The aims of this thesis were to determine the ability of human Cd34+ derived Langerin+ LCs to respond to different pattern recognition receptors (PRRs) through the MAMPs that they express by undergoing maturation and stimulating a T-cell mediated immunoproliferative response and moreover, to determine the ability of cytokine micro-environment to modulate this response. This is the one of the first few studies to look into immune responses of these LCs especially in the context of oral MAMPs. The studies also establish a model for immunomodulatory, viral-IL10, secreting epithelial microenvironment.
Langerhans cells were generated from CD34+ cord blood derived hematopoietic stem cells and highly purified by positive selection with the help of antibody to langerin a specific cell surface marker for Langerhans cells through fluorescence associated cells sorting. The phenotype of LC were further characterized and confirmed by flow cytometric analysis by the presence of cell surface markers like CD1a, HLADR, DEC205, E-cadherin, CLA etc.
The purified LC were then challenged with different doses of four different MAMPs namely, TLR2/TLR-4 ligand PGLPS, TLR4-ligand (Escherichia coli LPS) ECLPS; NOD1/NOD2 ligand Peptidoglycan (PGN) and DEC1/TLR2 ligand Zymosan for 24 hours. The resultant LC activation was determined by analyzing for co-stimulatory molecules CD86/CD80), activation markers (CD83) and HLADR through flow cytometric analysis. Cytokine response was measured through a flow cytometry based cytometric bead assay. The expression of PRRs were analyzed with help of real time PCR. Immunostimulatory capabilities of activated LC was measured with the help of CFSE based T cell proliferation assay.
LC were then pre-conditioned with hIL-10, subsequently challenged with four different MAMPs and again the immune and immuno-stimulatory responses were measured as described above. Genetically engineered viral IL-10 secreting epithelium was generated with the help of retro-virus mediated transduction using a MMLV viral vector carrying the vIL-10 gene. The ability of this conditioning micro-environment on LC immune response was also determined as described above.
The results suggest that, LCs express surface markers consistent with mucosal and epidermal LCs. LCs are capable of recognizing various MAMPs like TLR2&4-activating Porphyromonas gingivalis lipopolysaccharide, TLR2-activating peptidoglycan, TLR2 & DEC1-activating zymosan, and TLR4-activating Escherichia coli lipopolysaccharide. LC up regulate TLR4, NOD1 and NOD2 in response to various MAMPs but do not up regulate DEC-1 and TLR2. LCs also up-regulate co-stimulatory molecules and activation markers, including CD83, in response to these MAMPs. They also elicit a robust pro-inflammatory cytokine response against these MAMPs. Activated LCs are able to stimulate a proliferative response in both allogeneic and autogeneic CD4+-T-lymphocytes. Thus, contrary to current opinion, LCs are able to mount an immunostimulatory response. When LCs are pre-conditioned with recombinant human-IL10/viral-IL10, the immunostimulatory responses to MAMPs are abrogated i.e. up-regulation of co-stimulatory and activation markers are abrogated along with lack of a cytokine response.
CD40L conditioning produced unexpected and interesting result CD40L conditioning by itself produces a strong cytokine response from the LCs, but only produces a weak co-stimulatory effect. LC pre-conditioning with CD40 did not provide any additional effect to the T cell proliferating capabilities of LC. In fact CD40L reduced the cytokine secretory response as well as the co-stimulatory response of LCs to MAMP challenge.
Thus, in summary we show here that both hIL-10 and its viral equivalent, secreted by Epstein Barr virus, viral IL-10 (vIL-10) are able to down modulate the immune response of LC and make them unresponsive to activation to MAMPs both by themselves as well as in the context of an epithelial micro-environment. IL-10 immuno-modulation also abolishes immuno-stimulatory capabilities of MAMP activated LC on naïve T cells. Moreover, we show that CD40L activation of LC and subsequent attempt activation by MAMPs result in them either undergoing an endotoxin tolerance or make them semi-mature and hence resistant to further activation by LCs. (Abstract shortened by UMI.)
|School:||State University of New York at Stony Brook|
|School Location:||United States -- New York|
|Source:||DAI-B 71/04, Dissertation Abstracts International|
|Subjects:||Microbiology, Dentistry, Immunology|
|Keywords:||Immune responses, Langerhans cells, Microbial associated molecular patterns, Periodontal disease, Porphyromonas gingivalis|
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