challenged intraperitoneally with nonpathogenic antigens, which are purified microbe-associated molecular patterns (MAMPs) isolated from Staphylococcus aureus (LTA) and Escherichia coli (LPS).
Exposure to LPS is associated with the condition called endotoxemia, which results in overexpression of inflammatory mediators, leading to a septic shock and increased mortality (30). During intraperitoneal injection, the tested compound is deposited in the body cavity, from where it diffuses to the surrounding tissues as well as to the circulatory system (31). Since spleen filters the blood and samples the antigens from the bloodstream, it is the primary organ to mount immune responses and neutralize the antigens. On the other hand, caecal tonsils belong to GALT, which is also susceptible to endotoxemia-mediated injury (30). GALT is primed by the orally administered antigens rather than the peritoneal ones (32). Innate immune responses mounted in the GALT are either stimulatory (i.e., against oral pathogens) or tolerogenic (i.e., unresponsive to commensal or beneficial bacteria). Oral tolerance is developed in the GALT by early contact with intestinal microbiota and helps maintaining intestinal homeostasis. The cellular mechanism by which harmful bacteria are differentiated from beneficial ones is mediated by GALT-associated dendritic cells (CD103+ DCs), which cooperate with other cells in the gut (33). In this manner, acquired oral tolerance primes caecal tonsils to exert lower level of inflammatory responses.
LTA challenge exerted less pronounced effects in both tissues compared to LPS, which is a strong inflammatory stimuli. LTA is an exopolysaccharide sourced from cell walls of Gram-positive bacteria, including many commensal species, including Lactobacillus. Even though LTA induces acute inflammatory responses in the gut by activating TLR2, its major function is to enhance gut integrity via stimulating tight junctions in the intestinal epithelia (34). In vivo studies clearly indicate that mice treated orally with TLR2 ligand, Pam3CSK4 (synthetic LTA), were less susceptible to mucosal injury by increased integrity of intestinal epithelium (34). Recognition of LTA in the gut is mediated by DCs (e.g., CD103+ DCs subtype, mentioned earlier), and results in production of inflammatory and regulatory cytokines (35). For this reason, LTA activated gene expression signatures in caecal tonsils, but exerted no immunostimulation in the spleen.
In ovo stimulation
Effects of in ovo stimulation on immune-related gene expression signatures in chickens is of particular interest due to an early effect of intestinal microbiota development on immune system maturation in the neonatal chicks. According to the review of Taha-Abdelaziz, Hodgins (36), the beneficial effects of early dietary interventions on the immune system in chickens are expressed in three areas: development of lymphoid organs, gastrointestinal microbiome, and immune competence. Our previous and the current research indicates that in ovo-delivered prebiotics, probiotics, and synbiotics affect all three aspects of poultry immunology. Regarding the first aspect, which is lymphoid organs development, in ovo stimulation significantly influenced colonization of caecal tonsils and spleen with Bu-1+, CD4+, and CD8+ cells in the chickens analyzed in this paper (15). The influence depended on the bioactive compound, chicken genotype, and age. In broilers, in ovo stimulation increased CD4+ cells in caecal tonsils (Day 7), CD4+ and CD8+ cells in caecal tonsils and spleen (Day 21), as well as Bu-1+ cells in caecal tonsils and all type of lymphoid cells in spleen (Day 42). In native chickens, synbiotic increased CD4+ and CD8+ cells in spleen (but not in caecal tonsils) (Days 2, 21, and 42). Regarding the second aspect of early dietary interventions, i.e., gastrointestinal microbiome, to date we demonstrated that in ovo delivery of GOS prebiotic significantly increased counts of lactobacilli and bifidobacteria in feces of newly hatched chicks (37). These effects were long-lasting, and remained significant also on day of slaughter (11). Lactobacillus-based synbiotics delivered in ovo on day 12 of egg incubation increased Lactobacillus spp. and Enterococcus spp. in ileum of the Cobb broiler chickens (18). The current study reports that in ovo stimulation modulates immune competence (the third aspect of early dietary interventions) in broiler and native chickens.
The results presented in this paper strongly support our earlier findings, that in ovo stimulation with GOS prebiotic modulates splenic gene expression of IL-4 in fast-growing chicken broilers (12), and both, IL-2 and IL-4, in slow-growing chickens (13). We have also found that raffinose family oligosaccharides (RFOs) and RFO-based synbiotics, modulate IL-4 and IL-12p40 gene expression in caecal tonsils and spleen of chicken broilers (38) and native chickens (GP) (8). In this study, we have complemented the earlier data with additional treatments, i.e., immune challenge with LTA or LPS antigens. Results of the interactions between genotype, in ovo stimulation, and immune challenge in caecal tonsils and spleen are discussed below (section 3.4).
Mucosal immune responses in caecal tonsils
Caecal tonsils are the largest aggregates of lymphoid tissue present in the chicken GALT. As such, they represent a major site of mucosal immune responses. IL-1β and IL-6 are involved in acute inflammatory responses. Both cytokines are activated by microbes, including enteric infection with Eimeria or Salmonella (39). Increased gene expression of IL-1β and IL-6 in broiler chickens immunized with LPS reflects acute inflammatory responses mounted by GALT. Haghighi, Abdul-Careem (40) reported that the IL-6 gene expression was increased in caecal tonsils of broiler chickens infected with Salmonella, but the inflammatory effects of infection were mitigated by probiotics. In the current study, broiler chickens immunized with LPS (also present in Salmonella) increased IL-1β and IL-6 gene expression in caecal tonsils, but it was not influenced by in ovo-delivered bioactive compounds. IL-10 is anti-inflammatory cytokine and its expression counterbalances pro-inflammatory activity of IL-1β and IL-6 (41). Regulation of inflammation by the negative feedback is the supposed mechanism of IL-10 up-regulation in individuals expressing high abundance of pro-inflammatory mediators (IL-1β and IL-6).
The regulatory effects of bioactive compounds delivered in ovo were more pronounced in native chickens rather than in broiler chickens. Such results are not surprising, since in our early research on in ovo stimulation we found that the prebiotics and synbiotics applied on day 12 of egg incubation, down-regulate immune related gene expression signatures in caecal tonsils of native chickens (GP) (8) and chicken broilers (18, 38). However, this is the first time that we report effects of in ovo stimulation (day 12 of egg incubation) together with the immune challenge in two distinct chicken genotypes. Regulatory effect of GOS prebiotic (PRE) on IL-12p40 and IL-17 gene expression in GALT is considered beneficial, since it helps reducing LPS-induced inflammation in the intestines. Both cytokines are expressed by CD4+ cells (T lymphocytes) in response to inflammatory agents. The role of IL-12p40 cytokine is to drive pro-inflammatory Th1-type responses, while pleiotropic cytokine IL-17 (also known as IL-17A) stimulates pro-inflammatory Th17-type responses (42). Th17-type immune responses has been investigated recently due to their involvement in autoimmune diseases (43). Increased number of Th17-type cells as well as IL-17 cytokine eliminates therapeutic effects of oral tolerance in mice (44). Intestinal level of IL-17 varies depending on microbiota composition. For example, Lactobacillus fermentum IM12 suppressed LPS-activated IL-17 level in mice (45). On the other hand, dietary GOS increased IL-17 in caecum of Campylobacter-infected broiler chickens, but did not ameliorate the infection (46). We suppose, that decreased activity of IL-17 in prebiotic-supplemented and LPS-challenged native chickens might be a good biomarker of anti-inflammatory effects of prebiotics and probiotics delivered in ovo. But it requires more insight to determine the specific effects on immune responses.
Systemic immune responses in spleen
The pronounced difference between broilers and native chickens in inflammatory immune responses to in ovo stimulation were found in C group (mock-challenged). In chicken broilers, splenic gene expression of IL-1β and IL-6 was up-regulated, while in native chickens – it was down-regulated. In other words, chicken broilers mounted higher pro-inflammatory responses to in ovo stimulation than native chickens. As mentioned earlier, potent activation of the cellular and inflammatory immune responses is typical for the lymphoid system of chicken broilers (24). Broilers have been heavily selected for growth traits and feed efficiency (47). Such strong selection pressure affected immune system of the broiler chickens. Acquired immune responses takes weeks to develop functional antibodies against pathogens. Since, a typical lifespan of fast-growing broilers is only 35-42 days, strong innate immune responses are more likely to fight infection (48). Increased mRNA expression levels of pro-inflammatory cytokines are under genetic control. Thus, the chicken broilers analyzed in this study were more reactive to in ovo microbiota stimulation and activated stronger signatures of systemic, inflammatory immune responses than native chickens.
IL-12p40 encodes a subunit p40, which is shared between IL-12 and IL-23 cytokines in both avian and mammalian organisms (49). In spleen, IL-12p40 is produced by activated antigen presenting cells, such as macrophages or dendritic cells. It drives natural killer (NK) cells to secrete IFN-ɣ and directs immune responses towards Th1-type cell-mediated immunity (50). The most pronounced interaction in the systemic immune responses was down-regulation of the IL-12p40 mRNA abundance in the spleen of PRE/LPS/broilers (FC ~ -4.4). In our earlier studies we associated splenic mRNA abundance of IL-12p40 with responses to thermal stress in chicken broilers (12) and native chickens (13). Responses mounted by the immune system after acute or chronic exposition to heat stress resemble responses to LPS injected intraperitoneally into the body cavity. Both of those immune responses are aimed to neutralize circulating LPS molecules. In heat stress studies, the LPS molecules were sourced from intestinal bacteria, and in this study they were directly injected. In previously mentioned heat stress studies and in the current study, we found that GOS delivered in ovo successfully dampened LPS-stimulated IL-12p40 mRNA abundance. GOS has been known for its immunomodulatory properties in animal (51) and human models. In chickens, it promotes beneficial intestinal microbiota and alleviates the effects of the foodborne pathogens like Salmonella (52). In broiler chickens, GOS delivered in ovo has confirmed bifidogenic effects and beneficial effects on mucosal gene expression, enhancing gut barrier function (11).