Objectives: Breast milk transforming growth factor (TGF)-β2 is associated with healthy immune maturation and reduced risk of immune-mediated disease in infants. We sought to investigate whether conditioning with TGF-β2 may result in a more mature immune responder phenotype in immature human intestinal epithelial cells (IECs).
Methods: Primary human fetal IECs (hFIECs) and the human fetal small intestinal epithelial cell line (H4 cells) were conditioned with breast milk levels of TGF-β2, and an inflammatory response was subsequently induced. Inflammatory cytokine secretion and mRNA expression were measured by enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction, respectively. Alterations in activation of inflammatory signaling pathways were detected from IECs by immunoblotting and immunofluorescence. The effects of TGF-β2 conditioning on gene expression patterns in hFIECs were assessed by cDNA microarray analysis and quantitative PCR.
Results: Conditioning with TGF-β2 significantly attenuated subsequent interleukin (IL)-1β-, TNF-α-, and poly I:C-induced IL-8 and IL-6 responses in immature human IECs. Conditioning with TGF-β2 inhibited IL-1β-induced IκB-α degradation and NF-κB p65 nuclear translocation, which may partially result from TGF-β2-induced changes in the expression of genes in the NF-κB signaling pathway detected by cDNA microarray and qPCR.
Conclusions: Conditioning with TGF-β2 attenuates the subsequent inflammatory cytokine response in immature human IECs by inhibiting signaling in the NF-κB pathway. The immunomodulatory potential of breast milk may in part be mediated by TGF-β2, which may provide a novel means of supporting intestinal immune maturation in neonates.
Division of Pediatric Gastroenterology, Developmental Gastroenterology Laboratory, Massachusetts General Hospital for Children, Charlestown, MA.
Address correspondence and reprint requests to Samuli Rautava, MD, PhD, Division of Pediatric Gastroenterology, Developmental Gastroenterology Laboratory, Massachusetts General Hospital for Children, 114 16th St (114-3503), Charlestown, MA 02192-4404 (e-mail: firstname.lastname@example.org).
Received 22 April, 2011
Accepted 21 October, 2011
The present study was supported by grants NIH R01-HD12437; R01-DK70260; P01-DK33506; P30-DK40561 (W.A.W.), and NIH R01-HD059126 (N. N.N.). S.R. is supported by the Academy of Finland, Finnish Society for Pediatric Research, Foundation for Medical Research in Finland, and the Helsingin Sanomat Foundation.
The authors report no conflicts of interest.
Breast milk provides the newborn infant nutrients for growth and development in close to optimal quantities. It is well established that breast-feeding confers protection against infectious diseases, particularly those of the gastrointestinal tract, via antimicrobial molecules such as immunoglobulins, lysozyme, lactoferrin, defensins, and oligosaccharides (1). Accumulating evidence suggests that in addition to this passive immunoprotection, bioactive molecules in breast milk modulate the infant's mucosal and systemic immune responses and may thereby promote adequate and appropriate immune responsiveness against both potentially pathogenic and indigenous microbes and harmless environmental and dietary antigens (2). Data from well-conducted epidemiological studies suggest that breast-feeding may also have long-term immunological effects by reducing the risk of immune-mediated diseases such as celiac disease (3) or atopic disorders (4) in later life; however, the mechanisms of this immune conditioning by breast milk are poorly understood.
Transforming growth factor (TGF)-β is an immunomodulatory cytokine that is secreted in breast milk in significant quantities. Of the 3 human TGF-β isoforms (TGF-β1, 2, and 3), TGF-β2 is most abundant in breast milk. There are experimental data to suggest that breast milk TGF-β2 may be an important source of TGF-β during the neonatal period when endogenous production of TGF-β in the gut is still inadequate (5–7). A recent report indicates that intestinal expression of TGF-β2 is decreased in premature infants and especially in those experiencing necrotizing enterocolitis (NEC) as compared with term infants (5). Intestinal maturation results in an increase in TGF-β2 expression in the gut (5). Moreover, breast milk TGF-β2 may induce immune maturation in the immature intestine because epidemiological studies have demonstrated an association between breast milk TGF-β and both maturational changes in immune function and reduced risk of developing immune-mediated disease in infants and children (2). High concentrations of both TGF-β1 and TGF-β2 in colostrum have been reported to correlate with serum immunoglobulin A (IgA) concentrations and reduce the risk of developing atopic eczema during exclusive breast-feeding in high-risk infants (8).
We recently demonstrated that TGF-β2 administered at a concentration corresponding to that found in breast milk simultaneously with a proinflammatory stimulus attenuates inflammatory immune responses in the immature human intestinal epithelium (9). Given the potential of breast milk to induce long-term immune effects and the association between breast milk TGF-β2 and the infant immune responder phenotype discussed above, we suggest that breast milk TGF-β2 may provide a maturational stimulus to the immature intestinal epithelium and support an anti-inflammatory tone necessary for withholding from potentially detrimental inflammatory responses against colonizing microbes after birth. We specifically hypothesize that conditioning the neonatal gut with TGF-β2 may induce maturational changes in the immature intestinal epithelial cells’ (IECs’) inflammatory responses upon subsequent proinflammatory insult.
Dulbecco's modified Eagle's medium (DMEM)/F12 medium, Opti-MEM I medium, penicillin and streptomycin, Hepes buffer, and trypsin-ethylenediaminetetraacetic acid were obtained from Gibco-Invitrogen (Carlsbad, CA). Collagenase type IV, protease inhibitor cocktail, and phosphatase inhibitor cocktail I and II were obtained from Sigma-Aldrich (St Louis, MO). Fetal bovine serum (FBS) was obtained from Atlanta Biologicals (Lawrenceville, GA). Recombinant human insulin (Novolin R) was obtained from Novo Nordisk A/S (Bagsvaerd, Denmark). Extracellular matrix ECL was obtained from Upstate Biotechnology (Lake Placid, NY). The cytokines IL-1β, TNF-α, and TGF-β2 were obtained from R&D Systems (Minneapolis, MN). Rabbit anti-human NF-κB (p65) polyclonal antibody was obtained from Calbiochem (Gibbstown, NJ). Cy 3-conjugated F(ab′)2 fragment goat anti-rabbit IgG was obtained from Jackson ImmunoResearch (West Grove, PA). The BCA Protein Assay Kit was obtained from Thermo Scientific (Rockford, IL). The LDH Cytotoxicity Detection kit was obtained from Roche Applied Science (Mannheim, Germany). Human recombinant epidermal growth factor, TRIzol, and SuperScript III Platinum SYBR Green One-Step quantitative real-time polymerase chain reaction (qRT-PCR) kits were obtained from Invitrogen (Carlsbad, CA). All other reagents were of analytical or molecular biology grade and came from Sigma-Aldrich.
Human IEC Culture
The study was conducted according to National Institutes of Health guidelines and Partners Human Study Committee approval (IRB 1999p003833). Fetal intestinal tissue obtained from therapeutic abortions was used for isolation of primary human fetal intestinal epithelial cells (hFIECs) using a procedure modified from that reported by Quaroni and Beaulieu (10) as described previously (9). The cells were incubated for 3 hours and rinsed vigorously with phosphate-buffered saline. Adherent cells were maintained in tissue culture for 3 to 8 passages before use in experiments. Immunostaining for epithelial markers including E-cadherin, cytokeratin 18, mucin, and ZO-1 was performed to ensure that the cells were epithelial cells (data not shown). The hFIEC culture medium consisted of OptiMEM supplemented with 20 ng/mL human epidermal growth factor, 150 nmol//L hydrocortisone 21-hemisuccinate sodium salt, 0.2 U/mL human recombinant insulin, and 4% FBS. In addition, the nontransformed primary human fetal intestinal epithelial cell line H4 (11) was used in these studies. The H4 culture medium consisted of DMEM supplemented with 5% heat-inactivated FBS, 5% heat-inactivated neonatal bovine serum, 1% glutamine, 1% sodium pyruvate, 1% nonessential amino acids, 1% HEPES, 0.2 U/mL insulin, 50 U/mL penicillin, and 50 μg/mL streptomycin. To determine whether TGF-β2 modulates inflammatory responses in mature IECs, the adult IEC lines T84 and NCM460 were used in these studies. Both T84 and NCM460 cells are derived from the colon, which should be taken into consideration when interpreting the results. T84 culture medium consisted of DMEM supplemented with 5% heat-inactivated FBS, 1% glutamine, 2.5% HEPES, 50 U/mL penicillin, and 50 μg/mL streptomycin. NCM-460 culture medium consisted of M3D medium supplemented with 10% FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin. The cells were cultured in culture dishes at 37°C with 95% O2 and 5% CO2 atmosphere saturated with water vapor.
Effect of Conditioning With TGF-β2 on Subsequent Inflammatory Cytokine Secretion
Cells were grown to 70% confluence and treated with 3 ng/mL TGF-β2 for the indicated times before stimulation with 1 ng/mL IL-1β, 10 ng/mL TNF-α, or 50 ng/mL poly I:C. These proinflammatory stimulants were chosen after preliminary experiments showed that they induce consistent and significant inflammatory cytokine secretion in the cell culture models. They also provide a representative selection of proinflammatory insults experienced by the infant gut. TNF-α and IL-1β are powerful endogenous inflammatory mediators implicated, for example, in the pathogenesis of NEC (12). Poly I:C, however, is a synthetic ligand for Toll-like receptor (TLR)3, which binds viral antigens, whereas IL-1β, even though an endogenous molecule, shares an intracellular signaling pathway with TLRs recognizing bacterial molecular structures (13). Medium alone served as control. In some experiments, the cells were grown in medium for 2 to 24 hours after exposure to TGF-β2 before stimulation with IL-1β (“washout period”) as indicated. All of the experiments were performed in triplicate or quadruplicate. After 18 hours, the culture medium was collected, stored, and subjected to IL-8 and IL-6 measurement by enzyme-linked immunosorbent assay as described previously (9). IL-8 and IL-6 secretion in IEC culture experiments was normalized to total cellular protein content measured as described previously (9). The LDH Cytotoxicity Detection kit was used to assess cell viability among groups according to the manufacturer's instructions.
Effect of Conditioning With TGF-β2 on Subsequent Inflammatory Cytokine Gene Expression
hFIECs cells and H4 cells were grown to 70% confluence and treated with TGF-β2 for 48 hours before stimulation with IL-1β. Untreated cells served as control. All of the experiments were performed in triplicate. After 6 hours, total cellular RNA was extracted by the TRIzol-chloroform extraction method as described previously (14). This time point was chosen based on a preliminary time course experiment (data not shown). mRNA for IL-8 and IL-6 was measured in duplicate for each sample by qRT-PCR using the SuperScript III Platinum SYBR Green One-Step qRT-PCR kit with MJ Opticon 2 DNA engine (MJ Research Inc, Waltham, MA) according to the manufacturer's instructions. OpticonMONITOR analysis software version 2.01 (MJ Research Inc) was used to normalize the levels of IL-8 and IL-6 mRNA to the standard glyceraldehyde 3-phosphate dehydrogenase level for each sample.
Effect of Conditioning With TGF-β2 on Subsequent IL-1β-Induced NF-κB p65 Nuclear Translocation
H4 cells were grown to 70% confluency on coverslips and conditioned with TGF-β2 for 48 hours before stimulation with IL-1β for 10 minutes. Medium alone served as control. The cells were fixed in 4% paraformaldehyde for 20 minutes on ice. Once permeabilized with methanol (10 minutes on ice) and blocked with 10% goat serum in Tris-buffered saline (TBS) containing 0.25% bovine serum albumin (BSA), the cells were incubated overnight with a rabbit anti-human NF-κB p65 polyclonal antibody (1:2000) in TBS with 0.25% BSA. After washing, the cells were incubated with a Cy 3-conjugated goat anti-rabbit IgG (F[ab′]2) (1:2000) in TBS with 0.25% BSA and examined using a Zeiss Axiophot photomicroscope (Carl Zeiss, Oberkochen, Germany), and a digital image was obtained. Purified rabbit IgG was used as a control.
Involvement of Intracellular Signaling Pathways
H4 cells were grown to 70% confluency and conditioned with TGF-β2 for 48 hours before stimulation with IL-1β for 5, 15, 30, 60, 90, and 120 minutes. Untreated cells were used to determine baseline. The changing levels of activated kinases were determined by immunoblotting. Equal amounts of total cellular protein were fractionated by electrophoresis using NuPAGE 4% to 12% Bis-Tris gels with protein standards. Fractionated proteins were transferred onto a polyvinylidene fluoride membrane and blocked with 5% nonfat dry milk in TBS with 0.05% Tween 20 (TBST) before incubation overnight with the primary antibody at recommended concentration. The membrane was then washed with 5% milk in TBST and incubated with the appropriate horseradish peroxidase–conjugated secondary antibody. The amount of specific protein was visualized by enhanced chemiluminesence using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Rockford, IL). To confirm equal loading of the lanes, the membranes were stripped and reprobed with anti-glyceraldehyde 3-phosphate dehydrogenase antibody.
Inhibition of the ERK Pathway
To determine the role of ERK activation in IL-1β-induced secretion of IL-8 and IL-6, the specific MEK1/2-inhibitor PD 98059 was used. H4 cells were grown to 70% confluence and treated with 100 μmol/L PD 98059 1 hour before stimulation with IL-1β. Untreated cells served as control.
hFIECs were grown to 50% confluence and conditioned with TGF-β2 for 48 hours. Untreated cells served as control. The experiment was performed in quadruplicate. Total cellular RNA was extracted by the Trizol-chloroform extraction method as described previously (14) and purified using RNA extraction columns obtained from Qiagen as per the manufacturer's instructions. Affymetrix (Santa Clara, CA) cDNA microarray analysis was carried out by the genomic core facility at Harvard Medical School using Human Genome U133 Plus 2.0 Array. Differences in gene expression between hFIECs exposed and not exposed to TGF-β2 were analyzed using GeneGo Pathway Maps analysis software (Thompson Reuters, St Joseph, MO) with a false discovery rate filter significance level P < 0.01.
Secretion of IL-8 and IL-6 in cell culture experiments was expressed as mean ± standard error; comparisons between groups were performed using the 2-tailed Student t test. Gene expression data obtained by qRT-PCR are expressed as geometric mean with standard error after logarithmic transformation; comparisons between groups were performed using the Student 2-tailed t test after logarithmic transformation. P < 0.05 was considered statistically significant.
Conditioning With TGF-β2 Attenuates Subsequent Inflammatory Cytokine Secretion in Immature Human IEC
To investigate whether exposure to TGF-β2 modulates subsequent inflammatory responses in immature human IECs, H4 cells were conditioned 48 hours with and without 3 ng/mL TGF-β2 and then stimulated with IL-1β, and the inflammatory response was assessed by measuring secretion of IL-8 (Fig. 1A) and IL-6 (Fig. 1B). IL-1β increased IL-8 secretion from 0 ± 0 ng/mg protein to 3784 ± 41 ng/mg protein (P < 0.0001). Conditioning with TGF-β2 for 48 hours significantly attenuated subsequent IL-1β-induced IL-8 secretion to 1294 ± 106 ng/mg protein (P = 0.00012). In parallel, IL-1β increased IL-6 secretion from 100 ± 4 to 3184 ± 145 ng/mg protein (P < 0.0001), and this response was attenuated to 631 ± 52 ng/mg protein (P = 0.00091) after conditioning with TGF-β2. The anti-inflammatory effect of TGF-β2 was not confined to IL-1β-induced responses because a similar reduction in TNF-α-induced IL-8 (Fig. 1C) and poly I:C-induced IL-8 (Fig. 1D) and IL-6 (Fig. 1E) secretion as a consequence of TGF-β2 conditioning was observed in H4 cells. As previously reported (9), TGF-β2 administered simultaneously with IL-1β resulted in a reduction of IL-8 and IL-6 secretion. A similar modest reduction was observed in TNF-α-induced inflammatory cytokine secretion (Fig. 1C), whereas TGF-β2 administered simultaneously with poly I:C had no effect on inflammatory cytokine secretion (Fig. 1D and E). The anti-inflammatory effect on IL-1β– or TNF-α–induced cytokine secretion was significantly more pronounced after conditioning with TGF-β2 for 48 hours. In addition, a significant reduction in poly I:C-induced IL-6 secretion was observed after 48-hour conditioning (Fig. 1E).
The results obtained from H4 cells were confirmed by conducting similar experiments using primary hFIECs isolated from an 18-week-old fetus (Fig. 2). IL-1β induced a significant increase in IL-8 secretion from 43 ± 4 to 5731 ± 262 ng/mg protein (P < 0.0001). Conditioning with TGF-β2 for 48 hours attenuated IL-1β–induced IL-8 secretion to 3788 ± 133 ng/mg protein (P = 0.0027, Fig. 2A). IL-1β increased IL-6 secretion from 127 ± 11 to 5297 ± 306 ng/mg protein (P < 0.0001), which was reduced to 1754 ± 21 ng/mg protein by exposure to TGF-β2 (P = 0.00032, Fig. 2B). A significant reduction in TNF-α-induced IL-8 (Fig. 2C) and IL-6 (Fig. 2D) secretion as a consequence of TGF-β2 conditioning was detected in hFIECs.
TGF-β2 had no effect on inflammatory responsiveness in mature human colonic IECs. IL-1–induced IL-8 secretion in NCM460 cells (Fig. 3A) and TNF-α–induced IL-8 secretion in T84 cells (Fig. 3B) remained unaltered after conditioning with TGF-β2.
To investigate whether exposure to TGF-β2 induces a long-lasting change in IEC immune responder phenotype, H4 cells were conditioned with TGF-β2 for 24 hours and then stimulated with IL-1β after a 2- to 24-hour “washout period” with medium alone (Fig. 4). The 24-hour conditioning with TGF-β2 resulted in a significant attenuation of subsequent IL-1β–induced IL-8 (Fig. 4A) and IL-6 (Fig. 4B) secretion. It is of note that the presence of TGF-β2 at the time of stimulation with IL-1β was not necessary for the inhibitory effect. Moreover, the immunomodulatory effect of TGF-β2 conditioning remained unaltered after up to 24 hours in medium devoid of TGF-β2.
Conditioning With TGF-β2 Attenuates Subsequent IL-1β-Induced Inflammatory Cytokine Secretion in Immature IECs by Reducing mRNA Expression
The effect of conditioning with TGF-β2 on subsequent IL-1β–induced expression of IL-8 and IL-6 mRNA in H4 cells was assessed using qRT-PCR (Fig. 5A and B). Stimulation with IL-1β resulted in a 6849-fold increase in IL-8 mRNA (P < 0.0001) after 6 hours. This response was reduced 540-fold by previous conditioning with TGF-β2 (P = 0.00011). In a similar fashion, IL-6 mRNA expression was increased 691-fold by IL-1β (P < 0.0001), but the response was attenuated 39-fold after conditioning with TGF-β2 (P < 0.0001).
Conditioning With TGF-β2 Reduces IL-1β-Induced NF-κB Nuclear Translocation by Inhibiting IκB-α Degradation
Nuclear translocation of the transcription factor NF-κB is an important event in IL-1β–induced transcriptional activation of inflammatory gene expression. Stimulation with IL-1β was observed to induce nuclear translocation of NF-κB p65 within 10 minutes in H4 cells (Fig. 6A). Conditioning with TGF-β2 for 48 hours before stimulation with IL-1β partially inhibited IL-1β–induced nuclear translocation of NF-κB p65 (Fig. 6A).
Degradation of IκB-α is necessary for the release and subsequent nuclear translocation of NF-κB p65. To assess whether IL-1β–induced IκB-α degradation was reduced in H4 cells conditioned with TGF-β2, the rate of IκB-α degradation was monitored by immunoblotting. Stimulation with IL-1β led to rapid degradation of IκB-α in H4 cells within 15 minutes of stimulation (Fig. 6B). Conditioning of H4 cells with TGF-β2 for 48 hours partially inhibited subsequent IL-1β–induced degradation of IκB-α (Fig. 6B).
We recently reported that when administered simultaneously with IL-1β, TGF-β2 inhibits IL-1β–induced phosphorylation of ERK in H4 cells, and this inhibitory effect is causally related to attenuation of IL-1β–induced IL-8 and IL-6 secretion by TGF-β2 (9); however, in H4 cells conditioned with TGF-β2 48 hours before stimulation with IL-1β, a modest reduction in ERK phosphorylation not exceeding that resulting from exposure to TGF-β2 simultaneously with IL-1β was observed (Fig. 7A). In line with this observation, conditioning with TGF-β2 induced a significant further reduction in IL-1β–induced IL-8 (Fig. 7B) and IL-6 (Fig. 7C) secretion when ERK signaling was inhibited by the specific ERK kinase inhibitor PD 98059. Taken together, these data suggest that modulation of ERK signaling is not the primary mechanism by which conditioning with TGF-β2 attenuates IEC inflammatory responses.
Effect of TGF-β2 Conditioning on Expression of Genes in the IL-1β Signaling Pathway
Systematic analyses of functional gene groups and pathways contained within the well-annotated MetaCore database (Thompson Reuters) canonical gene pathway maps using our gene expression data obtained by cDNA microarray from hFIECs identified enrichment of genes involved in several signaling pathways or cellular processes. Our data indicate that conditioning with TGF-β2 for 48 hours had a significant effect on the expression of genes in a number of physiological processes including development and immune responses. The 10 most significantly affected pathways by conditioning with TGF-β2 for 48 hours are presented in Table 1. Of particular interest, the IL-1 signaling pathway was among the most significantly modulated pathways, which allowed further investigation into gene expression patterns within this pathway (Table 2). Among the downregulated genes, several proteins that are known to be positive regulators of IL-1 signaling and among the upregulated genes a number of proteins that are known to be negative regulators were identified (Table 2). Results obtained from cDNA microarray analyses were confirmed using qRT-PCR for selected key genes in the IL-1 signaling pathway. According to the qRT-PCR analyses, conditioning with TGF-β2 for 48 hours increased the expression of IκB-α 1.55-fold (P = 0.047) and decreased that of MyD88 2.70-fold (P = 0.058). These results correspond to those obtained by cDNA microarray analysis.
Our studies demonstrate that conditioning immature human IECs with TGF-β2 at a concentration corresponding to that in breast milk modulates their response to subsequent proinflammatory stimulation. Secretion of the inflammatory cytokines IL-8 and IL-6 in response to IL-1β, TNF-α, and the TLR3 ligand poly I:C was significantly reduced in human fetal IECs conditioned with TGF-β2 for 48 hours before the proinflammatory insult. The reduction in IEC inflammatory cytokine secretion after conditioning with TGF-β2 was more pronounced than that reported (9) when IECs were treated with TGF-β2 simultaneously with proinflammatory stimulation, and this anti-inflammatory effect was not dependent on TGF-β2 being present during stimulation with IL-1β. It is also of note that conditioning with TGF-β2 is necessary for significant attenuation of IL-8 secretion in response to poly I:C not seen with simultaneous stimulation. Taken together, these findings suggest that the anti-inflammatory effect of breast milk TGF-β2 on immature human IECs spans inflammatory responses elicited toward a wide range of endogenous and microbial signals and is mediated by several distinct mechanisms.
We have reported that TGF-β2 inhibits IL-1β-induced activation of the ERK signaling pathway in immature IECs if administered simultaneously with the proinflammatory stimulus and that intact ERK signaling is necessary for an optimal inflammatory cytokine mRNA expression and protein secretion (9). Conditioning with TGF-β2 48 hours before stimulation did not result in a greater inhibition of ERK activation as compared with when TGF-β2 was introduced simultaneously with IL-1β while the reduction in IL-1β-induced IL-8 and IL-6 response achieved by conditioning with TGF-β2 was greater than that observed after blocking ERK signaling by the specific inhibitor PD 98059 (Fig. 7). These data demonstrate that in contrast to the immediate effect of TGF-β2 on immature IEC inflammatory responsiveness, inhibition of the ERK signaling pathway is not the primary mechanism by which conditioning with TGF-β2 attenuates subsequent IL-1β-induced inflammatory cytokine secretion in immature human IECs.
According to a report by Choi et al (15), TGF-β1 inhibits IL-1β–induced nuclear translocation of the transcription factor NF-κB by reducing IκB-α degradation. In line with this observation, we detected a reduction in IL-1β–induced IκB-α degradation in H4 cells conditioned with TGF-β2 for 48 hours (Fig. 6B). Furthermore, the same treatment appeared to partially inhibit nuclear translocation of the p65 subunit of NF-κB as visualized by immunofluorescence (Fig. 6A), which may at least partially explain the reduction in IL-1β–induced IL-8 and IL-6 mRNA expression and IL-8 and IL-6 protein secretion observed in immature IECs conditioned with TGF-β2 (Fig. 5).
Significant changes in the expression of genes involved in the IL-1 signaling pathway were detected in immature human IECs conditioned with TGF-β2 for 48 hours according to a preliminary cDNA microarray analysis (Table 2). These data suggest that the observed decrease in IL-1β–induced IκB-α degradation and subsequent inflammatory cytokine gene expression and cytokine secretion in immature IECs conditioned with TGF-β2 may in part be explained by increased expression of mRNA for IκB-α and reduced expression of mRNA for MyD88 and IKK-β, the function of which is to degrade IκB-α when activated by, for example, IL-1β. Our laboratory has reported that the expression of IκB-α is developmentally underexpressed in immature IECs (16), and our present data may therefore be interpreted to suggest that breast milk TGF-β2 induces a maturational upregulation of this gene and thus promotes a more mature anti-inflammatory tone in the intestinal epithelium. The changes in the expression of genes relevant to the present investigation induced by TGF-β2 were not confined to these intracellular signaling molecules. The expression of IL-1 receptor mRNA was significantly decreased in IECs conditioned with TGF-β2, which may suggest that responsiveness to IL-1β in these cells may be diminished as a consequence of reduced expression of the receptor on the cell surface. Assessing cell surface protein expression and more detailed analyses of the microarray data are beyond the scope of the present report. Nonetheless, these expression data highlight the potential clinical importance of breast milk TGF-β2 in the modulation of expression of genes regulating innate immunity and inflammation.
It is well established that TGF-β is involved in maturation processes in immune cells (2). TGF-β induces IgA class switch in B cells (17) and alters antigen-presenting cell function (18) and thus modulates T cell maturation. In particular, TGF-β favors generation of regulatory T cells, which are essential in tolerance toward, for example, dietary antigens and indigenous intestinal microbes (19). Excessive expression of inflammatory cytokines in immature human intestinal macrophages has been shown to be suppressed as a result of maturation upon exposure to TGF-β2 (5). It is intriguing to speculate that TGF-β2 may also have a maturational effect on neonatal human IECs, which are in direct contact with breast milk TGF-β2. Based on our cDNA microarray data, conditioning with TGF-β2 induces changes in the expression of genes involved in cytoskeleton remodeling and development (Table 1), which may be interpreted to support the notion of TGF-β2–induced generalized maturation in immature IECs. According to our present observations and in contrast to our previous report (9), conditioning with TGF-β2 induces functional changes in IEC inflammatory responsiveness to IL-1β, which are not dependent on TGF-β2 being present at the time of the proinflammatory insult and remain unaltered after up to a 24-hour period devoid of TGF-β2 (Fig. 4). Unfortunately, the cell culture models used in the present series of experiments did not allow longer exposure or washout times to further assess the matter. Nonetheless, together with the fact that mature IEC responses were not affected by exposure to TGF-β2 (Fig. 3), we interpret our data to suggest that the neonatal gut immune responder may be modified by exposure to TGF-β2, and that breast milk TGF-β2 may act as an important maturational signal to the developing intestine. This notion is consistent with data from experimental animal studies demonstrating that TGF-β in breast milk is necessary for healthy immune maturation as indicated by defective formation of immune tolerance to inhaled (20) or dietary antigens (21) in newborn animals deprived of breast milk TGF-β function, and epidemiological data suggesting an inverse correlation between breast milk TGF-β2 and subsequent development of immune-mediated disease in infants and children (8).
We conclude that our data demonstrate that conditioning with TGF-β2 at a concentration comparable with that found in breast milk profoundly affects immature human IEC immune responsiveness. We hypothesize that breast milk TGF-β2 functions as a maturational stimulus to the infant's developing intestine and has a long-term effect on immune responsiveness. It is conceivable that supplementing premature infants with TGF-β2 may provide a novel means to support gut maturation and reduce the risk of disorders resulting from intestinal immaturity such as NEC. Further investigations into immune conditioning by breast milk TGF-β2 in more complex in vivo models, including experimental animals and eventually clinical studies, are warranted.
We thank Dr C. Pothoulakis and Dr P. Moyer for providing the NCM460 cells. The head of the Genomics Core for the Harvard Clinical Nutrition Research Center, Dr Ferederick M. Ausubel, is acknowledged for cDNA microarray analyses.
1. Newburg DS, Walker WA. Protection of the neonate by the innate immune system of developing gut and of human milk. Pediatr Res
2. Rautava S, Walker WA. Academy of Breastfeeding Medicine founder's lecture 2008: breastfeeding—an extrauterine link between mother and child. Breastfeed Med
3. Akobeng AK, Ramanan AV, Buchan I, et al. Effect of breast feeding on risk of coeliac disease: a systematic review and meta-analysis of observational studies. Arch Dis Child
4. Oddy WH, Holt PG, Sly PD, et al. Association between breast feeding and asthma in 6 year old children: findings of a prospective birth cohort study. BMJ
5. Maheshwari A, Kelly DR, Nicola T, et al. TGF-beta2 suppresses macrophage cytokine production and mucosal inflammatory responses in the developing intestine. Gastroenterology
6. Penttila IA, van Spriel AB, Zhang MF, et al. Transforming growth factor-beta levels in maternal milk and expression in postnatal rat duodenum and ileum. Pediatr Res
7. Zhang MF, Zola H, Read LC, et al. Localization of transforming growth factor-beta receptor types I, II, and III in the postnatal rat small intestine. Pediatr Res
8. Kalliomaki M, Ouwehand A, Arvilommi H, et al. Transforming growth factor-beta in breast milk: a potential regulator of atopic disease at an early age. J Allergy Clin Immunol
9. Rautava S, Nanthakumar NN, Dubert-Ferrandon A, et al. Breast milk-transforming growth factor-2 specifically attenuates IL-1β-induced inflammatory responses in the immature human intestine via an SMAD6- and ERK-dependent mechanism. Neonatology
10. Quaroni A, Beaulieu JF. Cell dynamics and differentiation of conditionally immortalized human intestinal epithelial cells. Gastroenterology
11. Sanderson IR, Ezzell RM, Kedinger M, et al. Human fetal enterocytes in vitro: modulation of the phenotype by extracellular matrix. Proc Natl Acad Sci U S A
12. Viscardi RM, Lyon NH, Sun CC, et al. Inflammatory cytokine mRNAs in surgical specimens of necrotizing enterocolitis and normal newborn intestine. Pediatr Pathol Lab Med
13. Rautava S, Walker WA. Commensal bacteria and epithelial cross talk in the developing intestine. Curr Gastroenterol Rep
14. Nanthakumar NN, Fusunyan RD, Sanderson I, et al. Inflammation in the developing human intestine: a possible pathophysiologic contribution to necrotizing enterocolitis. Proc Natl Acad Sci U S A
15. Choi KC, Lee YS, Lim S, et al. Smad6 negatively regulates interleukin 1-receptor-toll-like receptor signaling through direct interaction with the adaptor Pellino-1. Nat Immunol
16. Claud EC, Lu L, Anton PM, et al. Developmentally regulated IkappaB expression in intestinal epithelium and susceptibility to flagellin-induced inflammation. Proc Natl Acad Sci U S A
17. van Vlasselaer P, Punnonen J, de Vries JE. Transforming growth factor-beta directs IgA switching in human B cells. J Immunol
18. Takeuchi M, Alard P, Streilein JW. TGF-beta promotes immune deviation by altering accessory signals of antigen-presenting cells. J Immunol
19. Li MO, Wan YY, Sanjabi S, et al. Transforming growth factor-beta regulation of immune responses. Annu Rev Immunol
20. Verhasselt V, Milcent V, Cazareth J, et al. Breast milk-mediated transfer of an antigen induces tolerance and protection from allergic asthma. Nat Med
21. Penttila I. Effects of transforming growth factor-beta and formula feeding on systemic immune responses to dietary beta-lactoglobulin in allergy-prone rats. Pediatr Res
Keywords:Copyright 2012 by ESPGHAN and NASPGHAN
immature intestinal epithelium; modulation of immune response; TGF-β