Mycobacterial lipoarabinomannans modulate cytokine production in human T helper cells by interfering with raft/microdomain signalling

Abstract.: Lipoarabinomannans (LAMs) are major lipoglycans of the mycobacterial envelope and constitute immunodominant epitopes of mycobacteria. In this paper, we show that mannose-capped (ManLAM) and non-mannose- capped (PILAM) mycobacterial lipoglycans insert into T helper cell rafts without apparent binding to known receptors. T helper cells modified by the insertion of PILAM responded to CD3 cross-linking by decreasing type 1 (IL-2 and IFN-γ) and increasing type 2 (IL-4 and IL-5) cytokine production. Modification by the mannose-capped ManLAMs had similar, but more limited effects on T helper cell cytokine production. When incorporated into isolated rafts, PILAMs modulated membrane-associated kinases in a dose-dependent manner, inducing increased phosphorylation of Src kinases and Cbp/PAG in Th1 rafts, while decreasing phosphorylation of the same proteins in Th2 rafts. Mycobacterial lipoglycans thus modify the signalling machineries of rafts/microdomains in T helper cells, a modification of the membrane organization that eventually leads to an overall enhancement of type 2 and inhibition of type 1 cytokine productio

Immunological profiles of leprosy patients and Healthy family contacts towards Mycobacterium leprae antigens

In this study, we measured simultaneously the in vitro and in vivo T lymphocyte reactivities and the antibody responses of leprosy patients and healthy family contacts (HFC) toward Mycobacterium leprae antigens. The in vitro lymphoproliferative response of the HFC to leprosin A was comparable to that of tuberculoid leprosy patients. However, their skin-test reactivity to Dharmendra lepromin was considerably higher compared to the in vitro response to leprosin A. A significant number of HFC failed to respond to M. leprae antigens, both in vitro and in vivo, and the unresponsiveness to either test was not related to the type of leprosy patients in the household. A marginal correlation was observed between the skin-test reactivity of HFC and the age of the individuals. Even though a significant proportion of HFC showed positive anti-PGL-I IgM levels, none showed a positive titer in the serum antibody competition test toward the M. leprae-specific epitope My2. A positive anti-PGL-I IgM response together with a negative lepromin skin-test reactivity showed a clear downward trend from the lepromatous pole toward the tuberculoid pole. A small number of HFC, all contacts of lepromatous patients, were lepromin skin-test negative with positive anti-PGL-I IgM levels, but the majority among them showed T-cell reactivity to mycobacterial antigens in vitro. These results are discussed in relation to immunological correlates of the susceptibility to M. leprae infection

Selective Reduction of Post-Selection CD8 Thymocyte Proliferation in IL-15Rα Deficient Mice

Peripheral CD8+ T cells are defective in both IL-15 and IL-15Rα knock-out (KO) mice; however, whether IL-15/IL-15Rα deficiency has a similar effect on CD8 single-positive (SP) thymocytes remains unclear. In this study, we investigated whether the absence of IL-15 transpresentation in IL-15Rα KO mice results in a defect in thymic CD8 single positive (SP) TCRhi thymocytes. Comparison of CD8SP TCRhi thymocytes from IL-15Rα KO mice with their wild type (WT) counterparts by flow cytometry showed a significant reduction in the percentage of CD69− CD8SP TCRhi thymocytes, which represent thymic premigrants. In addition, analysis of in vivo 5-bromo-2-deoxyuridine (BrdU) incorporation demonstrated that premigrant expansion of CD8SP TCRhi thymocytes was reduced in IL-15Rα KO mice. The presence of IL-15 transpresentation-dependent expansion in CD8SP TCRhi thymocytes was assessed by culturing total thymocytes in IL-15Rα-Fc fusion protein-pre-bound plates that were pre-incubated with IL-15 to mimic IL-15 transpresentation in vitro. The results demonstrated that CD8SP thymocytes selectively outgrew other thymic subsets. The contribution of the newly divided CD8SP thymocytes to the peripheral CD8+ T cell pool was examined using double labeling with intrathymically injected FITC and intravenously injected BrdU. A marked decrease in FITC+ BrdU+ CD8+ T cells was observed in the IL-15Rα KO lymph nodes. Through these experiments, we identified an IL-15 transpresentation-dependent proliferation process selective for the mature CD8SP premigrant subpopulation. Importantly, this process may contribute to the maintenance of the normal peripheral CD8+ T cell pool

Effects of the glucolipid synthase inhibitor, P4, on functional and phenotypic parameters of murine myeloma cells

This study describes the effects of the glucolipid synthase inhibitor P4, (DL-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol), on various functional and phenotypic parameters of 5T33 murine myeloma cells. Cell recovery was reduced by >85% following incubation of the cells for 3 days in the presence of 4 μM P4 (the IC50 concentration). Both cytostatic and cytotoxic inhibition was observed with tumour cell metabolic activity and clonogenic potential reduced to 42% and 14% of controls, respectively, and viability reduced to 52%. A dose-dependent increase in cells undergoing apoptosis (from 7% to 26%) was also found. P4 induced a decrease in the number of cells expressing H-2 Class I and CD44, and a large increase in cells expressing H-2 Class II and the IgG2b paraprotein. It did not affect surface expression of CD45 or CD54 (ICAM-1). Based on these alterations in tumour cell growth, adhesion molecule expression and potential immunogenicity, it is anticipated that P4 will provide a novel therapeutic approach for the treatment of multiple myeloma. In addition, given that essentially all tumours rely heavily on overexpressed or abnormal glucosphingolipids for growth, development and metastasis, glucolipid synthase inhibitors may prove to be universally effective anti-cancer agents. © 1999 Cancer Research Campaig

NLRX1 inhibits the early stages of CNS inflammation and prevents the onset of spontaneous autoimmunity

Nucleotide-binding, leucine-rich repeat containing X1 (NLRX1) is a mitochondria-located innate immune sensor that inhibits major pro-inflammatory pathways such as type I interferon and nuclear factor-κB signaling. We generated a novel, spontaneous, and rapidly progressing mouse model of multiple sclerosis (MS) by crossing myelin-specific T-cell receptor (TCR) transgenic mice with Nlrx1−/− mice. About half of the resulting progeny developed spontaneous experimental autoimmune encephalomyelitis (spEAE), which was associated with severe demyelination and inflammation in the central nervous system (CNS). Using lymphocyte-deficient mice and a series of adoptive transfer experiments, we demonstrate that genetic susceptibility to EAE lies within the innate immune compartment. We show that NLRX1 inhibits the subclinical stages of microglial activation and prevents the generation of neurotoxic astrocytes that induce neuronal and oligodendrocyte death in vitro. Moreover, we discovered several mutations within NLRX1 that run in MS-affected families. In summary, our findings highlight the importance of NLRX1 in controlling the early stages of CNS inflammation and preventing the onset of spontaneous autoimmunity

Enhancement of Naringenin Bioavailability by Complexation with Hydroxypropoyl-β-Cyclodextrin

The abundant flavonoid aglycone, naringenin, which is responsible for the bitter taste in grapefruits, has been shown to possess hypolipidemic and anti-inflammatory effects both in vitro and in vivo. Recently, our group demonstrated that naringenin inhibits hepatitis C virus (HCV) production, while others demonstrated its potential in the treatment of hyperlipidemia and diabetes. However, naringenin suffers from low oral bioavailability critically limiting its clinical potential. In this study, we demonstrate that the solubility of naringenin is enhanced by complexation with β-cyclodextrin, an FDA approved excipient. Hydroxypropoyl-β-cyclodextrin (HPβCD), specifically, increased the solubility of naringenin by over 400-fold, and its transport across a Caco-2 model of the gut epithelium by 11-fold. Complexation of naringenin with HPβCD increased its plasma concentrations when fed to rats, with AUC values increasing by 7.4-fold and Cmax increasing 14.6-fold. Moreover, when the complex was administered just prior to a meal it decreased VLDL levels by 42% and increased the rate of glucose clearance by 64% compared to naringenin alone. These effects correlated with increased expression of the PPAR co-activator, PGC1α in both liver and skeletal muscle. Histology and blood chemistry analysis indicated this route of administration was not associated with damage to the intestine, kidney, or liver. These results suggest that the complexation of naringenin with HPβCD is a viable option for the oral delivery of naringenin as a therapeutic entity with applications in the treatment of dyslipidemia, diabetes, and HCV infection.National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (K01DK080241)Harvard Clinical Nutrition Research Center (P30-DK040561)European Research Council (Starting Grant (TMIHCV 242699))Massachusetts General Hospital (BioMEMS Resource Center (P41 EB-002503))Alexander Silberman Institute of Life Science

Characterization of the effects of cross-linking of macrophage CD44 associated with increased phagocytosis of apoptotic PMN

Control of macrophage capacity for apoptotic cell clearance by soluble mediators such as cytokines, prostaglandins and lipoxins, serum proteins, and glucocorticoids may critically determine the rate at which inflammation resolves. Previous studies suggested that macrophage capacity for clearance of apoptotic neutrophils was profoundly altered following binding of CD44 antibodies. We have used a number of different approaches to further define the mechanism by which CD44 rapidly and specifically augment phagocytosis of apoptotic neutrophils. Use of Fab ’ fragments unequivocally demonstrated a requirement for cross-linking of macrophage surface CD44. The molecular mechanism of CD44-augmented phagocytosis was shown to be opsonin-independent and to be distinct from the Mer/protein S pathway induced by glucocorticoids and was not functional for clearance of apoptotic eosinophils. CD44-cross-linking also altered macrophage migration and induced cytoskeletal re-organisation together with phosphorylation of paxillin and activation of Rac2. Investigation of signal transduction pathways that might be critical for CD44 augmentation of phagocytosis revealed that Ca 2+ signalling, PI-3 kinase pathways and altered cAMP signalling were not involved, but did implicate a key role for tyrosine phosphorylation events. Finally, although CD44 antibodies were able to augment phagocytosis of apoptotic neutrophils by murine peritoneal and bone marrow-derived macrophages, we did not observe a difference in the clearance of neutrophils following induction of peritonitis with thioglycollate in CD44-deficient animals. Together, these data demonstrate that CD4

Cyclodextrin Induces Calcium-Dependent Lysosomal Exocytosis

Cyclodextrins (CDs) have long been used to manipulate cellular cholesterol levels both in vitro and in vivo, but their direct effects at a cellular level are not well characterized. Recently, CDs have garnered much interest because of their ability to clear stored cholesterol from Niemann Pick Type C (NPC) cells and markedly prolong the life of NPC1 disease mice. Here, we investigate the hypothesis that treatment with 2-hydroxypropyl- β-cyclodextrin (HPB-CD) stimulates lysosomal exocytosis in a calcium-enhanced manner. We propose that this exocytosis is the mechanism by which HPB-CD ameliorates the endolysosomal cholesterol storage phenotype in NPC cells. These findings have significant implications for the use of HPB-CD in biochemical assays and data interpretation as well as for their use for the treatment for NPC and other disorders

Methyl-β-Cyclodextrins Preferentially Remove Cholesterol from the Liquid Disordered Phase in Giant Unilamellar Vesicles

Methyl-β-cyclodextrins (MβCDs) are molecules that are extensively used to remove and to load cholesterol (Chol) from artificial and natural membranes; however, the mechanism of Chol extraction by MβCD from pure lipids or from complex mixtures is not fully understood. One of the outstanding questions in this field is the capability of MβCD to remove Chol from lipid domains having different packing. Here, we investigated the specificity of MβCD to remove Chol from coexisting macrodomains with different lipid packing. We used giant unilamellar vesicles (GUVs) made of 1,2-dioleoylphosphatidylcholine:1,2-dipalmitoylphatidylcholine:free cholesterol, 1:1:1 molar ratio at 27°C. Under these conditions, individual GUVs present Chol distributed into lo and ld phases. The two phases can be distinguished and visualized using Laurdan generalized polarization and two-photon excitation fluorescence microscopy. Our data indicate that MβCD removes Chol preferentially from the more disordered phase. The process of selective Chol removal is dependent on the MβCD concentration. At high concentrations, MβCD also removes phospholipids

Targeting cholesterol-rich microdomains to circumvent tamoxifen-resistant breast cancer

Adjuvant treatment with tamoxifen substantially improves survival of women with estrogen-receptor positive (ER+) tumors. Tamoxifen resistance (TAMR) limits clinical benefit. RRR alpha tocopherol ether-linked acetic acid analogue (alpha-TEA) is a small bioactive lipid with potent anticancer activity. We evaluated the ability of alpha-TEA in the presence of tamoxifen to circumvent TAMR in human breast cancer cell lines. Methods: Two genotypically matched sets of TAM-sensitive (TAMS) and TAM-resistant (TAMR) human breast cancer cell lines were assessed for signal-transduction events with Western blotting, apoptosis induction with Annexin V-FITC/PI assays, and characterization of cholesterol-rich microdomains with fluorescence staining. Critical involvement of selected mediators was determined by using RNA interference and chemical inhibitors. Results: Growth-factor receptors (total and phosphorylated forms of HER-1 and HER-2), their downstream prosurvival mediators pAkt, pmTOR, and pERK1/2, phosphorylated form of estrogen receptor-alpha (pER-alpha at Ser-167 and Ser-118, and cholesterol-rich lipid microdomains were highly amplified in TAMR cell lines and enhanced by treatment with TAM. alpha-TEA disrupted cholesterol-rich microdomains, acted cooperatively with TAM to reduce prosurvival mediators, and induced DR5-mediated mitochondria-dependent apoptosis via an endoplasmic reticulum stress-triggered pro-death pJNK/CHOP/DR5 amplification loop. Furthermore, methyl-beta-cyclodextrin (M beta CD), a chemical disruptor of cholesterol rich microdomains, acted cooperatively with TAM to reduce prosurvival mediators and to induce apoptosis. Conclusions: Data for the first time document that targeting cholesterol-rich lipid microdomains is a potential strategy to circumvent TAMR, and the combination of alpha-TEA + TAM can circumvent TAMR by suppression of prosurvival signaling via disruption of cholesterol-rich lipid microdomains and activation of apoptotic pathways via induction of endoplasmic reticulum stress.Clayton Foundation for ResearchCenter for Molecular and Cellular Toxicology at the University of TexasNIEHS/NIH T32 ES07247Nutritional Science