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Upregulation of Oxidative Burst and Degranulation in Chicken Heterophils Stimulated with Probiotic Bacteria

M. B. Farnell^*,1, A. M. Donoghue, F. Solis de los Santos, P. J. Blore, B. M. Hargis, G. Tellez and D. J. Donoghue

^* Poultry Science Department, Texas A&M University, College Station 77843; Poultry Production and Product Safety Research Unit, Agricultural Research Service, USDA, Fayetteville, AR 72701; and Poultry Science Department, University of Arkansas, Fayetteville 72701

¹ Corresponding author: mfarnell{at}poultry.tamu.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The immune system of neonatal chicks is functionally immature during the first week of life. Researchers have previously demonstrated that the avian humoral response can be increased with probiotics. Although the humoral response provides the chick with an effective mechanism to combat pathogens, sufficient antibody titers are not attained until 7 to 10 d postinfection. However, the innate immune system (i.e., heterophils) can respond much more quickly to pathogens. The objective of this study was to determine whether probiotic bacteria can also upregulate heterophil function. Heterophils were isolated from the peripheral blood of neonatal chickens by using a discontinuous density gradient. Oxidative burst and degranulation are bactericidal mechanisms used by heterophils to kill pathogens and were used in this study as indicators of heterophil function. We found that each of the 10 "generally recognized as safe" probiotic isolates (designated G1 to G11) tested in vitro were capable of increasing (P < 0.05) heterophil oxidative burst and degranulation when compared with unstimulated controls. Bacillus subtilis (G3), Lactococcus lactis lactis (G6), and Lactobacillus acidophilus (G8) isolates were determined to elicit the greatest heterophil response in vitro and were subsequently fed to chicks. Phosphate-buffered saline or 1 of these 3 probiotic isolates (~2.5 x 10⁸ cfu/chick; 50 chicks/treatment) resuspended in PBS was administered by oral gavage on the day of hatch. Heterophils were isolated from chicks from each of these 4 treatment groups 24 h posttreatment. Significant increases in heterophil degranulation and oxidative burst were observed with the G3-, G6-, and G8-treated chicks when compared with heterophils isolated from birds with no probiotic treatment. These data suggest that probiotic bacteria can significantly improve heterophil oxidative burst and degranulation in broilers. To our knowledge, this is the first study demonstrating a relationship between probiotics and avian heterophil function.

Key Words: chicken • probiotic • heterophil • gastrointestinal tract • innate immunity

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Neonatal chickens have an immature immune system during the first week posthatch and are most susceptible to pathogens during this period (Barrow, 2000; Beal et al., 2004; Lowry et al., 2005). Heterophils, first responders of the avian innate immune system, can mount a rapid response to a bacterial infection within 30 min (Kogut et al., 1994; He et al., 2003). However, a sufficient adaptive immune response may take as long as 1 to 2 wk to clear an infection (Berndt and Methner, 2004). A broiler is harvested at approximately 6 wk of age and does not have much time to mount a suitable adaptive immune response and recover from any resulting loss in feed conversion. Therefore, it is important to concentrate on the fast-acting, innate immune response and to develop techniques that potentiate innate immune function. Recent reports demonstrate the importance of probiotics in stimulating the adaptive immune response in chickens (Koenen et al., 2004a,b) and they may also play a significant role in potentiating the innate immune response.

Probiotics are nonpathogenic bacteria that can promote bird health by reducing pathogen colonization (Mead, 2000, 2002). These reductions are attributed to competitive exclusion, increased volatile fatty acid production, and potentiation of the immune system (Nava et al., 2005, Donoghue et al., 2006). Probiotics can significantly increase the humoral immune response in chickens (Koenen et al., 2004a); however, significant increases in secretory IgA-producing cells are generally not observed until 8 d postchallenge (Berndt and Methner, 2004). A more rapid response is observed in phagocytes stimulated with similar microbial agonists, resulting in enhanced phagocytosis, killing, degranulation, and oxidative burst (Farnell et al., 2003; Galdeano and Perdigón, 2004; Lowry et al., 2005). It is feasible that modification of the avian gastrointestinal microflora with probiotic bacteria could affect the innate immune response similarly.

In this study, we evaluated the immunomodulating effects of probiotic isolates, previously shown to be bactericidal to Campylobacter jejuni, by quantitating oxidative burst and degranulation of chicken heterophils exposed to these bacteria. The objective of this study was to develop an in vitro screening procedure for probiotic bacteria that enhances heterophil function and to determine whether these bacterial isolates are immunostimulatory when administered in vivo.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
In Vitro Study Experimental Design
Heterophils were isolated from the pooled peripheral blood of neonatal chicks (100 chicks per collection day) to screen for probiotic bacteria that elicited a heterophil response in vitro. Each replicate was conducted on a separate collection day for a total of 3 replicate experiments. One milliliter of our pooled heterophil stock was aliquoted into each 1.5-mL microfuge tube. Each of these tubes also contained 125 µL of 1 of the 10 formalin-killed probiotic bacteria (G1 to G11) or Roswell Park Memorial Institute (RPMI) media alone and incubated for 30 min at 42°C before the addition of 2,7-dichlorofluorescein diacetate (DCF-DA). These tubes were then aliquoted into a 96-well plate (n = 8). Readings were collected 1 h after the addition of DCF-DA. The degranulation treatments were the same as the oxidative burst study, but heterophils were incubated for 1 h at 42°C before the addition of the degranulation substrate to the supernatant. Readings were collected immediately after the addition of the stop solution (n = 4).

In Vivo Study Experimental Design
Three probiotic bacteria (G3, G6, G8) were chosen from the in vitro study to determine whether these bacteria could also stimulate heterophil function in vivo. Fifty chicks per each treatment were orally gavaged with 0.5 mL of PBS or 1 of the 3 viable probiotic bacteria suspended in PBS on the day of hatch. Twenty-four hours postgavage, heterophils were collected from the pooled peripheral blood of each treatment group for a total of 4 separate heterophil preparations. One milliliter of each of these preparations was incubated with 125 µL of RPMI or phorbol myristate acetate for 30 min at 42°C before the addition of DCF-DA for the oxidative burst assay. These tubes were then aliquoted into a 96-well plate (n = 8). Readings were collected at 15 or 60 min after the addition of DCF-DA, according to the optimum readable response. A subset of heterophils was also used to determine degranulation in each of these treatment groups. One milliliter of each of these preparations was incubated with 125 µL of RPMI or opsonized zymosan for 1 h before the addition of the degranulation substrate to the supernatants (n = 4). Readings were collected immediately after the addition of the stop solution. Two replicates were conducted on different days for each assay.

Experimental Birds
Cobb-Vantress (Cobb-Vantress Inc., Fayetteville, AR) broiler chickens were obtained on the day of hatch from a local commercial hatchery and placed into floor pens with pine shavings and supplemental heat. Chicks were provided water and a balanced, unmedicated, corn-soybean ration ad libitum that met or exceeded the NRC (1994) guidelines for a broiler diet. These studies were conducted according to the guidelines of the University of Arkansas Animal Use Committee.

Heterophil Isolation
Blood was collected for heterophil isolation and for the in vitro and in vivo studies by decapitation, and EDTA (Sigma-Aldrich, St. Louis, MO) was used as an anticoagulant. Heterophils were isolated as previously described (Kogut et al., 1994, 1998). Briefly, blood was mixed with 1% methylcellulose (Sigma-Aldrich), dissolved in RPMI 1640 media (Mediatech Inc., Herndon, VA) at 1:1.5 and centrifuged at 250 x g for 30 min. The supernatant was removed and resuspended in Hanks’ balanced salt solution without Ca or Mg (Mediatech Inc.) at 1:1. The suspension was then layered over a 1.077/ 1.119 Histopaque gradient (Sigma-Aldrich) and centrifuged at 500 x g for 60 min. The interface of the 2 gradients was collected and washed with RPMI. Following the RPMI wash, cells were quantitated using a Neubauer hemacytometer, and the stock was adjusted to a working concentration of 2 x 10⁷ heterophils/mL for the oxidative burst and degranulation assays. Heterophil purity was typically >95% and was determined by using a commercially available Wright-Giemsa stain (Sigma-Aldrich). Heterophil viability was determined by a commercially prepared trypan blue solution (Sigma-Aldrich) and was always >95%.

Bacterial Preparation
Chicken cecal isolates were previously screened for bactericidal activity against C. jejuni and Campylobacter coli (Bhaskaran et al., 2003). Ten of these isolates were selected for this study and were identified as Pediococcus pentosaceus (G1), Bacillus licheniformis (G2), Bacillus subtilis (G3), Bacillus subtilis (G4), Lactococcus lactis lactis (G5), Lactococcus lactis lactis (G6), Lactobacillus acidophilus (G8), Bifidobacterium longum (G9), Streptococcus anginosus (G10) and Streptococcus anginosus (G11). These isolates were subcultured 3 times for 24 h in fresh tryptic soy broth (Becton, Dickinson and Co., Franklin Lakes, NJ) at 42°C. Each isolate was enumerated by serial dilution and spread-plated onto tryptic soy agar (Becton, Dickinson and Co.). Isolates used for the in vitro study were killed by a 24-h incubation in 1% formalin in PBS at 4°C, as previously described by Farnell et al. (2003). The killed bacteria were washed and resuspended to a working concentration of 10⁸ cfu/mL in PBS. Isolates used in the in vivo study (G3, G6, and G8) were subcultured individually into 100 mL of tryptic soy broth, washed twice with PBS, and resuspended at a concentration of approximately 5 x 10⁸ cfu of viable bacteria per milliliter of PBS for oral gavage. Chicks were orally gavaged with 0.5 mL of 1 of these 3 viable isolates or PBS 24 h before heterophil isolation for the in vivo study.

Oxidative Burst Assay
Oxidative activity of heterophils was measured using a Cytofluor 2300 fluorescent plate reader (Millipore Corp., Bedford, MA) and an indicator of reactive oxygen species, DCF-DA (Molecular Probes Inc., Eugene, OR), as previously described (Xie et al., 2002). Heterophils (2 x 10⁷/mL) were preincubated for 30 min with agonists or RPMI at 42°C in a heated orbital shaker plate (Thermo-Forma, Marrietta, OH). Phorbol-12-myristate-13-acetate (2 µg/mL of heterophils, Calbiochem, La Jolla, CA) was used as a heterophil agonist for the in vivo study and formalin-killed bacteria were used as agonists for the in vitro study. Alternatively, an equivalent volume of RPMI was added for the negative control treatments. Immediately after the preincubation period, DCF-DA (0.2 mg/mL) was added (125 µL), and samples were then mixed and aliquoted (8 replicates per sample) into a clear 96-well flat-bottomed plate. Oxidative burst was measured (excitation – 485/emission – 530) every 15 min for 90 min at 42°C in a fluorescent plate reader.

Degranulation Assay
Degranulation was monitored by quantifying ß-D glucuronidase released into the heterophil supernatant, as previously described (Dewald and Baggiolini, 1986; Lowry et al., 2005). Briefly, heterophils (2 x 10⁷/mL) were preincubated at 42°C for 60 min with agonists or RPMI in a heated orbital shaker plate (Thermo-Forma). Opsonized zymosan (2 mg/mL of heterophils; MP Biomedicals Inc., Aurora, OH) was used as an agonist for the in vivo study, and formalin-killed bacteria were used independently as agonists for the in vitro study. Alternatively, an equivalent volume of RPMI was added for the negative control treatments. After preincubation, cell suspensions were centrifuged and supernatants were collected and then aliquoted (4 replicates per sample) into a clear 96-well flat-bottomed plate. The degranulation substrate, 4-methylumbelliferyl-ß-D glucuronide (Calbiochem), was added to each sample and incubated in the dark at 42°C for 4 h. The reaction was stopped after the incubation period with a stop solution and immediately measured (excitation – 355/emission – 460) in a fluorescent plate reader (Cytofluor 2300).

Statistical Analysis
Relative fluorescence data were analyzed by ANOVA using the SAS (SAS Institute, 2000) GLM program. Treatment means were partitioned by LSMEANS analysis. A probability of P < 0.05 was required for statistical significance. Relative fluorescence data were presented in this paper as treatment means with SEM for each individual replicate.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The gastrointestinal tract is a selective barrier that allows for the efficient transfer of water and nutrients, but it must also prevent absorption of toxic substances and invasion by microbes. The gastrointestinal tract is a dynamic ecosystem containing up to 10¹¹ cfu of bacteria per gram of gut contents (Apajalahti et al., 2004). Microflora are continuously surveyed by M cells, present in the Peyer’s patches of the avian gastrointestinal tract (Kajiwara et al., 2003), to determine whether these microbes are pathogenic or nonpathogenic. Once this determination is made, the immune system can coordinate the appropriate immune response to pathogenic organisms that may be present. Neutrophils, the mammalian equivalent to an avian heterophil, have been demonstrated to sample bacteria within the lumen from a basolateral position by emitting pseudopodia (Sansonetti, 2004). It is possible that avian heterophils may function similarly. Heterophils are an important effector cell in avian immunity, because birds with impaired heterophil function have been shown to have significantly increased levels of organ invasion by Salmonella enteritidis when compared with challenged control birds with normal heterophil function (Kogut et al., 1993). Heterophil function can be increased by the oral administration of microbial products such as ß-glucan in chickens (Lowry et al., 2005). It is likely that avian heterophils may be similarly stimulated by changes in gut microflora initiated by changes in available nutrients (prebiotics) or the addition of pro-biotic bacteria.

The administration of probiotics has been previously shown to increase humoral and cell-mediated immunity in chickens (Koenen et al., 2004a,b). We hypothesized that probiotics would also stimulate avian heterophil function. The first study was designed to screen for pro-biotic isolates that increased heterophil oxidative burst and degranulation. We expected that only a few isolates would be immunostimulatory. Interestingly, each of our 10 probiotic isolates significantly increased oxidative burst and degranulation in 1 or more of the in vitro repetitions (Figure 1). However, the level of immune stimulation observed was inconsistent for some isolates among trials. These differences may be attributed to differences in the age (Peebles et al., 2001), genetics (Swaggerty et al., 2004), and environmental stressors (Mashaly et al., 2004) of breeder flocks producing the chicks. Additionally, immune factors passed on to the breeder hen’s progeny (Ward, 2004), changes in vaccination strategies (Barman et al., 2005), and stressors at the hatchery (Ernst et al., 1984) may have also affected our results.

View larger version (46K): [in this window] [in a new window]   Figure 1. Effect of formalin-killed probiotics on broiler heterophil function in vitro. Heterophils were isolated from the pooled peripheral blood of 100 unchallenged neonatal broiler chicks. These cells were aliquoted into individual groups and treated with 1 of 10 formalin-killed probiotic isolates (G1 to G11), or an equal volume of Roswell Park Memorial Institute 1640 media was used as a negative control (Con) treatment. Oxidative burst (panels A to C) and degranulation (panels D to F) assays were conducted to evaluate heterophil function. Each row of figures represents 1 collection day, i.e., panel A (oxidative burst) and panel D (degranulation) were conducted from the same pool of heterophils isolated from the same day. Heterophils were preincubated with their respective treatments for 30 (oxidative burst; n = 8) or 60 (degranulation; n = 4) min before being utilized in the functional assays. Each replicate was conducted on a different collection day for a total of 3 collection days. Data is presented as the mean relative fluorescence with the SEM. Treatments that were significantly different (P < 0.05) from the respective controls are indicated by an asterisk.

View larger version (43K): [in this window] [in a new window]   Figure 2. Effect of the oral administration of probiotics on broiler heterophil function. Broiler chicks (50 birds/isolate) were orally gavaged with approximately 2.5 x 108 cfu of 1 of 3 probiotic isolates (G3, G6, G8) selected from the in vitro screening procedure or PBS. Oxidative burst (panels A and B) and degranulation (Panels C and D) assays were conducted to evaluate heterophil function. Heterophils were isolated from the pooled peripheral blood of 50 neonatal broiler chicks from each group for the oxidative burst (n = 8) and degranulation (n = 4) assays. An equal volume of Roswell Park Memorial Institute (RPMI) 1640 media was used as a negative control (Con) treatment. Phorbol myristate acetate (PMA) and opsonized zymosan (OZ) were used as agonists for the oxidative burst and degranulation assays, respectively. Heterophils were preincubated with their respective treatments for 30 (oxidative burst) or 60 (degranulation) min before being utilized in the functional assays. Each replicate was conducted on a different collection day, for a total of 2 collection d. Each row of figures represents 1 collection day, i.e., panel A (oxidative burst) panel C (degranulation) were conducted with the same pool of heterophils isolated from the same day. Data is presented as the mean relative fluorescence with the SEM. Treatments that were significantly different (P < 0.05) from the respective controls are indicated by an asterisk.

The majority of probiotic research has focused on reductions in pathogen colonization and is often credited to competition for nutrients and binding sites (Clancy, 2003), but the actual mechanisms involved with ‘the Nurmi effect’ are still poorly understood (Nava et al., 2005, Donoghue et al., 2006). The importance of gut microflora in stimulating the mucosal immune system is only recently being studied in mammals, and little research is currently available for poultry. Researchers are making progress in elucidating the mechanisms involved, and immunostimulatory probiotics are now labeled as "immunobiotics" (Clancy, 2003). These data suggest that the oral administration of probiotics can stimulate heterophil oxidative burst and degranulation in chickens. To our knowledge, this is the first report of using probiotics to stimulate innate immunity in chickens. Heterophil oxidative burst and degranulation were focused on in this paper; however, other parameters need to be examined, such as phagocytosis, killing, cytokine expression, mucin secretion, and defensin secretion in the gut. These data and others (Koenen et al., 2004a,b) suggest that mucosal immunity can be enhanced by the oral administration of probiotics. The practical application of immunobiotics would be to enhance gut health, thereby decreasing disease and enhancing productivity.

	ACKNOWLEDGMENTS

 
We thank Wally McDonner and Sonia Tsai of the Poultry Production and Product Safety Research Unit for their excellent technical support. Additionally, this research was funded by a grant from the Arkansas Biosciences Institute, Jonesboro.

Received for publication May 2, 2006. Accepted for publication July 12, 2006.

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