J. Anim. Sci. 2005. 83:E48-E56
© 2005 American Society of Animal Science
Interactive responses in gut immunity, and systemic and local changes in the insulin-like growth factor system in nursery pigs in response to Salmonella enterica serovar Typhimurium1,2
B. J. Johnson3,
S. S. Dritz,
K. A. Skjolaas-Wilson,
T. E. Burkey and
J. E. Minton
Kansas State University, Manhattan 66506
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Abstract
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In recent years, we have sought to understand how disparate endocrine and immune signals converge in response to Salmonella enterica serovar Typhimurium (ST) to affect growth and the IGF system in the nursery pig. The enteric pathogen ST interacts with gut epithelium to rapidly upregulate the chemoattractive chemokines IL-8 and chemokine ligand-20, and to selectively affect toll-like receptors. Activation of these components of the innate immune system seems to confine the immune response largely to the gut mucosa and mesenteric lymph nodes, as evidenced by the lack of systemic elevation of proinflammatory cytokines. Despite the apparent restriction of proinflammatory signals to the gut-associated lymphoid tissue, ST provokes peripheral sequelae consistent with danger signaling, including the febrile response and activation of the adrenal axis. In addition, pigs undergoing ST-induced febrile responses experience a consistent period of inappetence that is independent of changes in leptin. Moreover, this period of decreased intake is invariably accompanied by an unmistakable decrease in serum IGF-I and, less consistently, with parallel reductions in circulating IGFBP-3. More recently, we characterized changes in expression of components of the IGF system within skeletal muscle of pigs undergoing ST-associated enteric disease. Despite the characteristic decrease in circulating IGF-I, the relative abundance of skeletal muscle IGF-I and IGFBP-3 mRNA was unaffected by ST. However, mRNA for IGFBP-5 was decreased in the skeletal muscle of ST-challenged pigs, suggesting a possible effect of the enteric disease on IGF availability. Taken together, oral challenge with ST engages elements of the mucosal innate immune system that seem to contain the spread of systemic proinflammatory cytokine signals. Even so, ST challenge is associated with parallel changes in both systemic and local IGF systems that may affect pig growth.
Key Words: Insulin-Like Growth Factor • Mucosal Immunity • Muscle Growth • Physiopathology • Pigs • Salmonella enterica
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Introduction
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Our group has been broadly interested in the search for alternatives to antibiotic feeding in pigs to stimulate growth. Currently, the dogmatic view is that the greatest growth response of nursery pigs to low-level antibiotic feeding is realized in nursery environments with the lowest hygiene (Dritz et al., 2002
). To that end, we developed a model of low hygiene by ensuring nursery pigs were carrying a burden of enteric pathogens by orally administering Salmonella enterica serovar Typhimurium (ST). This nursery model has allowed us to design experiments to empirically evaluate alternative feed additives for growth stimulation and to evaluate physiological and immunological correlates of the model. In addition, we have exploited the anorectic effects of acute enteric ST loading to evaluate characteristics of both systemic and local regulation of the IGF system. We are currently involved in using the model to gain insight into the molecular effects of chronic oral exposure to ST on gut immune cell trafficking and function, and on local components of the IGF system that affect skeletal muscle growth.
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Background for Development of the Salmonella enterica serovar Typhimurium Low-Hygiene Nursery Model
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Antibiotic Feeding and Growth Responses in Nursery Pigs
Subtherapeutic antibiotic feeding has been used to stimulate growth rate and efficiency of feed utilization of livestock for over 50 yr, and these effects have been summarized in comprehensive reviews (Zimmerman, 1986; Cromwell, 2002; Dritz et al., 2002). It is generally recognized that antibiotics as feed additives work to control or decrease to varying degrees certain species of enteric bacteria, depending on the spectral efficacy of the antibiotic used (Gaskins et al., 2002). Burkey (2003) evaluated summaries published in a recent review (Cromwell, 2002) indicating that ADG improved by 16.4, 10.6, and 4.2%, and feed efficiency by 6.9, 4.5 and 2.2% in the starting (nursery), growing, and growing-finishing phases, respectively. An even more recent compilation of trials conducted with over 21,000 nursery pigs and over 2,500 finishing pigs reported a 5.0% improvement in ADG for nursery pigs and no improvement in ADG for finishing pigs with varying antimicrobial regimens (Dritz et al., 2002). In that report, neither nursery nor finishing pigs demonstrated improvements in feed efficiency. Thus, it seems that a growth benefit to antibiotic feeding remains for nursery pigs, but not pigs in later stages of the production cycle. Among the more plausible explanations for the diminished performance responses to antibiotics over the years are 1) increased performance accounted for by changing genetics and improved management reflected in control animals; and 2) improved awareness of the importance of hygiene in production facilities (Dritz et al., 2002).
There is currently an active search in the United States for alternatives to low-level antibiotic inclusion in swine diets for growth promotion. This ongoing search has included, and continues to include, empirical studies designed to remove growth promoting levels of antibiotics and replace them with alternative additives hypothesized to improve mucosal immune function (e.g., Turner et al., 2002a,b; Davis et al., 2004), bind or kill pathogenic bacteria (e.g., Anderson et al., 2001; Burkey et al., 2004a; Davis et al., 2004), or otherwise alter gastrointestinal microbial ecology (e.g., Mathew et al., 1998; Barker, 2003) to partially capture the growth-promoting ability of low-level antibiotic feeding. To date, none of these approaches has consistently improved growth performance to the level of that achieved with antibiotic feeding. However, as pointed in recent reports (Gaskins, 2001; Gaskins et al., 2002), the precise mechanism(s) by which low-level antibiotic feeding stimulates growth are not known with certainty. Another related phenomenon that remains poorly understood is the dogmatic view that the growth response to antibiotic feeding is greater in production facilities with so-called low hygiene. Does this effect exist because pigs grown in facilities under low-hygiene conditions carry a greater enteric pathogen load, a greater and more diverse commensal bacterial load, or some combination of these circumstances?
Considerations for the Development of Our Low-Hygiene Nursery Model
Given the documented background of nursery pig growth responses to antibiotic, we set out to establish a low-hygiene nursery-pig model that could be exploited to better define the physiological correlates associated with pig growth responses to antibiotic feeding. For this model, we elected to take the approach of forcing an enteric pathogen burden in the form of oral ST to establish this model. Then, we compared responses of these animals with pigs with "normal" gastrointestinal microbiota. In some studies, we compared responses of those animals with pigs given only sterile bacterial growth media (Balaji et al., 2000; Turner et al., 2002a,b; Jenkins et al., 2004). For other studies, we provided the pathogen load orally to all animals and compared growth performance, physiology, bacterial shedding, and so on among a variety of dietary treatments (Barker, 2003; Burkey et al., 2004a).
Several points are relevant when placing our model in the context of practical swine production environments. First, we have made an effort to only vary dietary antibiotic, in our case, Carbadox at 55 ppm, and formulate diets otherwise free of growth-promoting levels of copper sulfate or zinc oxide. Second, we have generally provided a single oral dose of ST, usually on the order of 109 to 1010 cfu, although most recently as low as 106 to 107 cfu (Barker, 2003). Third, in all cases, pigs represented genotypes that were typical of those used in swine production facilities in the United States. In general, these animals have been purchased from high-health herds and have been cultured negative for fecal salmonellae organisms before each experiment. Fourth, in accord with requirements of the local Institutional Animal Care and Use Committee, animal pens were cleaned daily. This point may be relevant to the surprisingly rapid rebound in growth performance in pigs given ST as early as the second week following oral exposure to ST (Turner et al., 2002a,b; Burkey et al., 2004a). We speculate that, at least part of this rapid return to growth responses similar to control pigs, may be related to the relative cleanliness of the pens that likely decreases the potential for oral (re)exposure of pen mates to fecally shed bacteria that would be more typical of a production setting. In this regard, future studies are designed to provide repeated oral exposure to ST to set up an experimental paradigm that more closely resembles a production setting. Finally, despite this caveat, the model still provides an excellent experimental setting to study the growth response to antibiotic feeding. For example, in one of the more recent reports from our group, growth of pigs fed antibiotic was improved about 30% overall relative to controls (Burkey et al., 2004a).
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Physiopathologic Correlates of Oral Challenge with Salmonella enterica serovar Typhimurium
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The interval of the mid-1980s through the 1990s was a period of rapid expansion in the general understanding of the complex interactions between the neuroendocrine system and the immune system (Kelley, 1988; Kelley and Dantzer, 1990; Kelley et al., 1994; Sternberg, 1997; Dantzer et al., 2000). Additionally, during this same period, tremendous insight was gained into the role of circulating inflammatory cytokines, central expression of cytokines and contributions of the autonomic nervous system to the pathophysiology of systemic inflammatory mechanisms and the development of fever and so-called sickness behaviors (Kluger, 1991; Kluger et al., 1995, 1998; Romanovsky et al., 1997; Saper, 1998; Dantzer et al., 1998a,b). Many, although not all, of the early advances in the understanding of the interaction between inflammatory responses, inflammatory cytokine production, activation of the pituitary-adrenal axis, and fever induction were unraveled with the use of central or peripheral administration of lipopolysaccharide (LPS) to animals, mainly rodents. The signaling cascade for LPS, not well understood at that time, is now known to be mediated through engagement of toll-like receptor-4, the subsequent activation and nuclear action of nuclear factor 
b and the transcription of inflammatory cytokine genes (Akira et al., 2001; Takeda et al., 2003). In a broad sense, the inflammatory and systemic responses to peripheral administration of LPS were, at that time, generalized to more or less represent models of gram-negative bacterial infections.
Similar to investigations in laboratory rodents, much of the early understanding of interactions between systemic cytokinology, pathophysiology and sickness behavior in pigs were derived from peripheral or central injection of LPS (Johnson and von Borell, 1994; Johnson et al., 1994; Dritz et al., 1996; Hevener et al., 1997; Myers et al., 1997, 1999; Parrott et al., 1997, 1998; Webel et al., 1997; Parrott and Vellucci, 1998; Wright et al., 2000). Although dogma was developing regarding the pathophysiology of disease based on LPS challenge models, before the development of our oral ST challenge, low-hygiene nursery model in pigs, data from a bona fide enteric disease challenge model were generally lacking.
Consistent with LPS models in pigs, we confirmed that oral ST resulted in a febrile response that could be rather prolonged (Balaji et al., 2000) or, in some cases, more transient through the course of the week after ST (Turner et al., 2002a,b). Additionally, associated with oral ST, we found feeding behavior to be decreased in the first few days following exposure to the enteric pathogen, and that this decrease in intake could not be associated with an accompanying elevation in circulating leptin (Jenkins et al., 2004). However, daily feed intake of the treated pigs generally returned to that of the control pigs by the end of the first week after treatment (Balaji et al., 2000; Turner et al., 2002a,b; Burkey et al., 2004a; Jenkins et al., 2004). Furthermore, we found pigs receiving oral ST had a prolonged surge in circulating cortisol that lasted about 48 h (Balaji et al., 2000). Comparing the febrile (Wright et al., 2000), acute intake (Webel et al., 1997; Wright et al., 2000), and plasma cortisol responses (Webel et al., 1997; Wright et al., 2000) in pigs injected with LPS to pigs receiving an enteric disease challenge with live ST (Balaji et al., 2000), all these responses were far more prolonged than those achievable by LPS injection.
Beyond the differences cited above between our data with ST-challenged nursery pigs and data for pigs treated with LPS, we also found notable differences in systemic inflammatory cytokine secretion following challenge. Without exception, in the LPS studies conducted with pigs before our initial report (Balaji et al., 2000), LPS injection into pigs resulted in increased circulating inflammatory cytokines (Myers et al., 1997, 1999; Webel et al., 1997, 1998). In addition, studies in which LPS was injected into nursery pigs published since our initial report confirm the consistent ability of LPS to stimulate systemic inflammatory cytokine production in pigs provided a variety of nursery diet complexities (Touchette et al., 2002; Carroll et al., 2003; Gaines et al., 2003). Thus, we too hypothesized that ST challenge would increase circulating inflammatory cytokine production based on the studies conducted with LPS and the growing view that inflammatory cytokines might be systemic markers of disease associated with slow growth (Johnson, 1997; Spurlock, 1997; Fossum, 1998; Fossum et al., 1998). Yet, to our surprise, we found that neither tumor necrosis factor-
(TNF; Figure 1; Balaji et al., 2000) nor IL-6 (Burkey et al., 2004a) was increased systemically in pigs actively expressing other symptoms of active enteric disease. We confirmed that the lack of TNF response could not be accounted for by the inability of the swine specific ELISA to detect TNF because, under virtually identical experimental conditions, LPS provoked an unmistakable elevation in TNF (Figure 1; Wright et al., 2000). Our interpretation of these apparently conflicting results is that the mucosal immune system in the pig gastrointestinal tract seems to contain inflammatory signals provoked by the presence of ST locally. As a consequence, inflammatory chemokine and cytokine signals appear in the gut epithelium and gut wall (detailed below), but this does not result in a systemic elevation of cytokines. However, it is likely premature to assume that all enteric pathogens may fail to elevate systemic cytokine concentrations. For example, Salmonella enterica serovar Choleraesuis (SC) is known as a host-adapted swine pathogen and is reported to be more likely to cause systemic disease and sepsis than ST (Reed et al., 1986; Roof et al., 1992; Schwartz, 1999). Therefore, it seems plausible that the lack of systemic cytokine signaling characteristic of ST in our low-hygiene model is consistent with the view that ST produces colitis, enlarged ileocecal lymph nodes and self-limiting diarrhea, but not the septicemic salmonellosis that is characteristic of SC (Roof et al., 1992).
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Effects of Oral Challenge with Salmonella enterica serovar Typhimurium on Systemic Endocrine Regulators of Growth
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As noted above, we have observed consistently a marked decrease in feed intake during the first week following oral ST inoculation, and that intake generally had returned to that of control pigs or pigs not fed antibiotic by the end of the first week following bacterial inoculation (Balaji et al., 2000; Turner et al., 2002a,b; Burkey et al., 2004a; Jenkins et al., 2004). Because circulating IGF-I is associated with changes in intake in pigs fed dietary antibiotic (Hathaway et al., 1996), and pigs undergoing acute parasitic infection had decreased IGF-I in serum (Prickett et al., 1992), we also hypothesized that pigs challenged orally with ST would have reduced circulating IGF-I. This indeed proved to be the case as, in general, circulating IGF-I was decreased by about 48 h following oral ST, but it had returned to control (or preinoculation) levels by about 6 d following treatment (Balaji et al., 2000; Turner et al., 2002a; Burkey et al., 2004a; Jenkins et al., 2004). Of note is the observation in our initial study that changes in IGF-I were not associated with consistent changes in GH (Balaji et al., 2000), nor in a later report could we associate changes in IGF-I with changes in circulating leptin (Jenkins et al., 2004). Nonetheless, IGFBP-3 and total IGFBP were reduced in pigs inoculated orally with ST (Jenkins et al., 2004).
It should be pointed out that in all of our studies in which we have observed changes in circulating IGF, we could not determine the specific effect of enteric disease per se on IGF-I apart from the effect of decreased intake because the two were always confounded. That is, we have never evaluated intake in pair-fed, uninoculated control pigs; however, our interpretation has been that, in view of the close association of the depression in both intake and circulating IGF-I and their closely coupled rebound within the first week following inoculation of ST, most of the variation in circulating IGF-I we observe in our low-hygiene oral ST model can likely be accounted for by changes in feed intake.
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Changes in Local Regulation of Skeletal Muscle Growth During Disease States
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Pathological conditions, such as inflammatory disease, can result in a decrease in lean body mass. Elevated levels of cytokines, such as IL-1, IL-6, and TNF, are associated with these inflammatory conditions that can result in reductions in lean body mass. In domestic livestock such as the pig, the loss of lean mass is predominantly due to a loss of skeletal muscle, which represents the largest pool of protein (Casteneda, 2002). This loss of skeletal muscle mass during a diseased state involves interactions between increased muscle catabolism and reduced muscle hypertrophy including protein synthesis.
Much of the research in this area has involved the provision of LPS to mediate many of the inflammatory sequelae of infection. Intraperitoneal LPS administration resulted in rapid increased abundance the inflammatory cytokines TNF (9.5-fold) and IL-6 (106-fold) mRNA in the gastrocnemius muscle of mice (Frost et al., 2002). Similarly in rats, LPS administration provoked a rapid increase in skeletal muscle TNF mRNA levels (sevenfold) within 2 h following injection (Fernandez-Celemin et al., 2002). These changes in local cytokine expression in skeletal muscle were consistent with the observed changes in systemic cytokine levels following administration of LPS.
Insulin-like growth factor-I is an anabolic growth factor affecting growth and development of many tissues including skeletal muscle. Insulin-like growth factor-I is a critical mediator of skeletal muscle hypertrophy (Barton-Davis et al., 1999). Virally induced overexpression of IGF-I in skeletal muscle resulted in a 15% increase in overall muscle mass in young adult mice (Barton-Davis et al., 1998) and extended the proliferative lifespan of satellite cells (Chakravarthy et al., 2000). Because IGF-I is known to be a potent stimulator of both proliferation and differentiation of satellite cells (Florini et al., 1996), the locally produced IGF-I could act through autocrine and/or paracrine mechanisms to promote the proliferation and differentiation of muscle satellite cells, thus enhancing skeletal muscle hypertrophy. Additionally, IGF-I has been shown to decrease muscle proteolysis most likely through an inhibition of the ubiquitin-proteasome system (Chrysis and Underwood, 1999). In light of the potent anabolic effects of IGF-I on skeletal muscle, a reduction in local concentrations of this growth factor may be partially responsible for the muscle wasting that occurs during catabolic conditions such as infection and inflammation. In fact, there is a growing body of evidence that suggests that alterations in local skeletal muscle expression of cytokines results in subsequent changes in IGF-I mRNA levels in skeletal muscle.
Previous studies that observed rapid changes in local inflammatory cytokine production also noted cytokine-dependent decreases in IGF-I mRNA abundance (Fernandez-Celemin et al., 2002; Frost et al., 2003). In mice, LPS administration resulted in a 50% decrease in IGF-I mRNA levels in skeletal muscle 6 h after LPS injection (Frost et al., 2003). Similar results were obtained by directly adding LPS to C2C12 myoblast cultures. These reductions in IGF-I mRNA levels were often sustained for approximately 48 h in culture (Frost et al., 2003). Taken together, the rapid, but transient, increased TNF mRNA at 1 to 2 h following LPS administration followed by a decrease in IGF-I mRNA at 6 h that was sustained to 18 h, in vivo, suggests that TNF may be mediating the reduction in skeletal muscle IGF-I mRNA.
From previous data with LPS administration, elevations of local cytokines, such as TNF, in skeletal muscle have resulted in consistent reductions in local IGF-I mRNA. We failed to show changes in local IGF-I mRNA in skeletal muscle samples collected with a biopsy procedure at several time points during the first 2 wk following a single oral dose (1 x 1010 cfu) of ST. However, over this timeframe, we did report rapid decreases in circulating IGF-I, consistent with several other studies using our enteric disease model. Many of these changes in systemic IGF-I could be a result of decreased feed intake observed the week following challenge. Furthermore, these changes in circulating IGF-I occur in the absence of any alteration in circulating TNF. It is possible that alterations in local cytokines, such as TNF, are a prerequisite to observing changes in local IGF-I abundance, at least in skeletal muscle. However, we have only attempted to measure proinflammatory cytokines in circulation and have not estimated the levels of local cytokines in skeletal muscle following an oral challenge of ST.
In addition to evaluating changes in local IGF-I mRNA in skeletal muscle following an enteric disease challenge, we also assessed changes in mRNA concentrations for both IGFBP-3 and IGFBP-5 in muscle biopsy samples of nursery pigs. A significant treatment x day interaction was observed for IGFBP-5 mRNA in skeletal muscle of nursery pigs. Insulin-like growth factor binding protein-5 mRNA levels were 2.2-fold greater (P < 0.05) on d 14 following challenge, in muscle samples obtained from control pigs compared with muscle samples from pigs challenged with ST (Figure 2). Additionally, it is also interesting to note the relationship of IGFBP-5 mRNA levels to those of IGF-I mRNA in skeletal muscle of nursery pigs (Figure 2). No differences were noted for IGFBP-3 mRNA concentrations following the enteric disease challenge. Contrary to IGFBP-5, skeletal muscle IGFBP-3 mRNA levels had very little relationship with IGF-I mRNA.
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Figure 2. Relative abundance of IGFBP-5 mRNA by quantitative real-time PCR in skeletal muscle samples taken by biopsy of gluteus medius in pigs inoculated orally with Salmonella enterica serovar Typhimurium. *Difference (P < 0.05) between control and Salmonella enterica serovar Typhimurium treatment (top; taken from Kayser et al., 2003). Relationship between skeletal muscle IGFBP-5 mRNA and IGF-I mRNA concentrations (bottom).
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Effect of Salmonella enterica serovars Typhimurium and Choleraesuis on Immune Signaling in the Gut
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Although nursery pigs demonstrated unmistakable physiologic markers reflecting the presence of enteric pathogen following oral ST inoculation, we were surprised that these animals did not have a surge in plasma TNF (Balaji et al., 2000). Similarly, in an experiment conducted subsequently, we failed to observe significant ST treatment-associated changes in serum IL-6 (Burkey et al., 2004a). As noted above, the dogmatic view at the time of conduct of the study by Balaji et al. (2000) was that infection with gram-negative bacteria could be modeled by injection of pigs with LPS (Webel et al., 1997). Salmonella enterica serovar Typhimurium is recognized as a significant swine enteric pathogen, but this serovar is not noted for causing systemic disease, at least to the extent that SC causes septicemia in pigs (Roof et al., 1992). Thus, our interpretation has been that the failure to observe significant changes in circulating TNF reflected the ability of the pig gastrointestinal mucosal immune system to contain the pathogen locally. This interpretation, together with an increased appreciation for the immune signaling capability of intestinal epithelial cells (Kagnoff and Eckmann, 1997; Eckmann and Kagnoff, 2001; Gewirtz, 2003) led us to embark on a new line of investigation evaluating gut immune function in the pig. Published reports substantiating direct actions of salmonellae organisms on pig gut epithelial cells are not currently available, and the data presented in this report are preliminary in nature. Nevertheless, our data gathered to date suggest that ST can provoke immune chemokine signaling in the pig gut wall in vivo, and elicit highly polarized chemokine secretion in vitro.
Figure 3 illustrates the time course of IL-8 mRNA expression in ileal Peyer’s patches collected at various times after oral ST inoculation in pigs. Through 6 d after ST (144 h), IL-8 mRNA seems to gradually increase in Peyer’s patch samples relative to tissue from pigs sacrificed before ST. This observation is consistent with the neutrophil chemoattractant function of IL-8 (Thelen, 2001) and the well-known pathology of salmonellae organisms to provoke infiltration of inflammatory cells into the gut mucosa (Schwartz, 1999).
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Figure 3. Relative abundance of IL-8 mRNA by quantitative real-time PCR in ileal Peyer’s patch samples taken at sacrifice after oral in pigs inoculated orally with Salmonella enterica serovar Typhimurium. Each bar represents the mean ±SEM of two to four pigs. (K. A. Skjolaas-Wilson, T. E. Burkey, and J. E. Minton, unpublished data).
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Most recently, we have used a swine jejunal epithelial cell line (Rhoads et al., 1994) to determine effects of direct pathogen exposure on secretion and expression of chemoattractant molecules in polarized cell monolayers. When cultured on permeable membrane supports, this cell line, IPEC-J2, develops confluent polarized monolayers that maintain transepithelial resistance of approximately 1,000 ohm•cm2 within 10 to 14 d in culture (K. A. Skjolaas-Wilson, T. E. Burkey, and J. E. Minton, unpublished data). We have conducted a series of experiments in which these polarized cells were exposed to LPS, ST, or SC on their apical side for 1 h. Then, the cells were washed repeatedly and the media replaced with growth media containing 50 µg/mL gentamicin to kill bacteria that had not been internalized. Apical or basolateral media and/or total RNA were obtained at 1.5, 3, and 6 h after the initiation of LPS or bacterial exposure.
Utilizing this in vitro model, we have shown that apical exposure to ST, but not SC, provokes highly polarized IL-8 secretion (Figure 4) and enhanced expression of the dendritic cell chemoattractant, CC chemokine ligand-20 (Figure 5). Both of these observed effects of bacteria on swine intestinal epithelial cells are consistent with published effects of ST on other epithelial cell lines and model systems (Gewirtz et al., 2000, 2001; Rescigno et al., 2001; Sierro et al., 2001). More importantly, these experiments begin to shed light on the role of swine epithelial cells as immune signaling cells, and how important swine enteric pathogens with very different pathologies provoke very different patterns of inflammatory cell chemokines.
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Figure 4. Secretion of IL-8 into apical or basolateral media from cultured swine jejunal epithelial cells treated apically with media only (CON), 10 ng/mL lipopolysaccharide (LPS; 5 ng total in the apical well), and 108 Salmonella enterica serovars Choleraesuis (SC) or Typhimurium (ST) at 1.5, 3, and 6 h after treatment. ST increased IL-8 (P < 0.05) above control and LPS in the basolateral compartment at 3 h, and above all treatments at 6 h (P < 0.01). Similarly, only ST increased IL-8 in the AP compartment at 6 h (P < 0.01 relative to all other treatments). In wells treated with ST at 6 h, IL-8 was strongly, although not completely, polarized toward the BL compartment (P < 0.01; from Burkey et al., 2004b).
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Figure 5. Relative abundance of CC chemokine ligand 20 (CCL20) mRNA by quantitiative real-time PCR in cultured swine jejunal epithelial cells treated apically with media only (CON), 10 ng/mL lipopolysaccharide (LPS; 5 ng total in the apical well), and 108 Salmonella enterica serovars Choleraesuis or Typhimurium (ST). Superscripts denote times when CCL20 was increased by bacterial treatment (a: P = 0.07; b: P < 0.01; from Skjolaas-Wilson et al., 2004).
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Footnotes
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1 Presented at the ASAS Symposium: Animal Health: Integrative Aspects of Immunity, Nutrient Metabolism, and Production in Livestock, St. Louis, MO, July 29, 2004. 
2 Contribution No. 05-59-J of the Kansas Agric. Exp. Stn., Manhattan.
3 Correspondence: 126 Call Hall (phone: 785-532-3476; fax: 785-532-5681; e-mail: bjohnson{at}ksu.edu).
Received for publication August 11, 2004.
Accepted for publication November 18, 2004.
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Abstract
Introduction
