Non-pathogenic Bacterial Lipid Liposomes are Efficient Adjuvants that Induce Both Innate and...
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the number of Foxp3 + CD4, IL-17A + CD4, and Foxp3 + IL-17A CD4 T cells was significantly increased in Min mice. Taken together, our results show that the cytokine IL-17A largely derived from CD4 T cells promotes spontaneous intestinal tumorigenesis. doi:10.1016/j.clim.2010.03.191 T.80. Non-pathogenic Bacterial Lipid Liposomes are Efficient Adjuvants that Induce Both Innate and Adaptive Immune Responses Syed Faisal, Jen-wei Chen, Wei-wei Yan, Sean McDonough, Yung-Fu Chang. Cornell University, Ithaca, NY Novel liposomes prepared from total polar lipids of nonpathogenic bacteria viz. Leptospira biflexa serovar potac (designated leptosomes) and Mycobacterium smegmatis (designated smegmosomes) were evaluated for their adjuvant effects with various antigen presenting cells (APCs) viz. murine macrophages, J774A.1 and dendritic cells (BMDCs). These liposomes induced strong membrane fusion as evident from resonance energy transfer (RET) assays and effectively transferred the fluorescent probe to the membrane of these APCs. Moreover, both vesicles caused significant activation of APCs as revealed by release of proinflammatory cytokines (IL- 6, IL-12, and TNF-a) and enhanced expression of costimulatory signals/maturation markers (CD80, CD86, and MHCII) which was significantly higher for smegmosomes as compared to leptosomes. In addition, activation of APCs by liposomes correlated with their ability to stimulate allospecific T-cell proliferation and IFN-γ release. In contrast, conventional PC/ chol liposomes failed to fuse and induced only a very low level of APC activation. Interestingly, the stimulatory activity of these lipid vesicles was restricted to APCs as they did not cause any significant activation or mitogenic effect on lymphocytes (B and T cells) in vitro. Overall, the activation of APCs by both leptosomes and smegmosomes correlated with activation of strong humoral and cell-mediated immune responses in C57/BL6 mice to entrapped ovalbumin (OVA) and was significantly higher than those induced by conventional liposome and alum, which failed to activate cytotoxic T lymphocytes (CTLs). Taken together these results demon- strate the adjuvant potential of these novel lipid vesicles that may simultaneously induce both innate and adaptive immune responses due to their immune stimulatory and antigen delivery properties. doi:10.1016/j.clim.2010.03.192 T.81. Regulation of the Atypical Chemerin Receptor, CCRL2, on Activated Brain Endothelial Cells Justin Monnier, Brian Zabel, Eugene Butcher. Stanford University, Palo Alto, CA Chemerin is a nonchemokine chemoattractant for plas- macytoid dendritic cells, macrophages, and NK cells that acts through the GPCR CMKLR1. We recently identified a second chemerin receptor, CCRL2, which binds to the attractant with high affinity but does not result in cell migration, receptor internalization, or intracellular calcium mobilization. CCRL2 deficiency reduces ear swelling and leukocyte infiltration during cutaneous allergic reactions in vivo, but the mechanisms involved remained to be determined. Recent reports show that chemerin colocalizes with activated endothelial cells in brain and skin. We hypothesize that CCRL2 on activated endothelial cells binds and presents chemerin to CMKRL1 + circulating leukocytes, which could trigger integrin-dependent arrest and enhanced recruitment. We hypothesize that this colocalization is due to the expression of CCRL2 on activated endothelial cells. Here we show that CCRL2 RNA and protein is upregulated in both ex vivo brain microvascular endothelial cells and a mouse brain endothelial cell line, bEND3, treated with pro- inflammatory stimuli (TNFa, IFNg, LPS, IL-1b, PolyIC, and IFNb). Following CCRL2 upregulation by TNFa, bEND3 cells, which do not express CMKLR1, bind chemerin with high affinity. We further show that upregulation of CCRL2 by bEND3 cells is blocked by treating the cells with BAY-11- 7082, a selective inhibitor of the NK-κB pathway. Thus, pharmacoinhibition of CCRL2 induction by small molecules that target NF-κB may block chemerin-dependent anchoring of circulating CMKLR1 + leukocytes to inflamed endothelial cells and limit cell recruitment in vivo. Our findings provide a potential explanation for the role of CCRL2 in allergic reactions in vivo. doi:10.1016/j.clim.2010.03.193 T.82. Dynamic Changes in the Lesional Regulatory T Cells in LDLR-deficient Mice Induced by Changes in Diet Elena Maganto-Garcia, Maggie Tarrio, Dexiu Bu, Nir Grabie, Andrew Lichtman. Brigham and Women's Hospital, Boston, MA Regulatory T cells (Treg) are present in atherosclerotic lesions and can modulate disease. To follow Treg responses associated with hypercholesterolemia and ath- erosclerotic lesion progression, we used Ldlr-/-/FOXP3- GFP knock in mice where Treg are identified by fluorescence. Mice were fed a control or cholesterol-rich diet, and Treg were enumerated in lymphoid tissues and in collagen digests of the descending aorta by flow cytometry, and by en face confocal microscopic analysis in the lesion-prone lesser curvature. We found an increase in splenic Tregs after 4, 8, and 20 weeks in cholesterol- rich diet fed mice. In contrast, in hypercholesterolemic mice, the number of Treg in lesions and in aortic digests peaked at 4 weeks and decreased significantly at 8 and 20 weeks, concomitant with the increase of the lesion size over this time period. Interestingly, we found that hypercholesterolemia altered the phenotype of splenic Treg, decreasing expression of GITR, PSGL1, and ability to P-selectin and E-selectin, but these changes were not seen in the GFP-negative (non-Treg) cells. Reversal of S63 Abstracts
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Transcript of Non-pathogenic Bacterial Lipid Liposomes are Efficient Adjuvants that Induce Both Innate and...
S63Abstracts
the number of Foxp3+CD4, IL-17A+CD4, and Foxp3+IL-17A CD4T cells was significantly increased in Min mice. Takentogether, our results show that the cytokine IL-17A largelyderived from CD4 T cells promotes spontaneous intestinaltumorigenesis.
doi:10.1016/j.clim.2010.03.191
T.80. Non-pathogenic Bacterial Lipid Liposomes areEfficient Adjuvants that Induce Both Innate andAdaptive Immune ResponsesSyed Faisal, Jen-wei Chen, Wei-wei Yan, Sean McDonough,Yung-Fu Chang. Cornell University, Ithaca, NY
Novel liposomes prepared from total polar lipids ofnonpathogenic bacteria viz. Leptospira biflexa serovar potac(designated leptosomes) and Mycobacterium smegmatis(designated smegmosomes) were evaluated for their adjuvanteffects with various antigen presenting cells (APCs) viz.murine macrophages, J774A.1 and dendritic cells (BMDCs).These liposomes induced strong membrane fusion as evidentfrom resonance energy transfer (RET) assays and effectivelytransferred the fluorescent probe to the membrane of theseAPCs. Moreover, both vesicles caused significant activation ofAPCs as revealed by release of proinflammatory cytokines (IL-6, IL-12, and TNF-a) and enhanced expression of costimulatorysignals/maturation markers (CD80, CD86, and MHCII) whichwas significantly higher for smegmosomes as compared toleptosomes. In addition, activation of APCs by liposomescorrelated with their ability to stimulate allospecific T-cellproliferation and IFN-γ release. In contrast, conventional PC/chol liposomes failed to fuse and induced only a very low levelof APC activation. Interestingly, the stimulatory activity ofthese lipid vesicles was restricted to APCs as they did notcause any significant activation or mitogenic effect onlymphocytes (B and T cells) in vitro. Overall, the activationof APCs by both leptosomes and smegmosomes correlatedwith activation of strong humoral and cell-mediated immuneresponses in C57/BL6 mice to entrapped ovalbumin (OVA) andwas significantly higher than those induced by conventionalliposome and alum, which failed to activate cytotoxic Tlymphocytes (CTLs). Taken together these results demon-strate the adjuvant potential of these novel lipid vesicles thatmay simultaneously induce both innate and adaptive immuneresponses due to their immune stimulatory and antigendelivery properties.
doi:10.1016/j.clim.2010.03.192
T.81. Regulation of the Atypical Chemerin Receptor,CCRL2, on Activated Brain Endothelial CellsJustin Monnier, Brian Zabel, Eugene Butcher. StanfordUniversity, Palo Alto, CA
Chemerin is a nonchemokine chemoattractant for plas-macytoid dendritic cells, macrophages, and NK cells that
acts through the GPCR CMKLR1. We recently identifieda second chemerin receptor, CCRL2, which binds to theattractant with high affinity but does not result in cellmigration, receptor internalization, or intracellular calciummobilization. CCRL2 deficiency reduces ear swelling andleukocyte infiltration during cutaneous allergic reactionsin vivo, but the mechanisms involved remained to bedetermined. Recent reports show that chemerin colocalizeswith activated endothelial cells in brain and skin. Wehypothesize that CCRL2 on activated endothelial cells bindsand presents chemerin to CMKRL1+ circulating leukocytes,which could trigger integrin-dependent arrest and enhancedrecruitment. We hypothesize that this colocalization is dueto the expression of CCRL2 on activated endothelial cells.Here we show that CCRL2 RNA and protein is upregulated inboth ex vivo brain microvascular endothelial cells and amouse brain endothelial cell line, bEND3, treated with pro-inflammatory stimuli (TNFa, IFNg, LPS, IL-1b, PolyIC, andIFNb). Following CCRL2 upregulation by TNFa, bEND3 cells,which do not express CMKLR1, bind chemerin with highaffinity. We further show that upregulation of CCRL2 bybEND3 cells is blocked by treating the cells with BAY-11-7082, a selective inhibitor of the NK-κB pathway. Thus,pharmacoinhibition of CCRL2 induction by small moleculesthat target NF-κB may block chemerin-dependent anchoringof circulating CMKLR1+ leukocytes to inflamed endothelialcells and limit cell recruitment in vivo. Our findings provide apotential explanation for the role of CCRL2 in allergicreactions in vivo.
doi:10.1016/j.clim.2010.03.193
T.82. Dynamic Changes in the Lesional RegulatoryT Cells in LDLR-deficient Mice Induced by Changesin DietElena Maganto-Garcia, Maggie Tarrio, Dexiu Bu, NirGrabie, Andrew Lichtman. Brigham and Women's Hospital,Boston, MA
Regulatory T cells (Treg) are present in atheroscleroticlesions and can modulate disease. To follow Tregresponses associated with hypercholesterolemia and ath-erosclerotic lesion progression, we used Ldlr−/−/FOXP3-GFP knock in mice where Treg are identified byfluorescence. Mice were fed a control or cholesterol-richdiet, and Treg were enumerated in lymphoid tissues andin collagen digests of the descending aorta by flowcytometry, and by en face confocal microscopic analysisin the lesion-prone lesser curvature. We found an increasein splenic Tregs after 4, 8, and 20 weeks in cholesterol-rich diet fed mice. In contrast, in hypercholesterolemicmice, the number of Treg in lesions and in aortic digestspeaked at 4 weeks and decreased significantly at 8 and20 weeks, concomitant with the increase of the lesion sizeover this time period. Interestingly, we found thathypercholesterolemia altered the phenotype of splenicTreg, decreasing expression of GITR, PSGL1, and ability toP-selectin and E-selectin, but these changes were notseen in the GFP-negative (non-Treg) cells. Reversal of
