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Effects of dietary conjugated linoleic acid on the intestinal mucosal immunity of broiler chickens

Abstract The effects of dietary conjugated linoleic acid (CLA) on the intestinal mucosal immunity of broiler chickens were investigated. A total of 96 1-day-old Arbor Acres male broiler chickens were assigned to two dietary treatments (0 and 1% CLA) with eight replicates/each of six chickens. The intestinal mucosal immune status of broiler chickens was evaluated at 21-day old. The jejunal mucosa SIgA level increased by 43.58% (p < .01) while its mRNA expression increased by 22.95% (p < .01). The broilers receiving the CLA diet exhibited higher gene expression of jejunal peroxisome proliferator-activated receptor γ (PPARγ) (p < .01) while the mRNA expression level of transforming growth factor β4 (TGFβ4) was not different between two dietary treatments group (p > .05). After dietary CLA supplementation, the percent of Peyer's node CD8+ T lymphocytes increased (p < .01) and lymphocytes proliferation of Peyer's node increased (p < .05). In conclusion, the CLA diet enhanced jejunal SIgA expression, and increased lymphocyte transformation rate and the percent of CD8+ T lymphocyte in Peyer's node of broiler chickens.


Introduction
Conjugated linoleic acid (CLA) refers to a class of spatial and geometric isomers of linoleic acid containing conjugated double bond, belonging to the polyunsaturated fatty acids family members. CLA has many important properties that regulate physiological and metabolic responses, for example, immune modulation, anti-cancer effects, prevention of cardiovascular disease and diabetes and weight control. Studies on the effects of CLA on immune function have thus far been conducted in the normal physiological status or inflammatory challenge status (O'Shea et al. 2004;Lai et al. 2005a;Renner et al. 2012).
In poultry science, enrichment of chicken meat and egg with CLA by the addition of CLA in the diet has been successfully demonstrated by several researchers (Kawahara et al. 2009;Herzallah 2013;Shinn et al. 2015Shinn et al. , 2016. CLA has positively influences carcass traits, meat quality, antioxidant capacity, and fatty acid (FA) composition of poultry (Jiang et al. 2014;Fesler & Peterson 2013). In addition, little attention has been drawn to the beneficial immunomodulatory properties of CLA in broiler chickens (Takahashi et al. 2003;Zhang et al. 2005;He et al. 2007; Moraes et al. 2016).
Intestinal mucosal immune system is the largest and the most complicated immune system of animal organism. P erez-Cano et al. (2009) reported that dietary CLA increased intestinal secretory IgA (SIgA) level of young rats. Bontempo et al. (2004) found dietary CLA improved intestinal SIgA level and cellular immune function in late pregnancy and lactation period of sows and growth and fattening period of offsprings. When piglets were infected with B. hyodysenteriae, dietary CLA effectively enhanced the expression of colonal proliferator-activated receptor c (PPARc) and alleviated the intestinal mucosal inflammatory injury . These researches hinted a close relationship between dietary CLA and intestinal mucosal immune response. So it was possible that CLA affects the intestinal mucosal immunity of broiler chickens. Although it was accepted that conjugated linoleic acid (CLA) has immunomodulatory properties in broiler chickens, no studies were available on the roles of CLA in intestinal mucosal immunity of poultry. The purpose of this study was to determine the effects of dietary CLA on the intestinal mucosal immunity of broiler chickens fed with 1.0% CLA diet.

Experimental design
The animal management protocol for this research was approved by Animal Care and Use Committee of the Henan University of Animal Husbandry and Economy. A total of 96, one-day-old Arbor Acres male broiler chickens were purchased from Lvye Poultry Huabandry Co., Ltd. and were randomly distributed into two dietary treatments, fed either on the control or 1% CLA-supplemented diet. Each treatment group had eight replicates of six chickens each.
A maize soybean meal diet was used, and energy was adjusted using soybean oil. In CLA diet, 1% soybean oil was replaced by 1% CLA oil to keep both the CLA-supplemented diet and the control diet isoenergetic. The 1% adding amount of CLA was used in the experiment as described in the previous studies (Zhang et al. 2005;Jiang et al. 2014). The compositions of the control diet are presented in Table 1. The experimental diets were formulated to meet poultry requirements according to the National Research Council (1994) requirements. CLA was provided by the Qingdao Aohai Biology Technology Co., Ltd (Qingdao, China). and contained 80.80% conjugated dienes with 39.24% 9c, 11t-CLA and 38.93% 10t, 12c-CLA representing 96.74% of the contained isomers.
Feeding experiment was conducted in the scholastic experiment pasture. The broilers were housed in wire cages and maintained on a 16L:8D light programme. Temperature in the poultry house was set at 31-33 C for the first 5 d and was reduced by 2-3 C each week until 22 C. The broilers were vaccinated according to the routine immunisation programme for broilers, and birds had free access to feed and water throughout the experiment. The experiment lasted for 3 weeks.

Growth performance
The body weights and feed consumption of broiler chickens were registered at the end of the third week. Average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR) were calculated for this interval.

Sample collection and jejunal mucosa SIgA
On day 21, six birds (one per replication) were randomly chosen from each treatment and killed by cervical dislocation, manual exsanguination. The body cavity was opened and jejunum was removed. The jejunum was flushed with ice-cold KCl (1.5%, w/v). About a 2 cm portion of the jejunum was snap-frozen in liquid nitrogen for RNA isolation. The remaining jejunum was opened length-wide on an ice-cooled surface. The mucosa was gently scraped off using a blade and snap-frozen in liquid nitrogen for determining SIgA. The jejunum and mucosa were stored at À80 C until analysis.
After thawing, the mucosa was homogenised (10%, w/v) at 15,000 rpm for 2 min at 4 C in 0.88% sodium chloride water solution. Homogenate was centrifuged at 6000g for 5 min at 4 C. The supernatant was collected, sub-packed in 0.5 ml tubes and stored at À80 C until further analysis. The level of of jejunal mucosa SIgA was determined with a double antibody sandwich ELISA method according to the manufacturer's instructions (chicken IgA ELISA quantitation kit from Shanghai SHireike Biotechnology Co., Ltd.).

RNA isolation and real-time polymerase chain reaction analysis
Total RNA of jejunum samples was extracted using TRIZOL reagent according to the manufacturer's instruction (15596026, Introvgen Company, CA, USA). The quantity and quality were assessed by OD 260:280 . About 5 ll purified RNA was used as template for cDNA synthesis in the presence of 1.25 ll M-MLV RT reverse transcriptase (200 U) (28025-013, Introvgen Company, CA, USA), 1 ll of Oligo dT8 (30 mM), 3 ll of RNAse-free water, 1.25 ll of 10 mmol dNTP Mix, 1 ll of RNasin ribonuclease inhibitor (50 U/ll) (Vigorous Biotechnology Co., Ltd. Beijing, China). After incubation for 60 min at 37 C, the reverse ranscriptase was inactivated at 70 C for 15 min.
Lymphocytes separation, lymphocyte transformation rate and lymphocyte subsets in Peyer's node Six birds (one per replication) were randomly chosen from each treatment on day 21 and killed by cervical dislocation, manual exsanguination. Separation of Peyer's node was conducted according to the method of Vaughn et al. (2006). Briefly, the intestinal segment between the proximal of Mccoll's diverticulum and the distal of the ileocecal junction was excised, rinsed thoroughly with distilled water, ligated with tweezers near the junction of jejunum and ileum, perfused with 10-20 ml low concentration eosin solution, after holding for 1 min, and with 10-20 ml crystal violet solution. Peyer's node was removed, then stored in cold RPMI-1640 solution (Nanjing Jiancheng Bioengineering institute, Nanjing, China). After cutted and crushed, the fragments were ground with a glass syringe core on a stainless steel cell screen. The collected lymphocytes layered over equivalent lymphocyte Ficoll-Paque density gradient centrifugation medium (density ¼ 1.077; HaoYang Biological Manufacture Co. Ltd., Tianjin, China) and centrifuged at 2500g for 20 min. The white cell layer at the interface was collected and treated with haemolysis buffer to remove erythrocytes. Lymphocyte were counted and re-suspended in RPMI-1640 medium. The cell concentration was adjusted to 1 Â 10 6 /ml. A 3-[4,5-dimethylthiazol]-2,5-diphenyltetrazolium bromide (MTT, Sigma Chemical Co., St. Louis, MO) assay was used to determine lymphocyte transformation rate. Suspensions of lymphocytes (1 Â 10 6 /ml) were incubated in 96-well culture plate at 80 ll per well; each sample was seeded in 6 wells. Exactly 20 ll of RPMI-1640 nutrient solution containing concanavalin A (ConA; 30 lg/ml; Sigma Chemical Co., St. Louis, MO) was then added into three wells. As the control, the same volume of RPMI-1640 nutrient solution containing no ConA was added into other three wells. Cell cultures were incubated at 37 C in a humidified atmosphere at 5% CO 2 for 72 h. At 4 h before the end of incubation, a 10-ll MTT solution (5 mg/ml) was added to each well and the plates were incubated for another 4 h. Subsequently, 100 ll of 10% sodium dodecyl sulphate dissolved in 0.04 mol/l HCl solution was added into each well to lyse the cells and solubilise the MTT crystals. Thirty minutes later, the optical density was measured at a wavelength of 570 nm using a Microplate Reader (Flash spectrum Biotechnology Co., Ltd. Shanghai, China).
For detection of T lymphocyte subpopulations, the lymphocytes were incubated with an optimal concentration of fluorescence-labeled anti-CD monoclonal antibody for 30 min at 4 C in the dark, and later with a fluorescein isothiocyanate-conjugated goat antimouse IgG. Cells were washed and re-suspended in HBSS buffer, and analysed with a Beckman-Counter flow cytometer. A minimum of 10 4 viable cells from each experiment was analysed. PE-labeled rat antichicken CD4 þ and PE-labeled mouse anti-chicken CD8 þ antibody were obtained from Southern Biotechnology Associates, Inc (Birmingham, AL).

Statistical analysis
Data were expressed as the means ± SE. The means were compared by student's t-test. Table 2, the ADG, ADFI and FCR were not different between the 2 groups (p > .05).

As shown in
After dietary CLA supplementation, jejunal mucosa SIgA level increased by 43.58% (p < .01) while jejunal  Table 3. The broilers fed the CLA diet had higher expression of the jejunal PPARc than the broilers fed the control diet (p < .01). Dietary CLA supplementation did not change the mRNA level of TGFb4 (p >.05). Data are shown in Table 4.
As shown in Table 5, dietary CLA significantly increased the percent of CD8 þ T lymphocyte in Peyer's node (p < .01), however, did not change the percent of CD4 þ T lymphocytes (p > .05). After dietary CLA supplementation, lymphocytes transformation rate of Peyer's node increased by 7.0% (p < .05).

Discussion
We demonstrated that the CLA diet increased the jejunal mucosa SIgA level and the percent of Peyer's node CD8 þ T lymphocytesin broiler chickens. This is the first in vivo report, to our knowledge, showing an increase of intestinal mucosal immune responses in broiler chickens after feeding CLA.

Effects of dietary CLA on on the growth performance of broiler chickens
Feeding broilers with 1% CLA had no significant effects on the growth performance of broiler chickens. The result was in agreement with earlier observations from Zhang et al. (2006) who investigated the effects of increasing CLA concentration at 0, 2.5, 5.0, or 10.0 g pure CLA/kg for 6 weeks in broiler chickens and observed no alteration on the growth performances. In addition, Jiang et al. (2014) observed no significant effects on growth performance with 1% CLA supplementation. However, literature is inconsistent because several studies found that dietary supplementation with CLA at levels greater than 10 g/kg decreased the growth rate of broilers (Szymczyk et al. 2001;Suksombat et al. 2007). Moraes et al. (2016) found that CLA exhibited negative effects on performance that could be observed not only at 2% CLA inclusion, but also at 1% CLA inclusion. This effect could be explained with the negative effect of trans 10 cis12-CLA supplementation on the body weight and growth because CLA used in the present study is a blend of isomers where trans 10 cis 12-CLA representing 50% of the contained isomers. Therefore, the use of CLA in broiler diets has been kept low to avoid adverse effects on growth performance.

Effects of dietary CLA on the jejunal SIgA levels of broiler chickens
SIgA is the principal immunoglobulin on the surface of the mucosa and particularly important in preventing bacterial or viral infection, and acts as a potentiator of the immune response in intestinal tissue by uptaking antigen to dendritic cells. The present experiment found that 1% dietary CLA increased the expression of jejunal SIgA both at protein and gene level. This result is basically in line with P erez-Cano et al. (2009) who reported that dietery CLA supplementation in the maternal diet increased SIgA levels and its mRNA expression in the intestine and colon of newborn mouse. It has already been demonstrated that longterm feeding of diet supplemented with CLA increased the specific OVA-IgA production in mesenteric lymph node of mouse ). Moreover, the better humoral enhancing effects were observed after feeding CLA to young rodents, which reportedly increased concentrations of spleen IgG, IgM, and IgA (Yamasaki et al. 2000(Yamasaki et al. , 2003, although specific adaptive responses were not addressed in such studies. The specific mechanism by which CLA enhances IgA levels at mucosal sites remains unknown. It was well accepted that a great deal of CLA's metabolic effects   are mediated through alterations in gene expression, either directly or indirectly via PPARs. PPARs are novel members of the nuclear receptor superfamily with several isoforms (a, ß and c), of which PPARc is the predominant isoform in immune cells and enterocytes. PPARs were expressed by monocytes, macrophages, T cells, dendritic cells, and gastrointestinal epithelial cells in gut mucosa (Tontonoz et al. 1998;Thompson 2007). Transforming growth factor ß (TGFß)-mediated signalling has a pivotal role in the stimulation of IgA responses at mucosal sites (Borsutzky et al. 2004). TGFb attracts lymphocytes move to the intestinal epithelial layer, and plays important role in the course of the differentiation and proliferation of IgA þ B cells and then maturation into IgA-phlogocytes (Ehrhardt et al. 1992). We quantified TGFß and PPARc mRNA expression as possible mediators of CLA's immunomodulatory effects on the intestinal mucosal immunity response of broiler chickens. Here, we have shown that after dietary CLA supplementation, jejunal PPARc mRNA expression was enhanced, and simultaneously SIgA expression level at both transcription and protein level increased. Taken together, these results indicated that the increase of PPARc expression may promote the SIgA expression. No difference in TGFb gene expression was observed between two diet groups in the present experiment. Thus, the increase of sIgA expression at transcription and protein level could not be attributted to TGFb stimulation.

Effects of dietary CLA on lymphocytes subsets and lymphocytes transformation rate of Peyer's node
Peyer's node, known as the second lymphoid organ, is an important component of the intestinal mucosal immune system, characteried by a mediator and amplifier role in the process of intestinal immune response to external antigen. The present study found that proliferative responses of Peyer's node lymphocytes to ConA were also increased by dietary CLA supplementation. The result is consistent with previous experiments which found dietary CLA enhanced the proliferation activity of peripheral blood and spleen lymphocytes in animals such as rats and pigs (Chew et al. 1997;Bassaganya-Riera et al. 2001;Lai et al. 2005b). In addition, following hepatitis B vaccination, specific lymphocyte proliferation was higher in humans fed CLA 50:50 than in the control group (Yaqoob 2004). However, Zhang et al. (2006) reported dietary CLA significantly increased peripheral blood lymphocyte proliferation response to Con A in 42-dayold broiler chicks, but not on the 21-day-old broiler chickens. This might be due to different proliferation response to ConA between peripheral blood lymphocytes and Peyer's node lymphocytes. Alternatively, intestine is in direct contact with CLA, thus responses to CLA may be faster than peripheral blood lymphocytes.
Dietary CLA increased the percent of Peyer's node CD8 þ T lymphocytes in the present study. In the absence of exogenous stimulus, several studies in rat, pig and poultry have shown that dietary CLA increased CD8 þ T lymphocyte percentage in such tissues as peripheral blood, thymus, spleen (Chew et al. 1997;Yamasaki et al. 2003;Zhang et al. 2006). Moreover, the present data agrees with previous study that reported a higher specific proliferative response of CD8 þ T lymphocytes from pigs fed a CLA diet (50:50 isomers mix) .
CD8 þ T lymphocytes are the effectors of cellular immune, directly destroy infected cells and play an important role in the body's immune resistance to the virus infection. Therefore, the increase of Peyer's node CD8 þ T lymphocytes resulted from dietary CLA on may imply enhance of antiviral (bacteria) capacity in birds. Although this needs further challenge test to verify, this effect has been confirmed in the trials of swine enteritis induced by intestinal pathogenic bacteria B. hyodysenteriae in which dietary CLA was conducive to the maintenance of normal lymphocyte subsets (CD4 þ and CD8 þ ) and reduced the intestinal mucosal damage .
It is unclear why dietary CLA increased the percent of Peyer's node CD8 þ T lymphocytes. Maturation and lineage choice of T cells takes place in the thymus. Therefore, if the observed changes in the profile of Peyer's node CD8 þ T lymphocytes originated in the thymus, phenotypic shifts in this organ were expected. Bassaganya-Riera et al. (2001) found that an increase in CD8aß þ cells was detected earlier in the thymus than in peripheral blood, and that the increase in percentages of CD8aß þ thymocytes could be correlated with greater numbers of thymic lymphoid progenitor cells (CD4 À CD8 À or CD4 þ CD8 þ thymocytes) in pigs fed CLAsupplemented diets. We previously found that dietary CLA significantly increased the percentages of CD4 þ CD8 þ double-positive and CD8 þ single-positive T lymphocytes in the thymus of pigletsin the thymus after CsA injection (Liu et al. 2016). Thus, dietary CLA appears to initially modulate the cellular profiles within primary lymphoid pool (i.e. thymus).

Conclusions
Under the conditions of the experiment, 1% dietary CLA enhanced jejunal SIgA expression, and increased lymphocyte transformation rate and the percent of CD8 þ T lymphocyte in Peyer's node of broiler chickens. The results confirmed the previous findings that dietary CLA changed intestinal mucosal immune response in animal and human. In poultry production, this means that CLA might be used as a nutrient to improve the intestinal mucosal immunity of broiler chickens.