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Indiana University-Purdue University Indianapolis (IUPUI), CD1d is a cell surface glycolipid that, like Major Histocompatibility Complex (MHC) class I and MHC class II molecules, presents antigen. However, instead of peptides, CD1d presents lipids to Natural Killer (NK) T cells, a subset of T cells that express both NK cell markers and the T cell receptor and produces both T helper (Th) 1 and Th2 cytokines. Our lab focuses on the regulation CD1d-mediated antigen presentation. TGF beta is a known regulator of the immune system, such as controlling MHC class II antigen presentation. Further, TGF beta can activate the mitogen activated protein kinase (MAPK) p38, a known negative regulator of CD1d-mediated antigen presentation. Therefore, we hypothesized that TGF beta would be a negative regulator of CD1d-mediated antigen presentation, and our results showed a decrease in antigen presentation by CD1d in response to TGF beta treatment. However, this inhibition was not through p38 activation, as indicated by the absence of a rescue of CD1d-mediated antigen presentation in, TGF beta-treated, p38 dominant negative-expressing cells. Alternatively, the Smad pathway, the canonical pathway activated by TGF beta, was investigated through a lentivirus shRNA-mediated knockdown of Smad2, Smad3 and Smad4 proteins. Smad2 shRNA-expressing cells showed in an increase in CD1d-mediated antigen presentation, suggesting an inhibitory role for Smad2. In contrast, Smad3 shRNA-expressing cells did not differ from control cells. However, as in the case of Smad2, CD1d+ cells in which Smad4 was knocked down, were substantially better at CD1d-mediated antigen presentation than control cells, suggesting that it also negatively regulates antigen presentation. Overall, these studies demonstrate that the canonical TGF beta/Smad pathway regulates an important part of the host���s innate immune response, vis-��-vis CD1d-mediated antigen presentation.

Background: High mobility group box protein 1 (HMGB-1) is a chromatin binding protein that bends DNA thereby facilitating gene transcription. HMGB-1 has also been observed as an extracellular secreted protein in serum of patients with sepsis and has putative intracellular signalling effects regulating the production of interleukin-1 and tumour necrosis factor in a number of inflammatory conditions. Methods: We established a model of immune-mediated epithelial-mesenchymal transition (EMT) in human proximal tubular epithelial cells (PTECs). PTECs were cultured with conditioned medium containing supernatant from activated peripheral blood mononuclear cells (aPBMC). The model was characterised using phenotypic and transcriptomic approaches and suppression subtractive hybridisation was performed to identify differentially regulated genes. Results. Activation of PBMCs resulted in increased secretion of HMGB-1. In addition, treatment of PTECs with aPBMC-conditioned medium resulted in significant upregulation of HMGB-1 in PTECs. Direct treatment of PTECs with recombinant human HMGB-1 induced alterations in epithelial morphology consistent with EMT including reduced E-cadherin expression, increased α-SMA expression and enhanced cell migration. HMGB-1 effects were mediated at least in part by the receptor for advanced glycation end products (RAGE) and through induction of TGF-β1 secretion from PTECs. Conclusions. These results suggest that HMGB-1 is a key mediator of immune-mediated EMT of PTECs and a potentially important signalling molecule in the development of renal fibrosis. Science Foundation Ireland Higher Education Authority Health Research Board Other funder Enterprise Ireland Irish Nephrological Society Amgen sp, ab, ke, li - TS 15.05.12