Intestinal mucus acts is a maturation factor for dendritic cells

Abstract

BackgroundGastrointestinal (GI) epithelial cells are covered by a thick layer of mucus which acts as a barrier, shielding the host epithelium from the luminal contents. Secreted mucus is expelled from goblet cells scattered throughout the GI tract, while adherent mucins are found on all epithelial cells. Mucus consists of highly glycosylated mucin proteins, primarily MUC2. Beneath the mucus and epithelial layer lies the lamina propria which contains multiple immune cell types. Intestinal dendritic cells (DCs) are professional antigen presenting cells capable of extending projections through the epithelium to sample antigen from the mucus layer and intestinal lumen. In this way, DCs are capable of interacting with antigens and metabolites from the intestinal microbiome. DCs act as sentinels in the GI tract by capturing and processing antigen, undergoing maturation, and regulating immune responses via production of cytokines. Previous studies have demonstrated that DCs are capable of extending processes through the mucus layer or acquiring antigen from antigen‐presenting goblet cells. However, little is known about the effect of mucus recognition by DCs and how this affects interaction with the microbiome. We hypothesize that intestinal mucin glycans stimulate monocytes toward DC lineages, thereby acting on innate immunity and immune/microbiota cross talk.MethodsTo assess the role of mucus in DC maturation and cytokine production the human monocyte cell line THP‐1 were matured to DC by stimulation with IL‐4 and GM‐CSF. Alternatively THP‐1 cells were stimulated with human stool mucus, mucus producing human cell line HT29‐MTX secreted mucus, pig stomach mucus or O‐linked mucus glycans. Mucus‐stimulated THP‐1 cells or IL‐4, GM‐CSF DCs were subjected to bacterial lipopolysaccharide (100 ng/ml LPS) for 3 hrs. Cells were analyzed by flow cytometry for mature DC markers CD11c and HLA and supernatants were examined for TNF production by ELISA. Human blood monocytes were isolated from whole blood and processed in the same manner.ResultsFlow cytometry data demonstrated that THP‐1 cells and human blood monocytes could be stimulated towards a DC‐like phenotype with IL‐4, GM‐CSF based on increased expression of CD11c, CD83 and HLA. Introduction of LPS resulted in an enhanced secretion of TNF in DCs compared to unstimulated monocytes or THP‐1 cells (8‐fold higher TNF). Interestingly, the presence of stool, pig stomach or HT29‐MTX cell‐derived mucus produced an increase in the CD83 hi CD11chi and CD11chiHLAhi DC populations from unstimulated THP‐1 cells. This increased DC population was also observed in gross morphology as mucus stimulated THP‐1s presented with increased diameter and projections. The ability of DCs to perform bacterial uptake in the presence of mucus was assessed using fluorescent carboxyfluorescein diacetate succinimidyl ester (CFDA‐SE) labeled bacteria. Mucus did not alter TNF production in unstimulated or LPS‐treated THP‐1 or in unstimulated or LPS‐treated DCs. However human and HT29‐MTX mucus did increase the CD11chiHLAhi population among mucus and LPS‐treated cells.ConclusionsTogether, this data indicates that intestinal mucus may act as a DC maturation factor and works to prime DC for interaction with members of the gut microbiota. This action of mucins has far‐reaching implications for immune interactions with the host and the intestinal microbiomeSupport or Funding InformationT32 DK007664