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Poult Sci 2006. 85:1907-1911
© 2006 Poultry Science Association
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IMMUNOLOGY, HEALTH, AND DISEASE: Research Note

Cytokine Expression in Chicken Peripheral Blood Mononuclear Cells after In Vitro Exposure to Salmonella enterica serovar Enteritidis1

M. G. Kaiser*, J. H. Cheeseman*, P. Kaiser{dagger} and S. J. Lamont*,2

* Department of Animal Science, Iowa State University, Ames 50011; and {dagger} Institute for Animal Health, Compton, Berkshire RG20 7NN, UK

2 Corresponding author: sjlamont{at}iastate.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Cytokines are secreted proteins involved with cell recruitment and regulation of both innate and adaptive immune responses. They are essential for an effective host immune response to pathogens. The objective of this study was to determine the effect of Salmonella enterica serovar Enteritidis (S. Enteritidis) exposure and genetic line on cytokine mRNA expression level of cultured chicken peripheral blood mononuclear cells (PBMC). Interleukin-2, interleukin-6 (IL-6), CXCLi2, and transforming growth factor-ß4 (TGF-B4) messenger ribonucleic acid expression was measured by quantitative reverse transcription-PCR assays in PBMC from 3 chicken lines (broiler, Leghorn, Fayoumi) after in vitro exposure to S. Enteritidis. The PBMC were isolated from uninfected birds and cultured overnight. The next day, live pathogenic S. Enteritidis was added to half of the cultures. All cultures were harvested after 2 or 4 h of exposure. Exposure to S. Enteritidis downregulated IL-6, CXCLi2, and TGF-ß4 but not interleukin-2 mRNA expression. No significant genetic line or exposure time effects were detected. These findings demonstrate that exposure of chicken PBMC to S. Enteritidis can induce a rapid change in both proinflammatory (IL-6, CXCLi2) and antiinflammatory (TGF-ß4) cytokine gene expression.

Key Words: chicken • Salmonella Enteritidis • cytokine • expression • in vitro


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
In recent years, quantitative reverse transcription-PCR assays have been developed for the measurement of relative mRNA expression levels specific for many of the chicken cytokines (Kaiser et al., 2000; Jarosinski et al., 2002). These tools expand the ability to understand the host response to pathogens. As in mammals, chicken interleukin-2 (IL-2) is pivotal in T-cell activation and has recently been demonstrated to activate heterophils, which are comparable to mammalian neutrophils (Kogut et al., 2002). Chicken interleukin-6 (IL-6) has been confirmed to have similar functions as its mammalian counterparts, including a role in proinflammatory responses (Kaiser et al., 2000). An early stage of inflammation involves secretion of chemokine CXCLi2 (previously known as interleukin-8) as a chemotaxin for chicken heterophils (Kogut, 2002; Kaiser et al., 2005). Transforming growth factor-ß4 (TGF-ß4), the most commonly expressed transforming growth factor-ß isoform in the chicken, has gene sequence homology to mammalian transforming growth factor-ß1 (TGF-ß1; Jakowlew et al., 1997) and shares its antiinflammatory properties (Withanage et al., 2005). Chicken TGF-ß4 mRNA is expressed in response to exposure to Eimeria acervulina (Jakowlew et al., 1997; Choi et al., 1999), Salmonella enterica serovar Typhimurium (Beal et al., 2004; Withanage et al., 2005), and Salmonella enterica serovar Enteritidis (commonly known as S. Enteritidis; Kogut et al., 2003; Swaggerty et al., 2004).

The natural route of S. Enteritidis invasion occurs via oral exposure, after which the bacteria traverse the intestinal epithelial cell wall leading to an inflammatory response involving immune cell migration to the cecal lamina propria (van Immerseel et al., 2002). Either by invasion or through phagocytosis, intracellular S. Enteritidis in macrophages can lead to systemic infection (Gast and Benson, 1996; Desmidt et al., 1998). To begin to understand the complex immune response that occurs in systemic infections, the functions of isolated cells need to be examined (Kaiser et al., 2000; Swaggerty et al., 2004). In the present study, we evaluated effects of chicken genetic line, in vitro exposure to S. Enteritidis, and postexposure cell harvest time on peripheral blood mononuclear cell (PBMC) expression of IL-2, IL-6, CXCLi2, and TGF-ß4 mRNA.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Experimental Birds
Three adult chickens from each of 3 genetically diverse lines were used. These lines were a broiler line, which was established from an outbred breeder male line; a highly inbred (>99%) Leghorn (G-B2) line; and a highly inbred (>99%) Egyptian Fayoumi (M 15.2) line (Zhou and Lamont, 1999). All birds were given ad libitum access to water and feed that met or exceeded NRC (1994) requirements.

Cell Collection, Culture, and S. Enteritidis Exposure
Whole blood (20 mL) was collected via wing-vein puncture into an EDTA-containing tube. The blood was layered on 1077 Histopaque (1077-1, Sigma-Aldrich, St. Louis, MO) and spun at 400 x g for 30 min. Mononuclear cells were collected from the gradient interface, and plasma suspension combined and washed 3 times with antibiotic-free RPMI 1640 (R0883, Sigma-Aldrich). Cell viability and number were determined by trypan blue exclusion. Cells from each bird were plated into 12 individual wells of 6-well plates (3516, Corning Costar, Acton, MA) at 5 x 107 cells/mL in 5 mL of antibiotic-free RPMI 1640 supplemented with L-Gln (50 mM; G-5763, Sigma-Aldrich) and 10% fetal bovine serum (511150, Atlanta Biological, GA) and cultured overnight (41°C, 5% CO2). Salmonella Enteritidis phage type 13a, in log growth phase, was suspended in RPMI 1640 at 5 x 108 cfu/mL (Kaiser and Lamont, 2002). Either 100 µL of S. Enteritidis or culture medium was added to each well after overnight culture of the PBMC. The PBMC were harvested 2 and 4 h postexposure (Kaiser et al., 2000). There were 3 wells per bird per exposure treatment per postexposure cell harvest time.

RNA Isolation
Cells were harvested from each well by pipetting the medium up and down several times to suspend the non-adherent cells. The nonadherent cells and media were centrifuged at 800 x g for 2 min, and the resulting pellet was resuspended in 1 mL of Trizol (10296028, Invitrogen, Carlsbad, CA). Each Trizol-cell mixture was then added back to the same culture plate well to lyse and recover RNA from the adherent cells. The resulting Trizol mixture was extracted with chloroform then precipitated with isopropanol. The RNA pellet was briefly washed with 75% ethanol then resuspended in RNase-free water (10977-015, Invitrogen). The RNA concentration was determined by spectrophotometry and diluted to 50 ng/µL.

Quantitative Real-Time Reverse Transcription PCR
Expression levels of mRNA were determined for each individual bird in a 2 x 2 factorial design of postexposure times (2 and 4 h) and treatment (S. Enteritidis exposed and unexposed). Relative quantification of IL-2, IL-6, CXCLi2, and TGF-ß4 mRNA expression was conducted by real-time reverse transcriptase PCR (RT-PCR), using the QuantiTect SYBR Green RT-PCR system (204-243, Qiagen, Waltham, MA). The real-time RT-PCR protocol was modified from a previous report (Kaiser et al., 2000). The RT-PCR mixture consisted of 50 ng/µL of total RNA, 25 µL of QuantiTect SYBR Green Master Mix (Qiagen), primers, 0.5 µL of QuantiTect RT mix (Qiagen), and RNase-free water to a final volume of 50 µL. Primers were used at the following final concentrations: IL-2, 0.4 µM; IL-6, 0.2 µM; CXCLi2, 0.1 µM; TGF-ß4, 0.1 µM; and 28S, 0.6 µM. Primer sequences have been previously reported (Kaiser et al., 2000; Kogut et al., 2003), and primer sets were designed so that at least 1 primer annealed across an exon-exon boundary. Each PCR plate contained target genes and 28S rRNA (concentration standard) in triplicate, a serial dilution of target-specific RNA to generate a standard curve, and a no-template negative control. Real-time RT-PCR was carried out on an Icycler (Bio-Rad, Hercules, CA) with the following program: 1 cycle at 50°C for 30 min, 95°C for 15 min, followed by 40 cycles of 94°C for 15 s, 59°C for 30 s, and 72°C for 30 s. The cycle at which the sample amplicon reporter dye concentration crossed a preset threshold was recorded as the cycle threshold (Ct) value. To convey the inverse relationship between starting template concentration and Ct value, results were expressed and analyzed as a 40 – Ct value, and to adjust for template concentration and for PCR efficiency, the following equation was used:


Formula

where Mean 40 – Cttarget = the triplicate mean of 40 – Ct value; Slopetarget = the slope from the standard curve regression equation for the target gene; 28S df = the triplicate mean of 28S/overall mean for all 28S values within the experiment; and Slope28S = the slope from the standard curve regression equation for the 28S gene.

Statistical Analysis
The relative amount of mRNA expression of each gene (expressed as adjusted Ct value) was analyzed by GLM with JMP Version 5.1.1 software (SAS Institute, 2004). Fixed main effects included genetic line (broiler, Leghorn, Fayoumi), postexposure cell harvest time (2 h, 4 h), and treatment (S. Enteritidis exposure or nonexposure) with PCR plate (n = 6) as a random effect. Two-way interactions were included in the final model for a gene for all fixed main effects, for which the interaction P-value was ≤0.1. One data point (from TGF-ß4, a Fayoumi 2-h nonexposed sample) was excluded from analysis, because the adjusted Ct value exceeded twice the overall mean + SD. Tukey’s honestly statistical differences (HSD) test (SAS Institute, 2004) was performed for multiple comparisons of least square means if the main effect or interaction was significant. Differences were considered significant at P ≤ 0.05.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
There was no significant main effect of chicken genetic line or postexposure time of cell harvest on the cytokine mRNA levels. In vitro exposure of PBMC from the 3 chicken lines to S. Enteritidis significantly downregulated the mRNA expression levels of both proinflammatory IL-6 and CXCLi2 (Table 1Go). The least square mean of adjusted Ct value for IL-6 mRNA in nonexposed PBMC was 14.3, compared with 9.3 for S. Enteritidis-exposed PBMC, whereas values were 12.4 and 4.2, respectively, for CXCLi2 expression in nonexposed and exposed PBMC (Figure 1Go). The proinflammatory response, which includes expression of IL-6 and CXCLi2, is a key initial host immune defense against pathogens. The downregulation of mRNA for both of the proinflammatory cytokines may be a consequence of in vitro invasion of naïve PBMC by S. Enteritidis. Reduced mRNA expression may reduce protein expression, which would reduce the host inflammatory response during an in vivo infection. There are conflicting reports regarding proinflammatory cytokine responses to in vitro S. Enteritidis exposure. Expression of both IL-6 and CXCLi2 mRNA were upregulated in heterophils after S. Enteritidis exposure (Kogut et al., 2003), and exposure of primary chick kidney cells (CKC) to S. Enteritidis led to upregulation of IL-6 mRNA and downregulation of interleukin-1ß mRNA expression (Kaiser et al., 2000). The 2- and 4-h cell harvest times of the present study were based upon the detectable levels of cytokines observed at these times in the latter study. Upregulation of cecal CXCLi2 mRNA, along with other proinflammatory cytokines, has been observed as early as 6 h post-in vivo oral challenge with Salmonella Typhimurium (Withanage et al., 2004).


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  		Table 1. Genetic line, postexposure time, and treatment effects on peripheral blood mononuclear cell cytokine expression after in vitro exposure to Salmonella enterica serovar Enteritidis (S. Enteritidis; P-value)
 

Figure 1
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  		Figure 1. In vitro Salmonella enteritidis treatment effect on peripheral blood mononuclear cell cytokine mRNA expression levels (least square means). Bars within cytokine not sharing superscripts are significantly different, P ≤ 0.05 (GLM, SAS Institute, 2004). Ct = cycle threshold.

 
Salmonella Enteritidis-exposed PBMC expressed significantly less TGF-ß4 mRNA than nonexposed PBMC (Table 1Go), with adjusted Ct values of 11.3 and 12.6, respectively (Figure 1Go). Although TGF-ß4 mRNA levels may not necessarily equate to levels of bioactive TGF-ß4 protein, in the absence of bioassays specific for TGF-ß4, measuring TGF-ß4 mRNA levels is currently the only available method to reliably quantify this cytokine. Transforming growth factor-ß4 is the chicken homolog of mammalian TGF-ß1 (Jakowlew et al., 1997; Pan and Halper, 2003) and has antiinflammatory properties (Kogut et al., 2003; Secombes and Kaiser, 2003; Swaggerty et al., 2004). Because there is no evidence in the chicken genome sequence of, and there have been no confirming studies on, the originally reported chicken TGF-ß1, it is questioned as to whether this gene exists or if it was an erroneous assignment of gene identity. Mammals possess only 3 transforming growth factor-ß genes, and the established function of chicken TGF-ß4 as the mammalian TGF-ß1 counterpart would be better explained, because it is chicken TGF-ß1. Swaggerty et al. (2004) proposed a relationship between upregulation of TGF-ß4 mRNA expression and increased susceptibility to S. Enteritidis. Genetic line difference in TGF-ß4 mRNA expression has been reported in chickens exposed to the parasite E. acervulina, in which the resistant line had upregulated TGF-ß4 mRNA expression (Choi et al., 1999). There was also a significant random effect of PCR plate on TGF-ß4 expression levels, so PCR plate was included in the statistical model to adjust the analysis for this random effect.

There were no significant differences detected for expression of IL-2 mRNA in this study (Table 1Go). This does not preclude IL-2 playing a role in host immune response to S. Enteritidis at other times or in other tissues or cells. The experimental design of this study, with exposure of the PBMC to S. Enteritidis for a maximum of 4 h, was directed toward measuring an early innate immune response and not T-cell proliferative cytokines, such as IL-2. However, the IL-2 mRNA level was downregulated by in vitro S. Enteritidis exposure in primary CKC after 4 h of postexposure (Kaiser et al., 2000).

The only 2-way interaction at or near significance was genetic line X postexposure time, which affected IL-6 and TGF-ß4 RNA expression levels (Table 1Go). More statistically stringent analysis for multiple comparisons by Tukey’s HSD, however, found no significant differences among the 6 genetic line time x comparisons for IL-6 mRNA expression (data not shown). For combined data of exposed and nonexposed PBMC, TGF-ß4 mRNA expression levels from broilers at 2 h postexposure were significantly different from Fayoumi at 2 h postexposure, with no other genetic line X time comparisons differing as determined by Tukey HSD.

In the present study, both proinflammatory (IL-6, CXCLi2) and antiinflammatory (TGF-ß4) responses in PBMC were significantly downregulated as a result of S. Enteritidis exposure. The conventional paradigm for inflammatory responses is for inverse expression of proinflammatory and antiinflammatory cytokines (Stober et al., 1997), as was observed for in vitro experiments using heterophils isolated from commercial lines of birds that differed for resistance to Salmonella infection (Swaggerty et al., 2004). In vitro S. Enteritidis exposure of heterophils from outbred Rhode Island Red chickens, however, resulted in simultaneous upregulation of both proinflammatory (IL-6, CXCLi2) and antiinflammatory (TGF-ß4) cytokine mRNA expression (Kogut et al., 2003). Kogut et al. (2003) also reported opsonization-dependent regulation of proinflammatory interleukin-1ß mRNA expression, in which serum opsonization resulted in downregulation, and both nonopsonization and IgG opsonization resulted in upregulation. These varied findings underscore the usefulness of evaluating defined and diverse chicken genetic lines to understand the specific relationship of proinflammatory and antiinflammatory cytokine mRNA expression to pathogen exposure. They also suggest the value of measuring heterogeneous cell cultures in which cells produce cytokines within the context of the activity of other cell types. The current study suggests that the initial encounter with S. Enteritidis results in suppression of both proinflammation and antiinflammation immune response in PBMC.

The cytokines measured in the present study were expressed by PBMC at detectable levels without bacterial exposure. This baseline expression has been observed in heterophil (Kogut et al., 2003) and CKC cultures (Kaiser et al., 2000). It is not possible to determine, from these collective studies, if the cytokines are constitutively expressed by PBMC or if the expression is a result of the isolation and culture conditions.

Previous studies of cytokine mRNA expression in response to S. Enteritidis in vitro exposure used homogeneous cell cultures of isolated heterophils (Kogut et al., 2002, 2003; Swaggerty et al., 2004) or CKC (Kaiser et al., 2000). The present study evaluated cytokine mRNA expression of a heterogeneous PBMC cell population, providing an assessment of the combined response of diverse types of host cells to S. Enteritidis exposure and thereby expanding our understanding of cell interactions and avian cytokine function in host immunity.


   ACKNOWLEDGMENTS
 
We thank H. M. Opitz (University of Maine, Orono, ME) for providing the S. Enteritidis phage type 13a bacteria and J. McElroy (Iowa State University) for helpful discussion.


   FOOTNOTES
 
1 This work was supported by State of Iowa Funds, National Research Initiative Grant no. 2004-35205-14234 from the USDA Cooperative State Research, Education, and Extension Service and Research Grant US-3408-03 from the Binational Agriculture Research and Development Fund. Back

Received for publication December 16, 2005. Accepted for publication June 9, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
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Choi, K. D., H. S. Lillehoj, and D. S. Zalenga. 1999. Changes in local IFN-{gamma} and TGF-ß4 mRNA expression and intraepithelial lymphocytes after Eimeria acervulina infection. Vet. Immunol. Immunopathol. 71:263–275.[ISI][Medline]

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Jakowlew, S. B., A. Mathias, and H. S. Lillehoj. 1997. Transforming growth factor-ß isoforms in the developing chicken intestine and spleen: Increase in transforming growth factor- ß 4 with coccidian infection. Vet. Immunol. Immunopathol. 55:321–339.[ISI][Medline]

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