Maria E. C. Bruno

Secretory antibodies in breast milk promote long-term intestinal homeostasis by regulating the gut microbiota and host gene expression

Significance An experimental system was developed in mice to study the long-term benefits of early exposure to secretory antibodies of the IgA class (SIgA) in breast milk. We found that breast milk-derived SIgA promoted intestinal epithelial barrier function in suckling neonates, preventing systemic infection by potential pathogens. Long-term benefits of early exposure to SIgA included maintenance of a healthy gut microbiota and regulation of gene expression in intestinal epithelial cells. These findings suggest that maternal antibodies provide benefits to the intestinal immune system of the breast-fed infant, which persist into adulthood. Maintenance of intestinal homeostasis requires a healthy relationship between the commensal gut microbiota and the host immune system. Breast milk supplies the first source of antigen-specific immune protection in the gastrointestinal tract of suckling mammals, in the form of secretory IgA (SIgA). SIgA is transported across glandular and mucosal epithelial cells into external secretions by the polymeric Ig receptor (pIgR). Here, a breeding scheme with polymeric Ig receptor-sufficient and -deficient mice was used to study the effects of breast milk-derived SIgA on development of the gut microbiota and host intestinal immunity. Early exposure to maternal SIgA prevented the translocation of aerobic bacteria from the neonatal gut into draining lymph nodes, including the opportunistic pathogen Ochrobactrum anthropi. By the age of weaning, mice that received maternal SIgA in breast milk had a significantly different gut microbiota from mice that did not receive SIgA, and these differences were magnified when the mice reached adulthood. Early exposure to SIgA in breast milk resulted in a pattern of intestinal epithelial cell gene expression in adult mice that differed from that of mice that were not exposed to passive SIgA, including genes associated with intestinal inflammatory diseases in humans. Maternal SIgA was also found to ameliorate colonic damage caused by the epithelial-disrupting agent dextran sulfate sodium. These findings reveal unique mechanisms through which SIgA in breast milk may promote lifelong intestinal homeostasis, and provide additional evidence for the benefits of breastfeeding.

Targeted deletion of MyD88 in intestinal epithelial cells results in compromised antibacterial immunity associated with downregulation of polymeric immunoglobulin receptor, mucin-2, and antibacterial peptides.

Intestinal epithelial cells (IECs) form a physical and immunological barrier that separates the vast gut microbiota from host tissues. MyD88-dependent Toll-like receptor signaling is a key mediator of microbial–host cross-talk. We examined the role of epithelial MyD88 expression by generating mice with an IEC-targeted deletion of the Myd88 gene (MyD88ΔIEC). Loss of epithelial MyD88 signaling resulted in increased numbers of mucus-associated bacteria; translocation of bacteria, including the opportunistic pathogen Klebsiella pneumoniae, to mesenteric lymph nodes; reduced transmucosal electrical resistance; impaired mucus-associated antimicrobial activity; and downregulated expression of polymeric immunoglobulin receptor (the epithelial IgA transporter), mucin-2 (the major protein of intestinal mucus), and the antimicrobial peptides RegIIIγ and Defa-rs1. We further observed significant differences in the composition of the gut microbiota between MyD88ΔIEC mice and wild-type littermates. These physical, immunological, and microbial defects resulted in increased susceptibility of MyD88ΔIEC mice to experimental colitis. We conclude that MyD88 signaling in IECs is crucial for maintenance of gut homeostasis.

Regulation of the Polymeric Ig Receptor by Signaling through TLRs 3 and 4: Linking Innate and Adaptive Immune Responses

IgA Abs help to maintain homeostasis at mucosal surfaces by promoting defense mechanisms that protect against pathogens while suppressing inflammatory responses to commensal organisms and food Ags. The polymeric Ig receptor (pIgR) mediates transport of IgA across mucosal epithelial cells. We hypothesized that signaling through TLRs may up-regulate pIgR expression by intestinal epithelial cells and thus enhance IgA-mediated homeostasis. To test this hypothesis we treated the HT29 human intestinal epithelial cell line with dsRNA, a ligand for TLR3, or LPS, a ligand for TLR4. Both dsRNA and LPS up-regulated levels of pIgR mRNA and cell surface pIgR protein. By contrast, dsRNA but not LPS up-regulated expression of TLR3 and TLR4 mRNA. However, cell surface expression of both TLR3 and TLR4 was enhanced by treatment of HT29 cells with their respective ligands. Transfection of HT29 cells with wild-type and mutated promoter/enhancer plasmids suggested that TLR3 and TLR4 signal primarily through NF-κB to enhance transcription of pIgR mRNA. TLR3 signaling resulted in a more pronounced inflammatory response than did TLR4, as evidenced by up-regulation of the transcription factor IFN regulatory factor-1, chemokines IL-8 and RANTES, and the proinflammatory cytokine TNF. Signaling through LPS/TLR4 appears to up-regulate pIgR expression while minimizing proinflammatory responses, a mechanism that could promote IgA-mediated homeostasis in the presence of commensal Gram-negative bacteria.

Signature biomarkers in Crohn's disease: toward a molecular classification.

In an effort to develop a molecular classification scheme for Crohns disease (CD), mucosal biopsies from 69 CD patients and 28 normal controls were analyzed for expression of the RelA subunit of nuclear factor (NF)-κB, A20 (a negative regulator of NF-κB), polymeric immunoglobulin receptor (pIgR), tumor necrosis factor (TNF), and interleukin (IL)-8. Principal component analysis was used to classify individuals into three subsets based on patterns of biomarker expression. Set 1 included normal subjects and CD patients with mild disease and good responses to therapy, thus defining “normal” biomarker expression. CD patients in set 2, characterized by low expression of all five biomarkers, had moderate to severe disease and poor responses to immunosuppressive and anti-TNF therapy. Patients in set 3, characterized by low expression of RelA, A20, and pIgR, normal TNF and elevated IL-8, had acute inflammation that responded well to therapy. Classification of CD patients by these biomarkers may predict disease behavior and responses to therapy.

Regulation of the polymeric immunoglobulin receptor by the classical and alternative NF-κB pathways in intestinal epithelial cells

The polymeric immunoglobulin receptor (pIgR) transports IgA antibodies across intestinal epithelial cells (IECs). Expression of pIgR is upregulated by proinflammatory signaling pathways via activation of nuclear factor-κB (NF-κB). Here, we examined the contributions of the RelA-dependent classical and RelB-dependent alternative pathways of NF-κB to pIgR regulation in the HT-29 human IEC line following stimulation with tumor necrosis factor (TNF), lipopolysaccharide (LPS; Toll-like receptor 4 (TLR4) ligand), and polyinosinic: polycytidylic acid (pIC; TLR3 ligand). Whereas induction of proinflammatory genes such as interleukin-8 (IL-8) required only RelA, pIgR expression was regulated by complex mechanisms that involved both RelA and RelB. Upregulation of pIgR expression by ligation of the lymphotoxin-β receptor suggested a direct role for the alternative NF-κB pathway. Inhibition of mitogen-activated protein kinases reduced the induction of IL-8, but enhanced the induction of pIgR by TNF and TLR signaling. Regulation of pIgR through unique signaling pathways could allow IECs to sustain high levels of IgA transport while limiting the proinflammatory responses.

Long-Term Exposure of the HT-29 Human Intestinal Epithelial Cell Line to TNF Causes Sustained Up-Regulation of the Polymeric Ig Receptor and Proinflammatory Genes through Transcriptional and Posttranscriptional Mechanisms

Transport of IgA Abs across intestinal epithelial cells into gut secretions is mediated by the polymeric Ig receptor (pIgR). The cytokine TNF plays a central role in initiating and amplifying inflammatory reactions, and is implicated in the pathogenesis of inflammatory bowel diseases. Acute exposure of intestinal epithelial cell lines to TNF has been shown to up-regulate transcription of genes encoding pIgR and a number of proinflammatory factors, but the effects of chronic exposure to TNF have not been studied. We found that exposure of HT-29 human colon carcinoma cells to TNF for up to 20 days reduced the rate of cell proliferation, but did not cause gross morphological changes. Expression of mRNA encoding pIgR and several proinflammatory genes increased acutely, and then diminished but remained elevated above control levels throughout the experiment. Changes in gene expression were paralleled by increased expression of the transcription factors IFN regulatory factor-1 and the RelB subunit of NF-κB. HT-29 cells activated the endogenous TNF gene in response to TNF treatment, but the level of TNF production was insufficient to maintain pIgR and proinflammatory gene expression after withdrawal of exogenous TNF. Chronic exposure to TNF caused a marked increase in pIgR mRNA stability and a small but significant decrease in TNF mRNA stability, but no change in the half-lives of IL-8, c-Myc, and GAPDH. In summary, we observed different effects of acute vs chronic exposure to TNF on gene expression, and found evidence for transcriptional and posttranscriptional regulation of expression of the pIgR.

Regulation of the Polymeric Immunoglobulin Receptor in Intestinal Epithelial Cells by Enterobacteriaceae: Implications for Mucosal Homeostasis

The commensal microbiota of the human colon profoundly impacts host gene expression and mucosal homeostasis. Secretory IgA antibodies, which influence the composition of the intestinal microbiota and provide immunity against pathogens, are transported across intestinal epithelial cells (IEC) by the polymeric immunoglobulin receptor (pIgR). To compare the effects of different colonic bacteria on pIgR expression, the human IEC line HT-29 was stimulated with various species representing the 4 major phyla of colonic bacteria. Only bacteria from the family Enterobacteriaceae (phylum Proteobacteria) induced expression of pIgR and other target genes of bacterial pattern recognition receptors. HT-29 cells responded to purified ligands for Toll-like receptor (TLR)4 but not TLR2. Expression of pIgR and transport of IgA were significantly reduced in colons of mice deficient in the TLR adaptor MyD88, consistent with a role for TLR signaling in the regulation of pIgR by colonic bacteria. Induction of pIgR expression in HT-29 cells required NF-κB signaling but not MAPK signaling, in contrast to the requirement for both NF-κB and MAPK signaling for induction of pro-inflammatory genes. These results suggest that commensal Enterobacteriaceae may promote intestinal homeostasis by enhancing pIgR expression in IEC.

Secretory IgA is Concentrated in the Outer Layer of Colonic Mucus along with Gut Bacteria

Antibodies of the secretory IgA (SIgA) class comprise the first line of antigen-specific immune defense, preventing access of commensal and pathogenic microorganisms and their secreted products into the body proper. In addition to preventing infection, SIgA shapes the composition of the gut microbiome. SIgA is transported across intestinal epithelial cells into gut secretions by the polymeric immunoglobulin receptor (pIgR). The epithelial surface is protected by a thick network of mucus, which is composed of a dense, sterile inner layer and a loose outer layer that is colonized by commensal bacteria. Immunofluorescence microscopy of mouse and human colon tissues demonstrated that the SIgA co-localizes with gut bacteria in the outer mucus layer. Using mice genetically deficient for pIgR and/or mucin-2 (Muc2, the major glycoprotein of intestinal mucus), we found that Muc2 but not SIgA was necessary for excluding gut bacteria from the inner mucus layer in the colon. Our findings support a model whereby SIgA is anchored in the outer layer of colonic mucus through combined interactions with mucin proteins and gut bacteria, thus providing immune protection against pathogens while maintaining a mutually beneficial relationship with commensals.

Lessons from mother: Long-term impact of antibodies in breast milk on the gut microbiota and intestinal immune system of breastfed offspring

From birth to adulthood, the gut microbiota matures from a simple community dominated by a few major bacterial groups into a highly diverse ecosystem that provides both benefits and challenges to the host. Currently there is great interest in identifying environmental and host factors that shape the development of our gut microbiota. Breast milk is a rich source of maternal antibodies, which provide the first source of adaptive immunity in the newborns intestinal tract. In this addendum, we summarize our recent data demonstrating that maternal antibodies in breast milk promote long-term intestinal homeostasis in suckling mice by regulating the gut microbiota and host gene expression. We also discuss important unanswered questions, future directions for research in this field, and implications for human health and disease.

Multifactorial patterns of gene expression in colonic epithelial cells predict disease phenotypes in experimental colitis

Background: The pathogenesis of inflammatory bowel disease (IBD) is complex and the need to identify molecular biomarkers is critical. Epithelial cells play a central role in maintaining intestinal homeostasis. We previously identified five “signature” biomarkers in colonic epithelial cells (CEC) that are predictive of disease phenotype in Crohns disease. Here we investigate the ability of CEC biomarkers to define the mechanism and severity of intestinal inflammation. Methods: We analyzed the expression of RelA, A20, pIgR, tumor necrosis factor (TNF), and macrophage inflammatory protein (MIP)‐2 in CEC of mice with dextran sodium sulfate (DSS) acute colitis or T‐cell‐mediated chronic colitis. Factor analysis was used to combine the five biomarkers into two multifactorial principal components (PCs). PC scores for individual mice were correlated with disease severity. Results: For both colitis models, PC1 was strongly weighted toward RelA, A20, and pIgR, and PC2 was strongly weighted toward TNF and MIP‐2, while the contributions of other biomarkers varied depending on the etiology of inflammation. Disease severity was correlated with elevated PC2 scores in DSS colitis and reduced PC1 scores in T‐cell transfer colitis. Downregulation of pIgR was a common feature observed in both colitis models and was associated with altered cellular localization of pIgR and failure to transport IgA. Conclusions: A multifactorial analysis of epithelial gene expression may be more informative than examining single gene responses in IBD. These results provide insight into the homeostatic and proinflammatory functions of CEC in IBD pathogenesis and suggest that biomarker analysis could be useful for evaluating therapeutic options for IBD patients. (Inflamm Bowel Dis 2012;)