Type

Text

Type

Dissertation

Date

2009-08-01

Keywords

Langerhans cells | Periodontal therapy | Periodontitis

Language

en_US

Source

This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree.

Identifier

http://hdl.handle.net/11401/70851

Publisher

The Graduate School, Stony Brook University: Stony Brook, NY.

Format

application/pdf

Abstract

Langerhans cells (LCs) are dendritic cells that reside in the epidermis, but whose predominant function in immunity is still enigmatic. The overarching objective of the doctoral dissertation is to analyze responses of LCs to oral microbial associated molecular patterns (MAMPs) like the LPS of putative periodontal pathogen Porphyromonas gingivalis (PGLPS) and the importance of the cytokine micro-environment in determining the outcome of immune response iv (immunostimulatory vs. anergy / tolerance) mediated by LCs, especially in the context of oral inflammatory diseases like periodontitis. Most studies of Langerhans cells in the context of periodontal disease are histo-morphometric analysis of healthy and diseased tissue documenting the trafficking of Langerhans cells. Here we report on the immunological and immuno-stimulatory capabilities of oral mucosal equivalent Langerhans cells and our ability to modulate them in-vitro. Human IL-10 (hIL-10), an immunomodulatory cytokine has been shown to exert immuno-modulatory effect on immune cells. A viral equivalent of hIL-10 is secreted by Epstein Barr Virus, and is called viral IL-10 (vIL-10). vIL-10 exerts its immuno-modulatory effects on different immune cells without the negative effects of hIL-10 i.e., it’s immunostimulatory and growth factor like effects. Thus we explore here the immunomodulatory effects of both hIL-10 and vIL-10 on LC response to MAMPs. CD40L, expressed by T cell and traditionally used as an equivalent for T cell mediated activation of dendritic cells has been reported to be the signal necessary for final maturation of dendritic cells. Thus we explored the immunostimulatory effects of CD40L on LC and their response to MAMPs. The effects of hIL-10 and vIL-10 have been shown to be transient due to the extreme short half life of IL-10 molecules. Thus we wanted to develop a system which would continuously secrete high amounts of IL-10 into the microenvironment and thus may sustain the immune-modulation of the LC. v Thus 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. vi 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 vii response. When LCs are pre-conditioned with recombinant human-IL10/viralIL10, the immunostimulatory responses to MAMPs are abrogated i.e. upregulation of co-stimulatory and activation markers are abrogated along with lack of a cytokine response. The conditioned LCs fails to induce a proliferative response in allogeneic and autogenous T-lymphocytes. This effect was also observed when the LC are co-cultured with a viral-IL10 secreting epithelial micro-environment. 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 microenvironment. IL-10 immuno-modulation also abolishes immuno-stimulatory capabilities of MAMP activated LC on naïve T cells. viii 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. These results shed light on the immunobiological functions of LCs and on the importance of the cytokine micro-environment in initiation and, progression / quiescence of chronic inflammatory diseases. Moreover, it opens up the possibility of taking advantage of immuno-modulatory properties of viral-IL10 in periodontal therapy and treatment of other inflammatory conditions.

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