Induction of acquired immunological tolerance is the ultimate goal in transplantation. So far the acceptance of a mismatched graft is achieved through immunosuppression that requires long-term treatment and a variety of methods have been explored to prevent rejection and achieve transplant tolerance in mouse models. There are several factors that contribute to acquired tolerance. Recent studies have focused on the inhibition of costimulatory molecules and TLRs in Dendritic Cells (DCs), as a key to the mechanisms underlying the barrier to tolerance induction. Dendritic cells are the sentinels of the immune system. Immature Dendritic cells, which are characterized by low MHC Class II expression and weak T cell stimulation ability, reside in all organs of the body sampling the environment for antigens to bring back to the lymph nodes for T and B cell tolerization or activation, depending on the presence of danger signals. One of these danger signals is LPS from gram-negative Bacteria that can induce DC maturation by triggering TLR4, a surface PRR that is also stimulated by endogenous danger signals, like HMGB1, released during inflammation and tissue damage. Mature DCs highly express MHC II and costimulatory molecules and are potent T cell stimulators. However, LPS has multiple effects on DCs. Indeed, unpublished results from our lab also show that LPS induces DC cell death in vitro and in vivo. It has also been reported that DCs treated with LPS during their development remained in an immature state and they induced alloantigen-specific anergy of CD4+ T cells in vitro. The effects of the simultaneous exposure of DCs to LPS and endogenous danger signals requires further investigation. Therefore, we developed a mouse skin transplant model to determine the effects of LPS and endogenous danger signals, released during engraftment, on DC functions and the ability to induce rejection vs tolerance in transplantation. We used the spontaneous model of skin rejection of a single minor histocompatibility mismatch, the male-specific H-Y antigen. We performed skin grafts from the tail or ear of female or male C57BL/6 mice onto syngeneic female recipients. We administered 4 treatments of PBS 0.5ml or LPS 0.5ml at 25ug/mouse every other day starting from day 0. We observed that control mice transplanted with male skin completely rejected the graft between 24-34 days, while mice transplanted with male skin and treated with LPS did not show rejection of the graft until an average of 64 days and 50% of did not rejected at all. When we administered a different DC stimulator, the TLR9 ligand CpG, we found on the contrary that it induced acceleration of the graft rejection. To understand the mechanism underlying these results, we studied the DCs in vivo. Upon organ transplantation, DCs migrate out of the graft in the first 3 days. Studying the phenotype of the DCs migrating out of the skin graft, we found a sharp decrease of DCs in the skin graft as early as 48 hours post transplant and the loss of DCs was more severe with treatments of LPS. The analysis of the DCs in the epidermal sheets of the graft showed that mice treated with LPS treatment had strongly decreased numbers of DCs compared to mice injected with either PBS or with CpGs. Moreover, we analyzed the DCs from the graft-draining Lymph Nodes (Brachial and Inguinal), and from Spleen. We found again decreased numbers of DCs in both the Spleens and Lymph Nodes of grafted mice treated with LPS compared to mice injected with either PBS or CpGs. Based on these findings, we hypothesize that one of the mechanisms in which LPS prolongs graft survival is that it decreases the number of DCs leaving the graft to stimulate the immune response. LPS is either killing the DCs or holding them outside of the Lymph Nodes, not allowing for antigen presentation during the first week after transplantation when most of the DAMPS from the surgery and ischemia are released. / Microbiology and Immunology
| Identifer | oai:union.ndltd.org:TEMPLE/oai:scholarshare.temple.edu:20.500.12613/933 |
| Date | January 2012 |
| Creators | Chain, Robert Whatley |
| Contributors | Gallucci, Stefania, Monestier, Marc, Tsygankov, Alexander Y. |
| Publisher | Temple University. Libraries |
| Source Sets | Temple University |
| Language | English |
| Detected Language | English |
| Type | Thesis/Dissertation, Text |
| Format | 60 pages |
| Rights | IN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available., http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | http://dx.doi.org/10.34944/dspace/915, Theses and Dissertations |
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