Divisions of 1Gastroenterology and Nutrition and 2Neonatology, Department of Pediatrics; and 3Division of Gastroenterology, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California 90095
Submitted 20 March 2003 ; accepted in final form 5 June 2003
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ABSTRACT |
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facilitated glucose transporter; apical sodium-dependent bile acid transporter; polymeric IgA receptor; lactase-phlorizin hydrolase; Fc receptor of the neonate; Na+/glucose cotransporter
Developmental adaptation of small intestinal enterocytes is considered by most to be preprogrammed or hardwired. This conclusion is derived primarily from studies investigating the role that various hormones and luminal nutrients have in regulating the process of adaptation (13). However, despite evidence for the role of such an intrinsic clock in enterocyte ontogeny, its molecular basis and the factors that regulate it have not been defined (43). However, several studies have suggested that other external stimuli, such as diet, may also influence the precise timing and the level of expression of specific intestinal markers and therefore that environmental and hardwired central or local stimuli working together may be responsible for initiating the adaptive changes (10).
The systemic and mucosal adaptive immune systems also undergo dramatic changes during early postnatal development (1). The adaptive immune system is functionally naive and incapable of providing ample protection against the wide assortment of antigens and pathogens encountered during the early extrauterine period (13). Additionally, during postnatal development, mammals must rely on passive immunity provided by their mothers and their own innate immune system to compensate for the relative immaturity of their own adaptive immune system (1, 46).
During early development, passive immunity is provided by either transplacental flow of IgG or through breast milk, which is a rich source of both IgA and IgG. Breast milk from all species of animals is abundant in secretory IgA, which plays an important role in attenuating the influence that common pathogens (vibrio cholera and rotavirus) have on enterocyte function and is also believed to influence the establishment of the commensal microflora in the small and large intestine (9, 21). Maternal milk is also a rich source of the other proteins and cellular components of the adaptive and innate (i.e., oligosaccharides and lactoferrin) immune system, and it is well accepted that the various factors of breast milk play an essential role in ensuring the successful transition from the suckling to the weaned phases of development. The mucosal immune system of the suckling animal is also immature, because there is a paucity of functional T and B cells located in both the intraepithelial and lamina propria compartments (21). Furthermore, the polymeric IgA receptor (pIgR), which transports antibodies from basolateral to apical membranes, is not expressed in the intestine during the early preweaned phase of postnatal life (23).
In contrast, another major immunoglobulin in breast milk, IgG, unlike IgA is not limited to the intestinal lumen but in fact is transferred from the lumen to the systemic circulation by the intestinal Fc receptor of the neonate (FcRn) (34). During the latter stages of gestation in some mammals such as humans, FcRn expressed by syncytiotrophoblast carries IgG to the fetal compartment by a pH gradient in acidic endosomes. Serum levels of IgG in species such as cattle, pigs, sheep, and other developing ruminants depends on the passive transfer from breast milk, as FcRn expressed on enterocytes of the proximal intestine of developing pup transfers IgG from the luminal compartment to the systemic circulation (34). Recent data from humans suggest that enterocytes express FcRn, and it has been speculated to play a role in influencing systemic levels of IgG, because in premature infants serum IgG levels are higher in breast-fed compared with formula-fed infants (37).
There is also mounting evidence that both immune status and the commensal microflora of the gut play a role in the regulation of enterocyte gene expression (6, 22, 26, 41). Specifically, Hooper et al. (14) used eukaryotic microarray analysis to examine the influence that intestinal microflora has in shifting epithelial expression. In this study, Bacteroides thetaiotaomicron was introduced to germ-free adult mice and microarray analysis was performed on enterocytes isolated from the distal small bowel 10 days later. The study found that the steady-state levels of a large number of transcripts were altered by the introduction of a B. thetaiotaomicron microflora, including the developmentally regulated genes pIgR, lactase-phlorizin hydrolase (lactase), and Na+/glucose cotransporter (SGLT1) (15).
Together, these studies suggest that the presence of a normal maternal adaptive immune system (i.e., passive immunity) and the suckling animal's own adaptive immune system may play an important role in the developmental regulation of enterocyte-specific transcripts (13, 14). Therefore, we hypothesized that certain developmentally regulated genes expressed in the small bowel may not be hardwired but, in fact, may be controlled, at least in part, by the passive and adaptive immune system of the suckling pup. In this study, we set out to test the effect of altering the passive and adaptive immune system on the postnatal developmental expression pattern of six important genes expressed in the gastrointestinal tract of mice that code for proteins involved in antibody transport and nutrient assimilation. Our findings indicate that both passive and adaptive immunity influence enterocyte gene expression during the early extrauterine development of the gastrointestinal tract in a complex manner that appears to be specific to each gene.
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MATERIALS AND METHODS |
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Animals used in this study were obtained originally from Jackson Laboratories (Bay Harbor, ME) and bred and maintained at the University of California (Los Angeles, CA) vivarium. The mice used were either C57BL/6J (Jackson Laboratories) recombination-activating gene (Rag)-1 (/) (B6.129S7-Rag1) (former name C57BL6/6J-Rag1), previously backcrossed 10 times with C57BL/6J mice (Jackson Laboratories), or Rag2 (/) mice, obtained from Dr. Ellen Rothenberg, California Institute of Technology (Pasadena, CA) (11). Mice were housed in autoclaved caging and bedding with acidified water and irradiated chow available ad libitum. Pregnant mice were identified and monitored daily until delivery. The day of birth was identified as day 0 of life, and mice were culled to a litter size of 6 to 8. All mice were weaned at 21 days of age. All animal protocols were preapproved by the UCLA Animal Research Committee.
Breeding Scheme
To investigate the role that the presence or absence of a functional maternal and neonatal immune system has in inducing changes in the expression of various enterocyte-specific transcripts, we used a breeding scheme that produced four different sets of experimental conditions (Fig. 1). Targeted disruption of either the rag1 or rag2 genes results in the absence of an adaptive immune system, including an entire deficiency of T and B cell functions (7). Because the rag1 and rag2 phenotypes are autosomal recessive, heterozygote mice (rag/C57BL/6) are comparable with wild-type (WT) and have normal T and B cell function. We chose to use the rag model, because mice from this strain are easily genotyped and have a more profound (and consistent) immunodeficiency compared with the SCID model (17).
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Our control group, WT dam breastfeeding a WT pup (WT dam/WT pup), was accomplished by mating WT C57BL/6J (+/+) females and males. The first experimental group tested the influence that passive immunity has on enterocyte expression in mice with an intact adaptive immune system. This group, immunodeficient (ID) dam breastfeeding a WT pup (ID dam/WT pup), was generated by breeding homozygous ID (Rag/) dams with WT C57BL/6J (+/+) males. In the second experimental group, we tested the role the adaptive immune system has in altering gene expression in mice exposed to passive immunity. This experimental group, WT dam breastfeeding an ID pup (WT dam/ID pup), was accomplished by mating heterozygous WT females (Rag/+) with homozygous ID (Rag/) males to produce homozygous ID pups (Rag/). Pups were genetically selected for (Rag/) by PCR genotyping described in DNA Isolation and PCR Genotyping. The final experimental group simultaneously tested the role of both the adaptive and passive immune systems in controlling enterocyte gene expression. This group, ID dam breastfeeding an ID pup (ID dam/ID pup), was developed by breeding homozygous ID (Rag/) males and females.
DNA Isolation and PCR Genotyping
PCR genotyping was performed in the WT dam/ID pup group to determine which pups were homozygous ID Rag knockouts (Rag/). DNA was isolated by cutting 1 cm from the tail and placing it in a solution containing 50 mM Tris (pH 8), 100 mM EDTA, 0.5% SDS, and 350 µg of proteinase K. The sample was incubated at 55°C overnight, and then DNA was isolated by a standard phenol and chloroform extraction protocol and precipitated with ethanol. RNA was removed by adding 100 µg of RNase and then precipitated with 100% ethanol. The samples were dissolved in 100 µl of Tris-EDTA buffer, and PCR was performed with a Rag cDNA primer by mixing Rag-A primer (sense), Rag-B primer (antisense), and neomycin (NEO) A primer in standard PCR buffer using AmpliTaq polymerase (Perkin-Elmer, Norwalk, CT) (17). The amplification primers used for Rag-A, Rag-B, and Neo-A are listed in Table 1. PCR amplification was carried out (one initial denaturation cycle at 94°C for 15 s, then one annealing cycle at 70°C for 2 min) for a total of 45 cycles. The DNA samples were then run on an agarose gel to analyze PCR fragments and determine genotype.
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Sample Collection
Intestinal samples were obtained from mice at 9, 15, 18, 21, 25, and 30 days of age by dissecting the small intestine and dividing it into thirds. The first and last third were considered the proximal and distal small intestine, respectively, and the middle third was discarded. Liver was also dissected, and all samples were immediately placed in liquid nitrogen and stored at 80°C until processing. Intestinal and liver mRNA extracts for analysis were obtained from four to five different mice at each age in each of the four experimental groups.
RNA Isolation
Tissue was processed for RNA isolation immediately by using RNase-free equipment and techniques with guanidinium thiocyanate RNA extraction solution as previously described by our lab (24). The sample was then precipitated with isopropanol and washed with 75% ethanol, centrifuged, air-dried, and resuspended in formamide. RNA concentration was quantified by UV spectrophotometry at 260 nm (A260), and the purity was determined by the A260/A280 ratio (SPECTRAmax PLUS; Molecular Devices, Sunnyvale, CA). The RNA pellets were stored at 80°C until use.
RNA Protection Assay
Probe synthesis. The oligonucleotide primers used in the RNA protection assay for SGLT1, GLUT5, FcRn, pIgR, lactase, and apical sodium-dependent bile acid transporter (ASBT) are listed in Table 1. Chris Corpe at the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD) graciously gave the GLUT5 cDNA template to us. The ASBT cDNA template was generously provided to us by Paul Dawson (Wake Forest University School of Medicine, Wake Forest, NC). The ribosomal protein S2 RNA template was kindly provided by Jeffery Smith (School of Medicine, University of California, Los Angeles, CA) (38). Lactase and FcRn probes were developed by RT-PCR of RNA isolated from 15-day-old mouse jejunum, whereas SGLT1 and pIgR were synthesized by RT-PCR from RNA isolated from adult mouse jejunum. Probe templates were linearlized plasmids treated with proteinase K, followed by phenol/chloroform extraction.
In vitro transcription. Labeled antisense RNA probes were generated by adding 100 ng of cDNA template, 1 µl of 10x buffer, 0.5 µl of 10 mM mixed nucleotides (ATP, GTP, CTP), 2.5 µlof[32P]UTP (800 Ci/mmol), 1.25 µl of unlabeled 10 mM UTP, and 1 µl of T7 RNA polymerase (Ambion, Austin, TX), and then incubating at 37°C for 1 h. We then added 0.5 µl of DNase I solution (Ambion) and incubated the solution at 37°C for 15 min. Reaction was stopped with 1 µl of 0.5 M EDTA. Labeled probes were precipitated with ammonium acetate and ethanol, washed with 70% ethanol, and dissolved in 20 µl formamide. One microliter of the solution was then counted with a liquid scintillation counter (model LS 5000TD; Beckman Instruments, Fullerton, CA).
Hybridization. A total of 2.5 µg sample RNA was used for each hybridization reaction and mixed with the 32P-labeled probe in hybridization buffer. Samples were then coprecipitated in ethanol at 20°C for 15 min and centrifuged at 4°C. The supernatant was discarded, and the pellets were air dried. RNA pellets were resuspended in 10 µl of hybridization buffer, heated to 90°C, and incubated overnight at 80°C. A probe master mixture was prepared by combining 1 x 106 counts/min of 32P-labeled probe, 2 µl 5x hybridization buffer [200 mM PIPES (pH 6.4) 2 M NaCl, 5 mM EDTA], and formamide. Each RNA sample was combined with probe master mixture and formamide; the samples were heated in a water bath at 90°C for 3 min and transferred to a 42°C incubator for overnight hybridization. The next day, RNase digestion solution was added, and the tubes were incubated at 37°C for 30 min. RNase digestion solution was prepared by diluting 50x RNase A/T1 stock solution (Ambion) to 1x in digestion buffer (10 mM Tris · HCl, pH 7.5, 300 mM NaCl, 5 mM EDTA). RNase inactivation solution was added (Ambion), samples were incubated at 20°C for 15 min and centrifuged at 4°C, and the supernatant was removed. Pellets were resuspended in a gel-loading buffer (Ambion), centrifuged for 5 min, and incubated at 92°C for 3 min. The samples were stored on ice briefly, loaded onto a 0.75 mm 5% polyacrylamide gel, and run at a constant 250 volts for 2 h. The gel was then transferred to filter paper and dried. The autoradiographic signal intensities of protected fragment bands were quantified by using a PhosphoImager (Molecular Dynamics) and analyzed with IMAGE-QUANT software (Molecular Dynamics). Bands were corrected for variations in loading by using the ribosomal protein S2 RNA signal in each lane as described by others (30).
Statistical Analysis
Standardized mRNA levels for each animal were computed by subtracting the background intensity in the same column and then dividing by the corresponding S2 mRNA control intensity after removing its background.
We compared mean standardized RNA levels across the four experimental conditions and six time periods by using a 4 x 6 factorial ANOVA. Statistical significance was assessed by using post hoc t-tests and the Tukey-Fisher criterion under the ANOVA model. All data presented are means with SE bars shown.
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RESULTS |
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An increase in the levels of expression of pIgR mRNA was seen in the liver and distal and proximal small intestine of WT mice between the ages of 9 and 30 days that were born and reared from WT dams (Fig. 2A). The proximal small intestine showed the greatest levels of expression of the three tissues. Specifically, at the point of highest expression at 25 days of age, expression in the proximal bowel was twofold higher than the distal bowel and ninefold higher than the liver (P < 0.001). All tissues showed a developmental-specific regulation of pIgR expression, with the most dramatic change occurring between 15 and 25 days of age.
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In contrast, a significant decline in FcRn mRNA expression in the proximal small intestine was seen at the time of weaning (P < 0.001). The expression of FcRn in the distal small intestine decreased only slightly through weaning, whereas hepatic expression was not developmentally regulated and remained unchanged at all ages examined (Fig. 2B). The expression levels of FcRn mRNA in the proximal small intestine were the highest at 9 days of age and were sixfold higher than the distal intestine and 15-fold higher than the liver (P < 0.001) at this age. All three tissues after 18 days showed similar expression patterns at constant, very low levels of expression.
The expression of lactase mRNA was highest at 9 days of age and decreased through weaning in both the proximal and distal small intestine (Fig. 3A). In weaned mice, the levels of lactase mRNA expression declined by 50% in the proximal small intestine (P < 0.001). However, lactase levels in the distal small intestine, in which expression was 80% less than the proximal small intestine (P < 0.001), declined dramatically to undetectable levels after weaning.
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Expression of the fructose transporter GLUT5 remained nearly undetectable until 15 days of age, at which time a significant increase in expression occurred (P < 0.001). At the greatest level of expression, at 30 days of age, the proximal bowel had fivefold (P < 0.01) higher levels than the distal bowel (Fig. 3B). The greatest change in expression levels, similar to all other transcripts tested, occurred around the time of weaning (15 to 25 days of age) (P < 0.001).
In contrast, expression of the glucose-galactose transporter, SGLT1, increased throughout the weaning period in both the proximal and distal small intestine (Fig. 3C). SGLT1 was very similar to pIgR in that both the proximal and distal bowel showed increased levels of expression, with the largest increase occurring between 15 and 25 days of age (P < 0.001). Through all ages studied, the proximal small intestine had levels of SGLT1 mRNA expression that were approximately twofold higher than the expression of the distal small intestine at the same respective age (P < 0.001).
Finally, steady-state mRNA levels of ASBT were undetectable in the proximal small intestine at all ages. However, expression in the distal intestine was initially low at 9 and 15 days of age and increased fivefold after weaning, reaching the highest level at 30 days of age (Fig. 3D).
In the subsequent analysis, we focused on how both passive and adaptive immunity altered the expression of these various transcripts. We report here how the immune status alters pIgR, FcRn, lactase, GLUT5, and SGLT1 steady-state levels in the proximal small bowel and evaluated levels of ASBT in the distal small bowel.
Enterocyte Gene Expression in the Presence of Adaptive but not Passive Immunity: ID Dam/WT Pup vs. WT Dam/WT Pup
When compared with WT pups reared in the presence of passive immunity, steady-state FcRn mRNA levels were not altered at any age in pups reared in the absence of passive immunity (Fig. 4). However, WT pups reared in the absence of passive immunity had significantly higher pIgR mRNA levels (P < 0.05) at 15 days of age compared with pups breast-fed in the presence of passive immunity (Fig. 5). However, beyond 15 days of age, pIgR mRNA levels were unaltered by maternal immune status. The expression levels of GLUT5, SGLT1, ASBT, and lactase mRNA in WT pups of ID dams did not significantly differ at any of the ages studied compared with WT pups breast-fed in the presence of passive immunity (Figs. 6, 7, 8).
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Enterocyte Gene Expression in the Presence of Passive but not Adaptive Immunity: WT Dam/WT Pup vs. WT Dam/ID Pup
ID pups breast-fed in the presence of passive immunity had a much greater level of FcRn mRNA expression at 15 days of age compared with pups that had a functional immune system and were born to and breast-fed by dams that had a normal adaptive immune system (P < 0.05) (Fig. 4). This 100% increase in expression was not seen at 18 days and beyond when both groups of pups had barely detectable levels of FcRn mRNA expression. Additionally, in these same ID pups, the expression of pIgR mRNA was significantly less at every age after 9 days of age compared with WT pups fed in the presence of passive immunity (P < 0.05) (Fig. 5). This difference persisted, and the greatest difference, an almost 50% decrease (P < 0.05), occurred at 30 days of age in the ID pups reared in the presence of passive immunity.
ID pups that were born and reared by immunocompetent dams showed a significant increase (P < 0.05) in GLUT5 mRNA expression at 18 and 21 days of age compared with WT pups breast-fed with passive immunity (Fig. 7). The other transcripts SGLT1, ASBT, and lactase did not differ between the ID pups and the WT pups breast-fed in the absence of passive immunity (Figs. 6 and 8).
Enterocyte Gene Expression in the Absence of Adaptive and Passive Immunity: ID Dam/ID Pup vs. WT Dam/WT Pup
Levels of FcRn mRNA in ID pups that were born and reared by ID dams did not differ significantly from WT pups reared in the presence of passive immunity at any age studied (Fig. 4). In contrast, pIgR mRNA expression levels in ID pups breast-fed in the absence of passive immunity were significantly (P < 0.05) less at every age examined after 9 days of age compared with immunocompetent pups breast-fed in the presence of passive immunity (Fig. 5).
Levels of SGLT1 and ASBT mRNA in ID pups reared in the absence of passive immunity did not differ significantly compared with pups with an intact adaptive and passive immunity (Fig. 8). In contrast, during the weaning period at 18 and 21 days, the expression of GLUT5 mRNA was nearly fivefold greater than seen in comparably aged WT pups reared in the presence of passive immunity (P < 0.05) (Fig. 7). In addition, lactase mRNA levels in ID pups reared in the absence of passive immunity had significantly lower levels at 9, 15, and 21 days of age than WT pups reared in the presence of passive immunity (P < 0.05) (Fig. 6).
Comparison of How Passive Immunity Influences Enterocyte Gene Expression in Absence of Adaptive Immunity: ID Dam/ID Pup vs. WT Dam/ID Pup
Interestingly, there were no significant differences in pIgR mRNA levels between ID pups regardless of the dam's immune status (Fig. 5). In addition, steady-state FcRn mRNA levels in ID pups reared in the presence of passive immunity had almost twofold higher expression levels at 15 days of age (P < 0.05) compared with ID pups reared in the absence of passive immunity (Fig. 4). Immunodeficient pups reared in the presence of passive immunity had twofold higher levels of GLUT5 mRNA (P < 0.05) compared with ID pups reared in the absence of passive immunity at 15 days of age (Fig. 7). Furthermore, lactase mRNA levels in ID pups reared in the absence of passive immunity were 20% lower at 15 days of age (P < 0.05) compared with ID pups reared by immunocompetent dams (Fig. 6). In contrast, SGLT1 and ASBT mRNA levels were not significantly different among any of the groups studied.
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DISCUSSION |
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To perform these studies, we selected six enterocyte-specific genes established as being both developmentally regulated and critical in processing various components of maternal milk. The sodium-dependent glucose/galactose cotransporter is expressed primarily in the small intestine and is less abundant in the kidney. Its expression has been previously shown to increase in postweaned rats and is found at the highest levels in the proximal bowel. In our model of WT pups, we found that SGLT1 mRNA levels increased fourfold with aging and that the transporter was twice as abundant in proximal compared with distal intestine (Fig. 3C). Fructose is the primary substrate for the facilitative transporter GLUT5, which has been extensively studied in rats and is expressed primarily in the proximal intestine of postweaned mammals (3, 4, 8, 20). We found similar results in WT pups reared by normal dams with a prominent proximal-to-distal and developmental gradient (Fig. 3B). The monosaccharides glucose and galactose are hydrolyzed from lactose by brush-border enzyme lactase (44). Lactase mRNA expression has been extensively studied in rodent models, and our study confirms that in preweaned mice, lactase levels are most abundant in the proximal intestine in which levels declined by 50% in postweaned animals (Fig. 3A) (39). Lactase mRNA levels were undetectable in the distal bowel of postweaned mice, confirming the findings of several other investigators (28, 35). Finally, the apical ABST gene expression is limited to the ileum and is biphasic with a prenatal onset of expression, followed by decline in the early postnatal period and a subsequent reinduction at the time of weaning in rats (12). We found that murine expression of ASBT mRNA was initially barely detectable early in suckling life and then increased dramatically at weaning in the distal intestine (Fig. 3D).
Our general interest in understanding the role of passive and adaptive immunity in altering enterocyte gene expression stemmed from our earlier studies (24, 25) that focused on defining the transcriptional regulation of the two immunoglobulin receptors that play critical roles in passive immunity: FcRn and pIgR. As outlined earlier, the major histocompatability complex class I-related receptor FcRn's role in passive immunity is to mediate the mother-to-pup transfer of IgG. Our lab investigated the expression of FcRn in a rat model in which we found a dramatic decline in expression in postweaned pups as well as a proximal-to-distal gradient. Results shown in Fig. 2B confirm a similar pattern in WT mice, with the highest level of FcRn mRNA expression in the suckling mice and a rapid decline to barely detectable levels after weaning. Moreover, there was a dramatic proximal-to-distal gradient, with levels in the intestine and liver in postweaned animals being barely measurable. The IgA that is present in maternal milk is transferred by pIgR, which is expressed on mammary gland epithelial cells (18). The expression of pIgR in the developing pups has been studied in rats, in which it is limited to postweaned animals and expressed in both the liver and small intestine, and the results displayed in Fig. 2A confirm these same trends in WT mice (18).
In WT suckling mice that were reared in the absence of passive immunity (ID dam/WT pup), we found that only pIgR steady-state mRNA levels differed significantly from those reared by an immunocompetent dam (Fig. 5). The ligand for pIgR, secretory IgA, is present in the basolateral portion of the enterocytes only after weaning when functional plasma cells begin to migrate into the lamina propria and secrete large amounts of IgA (32, 33). The absence of plasma cells in the lamina propria and the expression of pIgR in developing mice appears to be due, at least in part, to the protection provided by passive immunity, because it has been shown that normal pups reared by ID dams (SCID) have premature migration of IgA-secreting plasma cells to the subepithelial compartment (21). Furthermore, in a germ-free environment, plasma cells fail to migrate into the lamina propria of postweaned mice and pIgR levels increase significantly with introduction of normal commensal bacteria (5, 15). The synthesis of pIgR has been shown to be upregulated by the proinflammatory cytokines such as TNF-, IFN-
, and IL-1 in the HT-29 human colon carcinoma cell line, and the cis-acting elements that mediate these cytokine-induced changes have been identified within the first exon and intron of the human pIgR gene (2, 36). Taken together, these studies suggest a model in which pIgR expression during the early stages of postnatal development is controlled, at least in part, by the process of passive immunity. In the absence of passive immunity, an alteration of the intestinal microflora (composition or quantity) induces an early migration of components of the adaptive immune system, which should alter proinflammatory cytokines that may influence enterocyte gene expression.
To decipher the influence that the developing pup's own adaptive immune system has in altering enterocyte gene expression, we compared ID to WT pups, which were both breast-fed in the presence of passive immunity (WT dam/ID pup) (Fig. 1). Overall, compared with WT pups, both FcRn (Fig. 4) and GLUT5 (Fig. 7) mRNA levels were increased and pIgR levels were significantly lower in ID pups (Fig. 5). Because FcRn expression coincides to the period when its ligand (IgG) is available for absorption, we speculated that FcRn levels would be lower in mice reared in the absence of passive immunity. In fact, our data demonstrate that passive immunity only influenced FcRn expression in mice with a defective adaptive immune system and not in WT preweaned mice (Fig. 4). Because the innate immune system remains intact in the ID model used in these studies, one may speculate that the chemokines and cytokines secreted by the cellular components of the innate immune system (macrophages and dendritic cells) may be directly influencing enterocyte gene expression. Bacterial overgrowth of the intestine has been reported to occur in children with defective adaptive immune systems; however, there is minimal information regarding the consequences of the intestinal microflora of ID mice (27, 31). We would speculate that under these circumstances, the microflora would be vastly different, because the luminal IgA levels in postweaned mice would be absent. The microflora must also compete with enterocytes for nutrients to sustain a robust growth pattern, and carbohydrates such as fructose may be an excellent carbon source (16). The increased expression of GLUT5 in ID host may be an adaptive response required to compete with an abundant microflora for the limited nutrients.
In the absence of both passive and adaptive immunity (ID dam/ID pup), our analysis found that lactase, pIgR, and GLUT5 expression were influenced during both the pre- and postweaning periods compared with WT controls (WT dam/WT pup). In fact, the simultaneous absence of adaptive immunity in the dam and pup appeared to more profoundly influence enterocyte gene expression compared with its absence in only the dam or pup (Figs. 5, 6, 7). Lactase mRNA levels were particularly low in preweaned animals, whereas GLUT5 expression was higher in weaned mice. Expression of pIgR mRNA levels was significantly lower in both pre- and postweaned mice. There are substantial data that suggest that the presence of dietary lactose does not alter the level of lactase mRNA or enzyme activity (19), and although the decline in lactase in weaning rats is generally considered to be at the transcriptional level (29), studies have shown that lactase expression in cell lines is not altered by several cytokines (45).
Passive immunity appeared to have more dramatic effects on enterocyte gene expression of mice with defects in adaptive immunity (WT dam/ID pup). For instance, lactase levels and GLUT5 mRNA were significantly different in ID pups that were reared by an immunocompetent compared with an ID dam (Figs. 6 and 7). These data would suggest that the suckling pup's own adaptive immune system may have buffered the influence that passive immunity has on enterocyte function. These data are rather surprising, because the adaptive mucosal immune system during the first2wk of life is believed to be largely ineffective (46). Yet it clearly plays an important role in postweaned mice that never received maternal immunologic support and are not capable of producing their own immunoglobulin. Passive immunity also influenced enterocyte expression in ID postweaned mice, in which it may have influenced either the innate immune system or intestinal colonization with microflora. Taken together, these data suggest that the intestinal microflora may be significantly influenced by passive immunity in ID pups and that these bacteria alter enterocyte function either directly or by influencing the underlying innate immune system.
Our studies also found that two transcripts (SGLT1 and ASBT) were not influenced by the immune status of either the dam or the developing pup. A study by Hooper et al. (15) found that SGLT1 mRNA levels increased in postweaned germ-free mice after the introduction of B. thetaiotaomicron. Our data would suggest that the influence a diverse microflora has on enterocyte function is more complex than what can be assessed with a monoassociated germ-free model. In fact, intestinal function in both normal physiological or pathological states is highly complex and has been defined by the trialogue hypothesis that emphasizes the interaction between the enterocyte, the mucosal immune system, and the intestinal microflora. Our study, however, would suggest that the intestine might be best depicted as a tetralogue that underscores the role passive immunity has in influencing these three compartments.
Overall, this study demonstrates the complex role that the passive and adaptive immune systems have in influencing enterocyte function in mice at all ages. We have documented that the maternal adaptive immune system has its most significant influence on enterocyte function of pups that lack adaptive immunity. The data suggest that the regulation of the developmental expression of intestinal mRNA is multifactorial. Clearly, the expression of each gene appears to be controlled by a unique developmental program, which for the most part has not been well defined and does not appear to be dramatically influenced by either passive or adaptive immunity. Nevertheless, the overall expression levels of the majority of the transcripts were significantly altered in both pre- and postweaned mice. Furthermore, cell differentiation in the intestinal epithelium is a continuous and gradual process involving both transcriptional and translational regulation of different sets of genes, and whether or not the differences we have seen serve as cues for developmental long-term changes in expression remains to be determined. Finally, we have shown that passive and adaptive immunity are required for normal developmental gene expression, and it is likely that they are a part of multiple factors that at specific times play at least a modest role in regulating and guiding multifactorial enterocyte differentiation.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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