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Effects of Glutamine on Lymphocyte Proliferation and Intestinal Mucosal Immune Response in Heat-Stressed Broilers

ABSTRACT

To investigate the protective effect of glutamine (Gln) on lymphocyte proliferation and the intestinal mucosal immune response in heat-stressed broilers, 360 21-day-old Arbor Acres (AA) broilers were assigned to 4 groups in a completely randomized design, each of which included 6 replicates with 15 birds per replicate for 21 days. The chickens were fed a basal diet under no stress (NS group), a basal diet under heat stress (HT group), or a basal diet under heat stress with the addition of either 0.5 % or 1.0 % Gln. The results showed that the broilers in the HT group exhibited fewer proliferating peripheral lymphocytes, a lower growth performance, phagocytic rate and index of neutrophils, fewer goblet cells in whole intestine and intraepithelial lymphocyte (IEL) cells in the ileum, a lower sIgA content in the duodenum and the jejunum, a lower immunoglobulin content of serum and intestinal mucosa, than those of the NS group (p<0.05). Diets supplemented with Gln increased growth performance, the number of proliferating peripheral lymphocytes, the phagocytic rate and phagocytic index of neutrophils, the number of whole intestine goblet cells and ileum IEL cells, the sIgA contents of the duodenum and the jejunum, and the immunoglobulin contents of serum and intestinal mucosa (p<0.05) in broilers exposed to HT. In conclusion, Gln can enhance intestinal immune function in broiler chickens by stimulating T and B lymphocyte proliferation, increasing the number of goblet cells and IEL cells, as well as increasing the content of sIgA and immunoglobulin secretion.

Keywords:
Broilers; Glutamine; Heat stress; Intestinal mucosa immune function

INTRODUCTION

Heat stress is a stress response induced by high environmental temperature-relative humidity. Currently, heat stress is a major health problem worldwide for the poultry industry. There is accumulating evidence that high temperature decreases the growth of poultry and the quality of products and causes a series of nonspecific physiological reactions (Crespo & Shivaprasad, 2013Crespo R, Shivaprasad HL. Diseases of poultry. 13th rev. ed. Iowa: John Wiley & Sons; 2013; Wang et al., 2010Wang JP, Bu DP, Wang JQ, Huo XK, Guo TJ. Effect of saturated fatty acid supplementation on production and metabolism indices in heat-stressed mid-lactation dairy cows. Journal of Dairy Science 2010;93(9):4121-4127.). These effects have been attributed to a decrease in the intestinal immune activity and to impairment of the normal intestinal tract immunologic barrier (Calefi et al., 2014Calefi AS, Honda BTB, Costola-de-Souza C, Siqueira Ade, Namazu LB, Quinteiro-Filho WM, et al. Effects of long-term heat stress in an experimental model of avian necrotic enteritis. Poultry Science 2014;93:1344-1353.). The gut is one of the most important and longest digestive organs, and its permeability has been reported to be strongly correlated with heat stress (Song et al., 2014Song J, Xiao K, Ke YL, Jiao LF, Hu CH, Diao QY, et al. Effect of a probiotic mixture on intestinal microflora, morphology, and barrier integrity of broilers subjected to heat stress. Poultry Science 2014;93:581-588.; Nanto et al., 2015Nanto F, Ito C, Kikusato M. Effects of whole-grain paddy rice on growth performance, oxidative stress and morphological alterations of the intestine in broiler chickens exposed to acute and chronic heat stress. Journal of Poultry Science 2015;52:109-118.). Many studies have evaluated the effects of biological barriers on intestinal permeability in heat stress animal models. These studies show that high temperature can affect nutrient transportation and absorption (Baumgard & Rhoads, 2013Baumgard LH, Rhoads RP. Effects of heat stress on post absorptive metabolism and energetics. Annual Review of Animal Biosciences 2013;(1):311-337.), cause a significant decline in the number of lymphocytes and goblet cells in the jejunum (Quinteiro-Filho et al., 2015Quinteiro-Filho WM, Rodrigues MV, Ribeiro A, Ferraz-de-Paula V, Pinheiro ML, Sá LRM, et al. Acute heat stress impairs performance parameters and induces mild intestinal enteritis in broiler chickens: role of acute hypothalamic-pituitary-adrenal axis activation. Journal of Animal Science 2015;90:1986-1994.), and decrease the specific IgA (sIgA) antibody secretion contents of intestinal mucosa lymphatic tissue (Tsujita & Morimoto, 1999Tsujita S, Morimoto K. Secretory IgA in saliva can be a useful stress marker. Environmental Health Preventive Medicine 1999;4:1-8.). Under these conditions, a functional imbalance of the immune system arises, making poultry susceptible to pathogenic organism colonization and invasion and deleterious functional and structural changes in many organs (including the intestine) (Chen et al., 2014Chen Z, Xie J, Wang B, Tang J. Effect of ?-aminobutyric acid on digestive enzymes, absorption function, and immune function of intestinal mucosa in heat-stressed chicken. Poultry Science 2014;93:2490-2500.), thus causing significant economic loss to poultry producers. Therefore, the intestinal mucosa immune barrier is an especially important target in the protection against heat-induced organ dysfunction and damage.

Glutamine (Gln) is traditionally considered a truly functional amino acid in nutrition. The compound is associated with beneficial effects for gut growth, development and health; is believed to provide ATP; and regulates cellular signaling pathways for fast-dividing enterocyte and lymphocyte cells (Wu, 1996Wu G, Meier SA, Knab DA. Dietary glutamine supplementation prevents jejunal atrophy in weaned pigs. Journal of Nutrition 1996;126:2578-2585.). Gln can effectively stimulate protein synthesis in intestinal cells, protect intestinal morphology, modulate the intestinal immunity response, and enhance the physiological function of the intestinal mucosal barrier (Wu et al., 2018). Although several beneficial effects of Gln on intestinal growth, development and health have been reported (Jiang et al., 2009Jiang ZY, Sun LH, Lin YC, Ma XY, Zheng CT, Zhou GL, et al. Effects of dietary glycyl-glutamine on growth performance, small intestinal integrity, and immune responses of weaning piglets challenged with lipopolysaccharide. Journal of Animal Science 2009;87(12):4050-4056.), detailed studies on the regulation of intestinal mucosal immune function by Gln, especially under stress conditions, have been reported only in recent years. These researchers have clearly indicated that the addition of Gln can reduce bacterial translocation, repair the damaged intestinal mucosa, and regulate intestinal expression and activation of immune response regulators (such as nuclear factor-κB (NF-κB), IKK, interleukin (IL) IgA, IgM and IgG antibody) (Mondello et al., 2010Mondello S, Italiano D, Giacobbe MS, Mondello P, Trimarchi G, Aloisi C, et al. Glutamine-supplemented total parenteral nutrition improve immunological status in anorectic patients. Nutrition 2010;26(6):677-681.), as well as improve gut barrier function under stress conditions, altogether suggesting a beneficial effect of Gln on gut immune function (Wang et al., 2015Wang B, Wu GY, Zhou ZG, Dai ZL, Sun YL, Ji Y, et al. Glutamine and intestinal barrier function. Amino Acids 2015;47(10):2143-2154.).

Previous studies have discussed the changes in intestinal mucosal immunity during heat stress (Ashraf et al., 2013Ashraf S, Zaneb H, Yousaf MS, Ijaz A, Sohail MU, Muti S, et al. Effect of dietary supplementation of prebiotics and probiotics on intestinal microarchitecture in broilers reared under cyclic heat Stress. The Journal of Animal Physiology and Animal Nutrition 2013;97:68-73.), but the underlying immunity mechanisms in the gut under heat stress and what ultimately happens to gut immunity are not clear. Therefore, changes in the number of intestinal mucosal immune cells and proliferating peripheral lymphocytes, the phagocytic function of neutrophils, and the contents of sIgA and immunoglobulin in broilers under heat stress were tested to investigate whether Gln could protect intestinal mucosal immune barrier function by mediating proliferation of intestinal immune cells and T cells of peripheral blood in heat-stressed broilers.

MATERIALS AND METHODS

Birds, Experimental design, and Diet

The study was carried out at Henan University of Science and Technology, following the requirements of the experimental protocol of the Institutional Animal Care and Use Committee of Henan University of Science and Technology. A total of 360 Arbor Acres (mixed male and half) 21-day-old broiler chicks were selected, individually weighed chicks were allocated to 4 groups of 90 birds each in a completely randomized design, each of which included 6 replicates with 15 birds per replicate, and each replicate was placed into a cage (140 cm × 70 cm × 28 cm). The four experimental groups included a nonstressed control group (NS), which was fed a corn-soybean meal basal diet and kept in a thermoneutral room at 22 ± 1ºC; a heat stress control group (HT), which was fed the same basal diet and kept at 33 ± 1 1ºC for 10 h/d from 08:00 to 18:00 h and at 22 ± 1 1ºC for the remaining time); and HT + Gln treatment groups, which were fed 0.5 % or 1.0 % Gln diet (i.e., HT-G1 and HT-G2, respectively) and stressed by heat. Gln was obtained from Henan Honda Biological Medicine Co., Ltd., China (99 % purity, pharmaceutical grade) and was added on top of the basal diet.

Broilers were fed corn and soybean meal-based diets for 3 weeks. The formulations and nutrient composition of basal diets for finisher (21- 42 days) broiler growth periods are presented in Table 1, which were necessary to meet or slightly exceed the minimum nutrient requirements of broiler chickens, as recommended by the NRC (National Research Council, 1994). The room temperature was continuously regulated with electric heaters. From 22 to 41 days, the broilers were exposed to 33 ± 1 1ºC and 55 ± 5 % relative humidity. All broilers were allowed free access to feed and water.

Table 1
Ingredient and nutrient levels of the experimental diets (as-fed basis).

Growth performance

During the experimental period, body weight, feed intake of the chicks was recorded, and average daily gain (ADG), average daily feed intake (ADFI), and feed/gain ratio (F/G) were measured.

Blood sample collection

Six broilers (one bird per replicate) from each treatment group were randomly selected, and serum was sampled via the wing vein and collected in a heparinized centrifuge tube on day 21 of the trial. Half of each blood sample was centrifuged at 3500 × g for 15 min, and serum samples were obtained and frozen at -80ºC for ELISA analysis. The remaining blood samples were used in the lymphocyte proliferation assay. Then, all of the broilers were euthanized immediately.

Collection and preparation of intestinal tissue and mucosa

After decapitation, small intestine tissue from the duodenum, jejunum, and ileum was dissected and carefully washed with normal saline and opened longitudinally. Approximately 3 cm of each intestinal segment was removed, fixed in 4 % paraformaldehyde, dehydrated and embedded in paraffin. Sections measuring 5 µm were cut, mounted, and stained with hematoxylin and eosin (H&E) stain.

The mucosa of the remaining intestinal segments was excised, homogenized, and centrifuged (4000 × g, 10 min, 4ºC). The supernatant was then harvested frozen and kept for further analysis.

Lymphocyte proliferation assay

The peripheral blood lymphocyte assay was conducted against concavalin A (Con A) using the MTT method. Lymphocyte-rich mononuclear cells were isolated from blood collected with 7.2 mg dipotassium EDTA and separated on Ficoll-Histopaque density system (Sigma-Aldrich Co.) as described by Böyum (1968) and Nathanson (1982). Then, separated cells were washed (twice) with cold RPMI-1640 medium (2.5 × 105 cells/ml), resuspended in the media, and counted by the trypan blue dye exclusion technique. MTT solution was added to each well, and the plates were incubated at 37ºC for 5 h. After this period, the plate was centrifuged at 1 000 × g for 5 min, and sodium dodecyl sulfate was then added to lyse the cells and solubilize the MTT crystals. Subsequently, the optical density was measured in a microspectrophotometer at 570 nm, and blood T and B lymphocyte proliferation was calculated by the corresponding equation and expressed as a mean stimulation index (SI).

Isolation and evaluation of phagocytic function of neutrophils

Broiler blood was layered onto 5 mL of Ficoll-Paque Plus and incubated at room temperature. Neutrophils were obtained using Histopaque 1077 (Sigma-Aldrich Co., Saint Louis, MO, USA) separating medium. Cells were centrifuged, withdrawn, detached, and resuspended in Hanks-Tris solution (5 ×105 neutrophils/mL), then were washed, suspended, smeared, and stained with acridine orange. All counts of phagocytosed neutrophils and dead bacteria were then performed. The phagocytic index (PI) was calculated as the average number of bacteria phagocytosed per 100 neutrophils. The phagocytosis percentage (PP) was calculated as the percentage of neutrophils taking part in phagocytosis.

Counting of intestinal intraepithelial lymphocytes and goblet cells

The numbers of intestinal intraepithelial lymphocytes (IEL) and goblet cells (GC) were counted on the H&E stained sections. The prepared glass slides carrying samples were observed under a Motic Med 6.0 CMIAS Image Analysis System (Motic China Group Co., Ltd., Guangzhou, China). A total of 3 fields per section and 5 sections per chicken were selected randomly. The mean values of IEL and GC among 100 enterocytes in different fields were then calculated for statistical analysis.

Determination of sIgA, IgA, IgG and IgM content by ELISA

Intestinal mucus concentrations of secretory IgA (sIgA) and immunoglobulins (IgA, IgG and IgM) were measured by enzyme-linked immunosorbent assay (ELISA, BlueGene, Shanghai, People’s Republic of China) kits specific for chicks, following the instructions of the kit’s manufacturer. The total protein concentration of each intestinal mucosa was determined simultaneously by the Bradford brilliant blue method.

Statistical analysis

Statistical analysis of all data was carried out using SPSS version 20.0. Data for all of the treatment groups were compared using Tukey’s range test. All data were expressed as means ± standard error of mean (SEM at levels of significance (p<0.05)).

RESULTS

Broiler production performance

During the feeding stages, the final weight, ADG, and ADFI were significantly lower in the HT group than in the NS group, and F/G were significantly higher in the HT group than in the NS group, (p<0.05) (Table 2). Supplementation with Gln significantly increased the final weight, ADG, and ADFI, and decreased the F/G compared to the number observed in the HT group (p<0.05).

Table 2
Effects of glutamine on the growth performance in heat-stressed broilers.

The number of proliferating peripheral blood T and B lymphocytes

The number of proliferating peripheral blood T and B lymphocytes was significantly lower in the HT group than in the NS group (p<0.05) (Fig. 1A and B). Supplementation with Gln significantly increased the number of proliferating peripheral blood T and B lymphocytes compared to the number observed in the HT group (p<0.05). The number of proliferating peripheral blood T and B lymphocytes approached nonheat stress levels (p>0.05). There was no significant difference between the HT-G1 group and the HT-G2 group (p>0.05).

Figure 1
Effects of Gln on the number of proliferating peripheral blood T and B lymphocytes in broilers under heat stress. Each column and the corresponding bar represent the mean and SEM, n=8, respectively. a-bMeans that possess different superscripts differ significantly (p<0.05). NS= birds were kept at 22 ± 1 ºC and fed the basal diet; HT= birds were kept at 33 ± 1 ºC for 10 h (08:00 to 18:00 h) and 22 ± 1 ºC for the remaining time and fed the basal diet; HT-G1 and G2 groups= birds were kept at 33 ± 1 ºC for 10 h (08:00 to 18:00 h) and 22 ± 1 ºC for the remaining time and fed the basal diet with 0.5 % and 1.0 % Gln, respectively.

Phagocytic rate and phagocytic index of neutrophils

Compared to the NS group, heat stress induced a significant downregulation of the phagocytic rate and phagocytic index of neutrophils (p<0.05) (Fig. 2A and B). Supplementation with Gln significantly increased the phagocytic rate and phagocytic index of neutrophils compared to those of the HT group (p<0.05). The phagocytic rate and phagocytic index of neutrophils gradually approached nonheat stress levels (p>0.05). There was no significant difference between the HT-G1 group and the HT-G2 group (p>0.05).

Figure 2
Effects of Gln on phagocytic rate and phagocytic index of neutrophils in broilers under heat stress. Each column and the corresponding bar represent the mean and SEM, n=8, respectively. a-bMeans that possess different superscripts differ significantly (p<0.05). NS= birds were kept at 22 ± 1 ºC and fed the basal diet; HT= birds were kept at 33 ± 1 ºC for 10 h (08:00 to 18:00 h) and 22 ± 1 ºC for the remaining time and fed the basal diet; HT-G1 and G2 groups= birds were kept at 33 ± 1 ºC for 10 h (08:00 to 18:00 h) and 22 ± 1 ºC for the remaining time and fed the basal diet with 0.5 % and 1.0 % Gln, respectively.

The number of intestinal goblet cells

Exposure to heat stress (HS) significantly reduced the number of goblet cells in the duodenum, jejunum, and ileum compared to the numbers observed in the NS group (p<0.05) (Fig. 3A, B and C). Supplementation with Gln significantly increased the number of goblet cells in the duodenum, jejunum, and ileum compared to the HT group (p<0.05). The number of intestinal goblet cells gradually approached nonheat stress levels (p>0.05), and there was no significant difference between the HT-G1 group and the HT-G2 group (p>0.05).

Figure 3
Effects of Gln on the number of intestinal goblet cells in broilers under heat stress. Each column and the corresponding bar represent the mean and SEM, n=8, respectively. a-bMeans that possess different superscripts differ significantly (p<0.05). NS= birds were kept at 22 ± 1 ºC and fed the basal diet; HT= birds were kept at 33 ± 1 ºC for 10 h (08:00 to 18:00 h) and 22 ± 1 ºC for the remaining time and fed the basal diet; HT-G1 and G2 groups= birds were kept at 33 ± 1 ºC for 10 h (08:00 to 18:00 h) and 22 ± 1 ºC for the remaining time and fed the basal diet with 0.5 % and 1.0 % Gln, respectively.

The number of IEL-secreting cells

In the ileum, the number of IEL-secreting cells was significantly lower in the HT group than in the control group (p<0.05) (Fig. 4A, B and C). Supplementation with Gln significantly increased the number of IEL-secreting cells in the ileum compared to the number in the HT group (p<0.05). The number of IEL-secreting cells gradually approached nonheat stress levels (p>0.05). There was no significant difference between the HT-G1 group and the HT-G2 group (p>0.05). In the duodenum and jejunum, the HT group had fewer IEL-secreting cells than the NS group did, but there was no significant difference between the NS group and the Gln group (p>0.05).

Figure 4
Effects of Gln on IEL cells of the intestine in broilers under heat stress. Each column and the corresponding bar represent the mean and SEM, n=8, respectively. a-bMeans that possess different superscripts differ significantly (p<0.05). NS= birds were kept at 22 ± 1 ºC and fed the basal diet; HT= birds were kept at 33 ± 1 ºC for 10 h (08:00 to 18:00 h) and 22 ± 1 ºC for the remaining time and fed the basal diet; HT-G1 and G2 groups= birds were kept at 33 ± 1 ºC for 10 h (08:00 to 18:00 h) and 22 ± 1 ºC for the remaining time and fed the basal diet with 0.5 % and 1.0 % Gln, respectively.

sIgA contents of the intestine mucosa

In the jejunum and ileum, the sIgA contents of the HT group was lower than that of the control group (p<0.05) (Fig. 5A, B and C). Supplementation with Gln significantly increased the sIgAs content in the jejunum and ileum compared to that of the HT group (p<0.05). The sIgA contents of the Gln group decreased, but there was no significant difference compared to the control group (p>0.05). In the duodenum, there was no significant difference among the groups (p>0.05).

Figure 5
Effects of Gln on sIgA contents of the intestine mucosa in broilers under heat stress. Each column and the corresponding bar represent the mean and SEM, n=8, respectively. a-bMeans that possess different superscripts differ significantly (p<0.05). NS= birds were kept at 22 ± 1 ºC and fed the basal diet; HT= birds were kept at 33 ± 1 ºC for 10 h (08:00 to 18:00 h) and 22 ± 1 ºC for the remaining time and fed the basal diet; HT-G1 and G2 groups= birds were kept at 33 ± 1 ºC for 10 h (08:00 to 18:00 h) and 22 ± 1 ºC for the remaining time and fed the basal diet with 0.5 % and 1.0 % Gln, respectively.

The immunoglobulin contents of serum and intestinal mucosa

In the serum, duodenum, jejunum and ileum, the immunoglobulin contents of the HT group were lower than that of the control group (p<0.05) (Fig. 6A-D). Supplementation with Gln significantly increased the immunoglobulin contents in the serum, duodenum, jejunum and ileum compared to that of the HT group (p<0.05). The immunoglobulin contents of the Gln group decreased, but there were no significant differences compared to the control group (p>0.05). Moreover, there was no significant difference between the HT-G1 group and the HT-G2 group (p>0.05).

Figure 6
Effects of Gln on the immunoglobulin contents of serum and intestinal mucosa in broilers under heat stress. Each column and the corresponding bar represent the mean and SEM, n=8, respectively. a-bMeans that possess different superscripts differ significantly (p<0.05). NS= birds were kept at 22 ± 1 ºC and fed the basal diet; HT= birds were kept at 33 ± 1 ºC for 10 h (08:00 to 18:00 h) and 22 ± 1 ºC for the remaining time and fed the basal diet; HT-G1 and G2 groups= birds were kept at 33 ± 1 ºC for 10 h (08:00 to 18:00 h) and 22 ± 1 ºC for the remaining time and fed the basal diet with 0.5 % and 1.0 % Gln, respectively.

DISCUSSION

As previously reported, the intestine is not only an organ for digestion and absorbing nutrients but it is also regarded as an important immunological organ (Steinert et al., 2016Steinert A, Radulovic K, Niess J. Gastro-intestinal tract: the leading role of mucosal immunity. Swiss Medical Weekly. 2016;146:w14293.). Fundamental immunological functions are provided by the intestinal mucosal immunity system, which is, in part, composed of gut-associated lymphoid tissue, IEL, GC, and abundant immunoglobulins. It has been shown that responsiveness of the intestinal mucosal immunity system to high temperature can reduce the number of goblet cells in the epithelia of the intestine and damage immune function of the intestinal mucosa and mesenteric lymphoid tissues, thus decreasing the growth performance (Liu et al., 2012Liu XX, Li HR, Lu A, Zhong YG, Hou XL, Wang N, et al. Reduction of intestinal mucosal immune function in heat-stressed rats and bacterial translocation. International Journal of Hyperthermia 2012;28(8):756-765.; Ashraf et al., 2013Ashraf S, Zaneb H, Yousaf MS, Ijaz A, Sohail MU, Muti S, et al. Effect of dietary supplementation of prebiotics and probiotics on intestinal microarchitecture in broilers reared under cyclic heat Stress. The Journal of Animal Physiology and Animal Nutrition 2013;97:68-73.). The peripheral blood T and B lymphocytes were used to evaluate the influence of heat stress on cell-mediated immunity in our present study. There was a significant decrease in the number of proliferating peripheral blood T and B lymphocytes in the heat stress groups. Similarly, exposure to heat stress has been shown to decrease the growth performance and inhibit lymphocyte cell proliferation in cattle (Sunilkumar & Kataria, 2011Sunilkumar BV, Kataria M. Effect of dietary supplementation on electrolyte profile and lymphocyte proliferation during heat stress in buffaloes. Cell and Tissue Research 2011;11(3):2977-2980.). The decrease in the growth performance and lymphocyte proliferative response might be due to excessive production of reactive oxygen species due to heat stress, which initiates a cascade of lipid peroxidation of biological membranes, thereby reducing the growth performance and cell-mediated immune response (Goldstone & Hunt, 1997Goldstone SD, Hunt NH. Redox regulation of the mitogen-activated protein kinase pathway during lymphocyte activation. Biochemica et Biophysica Acta 1997;1355:353-360.). In the present study, dietary supplementation with Gln significantly enhanced stress-stimulated lymphocyte proliferation and growth performance. These results might be observed because supplementation of Gln could enhance reactive oxygen species (such as GSH) production of lymphocyte cells and prevent lipid peroxidation by eliminating free active oxygen and lipid peroxides (Curi et al., 1999Curi R, Newsholme P, Pithon-Curi TC, Pires-de-Melo M, Garcia C, Homem-de-Bittencourt Jr. PI, et al. Metabolic fate of glutamine in lymphocytes, macrophages and neutrophils. Brazilian Journal of Medical & Biological Research 1999;32(1):15-21.), thereby increasing growth performance and preventing cell membrane disruption. Indeed, previous studies have demonstrated that glutamine is an important precursor of lipid synthesis under stress conditions (Curi et al., 1999). However, these results may also be related to alterations in the biosynthetic processes of lymphocyte proliferation and cell cycle propagation. Glutamine is a primary fuel for lymphocytes and is used at high rates during lymphocyte proliferation (Chang et al., 1999Chang WK, Yang KD, Shaio MF. Lymphocyte proliferation modulated by glutamine:involved in the endogenous redox reaction. Clinical and Experimental Immunology 1999;117:482-488.). Moreover, the influence of glutamine on lymphocyte proliferation can be explained by the requirement of glutamine for the production of IL-2 and IFN-γ (Rohde et al., 1996Rohde T, Maclean DA, Pedersen BK. Glutamine, lymphocyte proliferation and cytokine production. Scandinavian Journal of Immunology 1996;44:648-650.).

It is generally recognized that neutrophils constitute the first line of defense against invading pathogens, are involved in the initiation of innate immune responses, and promote the protective inflammation response through the clearance of proinflammatory necrotic tissue and cell residues. The above findings are similar to the findings of Tsukamoto & Machida (2012Tsukamoto K, Machida K. Effects of life events and stress on neutrophil functions in elderly men. Immunity & Ageing 2012;(9):13-17.); and Li et al. (2018) who found that the phagocytic capacity of neutrophils is significantly decreased under physiological stress. These results concluded that the general effect of physiological stress can cause neutrophil dysfunction, decrease the phagocytic ability of the broiler’s neutrophils, and destroy microbial invaders (Tsukamoto & Machida, 2012). Supplementation with Gln enhanced the phagocytic rate and phagocytic index of neutrophils compared with those of the HT group. Sasaki et al. (2013Sasaki E, Umeda T, Takahashi I, Arata K, Yamamoto Y, Tanabe M, et al. Effect of glutamine supplementation on neutrophil function in male judoists. Luminescence 2013;28(4):442-449.) also showed that Gln supplementation enhanced neutrophil activity in male judoists after high-intensity exercise. Gln supplementation contributes to the maintenance of the body’s immune functions (Wu et al., 2019Wu QJ, Jiao C, Liu ZH, Li SW, Zhu DD, Ma WF, et al. Effect of glutamine on the intestinal function and health of broilers challenged with Salmonella pullorum. Indian Journal of Animal Research 2019;53(9):1210-1216.). This result is likely associated with the antioxidant effect of Gln; it has been shown that Gln can promote reactive oxygen species production and an oxidative burst of neutrophils in broilers (Sasaki et al., 2013), thus explaining the good immune status of broilers in the present study. However, our study could not reveal the mechanism by which Gln affected the oxidative burst of neutrophils in detail; therefore, it is necessary to investigate the antioxidant capacity of glutamine during psychological stress.

Moreover, heat stress promoted a decrease in the number of goblet cells in broilers. A similar reduction of goblet cells has been observed in an animal model with prolonged heat stress (Shi et al., 2015Shi QM, Gao GS, Xing C, Liu H, Shen P, Pan FF, et al. Effects of traditional chinese medicine on numbers of lymphocytes and goblet cells in villus epithelia of layers under heat stress. Agriculture Science Technology 2015;16(2):311-316, 335). These results indicate that heat stress can slow intestinal peristalsis and cause digesta retention and thereby promote the production of a large number of oxygen free radicals, ultimately resulting in the vulnerability of these segments and the intestinal mucosa barrier. Supplementation with Gln in the Gln group promoted an increase in the number of goblet cells in these broilers. Therefore, Gln supplementation caused beneficial protective effects by strengthening the intestinal wall of broilers. The observed increase in the number of goblet cells due to supplementation with Gln can be attributed to their antioxidant capacity and acceleration of differentiation and immunostimulatory effects (Martins et al., 2016Martins HA, Sehaber CC, Hermes-Uliana C, Mariani FA, Guarnier FA, Vicentini GE, et al. Supplementation with l-glutamine prevents tumor growth and cancer-induced cachexia as well as restores cell proliferation of intestinal mucosa of Walker-256 tumor-bearing rats. Amino Acids 2016;48(12):2773-2784.). Because Gln is the main metabolic fuel for migrating and proliferating cells of the intestinal mucosa, it can improve the immune response and intestinal barrier function by impairing reactive oxygen species production and, in turn, reducing bacterial translocation (Alves et al., 2010Alves EPB, Alves AMP, Pereira RVF, Neto MH dM, Zanoni JN. Immunohistochemical study of vasoactive intestinal peptide (VIP) enteric neurons in diabetic rats supplemented with l-glutamine. Nutritional Neuroscience 2010;13(1):43-51.).

The IEL compartment is mainly composed of epithelial cells and dispersed lymphocytes and is considered to be the first compartment of the immune system that plays an important role in mucosal immunity by gut-derived antigens (Shi et al., 2015Shi QM, Gao GS, Xing C, Liu H, Shen P, Pan FF, et al. Effects of traditional chinese medicine on numbers of lymphocytes and goblet cells in villus epithelia of layers under heat stress. Agriculture Science Technology 2015;16(2):311-316, 335). Therefore, changes in the number of IELs can reflect the integrity status of the epithelial mucosa barrier in certain respects. The present study found that under heat stress conditions, the number of IELs in the ileum of broiler chickens was significantly decreased. Quinteiro-Filho et al. (2010Quinteiro-Filho WM, Ribeiro A, Ferraz-de-Paula V, Pinheiro ML, Sakai M, Sa LR, et al. Heat stress impairs performance parameters, induces intestinal injury, and decreases macrophage activity in broiler chickens. Poultry Science 2010;89:1905-1914.) reported similar effects under high-temperature conditions in birds, which reflect the potential of heat stress to impair the activity of intestinal mucosal immunity and initiate an inflammatory response. Our study indicates that the Gln-administered groups could increase the IEL number and ameliorate the inflammatory response triggered by heat stress. The increase in the IEL count in Gln-administered groups can be attributed to Gln-induced reduction in the level of proinflammatory cytokines (Isolauri et al., 2001Isolauri E, Sütas Y, Kankaanpää P, Ar vilommi H, Salminer S. Protiotics: effects on immunity. American Journal of Clinical Nutrition 2001;73:S444-450.), thereby maintaining the defense capability of intestinal mucosa to some extent under heat stress, a phenomenon observed in our previous study (Wu et al., 2018Wu QJ, Liu N, Wu XH, Wang GY, Lin L. Glutamine alleviates heat stress-induced impairment of intestinal morphology, intestinal inflammatory response, and barrier integrity in broilers. Poultry Science 2018;97(8):2675-2683.). The finding that Gln maintained epithelial barrier integrity and influenced the intestinal immune responses by mediating the proliferation of IELs (Horio et al., 2008Horio Y, Osawa S, Takagaki K, Hishida A, Furuta T, Ikuma M. Glutamine supplementation increases Th1-cytokine responses in murine intestinal intraepithelial lymphocytes. Cytokine 2008;44(1):92-95.) supports the findings of the current study. We also found that Gln increased ileal IEL numbers. The reason for the discrepancy might be that the ileum hosts more microorganisms than do the other segments of the small intestine.

Some reports have indicated that the concentration of sIgA in mucosa is an indicator of specific immunity against psychological stress, decreasing under stress (Zuo et al., 2014Zuo T, Cao L, Sun XH, Li XM, Wu J, Lu SY, et al. Dietary squid ink polysaccharide could enhance SIgA secretion in chemotherapeutic mice. Food Function 2014;5:3189-3196.; Wu et al., 2018Wu QJ, Liu N, Wu XH, Wang GY, Lin L. Glutamine alleviates heat stress-induced impairment of intestinal morphology, intestinal inflammatory response, and barrier integrity in broilers. Poultry Science 2018;97(8):2675-2683.). In this study, we found that intestinal sIgA levels were significantly reduced in the jejunal and ilial mucosa in the HT group. In some studies, secretory IgA has also been found to decrease significantly during stress (Matos-Gomes et al., 2010Matos-Gomes N, Katsurayama M, Makimoto FH, Santana LL, Paredes-Garcia E, Becker MA, et al. Psychological stress and its influence on salivary flow rate, total protein concentration and IgA, IgG and IgM titers. Neuroimmunomodulation 2010;17(6):396-404.). Moreover, a significant decrease in sIgA levels was found in the distal region of the small intestine; the underlying mechanism in the jejunum and ileum regions may be related to the regulatory effects of HT on redistribution and intestinal B cell homing via an adrenergic mechanism (Drago-Serrano et al., 2012Drago-Serrano ME, Godínez-Victoria M, Lara-Padilla E, Resendiz-Albor AA, Reyna-Garfias H, Arciniega-Martínez IM, et al. Moderate exercise enhances expression of SIgA in mouse ileum. International Journal of Sports Medicine 2012;33(12):1020-1025.). Supplementation with Gln can increase the levels of sIgA. These findings indicate that Gln supplementation enhances the secretion of IgA. This result is similar to that reported by Lai et al. (2004Lai YN, Yeh SL, Lin MT, Shang HF, Yeh CL, Chen WJ. Glutamine supplementation enhances mucosal immunity in rats with Gut-Derived sepsis. Nutrition 2004;20:286-291.), who showed that the addition of Gln to the rat diet can enhance sIgA secretion. Gln likely causes this effect by influencing the intestinal microbiota, the induction pathway (T-dependent and T-independent), IgA-secreting plasma cells, the expression of pIgR and even the transport of intestinal sIgA (Ren et al., 2016Ren WK, Wang K, Yin J, Chen S, Liu G, Tan B, et al. Glutamine-induced secretion of intestinal secretory immunoglobulin A: a mechanistic perspective. Frontiers Immunology 2016;7:503.).

In addition, IgA, IgG and IgM also participate in the intestinal mucosal immune response process. The low concentrations of serum and intestinal IgA, IgG and IgM in the HT group may indicate a decrease in the defensive capacity of the mucosal immune system. The results of this study agree with the data of Olfati et al. (2018Olfati A, Mojtahedin A, Sadeghi T, Sadeghi T, Martínez-Pastor F. Comparison of growth performance and immune responses of broiler chicks reared under heat stress, cold stress and thermoneutral conditions. Spanish Journal of Agricultural Research 2018;16(2):e0505.), which demonstrated that the IgA, IgG and IgM concentrations were markedly decreased under heat stress in broilers. These physiological changes also increase the penetration of bacteria through the intestinal lining. However, heat stress produces negative effects on redox balance, exacerbating the production of reactive oxygen species (Olfati et al., 2018) and contributing to the permeability of the gut lining to bacteria. This fact may be explained by the data reported by Quinteiro-Filho et al. (2015Quinteiro-Filho WM, Rodrigues MV, Ribeiro A, Ferraz-de-Paula V, Pinheiro ML, Sá LRM, et al. Acute heat stress impairs performance parameters and induces mild intestinal enteritis in broiler chickens: role of acute hypothalamic-pituitary-adrenal axis activation. Journal of Animal Science 2015;90:1986-1994.), which showed that heat stress increased serum corticosterone levels, the concentration of inflammation cytokines, and bacterial translocation in broiler chickens. This idea appears to be reinforced by the observed decreased percentage of T and B lymphocytes in chickens in the HS group, which also shows the suppressive effects of heat stress on humoral activity and its altering effects on the function of the intestine. Previous studies have reported modulatory actions of Gln on the humoral immune response, such as increased IgA, IgG and IgM production in humans and rats (Yeh et al., 2005Yeh SL, Lai YN, Shang HF, Lin MT, Chiu WC, Chen WJ, et al. Effects of glutamine supplementation on splenocyte cytokine mRNA expression in rats with septic peritonitis. World Journal of Gastroenterology 2005;11(12):1742-1746.). In our study, dietary supplementation with Gln increased the serum and intestinal mucosa IgA, IgG and IgM levels of broilers under heat stress and greatly improved immune functions in heat-stressed broilers compared to those of the control group. These results suggest that Gln may mediate protection of mucosal surfaces in the intestinal mucosa. The beneficial effects of Gln on mucosal immunity might be explained by its antioxidant activity and bacterial translocation.

In conclusion, dietary supplementation with Gln could modulate intestinal immune function in broilers under heat stress by enhancing lymphocyte proliferation, the phagocytic rate and phagocytic index of neutrophils, the number of goblet cells and IELs, sIgA secretion, and blood serum and intestinal mucosa IgA, IgG and IgM levels. The results of this study indicate that Gln supplementation can regulate immune responses at mucosal surfaces and improve the immune response and function of the intestinal barrier. The beneficial effects of Gln supplementation are associated with its antioxidant activity and bacterial translocation.

ACKNOWLEDGEMENTS

This research was supported by a project supported by the National Natural Science Foundation of China (Grant No. 31601971). The authors express special thanks to Qianqian Wang, Xiuqing Zhou, Yazhe Song, and Jing Zhou for skillful technical assistance with this research.

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Publication Dates

  • Publication in this collection
    01 Nov 2021
  • Date of issue
    2021

History

  • Received
    14 Jan 2021
  • Accepted
    22 June 2021
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