Cytokine 53 (2011) 363369

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Cytokine
journal homepage: www.elsevier.com/locate/issn/10434666

Modulation of chicken macrophage effector function by TH1/TH2 cytokines

Haiqi He , Kenneth J. Genovese, Michael H. Kogut
Southern Plain Agricultural Research Center, USDA-ARS, 2881 F&B Road, College Station, TX 77845, United States

a r t i c l e

i n f o

a b s t r a c t
Regulation of macrophage activity by TH1/2 cytokines is important to maintain the balance of immunity to provide adequate protective immunity while avoiding excessive inammation. IFN-c and IL-4 are the hallmark TH1 and TH2 cytokines, respectively. In avian species, information concerning regulation of macrophage activity by TH1/2 cytokines is limited. Here, we investigated the regulatory function of chicken TH1 cytokines IFN-c, IL-18 and TH2 cytokines IL-4, IL-10 on the HD11 macrophage cell line. Chicken IFN-c stimulated nitric oxide (NO) synthesis in HD11 cells and primed the cells to produce signicantly greater amounts of NO when exposed to microbial agonists, lipopolysaccharide, lipoteichoic acid, peptidoglycan, CpG-ODN, and poly I:C. In contrast, chicken IL-4 exhibited bi-directional immune regulatory activity: it activated macrophage NO synthesis in the absence of inammatory agonists, but inhibited NO production by macrophages in response to microbial agonists. Both IFN-c and IL-4, however, enhanced oxidative burst activity of the HD11 cells when exposed to Salmonella enteritidis. IL-18 and IL-10 did not affect NO production nor oxidative burst in HD11 cells. Phagocytosis and bacterial killing by the HD11 cells were not affected by the treatments of these cytokines. Infection of HD11 cells with S. enteritidis was shown to completely abolish NO production regardless of IFN-c treatment. This study has demonstrated that IFN-c and IL-4 are important TH1 and TH2 cytokines that regulate macrophage function in chickens. Published by Elsevier Ltd.

Article history: Received 10 June 2010 Received in revised form 18 October 2010 Accepted 6 December 2010 Available online 3 January 2011 Keywords: Cytokine Nitric oxide Oxidative burst Salmonella Chicken macrophage

1. Introduction Macrophages are phagocytic mononuclear cells that play a crucial role in both the innate and adaptive immune system. They are originated from bone marrow, entering the blood circulation as monocytes and subsequently differentiating into macrophages upon migration to various tissues. As the central component of the innate immune system, macrophages are procient in detecting, engulng, and ingesting invading pathogens and create an inammatory milieu through the secretion of various proinammatory mediators to control and resolve infections. Furthermore, as antigen-presenting cells (APCs), macrophages participate in and facilitate the development of adaptive immunity through antigen processing, presentation, and by expressing co-stimulatory molecules [1,2]. Macrophages express an array of innate immune receptors, also called pattern recognition receptors (PRRs), including the extensively studied Toll-like receptors (TLRs) [3]. Through these receptors, macrophages recognize pathogens and pathogen-associated molecular patterns (PAMPs) and activate cellular signaling cascades, leading to the production of antimicrobial and proinammatory immune mediators. These immune mediators include, but are

Corresponding author. Tel.: +1 979 260 3771; fax: +1 979 260 9332.
E-mail address: haiqi.he@ars.usda.gov (H. He). 1043-4666/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.cyto.2010.12.009

not limited to, reactive oxygen species (ROS), nitric oxide (NO), and pro-inammatory cytokines and chemokines. In addition to microbes and PAMPs, macrophages can also be activated by TH1-type cytokines, with interferon-c (IFN-c) being the principle macrophage-activating TH1 cytokine [4]. However, in the TH2 immune milieu typically seen in disease caused by parasitic and extracellular pathogen infections as well as in stages of the resolution of inammation and wound repair, macrophages undergo alternative activation, in which macrophages display anti-inammatory and tissue repair properties [57]. The TH2-type cytokines IL-4 and IL-13 are known to play major roles in macrophage alternative activation [6]. IL-10 has been described as a true macrophageinactivating TH2 cytokine that suppresses proinammatory cytokine secretion and inhibits the expression of MHC class II and co-stimulatory molecules [8]. However, recent evidence suggests that IL-10 plays a role as a master regulator for negative feedback control of both TH1 and TH2 immune response [9]. Clearly, regulation of macrophage activity by TH1/2 cytokines is critically important to maintain a balanced immunity to provide an adequate TH1-mediated protective immunity while avoiding detrimental, excessive inammation. As their mammalian counterpart, avian macrophages play a central role in the defense against microbial infection and pathogenesis of infectious diseases caused by viruses [1012], bacteria [1315], and parasites [16,17]. It is also well established that chicken macrophages, when exposed to pathogens and PAMPs,

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are activated to produce proinammatory cytokines and chemokines and antimicrobial ROS and NO [11,1821]. However, the regulatory effects of cytokines, particularly the TH2 cytokines, on the innate immune functions of chicken macrophages remain unclear. The objective of the present study was to evaluate the regulatory effect of IFN-c and IL-18, representing the TH1 cytokines, and IL4 and IL-10, representing the TH2 cytokines, on the effector functions of the chicken macrophage cell line HD11. These effector functions include phagocytosis, oxidative burst activity that produces ROS, and production of NO in response to PAMP stimulation in vitro. The effect of these cytokines on bacterial killing activity of the HD11 cells was also evaluated. 2. Materials and methods 2.1. Reagents Recombinant chicken cytokines IL-4, IL-10, IL-18, and IFN-c were obtained from Kingsher Biotech (St. Paul, MN) and used as instructed. Lipopolysaccharide (LPS, Salmonella minnesota), lipoteichoic acid (LTA, Staphylococcus aureus), peptidoglycan (PGN, Staphylococcus aureus), and synthetic dsRNA analog poly I:C were purchased from InVivoGen (San Diego, CA). Synthetic phosphorothioate-modied CpG-ODN (50 -GTC GTT GTC GTT GTC GTT-30 ) [19] were purchased from TriLink BioTechnologies (San Diego, CA, USA). All media and additives for cell culture were purchased from Sigma (St. Louis, MO). 2.2. Cell culture The HD11 cells, an avian macrophage cell line, were maintained in Dulbeccos Modied Eagles Medium (DMEM) containing 10% chicken serum, antibiotics (100 U penicillin/ml and 100 lg streptomycin/ml), and 1.5 mM L-glutamine at 39 C, 5% CO2, and 95% humidity. For cell stimulation assays, a 100 ll aliquot of cell suspension (2 106 cells/ml) was seeded into each well of a round-bottom 96-well plate and allowed to grow to about 85% conuence ($36 h). 2.3. Nitrite assay Prior to stimulation, the cells were replaced with fresh medium. To measure the direct stimulatory effects of cytokines on NO production in HD11 cells, the cells were stimulated without or with specied cytokines for 20 h. To evaluate the effects of cytokines on NO production of HD11 cells in response to microbial agonist stimulations, the cells were pre-incubated without or with specied cytokine at varying concentrations for 4 h and then stimulated with specied agonists for an additional 20 h in a nal volume of 200 ll culture per well. Nitrite, a stable metabolite of nitric oxide, produced by activated macrophages was measured by the Greiss assay [22]. Briey, an aliquot of 100 ll culture supernatant from each well was transferred to the wells of a new at-bottom 96-well plate and combined with 50 ll of 1% sulfanilamide and 50 ll of 0.1% naphthylenediamine (both were prepared in 2.5% phosphoric acid solution). After 10 min incubation at room temperature, the nitrite concentration was determined by measuring optical density (OD595) of each well using a SPECTRA MAX microplate reader (Molecular Devices, Sunnyvale, CA). Sodium nitrite (Sigma) was used as a standard to determine nitrite concentrations in the cell-free medium. 2.4. Oxidative burst assay Production of ROS by HD11 cells during oxidative burst was measured by oxidation of 20 ,70 -dichlorouorescin-diacetate

(DCFH-DA) to uorescent DCF as described [23]. HD11 (2 105 cells) was seeded into each well of a at-bottom 96-well black plate and allowed to grow for about 36 h. To determine the direct effect of cytokines on HD11 oxidative burst, the cells were replaced with fresh medium without chicken serum and incubated without or with specied cytokines at varying concentrations for 1 h in the presence of 10 lg/ml of DCFH-DA at 39 C in 5% CO2 and 95% humidity. To determine the effect of cytokines on HD11 cells oxidative response to Salmonella enteritidis (SE) stimulation, the cells were preincubated without or with specied cytokines for 4 h and then stimulated with SE for 1 h. The SE (NVSL#97-11771) used in the present study was a primary poultry isolate obtained from National Veterinary Services Laboratory (Ames, IA, USA). The relative uorescent units (RFU) were measured (485/530 nm) at the end of incubation using a uorescence microplate reader (Genios Plus Plate Reader, TECAN U.S. Inc., NC, USA).

2.5. Phagocytosis assay HD11 cells (2 105) were seeded into each well of a at-bottom 96-well black plate and allowed to grow for about 36 h. The cells were replaced with fresh medium and incubated without or with specied cytokines at varying concentrations for 6 h. Following pretreatment with cytokines, the phagocytosis activity of the cells was analyzed by measuring the uptake of uorescein-labeled Escherichia coli (K-12 strain) using the Vybrant Phagocytosis Assay Kit from Molecular Probes (Eugene, OR) according to the manufacturers instruction.

2.6. Bacterial killing assay The survival of SE in cytokine treated HD11 was determined by a gentamicin protection assay. HD11 cells (2 106) were seeded into each well of a 12-well plate and allowed to grow over to 85% conuence ($36 h). To evaluate bacteria survival in HD11 cells, four replicate wells of a 12-well plate were treated with or without specied cytokines for 6 h and then infected with SE for 1 h at 39 C in a 5% CO2 humidied incubator. At 1 h post-infection (pi), cells were washed with DMEM medium, treated with 100 lg/ ml of gentamicin sulfate for 1 h to kill extracellular bacteria, washed twice, and continued to culture for 24 h in the presence of 25 lg/ml of gentamicin sulfate. For enumeration of intracellular viable bacteria at 24 h pi, the infected cells were lysed in 1 ml of 1% Triton X-100. Aliquots of 100 ll of serial 1:10 dilutions were spread on Difco xylose-lysine tergitol 4 (XLT4) agar (BD, Franklin Lakes, NJ) plates, containing 25 lg/ml novobiocin and 20 lg/ml naladixic acid. The plates were incubated at 39 C for 24 h and colony forming units (CFU) were determined.

2.7. Comparison of live SE and heat-killed SE (HKSE) on macrophage NO production HD11 cells (5 105) were seeded into each well of a 24-well and allowed to grow for about 36 h. The cells were preincubated with IFN-c for 6 h or stimulated with CpG-ODN (2 lg/ml) for 3 h as indicated. Following pretreatments, the cells were replaced with medium containing no serum and antibiotics, equal amount ($1 108 CFU) of live SE or heat-killed SE (HKSE, prepared by incubating bacteria at 75 C for 15 min) were added to the designated wells, and incubated for 2 h. After incubation, the medium was removed and replaced with completed medium containing 100 lg/ml of gentamicin to kill extracellular live SE and the cells were incubated for an additional 20 h in the presence of CpGODN (2 lg/ml) or IFN-c (400 ng/ml) as indicated.

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2.8. Data analysis Data were representative of three independent experiments conducted on different dates. Within each experiment, there were four replicate assays for each treatment. Data were analyzed by one way ANOVA followed by multiple comparisons (Tukey test) using SigmaStat software (Jandel Scientic, San Rafael, CA). The value of p < 0.05 was considered to be signicant. 3. Results 3.1. Induction of NO production by IL-4 and IFN-c NO production by HD11 cells in response to cytokine treatments was determined to evaluate the stimulatory effect of the TH1 cytokines, IL-18 and IFN-c and TH2 cytokines, IL-4 and IL-10, on HD11 cells. Within the concentration range specied, both IFN-c and IL-4 induced dose-dependent production of NO, while IL-10 and IL-18 displayed no stimulatory activity in HD11 cells (Fig. 1). Surprisingly, IL-4 was found to be a more potent NO inducer than IFN-c, signicantly higher NO production was observed when IL-4 was used to treat the cells. 3.2. Up-regulation of microbial agonist induced NO production by IFNc, but not by IL-18 Preincubation with chicken IFN-c signicantly up-regulated NO production in response to stimulations with TLR agonists, LPS, LTA, PGN, CpG-ODN, and poly I:C in HD11 cells (Fig. 2). The priming effect of IFN-c on microbial agonist-induced NO production in HD-cells was clearly demonstrated and this effect was more significant when low concentrations of microbial agonists were used. These results indicate that HD11 cells were primed by IFN-c to become more sensitive and efcient in effector cell function. IL-18, however, showed no effect on TLR agonist stimulated NO production in HD11 cells (Data not shown). 3.3. Down-regulation of microbial agonists induced NO production by IL-4, but not by IL-10 In contrast to the above observation that IL-4 stimulated NO production of HD11 cells, IL-4 pretreatment demonstrated a strong inhibitory effect on microbial agonist stimulated NO production in

HD11 cells (Fig. 3). The reduction of NO production was more clearly exhibited at the treatments where low concentration of IL-4 was used; whereas, at high concentrations of IL-4, increased NO production can be attributed to the stimulatory effect of IL-4. The results clearly demonstrated the bi-directional regulatory activity of IL-4, in which IL-4 itself stimulates NO production while simultaneously inhibiting NO response of HD11 cells to microbial agonists. Furthermore, these observations strongly suggest that

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Fig. 1. Induction of NO production by IL-4 and IFN-c. HD11 cells were stimulated without or with specied cytokines at various concentrations as indicated for 20 h at 39 C in a 5% CO2 humidied incubator and then the nitrite accumulated in the medium was measured. The symbol () indicates signicant (p 6 0.5) difference between the untreated and the treated cells.

Fig. 2. Up-regulation of microbial agonist induced NO production by IFN-c. DH11 cells were preincubated without or with chicken recombinant IFN-c at concentrations of 100, 200, and 400 ng/ml for 4 h, and followed by stimulation with microbial agonists, LPS, LTA, PGN, CpG-ODN, and poly I:C, at concentration as indicated for an additional 20 h at 39 C in a 5% CO2 humidied incubator. Accumulated nitrite in the medium was then measured. The symbol () indicates signicant (p 6 0.5) difference between the un-treated and the IFN-c treated cells stimulated with agonists. The symbol (#) indicates signicant (p 6 0.5) difference between the untreated and the IFN-c treated cells without agonist stimulation.

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IL-4 and TLR agonists employ different cell signaling mechanisms for NO production. Surprisingly, IL-10 demonstrated no inhibitory effect on TLR agonist-induced NO production in HD11 cells (data not shown).

3.4. Enhancing oxidative burst response to bacteria by IL-4 and IFN-c The effect of these selected cytokines on the oxidative burst of HD11 cells in response to SE was evaluated after preincubation with cytokines. All cytokines used in this study did not show a direct stimulatory effect for oxidative burst in HD11 cells (Fig. 4a). However, signicantly enhanced oxidative burst activity when exposed to SE was observed in cells pretreated with IL-4 or IFN-c (Fig. 4b). Neither IL-10 nor IL-18 was found to affect oxidative burst response of HD11 cells to bacterial stimulation. These results again demonstrate the proinammatory property of IL-4. 3.5. No effect on Phagocytosis and intracellular SE survival in HD11 cells by IL-4. IL-10, IL-18, and INF-c treatments Under the present experimental conditions, pretreatment of HD11 cells with any of the cytokines altered neither the phagocytosis of dye-labeled E. coli nor the survival of the intracellular SE after 24 h post infection (Fig. 5). 3.6. Attenuation of NO production in HD11 cells by SE infection

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To investigate whether SE infection attenuates macrophage NO production, HD11 cells, untreated or pretreated by either CpG-ODN or IFN-c, were stimulated with either live SE or FKSE. Signicant

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Fig. 3. Down-regulation of microbial agonists induced NO production by IL-4. DH11 cells were preincubated without or with chicken recombinant IL-4 at concentrations of 50, 100, and 200 ng/ml for 4 h and followed by stimulation with microbial agonists, LPS, LTA, PGN, CpG-ODN, and poly I:C, at concentrations indicated for an additional 20 h at 39 C in a 5% CO2 humidied incubator. Accumulated nitrite in the medium was then measured. The symbol () indicates signicant (p 6 0.5) difference between the un-treated and the IFN-c treated cells stimulated with agonists. The symbol (#) indicates signicant (p 6 0.5) difference between the untreated and the IFN-c treated cells without agonist stimulation.

Fig. 4. Enhancing oxidative burst response to S. enteritidis by IL-4 and IFN-c. HD11 cells were preincubated without or with specied cytokines for 4 h and then stimulated with SE for 1 h at 39 C in a 5% CO2 humidied incubator. Production of ROS by HD11 cells during oxidative burst was measured by oxidation of DCFH-DA to uorescent DCF. The changes of relative uorescent units (RFU) were recorded (485/530 nm) using a uorescence microplate reader. [A]. Oxidative burst by HD11 cells stimulated with cytokines and 4-beta-phorbol 12-myristate 13-acetate (PMA) as positive control. [B]. Oxidative burst by HD11 cells stimulated with SE after pretreatment with cytokines. The symbol () indicates signicant (p 6 0.5) difference between the un-treated and the treated cells.

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Fig. 6. Attenuation of NO production in HD11 cells by SE infection. The HD11 cells were pretreated without or with CpG-ODN (2 lg/ml) for 3 h or IFN-c (400 ng/ml) for 6 h and then incubated with live SE or heat-killed SE for 2 h. After incubation, the cells were replaced with fresh medium containing 100 lg/ml of gentamicin sulfate to kill extracellular live SE and continued to incubate for an additional 20 h in the presence or absence of CpG-ODN (2 lg/ml) or IFN-c (400 g/ml) as indicated. The symbol () indicates signicant (p 6 0.5) difference between the untreated and the treated cells.

2
Interferon-c is a hallmark TH1cytokine and a key cytokine in the activation of macrophages. IFN-c was initially described as produced only by CD4+ TH1 lymphocytes, CD8+ cytotoxic lymphocytes, and NK cells [27]. However, increasing evidence suggests that the professional APCs, macrophages and dendritic cells, can also produce IFN-c and are regulated by IFN-c in an autocrine fashion [2831]. Similarly, upon exposure to microbial agonists, chicken HD11 cells also express IFN-c [32]. Chicken IFN-c expression, as seen in mammals, has been associated with viral, bacterial, and parasitic infections; and it is well established that IFN-c plays a critical role in control of infection and elimination of pathogens [3336]. Activation of macrophages by IFN-c plays a signicant role in the TH1 mediated protection. In the present study, chicken IFN-c was found to prime chicken macrophage cells and exerted a synergistic effect to produce signicantly higher levels of ROS and NO when exposed to bacteria and microbial molecules commonly found in both bacteria and virus. As the results indicate, chicken IFN-c itself is not a strong inducer for ROS and NO production in macrophages and therefore only acts as an immune modulator to prime macrophages, potentiating the ROS and NO response to microbial agonists. Although similar effect of IFN-c have been reported in CpG DNA- and LPS-activated mammalian macrophages [37,38] and LPS-activated chicken macrophages [14], our study further found that IFN-c programs the HD11 cells to produce signicantly higher NO when exposed to LTA and PGN from Gram-positive bacteria, synthetic microbial DNA CpG-ODN, and viral double-stranded RNA analog poly I:C. The results suggest that, by enhancing the production of antimicrobial, proinammatory ROS and NO, IFN-c plays a key role in macrophage-mediated protection against infections caused by viruses, bacteria, and parasites in chickens. In contrast to ROS and NO response, phagocytosis capacity of HD11 cells for bacteria was not affected by IFN-c treatment. Even though IFN-c up-regulated ROS and NO production, IFN-c activated HD11 cells were unable to kill intracellular SE. In line with these results, previous studies also found that IFN-c treatment did not affect either phagocytosis or survival of Salmonella bacteria in HD11 cells [14]. On the contrary, IFN-c activated primary macrophage cells are apparently able to more effectively kill intracellular SE [39]. The discrepancy of intracellular SE killing ability between primary macrophage and HD11 cells remains unclear. Despite it is well documented that IFN-c activates

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Fig. 5. Treatments with IL-4 IL-10, IL-18, and INF-c did not affect phagocytosis activity and intracellular SE survival in HD11 cells. The HD11 cells were pretreated without or with specied cytokines at varying concentrations for 6 h. [A]. Phagocytosis. The phagocytosis activity of the cells was analyzed by measuring the uptake of uorescein-labeled E. coli as described in the Section 2. The relative uorescent units (RFU) changes were recorded (485/530 nm) using a uorescence microplate reader. [B]. Intracellular viable SE. HD11 cells were pretreated without or with specied cytokines for 6 h and then infected with SE ($1 108 CFU) for 1 h, followed by washing with sterile PBS, and killing extracellular bacteria with 100 lg/ ml of gentamicin sulfate for 1 h at 39 C in a 5% CO2 humidied incubator. After gentamicin treatment, the cells were replaced with fresh medium containing 25 mg/ml of gentamicin and continued to culture for 24 h. At 24 h pi, the cells were lysed in 1% Triton X-100, viable SE was cultured at 39 C for 24 h, and colony forming units (CFU) were determined.

amounts of NO were produced in macrophages treated with CpG-ODN, IFN-c, and HKSE, with CpG-ODN and HKSE stimulating signicantly greater amount of NO production than IFN-c. However, live SE completely abolished NO production in macrophages regardless pretreatment with CpG-ODN or IFN-c (Fig. 6). 4. Discussion The HD11 cell line is an avian myelocytomatosis virus (MC29) transformed chicken macrophage-like cell line which has been characterized to strongly express Fc receptors, phagocytic capacity and macrophage cell surface antigens [24]. HD11 cells have been widely used for in vitro study of immune function as chicken macrophages [14,19,21,25,26]. In the present study, HD11 cells were used to elucidate the regulatory function of chicken TH1 and TH2 cytokines on the innate immune response of chicken macrophages to microbial agonists. Due to the importance of ROS and NO as antimicrobial arsenals that macrophage cells use to control and eliminate pathogens, this study has focused on the regulation of the ROS and NO response of chicken macrophage HD11 cells to microbes and their conserved PAMP by representative TH1 and TH2 cytokines.

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mammalian macrophage cells to kill intracellular Salmonella [40,41], evidence also indicates that S. typhimurium is able to resist antimicrobial activity and survive within IFN-c activated macrophages [42]. The ability of Salmonella to survive in the hostile intracellular environment depends on complex mechanisms to evade ROS, NO, and antimicrobial peptide mediated destruction by macrophages [4245]. It was no surprise to nd that live SE infection completely abolished NO production in HD11 cells regardless IFN-c treatment, while heat-killed SE induced signicant amount of NO production. Additionally, our study found that SE infection also suppressed microbial agonist stimulated NO production in HD11 cells. These results provide evidence supporting that SE is able to survive in IFN-c activated HD11 cells by neutralizing macrophage antimicrobial activity. Interleukin-18 structurally belongs to the IL-1 family and exerts its TH1 immune function in the host defense against infection through the induction of IFN-c [46]. IL-18 is produced by various immune and non-immune cells, but primarily by macrophages [47]. Previous studies have shown that recombinant chicken IL-18 stimulates NO production in chicken splenic lymphocytes [48] and enhanced IFN-c mRNA expression, which in turn leads to IFN-c-mediated induction of NO by macrophages [49]. However, results from the present study indicate chicken IL-18 does not directly induce nor prime ROS and NO synthesis in chicken macrophage cells. There is no direct evidence to suggest IL-18 can induce ROS and NO synthesis in mammalian macrophages, despite data showing that IL-18 stimulates NO production in peripheral blood mononuclear cells [50]. Therefore, IL-18 may exert effector function through IFN-c, but not directly affect chicken macrophage innate function as described in this study. Interleukin-4 is produced mainly by CD4+ TH2, CD8+ T cells, NKT cells, and granulocyte, basophils, eosinophils, and mast cells. Elevated levels of IL-4 are typically associated with tissue injury and TH2 diseases caused by infection with parasites or extracellular pathogens [7]. IL-4, along with IL-13, is a major player in macrophage alternative activation, in which IL-4 antagonizes IFN-c function and suppresses inammatory immune response [57]. In chickens, the immune regulatory function of IL-4 is unknown, although increased expression of IL-4 has been reported in infections caused by Mareks disease virus [51] and avian parasite coccidia [52]. In these cases, increased IL-4 expression most likely serves to suppress the TH1 immune response. In the current study, chicken IL-4 surprisingly stimulated NO production in chicken macrophages. Chicken IL-4 exhibited greater NO stimulatory capability than IFN-c in the absence of microbial PAMPs that are known to stimulate NO synthesis in HD11 cells. IL-4 has not been reported to stimulate NO production in mammalian macrophages, but has been documented in other types of immune-related cells including rat dendritic cell [53], mouse eosinophils [54] and equine pulmonary artery endothelial cells [55]. The current nding of the stimulatory activity of chicken IL-4 for NO production in macrophage HD11 cells was a new addition to the IL-4 function. In this study, IL-4 was also found to strongly suppress macrophage NO response to stimulation with microbial agonists found in both bacteria and viruses. This inhibitory activity is the classical function of IL-4, which has been extensively documented in mammalian macrophages. Together, these results demonstrate that chicken recombinant IL-4 is a dual-function cytokine that activates macrophage NO synthesis in the absence of inammatory agonists, but inhibits NO production in response to microbial agonist stimulations. This seemly conict effect on NO production suggest that IL-4 induce NO production via signaling pathway differing from the one mediating NO production stimulated by microbial agonists, otherwise the inhibitory effect of IL-4 would self nullify the stimulatory effect. The ability to induce NO while concomitantly suppressing NO production caused by inammatory microbial agonists

underscores the important role of IL-4 in maintaining immune homeostasis and control of inammation. However, the phenomenon of chicken IL-4 with both stimulatory and inhibitory effect on NO production in the macrophage cells cannot be readily explained and its mechanism warrants further investigation. In contrast to down-regulation of NO response, IL-4 potentiates ROS production by macrophages when exposed to bacteria. This new nding further demonstrates bi-directional pro- and anti-inammatory characteristics of chicken IL-4 on chicken macrophage cells. IL-10 was initially described as a cytokine synthesis inhibiting factor, produced mostly by CD4+ TH2, macrophages, dendritic cells, B cells and CD8+ T cells. It acts in an autocine fashion on macrophages to suppress proinammatory cytokine secretion and inhibits the expression of MHC class II and co-stimulatory molecules [8]. New evidence indicates that IL-10 is a master regulator acting as a negative feedback control mechanism to prevent over expression of both TH1 and TH2 immune response [9]. Chicken IL-10 has recently been identied and was found to inhibit IFN-gamma synthesis by mitogen-activated lymphocytes [56]. High levels of IL-10 expression have been associated with chickens susceptible to Eimeria tenella infection [56]. Chickens infected with Mareks disease virus were also found to express signicantly elevated level of IL-10 in addition to IL-4 and IL-13 during the lytic phase of infection [48]. These results provide clear evidence that IL-10 plays a signicant role in viral and parasitic pathogenesis by suppressing the TH1 protective immune response. However, unlike mammalian IL-10, which down-regulates iNOS activity in macrophages [57,58], chicken IL-10 has no inhibitory activity on NO and ROS production in HD11 cells in response to microbial stimulation. The discrepancy between the current results and well-documented anti-inammatory activity of mammalian IL-10 cannot be readily explained and should be further investigated. In summary, we have demonstrated the immune regulatory function of TH1 cytokine IFN-c and TH2 cytokine IL-4 on chicken macrophage effector function. Chicken IFN-c stimulates NO synthesis in chicken macrophage HD11 cells and potentiates HD11 cells to produce greater amounts of NO and ROS in response to microbial agonist stimulations. Chicken IL-4 exhibited a bi-directional regulatory function; activating macrophage NO synthesis in the absence of inammatory agonists, but inhibiting NO production in response to microbial agonist stimulations. Unlike IFN-c and IL-4, chicken cytokines IL-18 and IL-10 have no effect on both NO and ROS production in HD11 cells. None of the cytokines tested changed the phagocytosis activity or the intracellular survival of SE in HD11 cells. Additionally, we also demonstrated that SE is able to suppress NO production and survive in IFN-c activated HD11 cells.

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