For comparison, BM-DC that have not taken up FITC-dextran are also shown (red rectangular gate, fig

For comparison, BM-DC that have not taken up FITC-dextran are also shown (red rectangular gate, fig.3c,d). found thatMuc1/DC were constitutively activated, as determined by higher expression of co-stimulatory molecules (CD40, CD80 and CD86), greater secretion of immunoregulatory cytokines (TNF- and VEGF), and better stimulation of Optovin allogeneic nave CD4+ T cell proliferation. After activation by either LPS or flagellin and co-culture with allogeneic CD4+ T cells, Muc1/DC also induced greater secretion of TNF- and IFN- compared to similarly activatedMuc1+/+DC. Taken together, our results indicate that deletion Optovin of Muc1 promotes a heightened functional response of DC in response to TLR4 and TLR5 signaling pathways, and suggests a previously under-appreciated role for Muc1 in regulating innate immune responses of DC. Key Words:Inflammation, Dendritic cells, Muc1, Toll-like receptor, Innate immunity, Immunomodulation, Host defence, Optovin Cytokines == Introduction == Dendritic cells (DC) evolved to respond rapidly to infections or cellular damage. Conserved microbial-derived proteins and cellular fragments (danger signals) are recognized by cells of the innate immune system including DC and promote their ability to orchestrate adaptive T cell-mediated immunity [1,2,3]. Danger signals are transduced by different pattern recognition receptors including the toll-like receptor (TLR) family of innate immune receptors [4,5]. Microbial recognition by DC is achieved predominantly by occupation of cell membrane-expressed TLRs, particularly TLR2, TLR4 and TLR5 [4,6,7]. For example, binding of TLR4 by lipopolysaccharide (LPS) stimulates the release of pro-inflammatory cytokines (e.g. TNF-, IL-12), enhances cell surface expression of co-stimulatory molecules (e.g. CD80 and CD86) and MHC class II receptors by activated DC and airway epithelial cells [8,9]. These responses Mdk of appropriately activated DC assist in the activation and clonal proliferation of naive CD4+ T cells [1,2,3]. Thus, DC provide a rapid functional response to diverse environmental cues that link innate and adaptive immune responses. Muc1 is a membrane-tethered transmembrane mucin-like glycoprotein that is expressed on a variety of tumor cells as well as T lymphocytes and dendritic cells [10,11,12,13]. Unique among mucin-like molecules, Muc1 possesses signal transduction activity that is mediated in part by conserved tyrosine residues in its cytoplasmic tail [14]. Interestingly, Muc1 was recently shown to be a receptor forPseudomonas Optovin aeruginosaand the flagella proteins [15]. Flagellin is the major immunogenic protein of bacterial flagellum, and is a major virulence factor of Gram-positive and Gram-negative bacteria. Occupation of Muc1 by flagellin was recently shown to induce phosphorylation of the cytoplasmic domain and subsequent activation of the mitogen-activated protein kinases ERK1/2 [14]. Flagellin is known to also bind TLR5 [16,17] whose extracellular surface domain can recognize and discriminate different flagellins in mouse and human systems [18,19]. Since both Muc1 and TLR5 recognize and transduce signals following occupation by flagellin, it was recently postulated that there was cross-talk between TLR-dependent and Muc1-signaling pathways [20,21]. Indeed, Muc1 was recently shown to negatively regulate activation in response to diverse TLR ligands, including flagellin in airway epithelial cells and HEK 293T cells [22]. The objective of this study was to investigate the role of Muc1 in innate immune responses, and specifically test the hypothesis that Muc1 may regulate TLR-dependent DC activation. Here we compared different aspects of DC activation including uptake of exogenous antigen, costimulatory molecule expression, cytokine secretion and naive T cell stimulation using bone marrow-derived DC (BM-DC) fromMuc1/andMuc1+/+mice as well as primary DC obtained from the lung and spleen of Muc1 knockouts as compared with their wild-type counterparts. We found that deletion of Muc1 resulted in both constitutive and TLR-inducible enhancement of DC function, including a greater ability to stimulate allogeneic naive CD4+ T cell activation in co-culture assays. Moreover, we found marked concordance in functional responsiveness of in vitro generated BM-DC as well as primary DC isolated from the lung and spleen. == Materials and Methods == == Generation of Murine Dendritic Cells and Stimulation == Conventional myeloid DC were generated from bone marrow-derived precursors (BM-DC) of naiveMuc1+/+andMuc1/mice on the FVB genetic background and expressing theH-2qMHC class I haplotype, as previously described [23,24].Muc1/mice [22] and theirMuc1+/+littermates were generously provided by Dr. K. Chul Kim (Temple University School of Medicine). Mice were euthanized by CO2asphyxiation according to the animal experimentation policies of the Johns Hopkins University institutional animal care and use committee. Bone marrow suspensions were collected from the femurs and tibiae of 4 mice per pooled group by flushing the bone marrow cavity with 2 ml of ice-cold complete culture medium at the femoral and tibial epiphysis. Cells were centrifuged at 400g, 8C for 10 min to collect the cells, and.