TLR-Mediated Cytokine Gene Expression in Chicken Peripheral Blood Mononuclear Cells as a Measure to Characterize Immunobiotics

Immunobiotics are probiotics that promote intestinal health by modulating immune responses. Immunobiotics are recognized by Toll-like receptors (TLRs) and activate cytokine gene expression. This study aimed to characterize cytokine gene expression in the chicken peripheral blood mononuclear cells (PBMC) stimulated with purified TLR ligands and live probiotics. PBMC were isolated from the whole blood. PBMC were stimulated with: lipopolysaccharide (LPS), CpG ODN, Pam3CSK4, Zymosan, galactooligosaccharides (GOS), Lactococcus lactis subsp. cremoris (L. lactis), and Saccharomyces cerevisiae at 42.5 °C and 5% CO2 for 3 h, 6 h, and 9 h. After each time-point, PBMC were harvested for RNA isolation. Relative gene expression was analyzed with RT-qPCR for cytokine genes (IL-1β, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12p40, and IFN-ɣ) and reference genes (ACTB and G6PDH). Genes were clustered into pro-inflammatory genes, Th1/Th2 genes, and Th1-regulators. The gene expression differed between treatments in IL1-β, IL-6, IL-8, IL-10, and IL-12p40 (p < 0.001). The genes IL-1β, IL-6, and IL-8 had the highest fold change of mRNA expression at 3 h in response to TLR ligands. L. lactis up-regulated the pro-inflammatory genes at the 6 h time-point. L. lactis did not activate the anti-inflammatory IL-10 gene, but activated IL-12p40 at 6 h. Hereby, L. lactis was proven to exert immunostimulatory properties in PBMC.

and S. cerevisiae. Each of those compou chicken PBMC in a different manner. GO inflammatory cytokine genes, especially and IL-6 (58 fold change) at 3 h post-st trigger immune responses both in vitro GOS dose-dependently enhanced the PBMC upon LPS challenge. Quite rever lenged with LPS secreted a lower amo compared to LPS challenge alone [48]. T with the low molecular weight molecul L. lactis triggered the strongest up-r (IL-1β, IL-6, and IL-8) at 6 h post-stimula ɣ) at 9 h post-stimulation. Foligne et al. [5 the probiotic strains that promote the r biotics were ranked based on the IL-10/ tenuate inflammatory bowel disease in poorly, with an IL-10/IL-12 cytokine ra strain of L. lactis activated a higher abu inflammatory cytokine gene rather than it as an immunostimulatory probiotic. study did not activate immune-related cerevisiae (yeast) has been successfully u have also been known for their potentia or bind pathogenic enterobacteria [53]. are cell wall polysaccharides, such as m such as zymosan. Other bioactive comp products such as XPC [54]. In this study lation, which was not an efficient mode availability of the TLR ligands.
In comparison to the purified TLR live L. lactis bacteria were shifted in tim expression up-regulation of the pro-infl stimulation, whereas, L. lactis activated tion. The beneficial effects of L. lactis tributed to the activity of exopolysaccha either loosely bound to the cell wall in a Among Gram-positive bacteria, there strains of L. lactis subsp. cremoris [57,58 cific immunomodulatory effects in the h macrophages [59], induction of macroph phocytes [61]. Hereby, we hypothesize lactis was mediated indirectly by EPS rath

Conclusions
In conclusion, we have determined ulated with an array of purified TLR lig inflammatory Th1/Th2 and Th1-regulato est stimuli of PBMC were LPS and CpG O

Introduction
The immune responses include two major components: innate and adaptive immunity. Innate immunity identifies the general threats, whereas adaptive immunity occurs after exposure to the specific antigen, such as pathogen or vaccination. Lymphatic cells involved in both types of immune responses produce cytokines, which are small soluble proteins that direct the immune responses. All activated lymphatic cells can secrete cytokines, but predominantly cytokines are produced by different classes of Th lymphocytes involved in cell-mediated immunity [1]. The role and structure of the cytokines as well as their genetic makeup has been well characterized in mammals [2]. In chicken, the cytokine repertoire resembles the mammalian one, including the genetic homology of the cytokine genes [3][4][5]. The transcription of the cytokine genes is initiated when the signal from the antigen is recognized by the receptor and transduced to the nucleus of the lymphatic cell.
The most conserved group of antigens is called pathogen-associated molecular patterns (PAMPs) or microbe-associated molecular patterns (MAMPs). There are several types of PAMPs and MAMPs, which are expressed on the surface of certain classes of microorganisms. For example, lipopolysaccharide (LPS) is present on the membrane of Gram-negative bacteria, lipoteichoic acid (LTA) is present on the membrane of Gram-positive bacteria, and flagellin is characteristic for flagellated bacteria [6]. PAMPs and MAMPs are structurally of 100 µL of the respective stimulus was added to the well and mixed by gentle pipetting. The control wells were mock-stimulated with the media. The list of the stimuli is shown in Table 1. PBMC stimulation was conducted in a time-course manner; the cells were harvested at 3 h, 6 h, and 9 h post-stimulation. The experiment was repeated three times (biological replicates) at one-week intervals.

RNA Isolation and RT-qPCR
PBMC were harvested from individual wells in each experiment (biological replicate). Total RNA was isolated from PBMC cultures using a Universal RNA Purification Kit (EURx, Gdansk, Poland). The concentration and purity of RNA isolates were measured with NanoDrop 2000 spectrophotometer (Scientific Nanodrop Products, Wilmington, NC, USA). rRNA bands integrity were analyzed in 2% agarose gel stained with Simply Safe (EURx, Gdansk, Poland). Cytokine gene expression was determined at mRNA level using the two-step reverse transcription-quantitative PCR (RT-qPCR) method. The amount of 2.5 µg of total RNA was reversely transcribed using Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific/Fermentas, Vilnius, Lithuania). RT-qPCR gene expression was determined for ten target cytokine genes and two reference genes, based on the oligonucleotide primers reported in Table 2.

Quantitative Reverse Transcription PCR (RT-qPCR)
Complementary DNA (cDNA) was synthesized by using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific/Fermentas, Vilnius, Lithuania), following the manufacturer's recommendations. Obtained cDNA was diluted to 70 ng/µL working solutions and stored at −20 • C. Each RT-qPCR reaction was conducted in two technical replicates. Cytokine gene panel included the following genes: IL-1β IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12p40, and IFN-Genes 2021, 12, x FOR PEER REVIEW and S. cerevisiae. Each of those compounds stimulated c chicken PBMC in a different manner. GOS induced the stro inflammatory cytokine genes, especially IL-1β (~214 fold c and IL-6 (58 fold change) at 3 h post-stimulation. Oligosa trigger immune responses both in vitro and in vivo. Vend GOS dose-dependently enhanced the pro-inflammatory PBMC upon LPS challenge. Quite reversely, human PBM lenged with LPS secreted a lower amount of immune m compared to LPS challenge alone [48]. The primary stimul with the low molecular weight molecules, such as tri-and L. lactis triggered the strongest up-regulation of the pr (IL-1β, IL-6, and IL-8) at 6 h post-stimulation and Th1-regu ɣ) at 9 h post-stimulation. Foligne et al. [50] proposed a scre the probiotic strains that promote the release of anti-infla biotics were ranked based on the IL-10/IL-12 cytokine rat tenuate inflammatory bowel disease in vivo. In this rank poorly, with an IL-10/IL-12 cytokine ratio around 1 [50]. strain of L. lactis activated a higher abundance of mRNA inflammatory cytokine gene rather than the IL-10 anti-infla it as an immunostimulatory probiotic. On the other han study did not activate immune-related gene expression s . Reference genes used to normalize the samples were ACTB and G6PDH. The oligonucleotide sequences of the primers are presented in Table 2. RT-qPCR reactions were conducted with a total volume of 10 µL. The reaction mixture included Maxima SYBR Green qPCR Master Mix (Thermo Scientific/Fermentas, Vilnius, Lithuania), 1 µM of each primer (Sigma-Aldrich, Schnelldorf, Germany), and 2 µL of diluted cDNA (70 ng/µl). Thermal cycling was performed in a LightCycler II 480 (Roche Diagnostics, Basel, Switzerland). The thermal program included a step of initial denaturation (15 min at 95 • C), followed by 40 cycles of denaturation (10 s at 95 • C), annealing (15 s at 58 • C), and extension (30 s at 72 • C). Fluorescence was measured at the end of each extension step. After completing the thermal program, the melting curve was generated, which indicated amplification specificity. The thermal program for the melting curve included a gradual increase in the temperature up to 98 • C and measuring the fluorescence of the melting amplicon.  [48]. The primary stimulant of GOS has been associated with the low molecular weight molecules, such as tri-and tetrasaccharide fractions [49]. L. lactis triggered the strongest up-regulation of the pro-inflammatory cytokine genes (IL-1β, IL-6, and IL-8) at 6 h post-stimulation and Th1-regulators (IL-12p40, IL-10, and IFNɣ) at 9 h post-stimulation. Foligne et al. [50] proposed a screening test using PBMC to select the probiotic strains that promote the release of anti-inflammatory cytokines. These probiotics were ranked based on the IL-10/IL-12 cytokine ratio, indicating their ability to attenuate inflammatory bowel disease in vivo. In this ranking, L. lactis performed rather poorly, with an IL-10/IL-12 cytokine ratio around 1 [50]. In the current study, the tested strain of L. lactis activated a higher abundance of mRNA expression of the IL-12p40 proinflammatory cytokine gene rather than the IL-10 anti-inflammatory gene, which classifies it as an immunostimulatory probiotic. On the other hand, S. cerevisiae analyzed in this study did not activate immune-related gene expression signatures in chicken PBMC. S. cerevisiae (yeast) has been successfully used as probiotics for poultry for years [51]. They have also been known for their potential to activate the immune system of the animal [52] or bind pathogenic enterobacteria [53]. The immunomodulatory components of the yeast are cell wall polysaccharides, such as mannan-oligosaccharides and β-glucan, or MAMP, such as zymosan. Other bioactive compounds that can stimulate PBMC are fermentation products such as XPC [54]. In this study, we used purified yeast culture for PBMC stimulation, which was not an efficient mode of PBMC stimulation, most likely due to the low availability of the TLR ligands.
In comparison to the purified TLR agonists, the immune responses elicited by the live L. lactis bacteria were shifted in time. LPS or CpG ODN triggered the strongest gene expression up-regulation of the pro-inflammatory cytokines or Th1-regulators at 3 h poststimulation, whereas, L. lactis activated the respective genes at 6 h and 9 h post-stimulation. The beneficial effects of L. lactis and other immunobiotics have been widely attributed to the activity of exopolysaccharides (EPS) [55]. EPS are polysaccharides that are either loosely bound to the cell wall in a capsular form or secreted to the environment [56]. Among Gram-positive bacteria, there are several EPS-producers, including different strains of L. lactis subsp. cremoris [57,58]. Many of them released EPS that conferred specific immunomodulatory effects in the host, such as induction of IL-1α and IFN-ɣ in spleen macrophages [59], induction of macrophage cytotoxicity [60], and mitogen activity in lymphocytes [61]. Hereby, we hypothesize that the cytokine gene expression activated by L. lactis was mediated indirectly by EPS rather than the by direct contact with bacterial PAMPs.

Relative Quantification of Gene Expression and Statistical Analysis
The normalization of the expression levels (Ct-cycle threshold) of the target genes was performed with a geometric mean of the two reference genes (ACTB and G6PDH). ∆Ct was calculated by subtracting the Ct of the reference genes from the Ct of the target genes (Ct target-Ct reference). All statistical analyses were based on ∆Ct values. One-way analysis of variance (ANOVA) was performed for each time-point independently, using stimuli as the independent variable. The factor was considered significant at p < 0.05, p < 0.01, or p < 0.001. ANOVA was calculated in SAS Enterprise Guide 9.4 (SAS Institute, Cary, NC, USA). A hierarchical cluster tree was constructed in the Multiexperiment Viewer (MeV) version 4.9 [20]. The relative gene expression was calculated with the ∆∆Ct algorithm [21]. The fold change (FC) of the target gene in the experimental group vs. the control group was calculated according to the formula: 2 −∆∆Ct [22]. The calculations were performed in MS Excel. and graphs were drawn by using Graph Pad Prism 7 (GraphPad, La Jolla, CA, USA).

Variance Analysis (One-Way ANOVA)
Supplementary File S1 presents ∆Ct values used for statistical evaluation. Table 3 presents results of variance analysis in which the significance of the stimuli (i.e., TLR ligands or live probiotics) was tested on the cytokine gene expression in the PBMC cultures. The gene expression, measured as dCt values, differed significantly between treatments in IL1-β, IL-6, IL-8, IL-10, and IL-12p40 (p < 0.001). and S. cerevisiae. Each of those compounds stimulated cytokine gene expression in chicken PBMC in a different manner. GOS induced the strongest up-regulation of the inflammatory cytokine genes, especially IL-1β (~214 fold change), IL-8 (~62 fold chan and IL-6 (58 fold change) at 3 h post-stimulation. Oligosaccharides have been know trigger immune responses both in vitro and in vivo. Vendrig, et al. [47] determined GOS dose-dependently enhanced the pro-inflammatory immune responses of eq PBMC upon LPS challenge. Quite reversely, human PBMC cultured with GOS and c lenged with LPS secreted a lower amount of immune mediators (e.g., IL-1α and IL compared to LPS challenge alone [48]. The primary stimulant of GOS has been associ with the low molecular weight molecules, such as tri-and tetrasaccharide fractions [4 L. lactis triggered the strongest up-regulation of the pro-inflammatory cytokine g (IL-1β, IL-6, and IL-8) at 6 h post-stimulation and Th1-regulators (IL-12p40, IL-10, and ɣ) at 9 h post-stimulation. Foligne et al. [50] proposed a screening test using PBMC to s the probiotic strains that promote the release of anti-inflammatory cytokines. These biotics were ranked based on the IL-10/IL-12 cytokine ratio, indicating their ability t tenuate inflammatory bowel disease in vivo. In this ranking, L. lactis performed ra poorly, with an IL-10/IL-12 cytokine ratio around 1 [50]. In the current study, the te strain of L. lactis activated a higher abundance of mRNA expression of the IL-12p40 inflammatory cytokine gene rather than the IL-10 anti-inflammatory gene, which class it as an immunostimulatory probiotic. On the other hand, S. cerevisiae analyzed in study did not activate immune-related gene expression signatures in chicken PBMC cerevisiae (yeast) has been successfully used as probiotics for poultry for years [51]. T have also been known for their potential to activate the immune system of the animal or bind pathogenic enterobacteria [53]. The immunomodulatory components of the y are cell wall polysaccharides, such as mannan-oligosaccharides and β-glucan, or MA such as zymosan. Other bioactive compounds that can stimulate PBMC are fermenta products such as XPC [54]. In this study, we used purified yeast culture for PBMC sti lation, which was not an efficient mode of PBMC stimulation, most likely due to the availability of the TLR ligands.
In comparison to the purified TLR agonists, the immune responses elicited by live L. lactis bacteria were shifted in time. LPS or CpG ODN triggered the strongest g expression up-regulation of the pro-inflammatory cytokines or Th1-regulators at 3 h p stimulation, whereas, L. lactis activated the respective genes at 6 h and 9 h post-stim tion. The beneficial effects of L. lactis and other immunobiotics have been widely tributed to the activity of exopolysaccharides (EPS) [55]. EPS are polysaccharides tha either loosely bound to the cell wall in a capsular form or secreted to the environment Among Gram-positive bacteria, there are several EPS-producers, including diffe strains of L. lactis subsp. cremoris [57,58]. Many of them released EPS that conferred cific immunomodulatory effects in the host, such as induction of IL-1α and IFN-ɣ in sp macrophages [59], induction of macrophage cytotoxicity [60], and mitogen activity in l phocytes [61]. Hereby, we hypothesize that the cytokine gene expression activated b lactis was mediated indirectly by EPS rather than the by direct contact with bacterial PAM

Conclusions
In conclusion, we have determined cytokine gene expression in chicken PBMC s ulated with an array of purified TLR ligands and potential immunobiotics. We found inflammatory Th1/Th2 and Th1-regulator gene clusters. Among the TLR ligands, the str est stimuli of PBMC were LPS and CpG ODN. Among bioactive compounds, L. lactis su cremoris triggered more abundant cytokine gene expression, but S. cerevisiae seemed in cient in triggering immune-related gene expression. Immune responses of PBMC to L. l had a pro-inflammatory character and was expressed by increased mRNA abundance o 1β, IL-6, IL-8, and IL-12p40. In this manner, L. lactis subsp. cremoris has immunostimula properties in chicken PBMC and therefore can be considered an immunobiotic.

Hierarchical Clustering
The gene expression data were clustered into five clusters based on correlation distance ( Figure 1). The two largest clusters included: (1) Pro-inflammatory genes (IL-1β, IL-6, and IL-8), which were up-regulated (bright green bars), and (2) Th1/Th2 cytokine genes (IL-2, IL-3, and IL-4), which were down-regulated or unchanged relative to the control (black or red bars). With a large correlation distance from each other were (3) Th1-regulating cytokine genes: IL-12p40 pro-inflammatory gene and IL-10 anti-inflammatory gene, with IL-10 being up-regulated (bright green) vs. IL-12p40 moderately up-regulated (dark green) or unchanged relative to the control (black bars). The gene IFN- the probiotic strains that promote biotics were ranked based on the I tenuate inflammatory bowel disea poorly, with an IL-10/IL-12 cytokin strain of L. lactis activated a higher inflammatory cytokine gene rather it as an immunostimulatory probi study did not activate immune-rel cerevisiae (yeast) has been successfu have also been known for their pot or bind pathogenic enterobacteria [ are cell wall polysaccharides, such such as zymosan. Other bioactive c products such as XPC [54]. In this s lation, which was not an efficient m availability of the TLR ligands.
In comparison to the purified live L. lactis bacteria were shifted i expression up-regulation of the pro stimulation, whereas, L. lactis activ tion. The beneficial effects of L. l tributed to the activity of exopolys either loosely bound to the cell wall Among Gram-positive bacteria, t strains of L. lactis subsp. cremoris [5 cific immunomodulatory effects in macrophages [59], induction of mac phocytes [61]. Hereby, we hypothe lactis was mediated indirectly by EP

Conclusions
In conclusion, we have determ ulated with an array of purified TL inflammatory Th1/Th2 and Th1-reg was an outlier, located in the node opposite to IL-12p40.

Pro-Inflammatory Cytokines (IL-1β, IL-6, and IL-8)
The cytokine gene expression analysis in the chicken PBMC stimulated with various TLR agonists and live probiotics in three different time-points post-stimulation is shown in Figure  2. The first three genes show the pro-inflammatory cluster (IL-1β, IL-6, and IL-8). These cytokines were the most up-regulated, and early responders (3 h), especially to LPS (black line), CpG ODN (green line), Pam3CSK4 (red line), GOS (purple line), and zymosan (orange line). Interestingly, the pro-inflammatory immune response to live probiotics, L. lactis (sky blue line) and S. cerevisiae (navy blue line) were quite different. Pro-inflammatory immune response to L. lactis expressed by mRNA abundance of IL-1β, IL-6, and IL-8 genes grew in time, peaked at and S. cerevisiae. Each of those compounds stim chicken PBMC in a different manner. GOS induce inflammatory cytokine genes, especially IL-1β (~ and IL-6 (58 fold change) at 3 h post-stimulation trigger immune responses both in vitro and in v GOS dose-dependently enhanced the pro-infla PBMC upon LPS challenge. Quite reversely, hum lenged with LPS secreted a lower amount of im compared to LPS challenge alone [48]. The prima with the low molecular weight molecules, such a L. lactis triggered the strongest up-regulation (IL-1β, IL-6, and IL-8) at 6 h post-stimulation and ɣ) at 9 h post-stimulation. Foligne et al. [50] propo the probiotic strains that promote the release of biotics were ranked based on the IL-10/IL-12 cyt tenuate inflammatory bowel disease in vivo. In poorly, with an IL-10/IL-12 cytokine ratio aroun strain of L. lactis activated a higher abundance o inflammatory cytokine gene rather than the IL-10 it as an immunostimulatory probiotic. On the o study did not activate immune-related gene exp cerevisiae (yeast) has been successfully used as p have also been known for their potential to activa or bind pathogenic enterobacteria [53]. The imm are cell wall polysaccharides, such as mannan-ol such as zymosan. Other bioactive compounds th products such as XPC [54]. In this study, we used genes. Gene expression analysis performed using RT-qPCR with ACTB and G6PDH reference genes. Hierarchical Cluster Tree constructed in the Multiexperiment Viewer (MeV) version 4.9. The cytokine gene expression analysis in the chicken PBMC stimulated with various TLR agonists and live probiotics in three different time-points post-stimulation is shown in Figure 2. The first three genes show the pro-inflammatory cluster (IL-1β, IL-6, and IL-8). These cytokines were the most up-regulated, and early responders (3 h), especially to LPS (black line), CpG ODN (green line), Pam3CSK4 (red line), GOS (purple line), and zymosan (orange line). Interestingly, the pro-inflammatory immune response to live probiotics, L. lactis (sky blue line) and S. cerevisiae (navy blue line) were quite different. Pro-inflammatory immune response to L. lactis expressed by mRNA abundance of IL-1β, IL-6, and IL-8 genes grew in time, peaked at 6 h, and remained at a high level at 9 h post-stimulation. On the other hand, chicken PBMC did not respond to stimulation with S. cerevisiae by activating the pro-inflammatory pathway.

Th1/Th2 Cytokines (IL-2, IL-3, and IL-4)
The second cluster included Th1 (IL-2 and IL-3) and Th2 (IL-4) cytokine genes. They were up-regulated with much lower potency compared to pro-inflammatory genes. They were also slower responders as they peaked at 6 h. The Th1/Th2 cytokine gene expression was triggered by LPS and zymosan. Live probiotics (L. lactis and S. cerevisiae) triggered lower mRNA abundance and towards later time-point of stimulation (9 h).

Th1-Regulators (IL-10, IL12p40, and IFN-ɣ)
Pro-inflammatory IL-12p40 and anti-inflammatory IL-10 were clustered on two opposite sides of the cluster tree. It is because those two cytokines have the opposite function. It is quite well represented by mRNA abundance of IL-10 and IL-12p40 stimulated by CpG ODN, which initially (3 h) triggered the high gene expression of pro-inflammatory  [48]. The prima with the low molecular weight molecules, such a L. lactis triggered the strongest up-regulation (IL-1β, IL-6, and IL-8) at 6 h post-stimulation and ɣ) at 9 h post-stimulation. Foligne et al. [50] propo the probiotic strains that promote the release of biotics were ranked based on the IL-10/IL-12 cyt tenuate inflammatory bowel disease in vivo. In poorly, with an IL-10/IL-12 cytokine ratio aroun strain of L. lactis activated a higher abundance o inflammatory cytokine gene rather than the IL-10 it as an immunostimulatory probiotic. On the o study did not activate immune-related gene exp cerevisiae (yeast) has been successfully used as p have also been known for their potential to activa or bind pathogenic enterobacteria [53]. The imm are cell wall polysaccharides, such as mannan-ol such as zymosan. Other bioactive compounds th products such as XPC [54]. In this study, we used genes. Gene expression analysis performed using RT-qPCR with ACTB and G6PDH reference genes.

Th1/Th2 Cytokines (IL-2, IL-3, and IL-4)
The second cluster included Th1 (IL-2 and IL-3) and Th2 (IL-4) cytokine genes. They were up-regulated with much lower potency compared to pro-inflammatory genes. They were also slower responders as they peaked at 6 h. The Th1/Th2 cytokine gene expression was triggered by LPS and zymosan. Live probiotics (L. lactis and S. cerevisiae) triggered lower mRNA abundance and towards later time-point of stimulation (9 h). 3.3.3. Th1-Regulators (IL-10, IL12p40, and IFN-trigger immune responses both in vitro and in viv GOS dose-dependently enhanced the pro-inflamm PBMC upon LPS challenge. Quite reversely, huma lenged with LPS secreted a lower amount of imm compared to LPS challenge alone [48]. The primary with the low molecular weight molecules, such as t L. lactis triggered the strongest up-regulation o (IL-1β, IL-6, and IL-8) at 6 h post-stimulation and Th ɣ) at 9 h post-stimulation. Foligne et al. [50] proposed the probiotic strains that promote the release of an biotics were ranked based on the IL-10/IL-12 cytok tenuate inflammatory bowel disease in vivo. In th poorly, with an IL-10/IL-12 cytokine ratio around 1 strain of L. lactis activated a higher abundance of m inflammatory cytokine gene rather than the IL-10 an it as an immunostimulatory probiotic. On the oth study did not activate immune-related gene expre cerevisiae (yeast) has been successfully used as prob have also been known for their potential to activate or bind pathogenic enterobacteria [53]. The immun are cell wall polysaccharides, such as mannan-oligo such as zymosan. Other bioactive compounds that products such as XPC [54]. In this study, we used p lation, which was not an efficient mode of PBMC s availability of the TLR ligands.
In comparison to the purified TLR agonists, t live L. lactis bacteria were shifted in time. LPS or C expression up-regulation of the pro-inflammatory c stimulation, whereas, L. lactis activated the respect tion. The beneficial effects of L. lactis and other tributed to the activity of exopolysaccharides (EPS) either loosely bound to the cell wall in a capsular for Among Gram-positive bacteria, there are severa strains of L. lactis subsp. cremoris [57,58]. Many of t cific immunomodulatory effects in the host, such as macrophages [59], induction of macrophage cytotox phocytes [61]. Hereby, we hypothesize that the cyt lactis was mediated indirectly by EPS rather than the

Conclusions
In conclusion, we have determined cytokine g ulated with an array of purified TLR ligands and po inflammatory Th1/Th2 and Th1-regulator gene cluste est stimuli of PBMC were LPS and CpG ODN. Amon cremoris triggered more abundant cytokine gene exp cient in triggering immune-related gene expression. had a pro-inflammatory character and was expressed 1β, IL-6, IL-8, and IL-12p40. In this manner, L. lactis s properties in chicken PBMC and therefore can be con ) Pro-inflammatory IL-12p40 and anti-inflammatory IL-10 were clustered on two opposite sides of the cluster tree. It is because those two cytokines have the opposite function. It is quite well represented by mRNA abundance of IL-10 and IL-12p40 stimulated by CpG ODN, which initially (3 h) triggered the high gene expression of pro-inflammatory IL-12p40 together with anti-inflammatory IL-10, both at a similar level of mRNA abundance. However, at the 6 h time-point, the relative gene expression of IL-10 already surpassed IL-12p40, leading to its complete silencing at the 9 h time-point. The same pattern of gene expression was expressed by IFN-Genes 2021, 12, x FOR PEER REVIEW and S. cerevisiae. Each of those compounds stimulated cytokin chicken PBMC in a different manner. GOS induced the strongest inflammatory cytokine genes, especially IL-1β (~214 fold change and IL-6 (58 fold change) at 3 h post-stimulation. Oligosacchari trigger immune responses both in vitro and in vivo. Vendrig, e GOS dose-dependently enhanced the pro-inflammatory immu PBMC upon LPS challenge. Quite reversely, human PBMC cult lenged with LPS secreted a lower amount of immune mediato compared to LPS challenge alone [48]. The primary stimulant of with the low molecular weight molecules, such as tri-and tetras L. lactis triggered the strongest up-regulation of the pro-infla (IL-1β, IL-6, and IL-8) at 6 h post-stimulation and Th1-regulators ɣ) at 9 h post-stimulation. Foligne et al. [50] proposed a screening the probiotic strains that promote the release of anti-inflammato biotics were ranked based on the IL-10/IL-12 cytokine ratio, ind tenuate inflammatory bowel disease in vivo. In this ranking, L poorly, with an IL-10/IL-12 cytokine ratio around 1 [50]. In the strain of L. lactis activated a higher abundance of mRNA expres inflammatory cytokine gene rather than the IL-10 anti-inflammat it as an immunostimulatory probiotic. On the other hand, S. c study did not activate immune-related gene expression signatu cerevisiae (yeast) has been successfully used as probiotics for po have also been known for their potential to activate the immune or bind pathogenic enterobacteria [53]. The immunomodulatory are cell wall polysaccharides, such as mannan-oligosaccharides such as zymosan. Other bioactive compounds that can stimulate products such as XPC [54]. In this study, we used purified yeast lation, which was not an efficient mode of PBMC stimulation, m availability of the TLR ligands.
In comparison to the purified TLR agonists, the immune live L. lactis bacteria were shifted in time. LPS or CpG ODN trig expression up-regulation of the pro-inflammatory cytokines or T stimulation, whereas, L. lactis activated the respective genes at tion. The beneficial effects of L. lactis and other immunobioti tributed to the activity of exopolysaccharides (EPS) [55]. EPS are either loosely bound to the cell wall in a capsular form or secreted Among Gram-positive bacteria, there are several EPS-produ strains of L. lactis subsp. cremoris [57,58]. Many of them released cific immunomodulatory effects in the host, such as induction of macrophages [59], induction of macrophage cytotoxicity [60], and phocytes [61]. Hereby, we hypothesize that the cytokine gene e lactis was mediated indirectly by EPS rather than the by direct cont

Conclusions
In conclusion, we have determined cytokine gene expressio ulated with an array of purified TLR ligands and potential immu inflammatory Th1/Th2 and Th1-regulator gene clusters. Among th est stimuli of PBMC were LPS and CpG ODN. Among bioactive co cremoris triggered more abundant cytokine gene expression, but S cient in triggering immune-related gene expression. Immune resp had a pro-inflammatory character and was expressed by increased 1β, IL-6, IL-8, and IL-12p40. In this manner, L. lactis subsp. cremori properties in chicken PBMC and therefore can be considered an im  [47] determined that GOS dose-dependently enhanced the pro-inflammatory immune responses of equine PBMC upon LPS challenge. Quite reversely, human PBMC cultured with GOS and challenged with LPS secreted a lower amount of immune mediators (e.g., IL-1α and IL-1β), compared to LPS challenge alone [48]. The primary stimulant of GOS has been associated with the low molecular weight molecules, such as tri-and tetrasaccharide fractions [49]. L. lactis triggered the strongest up-regulation of the pro-inflammatory cytokine genes (IL-1β, IL-6, and IL-8) at 6 h post-stimulation and Th1-regulators (IL-12p40, IL-10, and IFNɣ) at 9 h post-stimulation. Foligne et al. [50] proposed a screening test using PBMC to select the probiotic strains that promote the release of anti-inflammatory cytokines. These probiotics were ranked based on the IL-10/IL-12 cytokine ratio, indicating their ability to attenuate inflammatory bowel disease in vivo. In this ranking, L. lactis performed rather poorly, with an IL-10/IL-12 cytokine ratio around 1 [50]. In the current study, the tested strain of L. lactis activated a higher abundance of mRNA expression of the IL-12p40 proinflammatory cytokine gene rather than the IL-10 anti-inflammatory gene, which classifies it as an immunostimulatory probiotic. On the other hand, S. cerevisiae analyzed in this study did not activate immune-related gene expression signatures in chicken PBMC. S. cerevisiae (yeast) has been successfully used as probiotics for poultry for years [51]. They have also been known for their potential to activate the immune system of the animal [52] or bind pathogenic enterobacteria [53]. The immunomodulatory components of the yeast are cell wall polysaccharides, such as mannan-oligosaccharides and β-glucan, or MAMP, such as zymosan. Other bioactive compounds that can stimulate PBMC are fermentation products such as XPC [54]. In this study, we used purified yeast culture for PBMC stimulation, which was not an efficient mode of PBMC stimulation, most likely due to the low availability of the TLR ligands.
In comparison to the purified TLR agonists, the immune responses elicited by the live L. lactis bacteria were shifted in time. LPS or CpG ODN triggered the strongest gene expression up-regulation of the pro-inflammatory cytokines or Th1-regulators at 3 h poststimulation, whereas, L. lactis activated the respective genes at 6 h and 9 h post-stimulation. The beneficial effects of L. lactis and other immunobiotics have been widely attributed to the activity of exopolysaccharides (EPS) [55]. EPS are polysaccharides that are either loosely bound to the cell wall in a capsular form or secreted to the environment [56]. Among Gram-positive bacteria, there are several EPS-producers, including different strains of L. lactis subsp. cremoris [57,58]. Many of them released EPS that conferred specific immunomodulatory effects in the host, such as induction of IL-1α and IFN-ɣ in spleen macrophages [59], induction of macrophage cytotoxicity [60], and mitogen activity in lymphocytes [61]. Hereby, we hypothesize that the cytokine gene expression activated by L. lactis was mediated indirectly by EPS rather than the by direct contact with bacterial PAMPs.

Conclusions
In conclusion, we have determined cytokine gene expression in chicken PBMC stimulated with an array of purified TLR ligands and potential immunobiotics. We found proinflammatory Th1/Th2 and Th1-regulator gene clusters. Among the TLR ligands, the strongest stimuli of PBMC were LPS and CpG ODN. Among bioactive compounds, L. lactis subsp. cremoris triggered more abundant cytokine gene expression, but S. cerevisiae seemed inefficient in triggering immune-related gene expression. Immune responses of PBMC to L. lactis had a pro-inflammatory character and was expressed by increased mRNA abundance of IL-1β, IL-6, IL-8, and IL-12p40. In this manner, L. lactis subsp. cremoris has immunostimulatory properties in chicken PBMC and therefore can be considered an immunobiotic.
, and (to a lesser extent) IL-10 at the 9 h time-point, but S. cerevisiae seemed inefficient in triggering the expression of those genes.

Discussion
In this paper, we determined the kinetics of the cytokine gene expression in the PBMC cells stimulated with (1) well-known TLR ligands, which often serve as vaccine adjuvants (e.g., Pam3CSK4 or CpG ODN), and (2) potential immunobiotics, relatively uncharacterized bioactive compounds (oligosaccharides, prokaryotic and eukaryotic probiotics) used in poultry nutrition and/or veterinary applications. We focused on the first nine hours poststimulation, which allowed us to pinpoint primary response genes (3 h) and secondary response genes (6 h and 9 h). This way, we characterized the overall cytokine gene expression triggered in PBMC by the respective stimuli.

Hierarchical Clustering
The gene expression data were first clustered using correlation distance. The most pronounced cluster included strongly up-regulated pro-inflammatory cytokines (IL-1β, IL-6, and IL-8). Pro-inflammatory cytokines orchestrate inflammation, which is a complex response of the immune system to the loss of homeostasis due to tissue stress, injury, and infection [23]. The inflammatory response is controlled by transcriptional activation of the gene sequences by three classes of transcriptional factors, activating primary response genes (Class I transcription factors), secondary response genes (Class II transcription factors), and macrophage-specific gene expression (Class III transcription factors) [24]. Typically, Class I transcription factors, including NF-kB and IRF, are activated post-translationally by the receptors of the innate immune system [24]. For example, the acute inflammatory responses are triggered by the microbial components (e.g., LPS) recognized by the respective TLR (e.g., TLR4). Acute inflammatory responses have also been regulated by other molecular mechanisms. Shen, et al. [21] demonstrated that the LPS-activated pro-inflammatory cascade in PBMC isolated from broiler chickens had epigenetic character. In particular, LPS stimulation triggered demethylation of some CpG islands within promoters of IL-6 and TNF-α genes, as well as increased availability of IL-1β gene promoter due to decreased chromatin compactness.
The second large cluster of the genes was classified as Th1/Th2 cytokine genes and was correlated based on a much lower gene expression pattern compared to pro-inflammatory genes discussed above. The second gene cluster included three cytokine genes: IL-2, IL-3, and IL-4. Those cytokine genes participate in the immunological decision-making process [25]. In particular, the Th1 and Th2 cytokines skew naïve T cells into mounting either Th1 or Th2 immune responses. Th1 cells secrete IL-2 and Th2 cells secrete IL-3 and IL-4 cytokines. Polarization of the naïve T cells into any of the Th phenotypes is associated with the adaptive (specific) immune responses attributed to the affinity of the T-cell receptor (TCR) to the antigen. Activation of the naïve T cells requires the presence of the particular antigen-presenting cells, which are called Dendritic Cells (DC). Upon infection, DC recognizes PAMPs with the conserved array of various TLRs present on their surface. DC engulfs the antigens and presents their digested fragment via the MHC class II region to the TCR. Together with the additional signals required for the first activation of the naïve T cells, primed DC can activate T cells into the development of the adaptive immune responses [26]. This way, DC bridge innate and adaptive immune responses.
Th1/Th2 cytokine gene expression can be detected in PBMC sourced from different organisms, including chicken [27], pig [28], and cattle [26]. T cell activation depends to a large extent on the stimuli used, time-point post-stimulation analyzed, and the immune competence of the organism that sourced the blood for PBMC. Kirthika et al. [28] compared cytokine gene expression in PBMC sourced from two distinct swine genotypes stimulated in vitro with phytohemagglutinin (PHA). PHA is a mitogen that stimulates the proliferation of the T cells. PBMC sourced from indigenous, well-adapted pig breed from India responded in much higher Th1 and Th2 gene expression signatures compared to PBMC sourced from a conventional commercial pig breed. Porcine PMBC stimulated with PHA expressed the highest abundance of IL-2 mRNA at 24 h post-stimulation and IL-4 at 2 h post-stimulation [29]. In the current study, a Th1/Th2 cluster expressed low mRNA abundance, but the only mitogen used was LPS, which stimulates the proliferation of B cells but not the T cells. PBMC analyzed in this study were sourced from GP chickens, which belong to a native, dual-purpose breed, known for its resilience and distinct immune responses [30,31].
The remaining genes were clustered as follows: IL-10 was clustered on the side of Th1/Th2 cluster and the remaining cytokines (IL-12p40 and IFN-Genes 2021, 12, x FOR PEER REVIEW and S. cerevisiae. Each of those chicken PBMC in a different ma inflammatory cytokine genes, e and IL-6 (58 fold change) at 3 h trigger immune responses both GOS dose-dependently enhan PBMC upon LPS challenge. Qu lenged with LPS secreted a low compared to LPS challenge alon with the low molecular weight L. lactis triggered the strong (IL-1β, IL-6, and IL-8) at 6 h post ɣ) at 9 h post-stimulation. Folign the probiotic strains that promo biotics were ranked based on th tenuate inflammatory bowel d poorly, with an IL-10/IL-12 cyt strain of L. lactis activated a hig inflammatory cytokine gene rat it as an immunostimulatory pr study did not activate immune cerevisiae (yeast) has been succe have also been known for their or bind pathogenic enterobacte are cell wall polysaccharides, su such as zymosan. Other bioacti products such as XPC [54]. In th lation, which was not an efficie availability of the TLR ligands.
In comparison to the puri live L. lactis bacteria were shifte expression up-regulation of the stimulation, whereas, L. lactis a tion. The beneficial effects of tributed to the activity of exopo either loosely bound to the cell w Among Gram-positive bacteri strains of L. lactis subsp. cremor cific immunomodulatory effects macrophages [59], induction of phocytes [61]. Hereby, we hypo lactis was mediated indirectly by

Conclusions
In conclusion, we have det ulated with an array of purified inflammatory Th1/Th2 and Th1est stimuli of PBMC were LPS an cremoris triggered more abunda cient in triggering immune-relat had a pro-inflammatory characte 1β, IL-6, IL-8, and IL-12p40. In th ) were outliers on the heat map. However, due to the functional relationship, those genes need to be analyzed together. We called the last cluster Th1-regulating cytokine genes because IL-12p40 skews lymphocytes T towards Th1 immune responses, whereas IL-10 negatively regulates Th1 activation [32]. As such, IL-10 also down-regulates INF-Genes 2021, 12, x FOR PEER REVIEW and S. cerevisiae. Each of those compound chicken PBMC in a different manner. GOS inflammatory cytokine genes, especially IL and IL-6 (58 fold change) at 3 h post-stim trigger immune responses both in vitro an GOS dose-dependently enhanced the pr PBMC upon LPS challenge. Quite reversel lenged with LPS secreted a lower amoun compared to LPS challenge alone [48]. The with the low molecular weight molecules, L. lactis triggered the strongest up-reg (IL-1β, IL-6, and IL-8) at 6 h post-stimulatio ɣ) at 9 h post-stimulation. Foligne et al. [50] the probiotic strains that promote the rele biotics were ranked based on the IL-10/ILtenuate inflammatory bowel disease in vi poorly, with an IL-10/IL-12 cytokine ratio strain of L. lactis activated a higher abund inflammatory cytokine gene rather than the it as an immunostimulatory probiotic. On study did not activate immune-related ge cerevisiae (yeast) has been successfully use have also been known for their potential to or bind pathogenic enterobacteria [53]. Th are cell wall polysaccharides, such as man such as zymosan. Other bioactive compou products such as XPC [54]. In this study, w lation, which was not an efficient mode of availability of the TLR ligands.
In comparison to the purified TLR a live L. lactis bacteria were shifted in time. expression up-regulation of the pro-inflam stimulation, whereas, L. lactis activated th tion. The beneficial effects of L. lactis an tributed to the activity of exopolysaccharid either loosely bound to the cell wall in a cap Among Gram-positive bacteria, there ar strains of L. lactis subsp. cremoris [57,58]. M cific immunomodulatory effects in the host macrophages [59], induction of macrophag phocytes [61]. Hereby, we hypothesize tha lactis was mediated indirectly by EPS rather

Conclusions
In conclusion, we have determined cy ulated with an array of purified TLR ligand inflammatory Th1/Th2 and Th1-regulator g est stimuli of PBMC were LPS and CpG OD cytokine gene expression, which is secreted by Th1 cells [33]. If we consider inflammatory processes described earlier, there are both physiological and pathological consequences to inflammatory immune responses. Physiologically, inflammation aims to protect the host organism against the microbial burden and restore homeostasis. On the other hand, the excessive or uncontrolled inflammatory responses promote tissue damage and autoimmune responses, which unbalance the homeostasis [23]. For this reason, the anti-inflammatory IL-10 cytokine gene expression was activated, which counter-balances pro-inflammatory cytokine IL-12p40, and plays a protective role in the PBMC population against excessive inflammation.

Pro-Inflammatory Cytokines (IL-1β, IL-6, and IL-8)
More detailed analysis of the gene expression patterns activated in chicken PBMC in vitro shows that the pro-inflammatory cytokine storm was activated by two major PAMPs: LPS (IL-1β, IL-6, and IL-8) and CpG ODN (IL-1β, IL-6, IL-12p40, and IFN-Genes 2021, 12, x FOR PEER REVIEW and S. cerevisiae. E chicken PBMC in a inflammatory cyto and IL-6 (58 fold c trigger immune re GOS dose-depend PBMC upon LPS c lenged with LPS s compared to LPS c with the low mole L. lactis trigge (IL-1β, IL-6, and ILɣ) at 9 h post-stimu the probiotic strain biotics were ranke tenuate inflammat poorly, with an IL strain of L. lactis ac inflammatory cyto it as an immunost study did not acti cerevisiae (yeast) ha have also been kno or bind pathogenic are cell wall polysa such as zymosan. O products such as X lation, which was availability of the T In comparison live L. lactis bacter expression up-regu stimulation, where tion. The beneficia tributed to the acti ). In both cases, the acute inflammatory responses were most pronounced at 3 h post-stimulation, with a gradual decrease at 6 h and 9 h post-stimulation. Even though both PAMPs stimulated the mRNA abundance of the effector cytokines similarly, the underlying molecular mechanism is quite distinct. LPS is a major constituent of the outer membrane of all Gramnegative bacteria. LPS is also a biologically active endotoxin, which stimulates immune responses in a dose-dependent manner. It contains two components: lipid A, associated with the toxic effect, and a polysaccharide, eliciting inflammatory immune responses [34]. LPS binds TLR4 and activates the NF-kB transcription factor, which stimulates downstream production of pro-inflammatory cytokines and B cell proliferation [35]. LPS is one of the most potent immunomodulators. In small doses, it triggers acute inflammatory responses in vitro and in vivo, but in large doses, it is lethal [36]. As LPS is the model stimuli, it's application in research can be considered as a reference point for less characterized bioactive compounds. For example, LPS induced a fold change of 637 in IL-1β mRNA abundance at 3 h post-stimulation. Zymosan was comparable with GOS (>200 fold change at 3 h), but L. lactis peaked at 6 h with~140 fold change and S. cerevisiae induced much lower up-regulation of IL-1β mRNA (~15 at 3 h and 6 h).

Th1/Th2 Cytokines (IL-2, IL-3, and IL-4)
Th1/Th2 polarization cytokines have been activated on a much lower level compared to pro-inflammatory cytokines. The peaks of IL-2, IL-3, and IL-4 mRNA abundance were at 6 h post-stimulation and the two stimuli that activated those cytokine genes were LPS and zymosan. IL-2 is a cytokine that is produced during the primary immune response by naïve Th cells. Upon differentiation of Th cells into Th1 or Th2 phenotypes, they reduce IL-2 secretion and take up production of the respective Th1-type (IFN-and S. cerevisiae. Each of th chicken PBMC in a different inflammatory cytokine gene and IL-6 (58 fold change) at trigger immune responses b GOS dose-dependently enh PBMC upon LPS challenge. lenged with LPS secreted a compared to LPS challenge a with the low molecular weig L. lactis triggered the str (IL-1β, IL-6, and IL-8) at 6 h p ɣ) at 9 h post-stimulation. Fo the probiotic strains that pro biotics were ranked based o tenuate inflammatory bowe poorly, with an IL-10/IL-12 strain of L. lactis activated a inflammatory cytokine gene it as an immunostimulatory study did not activate imm cerevisiae (yeast) has been su have also been known for th or bind pathogenic enteroba are cell wall polysaccharides such as zymosan. Other bioa products such as XPC [54]. I lation, which was not an eff availability of the TLR ligan In comparison to the p live L. lactis bacteria were sh expression up-regulation of stimulation, whereas, L. lact tion. The beneficial effects tributed to the activity of exo either loosely bound to the c Among Gram-positive bact strains of L. lactis subsp. crem cific immunomodulatory eff macrophages [59], induction phocytes [61]. Hereby, we h lactis was mediated indirectly

Conclusions
In conclusion, we have ulated with an array of purif inflammatory Th1/Th2 and T est stimuli of PBMC were LP cremoris triggered more abun cient in triggering immune-r had a pro-inflammatory char 1β, IL-6, IL-8, and IL-12p40. In properties in chicken PBMC ) or Th2-type (IL-4) cytokines [37]. In contrast, IL-3 is a T cell-derived multilineage hematopoietic growth factor, that is required for survival, proliferation, and differentiation of the primitive hematopoietic progenitor cells [38]. Zymosan is a crude component of the yeast's cell wall, containing glucans (55%), mannans (19%), and chitins [39]. It elicits unique immunomodulatory effects in vitro, associated with the glucan fraction. and S. cerevisiae. Each of those compounds stimul chicken PBMC in a different manner. GOS induced inflammatory cytokine genes, especially IL-1β (~21 and IL-6 (58 fold change) at 3 h post-stimulation. O trigger immune responses both in vitro and in viv GOS dose-dependently enhanced the pro-inflamm PBMC upon LPS challenge. Quite reversely, huma lenged with LPS secreted a lower amount of imm compared to LPS challenge alone [48]. The primary with the low molecular weight molecules, such as t L. lactis triggered the strongest up-regulation o (IL-1β, IL-6, and IL-8) at 6 h post-stimulation and Th ɣ) at 9 h post-stimulation. Foligne et al. [50] proposed the probiotic strains that promote the release of an biotics were ranked based on the IL-10/IL-12 cytok tenuate inflammatory bowel disease in vivo. In th poorly, with an IL-10/IL-12 cytokine ratio around 1 strain of L. lactis activated a higher abundance of m inflammatory cytokine gene rather than the IL-10 an it as an immunostimulatory probiotic. On the oth study did not activate immune-related gene expre cerevisiae (yeast) has been successfully used as prob have also been known for their potential to activate or bind pathogenic enterobacteria [53]. The immun are cell wall polysaccharides, such as mannan-oligo such as zymosan. Other bioactive compounds that products such as XPC [54]. In this study, we used p lation, which was not an efficient mode of PBMC s availability of the TLR ligands.
In comparison to the purified TLR agonists, t live L. lactis bacteria were shifted in time. LPS or C expression up-regulation of the pro-inflammatory c stimulation, whereas, L. lactis activated the respect tion. The beneficial effects of L. lactis and other tributed to the activity of exopolysaccharides (EPS) either loosely bound to the cell wall in a capsular for Among Gram-positive bacteria, there are severa strains of L. lactis subsp. cremoris [57,58]. Many of t cific immunomodulatory effects in the host, such as macrophages [59], induction of macrophage cytotox phocytes [61]. Hereby, we hypothesize that the cyt lactis was mediated indirectly by EPS rather than the

Conclusions
In conclusion, we have determined cytokine g ulated with an array of purified TLR ligands and po inflammatory Th1/Th2 and Th1-regulator gene cluste est stimuli of PBMC were LPS and CpG ODN. Amon cremoris triggered more abundant cytokine gene exp cient in triggering immune-related gene expression. had a pro-inflammatory character and was expressed 1β, IL-6, IL-8, and IL-12p40. In this manner, L. lactis s properties in chicken PBMC and therefore can be con ) CpG ODN was the stimuli that activated the most potent response of the pro-inflammatory (Th1-type) and the anti-inflammatory (Th2-type) cytokines ( Figure 2). CpG ODN refers to bacterial oligonucleotides rich in unmethylated CpG dinucleotides. In mammals, CpG ODN is an agonist of TLR9, which in avian species is represented by a functional orthologue, TLR21 [40]. Unlike other TLRs, which are expressed on the cell surface, TLR21 is an endosomal receptor. It recognizes a broad range of CpG ODN motifs from internalized bacteria and triggers NF-kBmediated expression of pro-inflammatory (Th1-type) cytokines, including IL-1β, IL-6, IL-12p40, and IFN- compared to LPS challenge alone [48]. The primary stimulant of GOS has been associated with the low molecular weight molecules, such as tri-and tetrasaccharide fractions [49]. L. lactis triggered the strongest up-regulation of the pro-inflammatory cytokine genes (IL-1β, IL-6, and IL-8) at 6 h post-stimulation and Th1-regulators (IL-12p40, IL-10, and IFN ɣ) at 9 h post-stimulation. Foligne et al. [50] proposed a screening test using PBMC to selec the probiotic strains that promote the release of anti-inflammatory cytokines. These pro biotics were ranked based on the IL-10/IL-12 cytokine ratio, indicating their ability to at tenuate inflammatory bowel disease in vivo. In this ranking, L. lactis performed rather poorly, with an IL-10/IL-12 cytokine ratio around 1 [50]. In the current study, the tested strain of L. lactis activated a higher abundance of mRNA expression of the IL-12p40 pro inflammatory cytokine gene rather than the IL-10 anti-inflammatory gene, which classifies it as an immunostimulatory probiotic. On the other hand, S. cerevisiae analyzed in this study did not activate immune-related gene expression signatures in chicken PBMC. S cerevisiae (yeast) has been successfully used as probiotics for poultry for years [51]. They have also been known for their potential to activate the immune system of the animal [52 or bind pathogenic enterobacteria [53]. The immunomodulatory components of the yeas are cell wall polysaccharides, such as mannan-oligosaccharides and β-glucan, or MAMP such as zymosan. Other bioactive compounds that can stimulate PBMC are fermentation products such as XPC [54]. In this study, we used purified yeast culture for PBMC stimu lation, which was not an efficient mode of PBMC stimulation, most likely due to the low availability of the TLR ligands.
In comparison to the purified TLR agonists, the immune responses elicited by the live L. lactis bacteria were shifted in time. LPS or CpG ODN triggered the strongest gene expression up-regulation of the pro-inflammatory cytokines or Th1-regulators at 3 h post stimulation, whereas, L. lactis activated the respective genes at 6 h and 9 h post-stimula tion. The beneficial effects of L. lactis and other immunobiotics have been widely at tributed to the activity of exopolysaccharides (EPS) [55]. EPS are polysaccharides that are either loosely bound to the cell wall in a capsular form or secreted to the environment [56] Among Gram-positive bacteria, there are several EPS-producers, including differen strains of L. lactis subsp. cremoris [57,58]. Many of them released EPS that conferred spe cific immunomodulatory effects in the host, such as induction of IL-1α and IFN-ɣ in spleen macrophages [59], induction of macrophage cytotoxicity [60], and mitogen activity in lym phocytes [61]. Hereby, we hypothesize that the cytokine gene expression activated by L lactis was mediated indirectly by EPS rather than the by direct contact with bacterial PAMPs

Conclusions
In conclusion, we have determined cytokine gene expression in chicken PBMC stim ulated with an array of purified TLR ligands and potential immunobiotics. We found pro inflammatory Th1/Th2 and Th1-regulator gene clusters. Among the TLR ligands, the strong est stimuli of PBMC were LPS and CpG ODN. Among bioactive compounds, L. lactis subsp cremoris triggered more abundant cytokine gene expression, but S. cerevisiae seemed ineffi as well as anti-inflammatory (Th2-type) cytokine IL-10. The broad spectrum of immune activation by different sequences of CpG ODN made them promising candidates for versatile vaccine adjuvants, used to boost the effectiveness of protein-based vaccines against allergies, infectious diseases, and cancer [41]. In poultry, immunostimulatory CpG ODN was applied orally to day-old chickens and followed by Salmonella enteritidis challenge, reduced pathogen invasion, and chicken mortality [42].
In this study, CpG ODN induced potent activation of Th1-type stimulating cytokine genes, IL-12p40 and IFN-  [47] d GOS dose-dependently enhanced the pro-inflammatory immune respo PBMC upon LPS challenge. Quite reversely, human PBMC cultured with lenged with LPS secreted a lower amount of immune mediators (e.g., I compared to LPS challenge alone [48]. The primary stimulant of GOS has with the low molecular weight molecules, such as tri-and tetrasaccharide L. lactis triggered the strongest up-regulation of the pro-inflammatory (IL-1β, IL-6, and IL-8) at 6 h post-stimulation and Th1-regulators (IL-12p40 ɣ) at 9 h post-stimulation. Foligne et al. [50] proposed a screening test using the probiotic strains that promote the release of anti-inflammatory cytok biotics were ranked based on the IL-10/IL-12 cytokine ratio, indicating th tenuate inflammatory bowel disease in vivo. In this ranking, L. lactis pe poorly, with an IL-10/IL-12 cytokine ratio around 1 [50]. In the current s strain of L. lactis activated a higher abundance of mRNA expression of th inflammatory cytokine gene rather than the IL-10 anti-inflammatory gene, it as an immunostimulatory probiotic. On the other hand, S. cerevisiae a study did not activate immune-related gene expression signatures in ch cerevisiae (yeast) has been successfully used as probiotics for poultry for have also been known for their potential to activate the immune system o or bind pathogenic enterobacteria [53]. The immunomodulatory compon are cell wall polysaccharides, such as mannan-oligosaccharides and β-glu such as zymosan. Other bioactive compounds that can stimulate PBMC a products such as XPC [54]. In this study, we used purified yeast culture fo lation, which was not an efficient mode of PBMC stimulation, most likely availability of the TLR ligands.
In comparison to the purified TLR agonists, the immune responses live L. lactis bacteria were shifted in time. LPS or CpG ODN triggered the expression up-regulation of the pro-inflammatory cytokines or Th1-regula stimulation, whereas, L. lactis activated the respective genes at 6 h and 9 tion. The beneficial effects of L. lactis and other immunobiotics have b tributed to the activity of exopolysaccharides (EPS) [55]. EPS are polysacc either loosely bound to the cell wall in a capsular form or secreted to the en Among Gram-positive bacteria, there are several EPS-producers, incl strains of L. lactis subsp. cremoris [57,58]. Many of them released EPS tha cific immunomodulatory effects in the host, such as induction of IL-1α and macrophages [59], induction of macrophage cytotoxicity [60], and mitogen phocytes [61]. Hereby, we hypothesize that the cytokine gene expression lactis was mediated indirectly by EPS rather than the by direct contact with b at 3 h post-stimulation, followed by their rapid down-regulation at 6 h and 9 h. Reversely, mRNA abundance of IL-10 cytokine gene peaked at 6 h poststimulation and remained high at 9 h post-stimulation. The IL-12p40 and IL-10 cytokines indicate the subtle balance between the stimulatory (pro-inflammatory) and regulatory (anti-inflammatory) effect of the given immunobiotic on the antigen-presenting cells (APC), such as macrophages, DC, and B cells [32]. Upon antigen recognition, engulfing, and presentation via MHC class II, the respective APC secretes cytokines that direct Th development toward Th1-type or Th2-type immune responses. CpG ODN induces B cell activation and prime macrophages to secrete IL-12 cytokine. Indirectly, IL-12 cytokine secreted by activated macrophages acts on natural killer (NK) cells to secrete IFN-Genes 2021, 12, x FOR PEER REVIEW and S. cerevisi chicken PBMC inflammatory and IL-6 (58 fo trigger immun GOS dose-dep PBMC upon L lenged with L compared to L with the low m L. lactis tr (IL-1β, IL-6, an ɣ) at 9 h post-s the probiotic s biotics were ra tenuate inflam poorly, with a strain of L. lac inflammatory it as an immu study did not cerevisiae (yeas have also been or bind pathog are cell wall p such as zymos products such lation, which w availability of In compa live L. lactis ba expression up stimulation, w tion. The ben [43]. The pleiotropic IL-10 cytokine produced by primed macrophages and DC cells (but not B cells) inhibits stimulation of IFN-Genes 2021, 12, x FOR PEER REVIEW and S. cerevisiae. Each of those compounds stimulated cytok chicken PBMC in a different manner. GOS induced the stronge inflammatory cytokine genes, especially IL-1β (~214 fold chan and IL-6 (58 fold change) at 3 h post-stimulation. Oligosaccha trigger immune responses both in vitro and in vivo. Vendrig, GOS dose-dependently enhanced the pro-inflammatory imm PBMC upon LPS challenge. Quite reversely, human PBMC cu lenged with LPS secreted a lower amount of immune media compared to LPS challenge alone [48]. The primary stimulant o with the low molecular weight molecules, such as tri-and tetra L. lactis triggered the strongest up-regulation of the pro-in (IL-1β, IL-6, and IL-8) at 6 h post-stimulation and Th1-regulator ɣ) at 9 h post-stimulation. Foligne et al. [50] proposed a screenin the probiotic strains that promote the release of anti-inflamma biotics were ranked based on the IL-10/IL-12 cytokine ratio, in tenuate inflammatory bowel disease in vivo. In this ranking, poorly, with an IL-10/IL-12 cytokine ratio around 1 [50]. In th strain of L. lactis activated a higher abundance of mRNA expr inflammatory cytokine gene rather than the IL-10 anti-inflamm it as an immunostimulatory probiotic. On the other hand, S. study did not activate immune-related gene expression signa cerevisiae (yeast) has been successfully used as probiotics for p have also been known for their potential to activate the immun or bind pathogenic enterobacteria [53]. The immunomodulato are cell wall polysaccharides, such as mannan-oligosaccharide such as zymosan. Other bioactive compounds that can stimula products such as XPC [54]. In this study, we used purified yea lation, which was not an efficient mode of PBMC stimulation, availability of the TLR ligands.
In comparison to the purified TLR agonists, the immun live L. lactis bacteria were shifted in time. LPS or CpG ODN tr expression up-regulation of the pro-inflammatory cytokines or production by Th1 lymphocytes [44]. As such, IL-10 serves as a feedback regulator and protects from excessive pro-inflammatory responses mediated by the primed APC.
In contrast, zymosan showed distinct stimulatory properties towards cytokines in the group of Th-1 regulators. Stimulation with zymosan increased IL-10 and IFN-Genes 2021, 12, x FOR PEER REVIEW and S. cerevisiae. chicken PBMC in inflammatory cyt and IL-6 (58 fold trigger immune r GOS dose-depen PBMC upon LPS lenged with LPS compared to LPS with the low mol L. lactis trigg (IL-1β, IL-6, and IL ɣ) at 9 h post-stim the probiotic stra biotics were rank tenuate inflamma poorly, with an I strain of L. lactis a inflammatory cyt it as an immunos study did not act cerevisiae (yeast) h have also been kn or bind pathogen are cell wall poly such as zymosan. products such as lation, which was mRNA abundance, but only at the 6 h time-point, while IL-12p40 mRNA abundance remained unchanged. A different pattern of gene expression regulation triggered by zymosan can be explained by its molecular recognition. It is known that zymosan activates macrophages and DC via TLR2 and Dectin-1 [45]. In an elegant study on human DC, Dillon et al. [46] demonstrated that zymosan promoted immune tolerance by inducing DC to secrete regulatory IL-10 and suppressing pro-inflammatory IL-12. As such, zymosan is now considered a suppressive molecule that increases immune tolerance via maintaining high levels of IL-10 cytokine during infection. Due to this property, zymosan is a promising target for autoimmune, allergy, and transplantation therapies.

Cytokine Gene Stimulated by Immunobiotics
Based on presented cytokine gene expression patterns in chicken PBMC, we characterized the immunomodulatory role of GOS prebiotic and two live probiotics, i.e., L. lactis and S. cerevisiae. Each of those compounds stimulated cytokine gene expression in the chicken PBMC in a different manner. GOS induced the strongest up-regulation of the proinflammatory cytokine genes, especially IL-1β (~214 fold change), IL-8 (~62 fold change), and IL-6 (58 fold change) at 3 h post-stimulation. Oligosaccharides have been known to trigger immune responses both in vitro and in vivo. Vendrig, et al. [47] determined that GOS dose-dependently enhanced the pro-inflammatory immune responses of equine PBMC upon LPS challenge. Quite reversely, human PBMC cultured with GOS and challenged with LPS secreted a lower amount of immune mediators (e.g., IL-1α and IL-1β), compared to LPS challenge alone [48]. The primary stimulant of GOS has been associated with the low molecular weight molecules, such as tri-and tetrasaccharide fractions [49].
L. lactis triggered the strongest up-regulation of the pro-inflammatory cytokine genes (IL-1β, IL-6, and IL-8) at 6 h post-stimulation and Th1-regulators (IL-12p40, IL-10, and IFN- and S. cerevisiae. Each of those compounds stimulated cytokine gene expression in the chicken PBMC in a different manner. GOS induced the strongest up-regulation of the proinflammatory cytokine genes, especially IL-1β (~214 fold change), IL-8 (~62 fold change), and IL-6 (58 fold change) at 3 h post-stimulation. Oligosaccharides have been known to trigger immune responses both in vitro and in vivo. Vendrig, et al. [47] determined that GOS dose-dependently enhanced the pro-inflammatory immune responses of equine PBMC upon LPS challenge. Quite reversely, human PBMC cultured with GOS and challenged with LPS secreted a lower amount of immune mediators (e.g., IL-1α and IL-1β), compared to LPS challenge alone [48]. The primary stimulant of GOS has been associated with the low molecular weight molecules, such as tri-and tetrasaccharide fractions [49]. L. lactis triggered the strongest up-regulation of the pro-inflammatory cytokine genes (IL-1β, IL-6, and IL-8) at 6 h post-stimulation and Th1-regulators (IL-12p40, IL-10, and IFNɣ) at 9 h post-stimulation. Foligne et al. [50] proposed a screening test using PBMC to select the probiotic strains that promote the release of anti-inflammatory cytokines. These probiotics were ranked based on the IL-10/IL-12 cytokine ratio, indicating their ability to attenuate inflammatory bowel disease in vivo. In this ranking, L. lactis performed rather poorly, with an IL-10/IL-12 cytokine ratio around 1 [50]. In the current study, the tested strain of L. lactis activated a higher abundance of mRNA expression of the IL-12p40 proinflammatory cytokine gene rather than the IL-10 anti-inflammatory gene, which classifies it as an immunostimulatory probiotic. On the other hand, S. cerevisiae analyzed in this study did not activate immune-related gene expression signatures in chicken PBMC. S. cerevisiae (yeast) has been successfully used as probiotics for poultry for years [51]. They have also been known for their potential to activate the immune system of the animal [52] or bind pathogenic enterobacteria [53]. The immunomodulatory components of the yeast are cell wall polysaccharides, such as mannan-oligosaccharides and β-glucan, or MAMP, such as zymosan. Other bioactive compounds that can stimulate PBMC are fermentation products such as XPC [54]. In this study, we used purified yeast culture for PBMC stimulation, which was not an efficient mode of PBMC stimulation, most likely due to the low availability of the TLR ligands.
In comparison to the purified TLR agonists, the immune responses elicited by the live L. lactis bacteria were shifted in time. LPS or CpG ODN triggered the strongest gene expression up-regulation of the pro-inflammatory cytokines or Th1-regulators at 3 h poststimulation, whereas, L. lactis activated the respective genes at 6 h and 9 h post-stimulation. The beneficial effects of L. lactis and other immunobiotics have been widely attributed to the activity of exopolysaccharides (EPS) [55]. EPS are polysaccharides that are either loosely bound to the cell wall in a capsular form or secreted to the environment [56]. Among Gram-positive bacteria, there are several EPS-producers, including different strains of L. lactis subsp. cremoris [57,58]. Many of them released EPS that conferred specific immunomodulatory effects in the host, such as induction of IL-1α and IFN-ɣ in spleen macrophages [59], induction of macrophage cytotoxicity [60], and mitogen activity in lymphocytes [61]. Hereby, we hypothesize that the cytokine gene expression activated by L. lactis was mediated indirectly by EPS rather than the by direct contact with bacterial PAMPs.

Conclusions
In conclusion, we have determined cytokine gene expression in chicken PBMC stimulated with an array of purified TLR ligands and potential immunobiotics. We found proinflammatory Th1/Th2 and Th1-regulator gene clusters. Among the TLR ligands, the strongest stimuli of PBMC were LPS and CpG ODN. Among bioactive compounds, L. lactis subsp. cremoris triggered more abundant cytokine gene expression, but S. cerevisiae seemed inefficient in triggering immune-related gene expression. Immune responses of PBMC to L. lactis had a pro-inflammatory character and was expressed by increased mRNA abundance of IL-1β, IL-6, IL-8, and IL-12p40. In this manner, L. lactis subsp. cremoris has immunostimulatory properties in chicken PBMC and therefore can be considered an immunobiotic.
) at 9 h post-stimulation. Foligne et al. [50] proposed a screening test using PBMC to select the probiotic strains that promote the release of anti-inflammatory cytokines. These probiotics were ranked based on the IL-10/IL-12 cytokine ratio, indicating their ability to attenuate inflammatory bowel disease in vivo. In this ranking, L. lactis performed rather poorly, with an IL-10/IL-12 cytokine ratio around 1 [50]. In the current study, the tested strain of L. lactis activated a higher abundance of mRNA expression of the IL-12p40 proinflammatory cytokine gene rather than the IL-10 anti-inflammatory gene, which classifies it as an immunostimulatory probiotic. On the other hand, S. cerevisiae analyzed in this study did not activate immune-related gene expression signatures in chicken PBMC. S. cerevisiae (yeast) has been successfully used as probiotics for poultry for years [51]. They have also been known for their potential to activate the immune system of the animal [52] or bind pathogenic enterobacteria [53]. The immunomodulatory components of the yeast are cell wall polysaccharides, such as mannan-oligosaccharides and β-glucan, or MAMP, such as zymosan. Other bioactive compounds that can stimulate PBMC are fermentation products such as XPC [54]. In this study, we used purified yeast culture for PBMC stimulation, which was not an efficient mode of PBMC stimulation, most likely due to the low availability of the TLR ligands.
In comparison to the purified TLR agonists, the immune responses elicited by the live L. lactis bacteria were shifted in time. LPS or CpG ODN triggered the strongest gene expression up-regulation of the pro-inflammatory cytokines or Th1-regulators at 3 h poststimulation, whereas, L. lactis activated the respective genes at 6 h and 9 h post-stimulation. The beneficial effects of L. lactis and other immunobiotics have been widely attributed to the activity of exopolysaccharides (EPS) [55]. EPS are polysaccharides that are either loosely bound to the cell wall in a capsular form or secreted to the environment [56]. Among Gram-positive bacteria, there are several EPS-producers, including different strains of L. lactis subsp. cremoris [57,58]. Many of them released EPS that conferred specific immunomodulatory effects in the host, such as induction of IL-1α and IFN-Genes 2021, 12, x FOR PEER REVIEW and S. cerevisiae. Ea chicken PBMC in a d inflammatory cytok and IL-6 (58 fold ch trigger immune res GOS dose-depende PBMC upon LPS ch lenged with LPS se compared to LPS ch with the low molecu L. lactis triggere (IL-1β, IL-6, and IL-8 ɣ) at 9 h post-stimul the probiotic strains biotics were ranked tenuate inflammato poorly, with an IL-1 strain of L. lactis act inflammatory cytok it as an immunosti study did not activ cerevisiae (yeast) has have also been know or bind pathogenic are cell wall polysa such as zymosan. O products such as XP lation, which was n availability of the T In comparison in spleen macrophages [59], induction of macrophage cytotoxicity [60], and mitogen activity in lymphocytes [61]. Hereby, we hypothesize that the cytokine gene expression activated by L. lactis was mediated indirectly by EPS rather than the by direct contact with bacterial PAMPs.

Conclusions
In conclusion, we have determined cytokine gene expression in chicken PBMC stimulated with an array of purified TLR ligands and potential immunobiotics. We found pro-inflammatory Th1/Th2 and Th1-regulator gene clusters. Among the TLR ligands, the strongest stimuli of PBMC were LPS and CpG ODN. Among bioactive compounds, L. lactis subsp. cremoris triggered more abundant cytokine gene expression, but S. cerevisiae seemed inefficient in triggering immune-related gene expression. Immune responses of PBMC to L. lactis had a pro-inflammatory character and was expressed by increased mRNA abundance of IL-1β, IL-6, IL-8, and IL-12p40. In this manner, L. lactis subsp. cremoris has immunostimulatory properties in chicken PBMC and therefore can be considered an immunobiotic.