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Mucosal DCs play a critical role in tissue homeostasis. Several stimuli can induce a mucosal phenotype; however, molecular pathways that regulate development of mucosal DC function are relatively unknown. This study sought to determine whether PPARγ contributes to the development of the "mucosal" phenotype in mouse DCs. Experiments demonstrated that PPARγ activation in BMDCs induced an immunosuppressive phenotype in which BMDCs had reduced expression of MHC class II and costimulatory molecules, increased IL-10 secretion, and reduced the ability to induce CD4 T cell proliferation. Activation of PPARγ enhanced the ability of BMDC to polarize CD4 T cells toward iTregs and to induce T cell expression of the mucosal homing receptor, CCR9. Activation of PPARγ increased the ability of BMDCs to induce T cell-independent IgA production in B cells. BMDCs from PPARγ(ΔDC) mice displayed enhanced expression of costimulatory molecules, enhanced proinflammatory cytokine production, and decreased IL-10 synthesis. Contrary to the inflammatory BMDC phenotype in vitro, PPARγ(ΔDC) mice showed no change in the frequency or phenotype of mDC in the colon. In contrast, mDCs in the lungs were increased significantly in PPARγ(ΔDC) mice. A modest increase in colitis severity was observed in DSS-treated PPARγ(ΔDC) mice compared with control. These results indicate that PPARγ activation induces a mucosal phenotype in mDCs and that loss of PPARγ promotes an inflammatory phenotype. However, the intestinal microenvironment in vivo can maintain the mucosal DC phenotype of via PPARγ-independent mechanisms.

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 Crohn's 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., (Copyright © 2012 Crohn's & Colitis Foundation of America, Inc.)

Neonatal humans and rodents are susceptible to infection with encapsulated bacteria as a result of an inability to make antibodies to capsular polysaccharides. This is partly a result of decreased production of proinflammatory cytokines by splenic macrophages (MPhi) from neonates. In this study, we show that when stimulated with a variety of agonists to TLR2, -4, and -9, neonatal MPhi make less proinflammatory cytokines and more IL-10 than adult MPhi. IL-10 appears to have a role in the decreased proinflammatory cytokine production, as neonatal MPhi treated with anti-IL-10 receptor antibody or from IL-10(-/-) mice produced levels of proinflammatory cytokines at a level comparable with that produced by adult MPhi. A microarray analysis of RNA from resting and LPS-stimulated MPhi from neonatal and adult mice showed that expression of a large number of genes encoding cytokines, chemokines, and their receptors was decreased dramatically in the neonatal MPhi, although some cytokines, including IL-10 and IL-16, were enhanced. Several genes in the TLR signaling pathway leading to NF-kappaB activation were down-regulated, which may account for the decreased chemokine and cytokine synthesis. It is surprising that p38alpha MAPK, known to be required for TLR-induced cytokine secretion, was enhanced in the neonatal MPhi. Our studies with the p38 MAPK inhibitor SB203580 suggested that excess p38 MAPK activity can be inhibitory for TLR2-, -4-, and -9-induced production of proinflammatory cytokines but not IL-10. The anti-inflammatory phenotype of the neonatal Mphi may be unique to the developing organism, although it compromises the neonate's ability to respond to encapsulated bacteria.

Aged humans and rodents are susceptible to infection with Streptococcus pneumoniae bacteria as a result of an inability to make antibodies to capsular polysaccharides. This is partly a result of decreased production of proinflammatory cytokines and increased production of interleukin (IL)-10 by macrophages (Mphi) from aged mice. To understand the molecular basis of cytokine dysregulation in aged mouse Mphi, a microarray analysis was performed on RNA from resting and lipopolysaccharide (LPS)-stimulated Mphi from aged and control mice using the Affymetrix Mouse Genome 430 2.0 gene chip. Two-way ANOVA analysis demonstrated that at an overall P < 0.01 level, 853 genes were regulated by LPS (169 in only the young, 184 in only the aged, and 500 in both). Expression analysis of systematic explorer revealed that immune response (proinflammatory chemokines, cytokines, and their receptors) and signal transduction genes were specifically reduced in aged mouse Mphi. Accordingly, expression of Il1 and Il6 was reduced, and Il10 was increased, confirming our previous results. There was also decreased expression of interferon-gamma. Genes in the Toll-like receptor-signaling pathway leading to nuclear factor-kappaB activation were also down-regulated but IL-1 receptor-associated kinase 3, a negative regulator of this pathway, was increased in aged mice. An increase in expression of the gene for p38 mitogen-activated protein kinase (MAPK) was observed with a corresponding increase in protein expression and enzyme activity confirmed by Western blotting. Low doses of a p38 MAPK inhibitor (SB203580) enhanced proinflammatory cytokine production by Mphi and reduced IL-10 levels, indicating that increased p38 MAPK activity has a role in cytokine dysregulation in the aged mouse Mphi.

A reduction in macrophage (MPhi) function with aging makes mice less responsive to bacterial capsular polysaccharides, such as those present in the pneumococcal polysaccharide vaccine, a model of thymus independent (TI) antigen (Ag). Using trinitrophenol (TNP)-lipopolysaccharide (LPS) and TNP-Ficoll, two other well-studied TI Ag, we studied the mechanistic basis of reduced MPhi function in the aged. We show that aged mice are profoundly hyporesponsive to these TI Ag. As a result of a requirement for MPhi, highly purified B cells from young-adult mice do not respond to TI Ag. When purified, young B cells were immunized with TNP-Ficoll, the antibody production from those cultures reconstituted with MPhi from aged mice was significantly lower than that seen with young MPhi. Consequently, this unresponsiveness can be overcome by a mixture of interleukin (IL)-1beta and IL-6. Upon stimulation with LPS, in comparison with young MPhi, aged MPhi secreted reduced amounts of IL-6, tumor necrosis factor alpha, IL-1beta, and IL-12, cytokines necessary for B cells to respond to TI Ag. LPS also induced aged MPhi to produce an excess of IL-10. Neutralization of IL-10 enhanced the production of proinflamatory cytokines by MPhi upon LPS stimulation and also induced Ab production by aged splenocytes. Thus, the inability of aged MPhi to help the B cell response appears to be caused by an excess of IL-10. As aged MPhi have a reduced number of cells expressing Toll-like receptor 4 and CD14, the imbalance in cytokine production might be partly a result of fewer cells expressing key components of the LPS receptor complex.

Neonates do not respond to thymus-independent (TI) antigens (Ag), making them vulnerable to infection with encapsulated bacteria. The antibody (Ab) response of adult and neonatal B cells to TI Ag requires certain cytokines, which are provided by T cells or macrophages (MPhi). Lipopolysaccharide (LPS) failed to induce neonatal MPhi to produce interleukin (IL)-1beta and tumor necrosis factor alpha (TNF-alpha) mRNA and to secrete IL-1beta, IL-12, and TNF-alpha. However, LPS induced neonates to secrete some IL-6 and three- to fivefold more IL-10 than adults. Accordingly, adding adult but not neonatal MPhi could restore the response of purified adult B cells to trinitrophenol (TNP)-LPS, a TI Ag. Increased IL-10 is causally related to decreased IL-1beta and IL-6 production, as IL-10(-/-) neonatal MPhi responded to LPS by secreting more IL-1beta and IL-6 than wild-type (WT) neonatal MPhi. When cultures were supplemented with a neutralizing Ab to IL-10, WT neonatal MPhi secreted increased amounts of IL-6 and allowed neonatal MPhi to promote adult B cells to mount an Ab response against TNP-LPS. Thus, neonates do not respond to TI Ag as a result of the inability of neonatal MPhi to secrete cytokines, such as IL-1beta and IL-6, probably as a result of an excess production of IL-10. This dysregulated cytokine secretion by neonatal MPhi may be a result of a reduction in expression of Toll-like receptor-2 (TLR-2) and TLR-4 and CD14.