Macrophage Phosphoproteome Analysis Reveals MINCLE-dependent and -independent Mycobacterial Cord Factor Signaling.

Immune sensing of Mycobacterium tuberculosis relies on recognition by macrophages. Mycobacterial cord factor, trehalose-6,6'-dimycolate (TDM), is the most abundant cell wall glycolipid and binds to the C-type lectin receptor (CLR) MINCLE. To explore the kinase signaling linking the TDM-MINCLE interaction to gene expression, we employed quantitative phosphoproteome analysis. TDM caused upregulation of 6.7% and suppressed 3.8% of the 14,000 phospho-sites identified on 3727 proteins. MINCLE-dependent phosphorylation was observed for canonical players of CLR signaling (e.g. PLCγ, PKCδ), and was enriched for PKCδ and GSK3 kinase motifs. MINCLE-dependent activation of the PI3K-AKT-GSK3 pathway contributed to inflammatory gene expression and required the PI3K regulatory subunit p85α. Unexpectedly, a substantial fraction of TDM-induced phosphorylation was MINCLE-independent, a finding paralleled by transcriptome data. Bioinformatics analysis of both data sets concurred in the requirement for MINCLE for innate immune response pathways and processes. In contrast, MINCLE-independent phosphorylation and transcriptome responses were linked to cell cycle regulation. Collectively, our global analyses show substantial reprogramming of macrophages by TDM and reveal a dichotomy of MINCLE-dependent and -independent signaling linked to distinct biological responses.

Differential control of Mincle-dependent cord factor recognition and macrophage responses by the transcription factors C/EBPß and HIF1α.

Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, and its synthetic analog Trehalose-6,6-dibehenate (TDB) bind to the C-type lectin receptors macrophage-inducible C-type lectin (Mincle) and Mcl to activate macrophages. Genetically, the transcriptional response to TDB/TDM has been defined to require FcRγ-Syk-Card9 signaling. However, TDB/TDM-triggered kinase activation has not been studied well, and it is largely unknown which transcriptional regulators bring about inflammatory gene expression. In this article, we report that TDB/TDM caused only weak Syk-phosphorylation in resting macrophages, consistent with low basal Mincle expression. However, LPS-priming caused MYD88-dependent upregulation of Mincle, resulting in enhanced TDB/TDM-induced kinase activation and more rapid inflammatory gene expression. TLR-induced Mincle expression partially circumvented the requirement for Mcl in the response to TDB/TDM. To dissect transcriptional responses to TDB/TDM, we mined microarray data and identified early growth response (Egr) family transcription factors as direct Mincle target genes, whereas upregulation of Cebpb and Hif1a required new protein synthesis. Macrophages and dendritic cells lacking C/EBPß showed nearly complete abrogation of TDB/TDM responsiveness, but also failed to upregulate Mincle. Retroviral rescue of Mincle expression in Cebpb-deficient cells restored induction of Egr1, but not of G-CSF. This pattern of C/EBPß dependence was also observed after stimulation with the Dectin-1 ligand Curdlan. Inducible expression of hypoxia-inducible factor 1α (HIF1α) also required C/EBPß. In turn, HIF1α was not required for Mincle expression, kinase activation, and Egr1 or Csf3 expression, but critically contributed to NO production. Taken together, we identify C/EBPß as central hub in Mincle expression and inflammatory gene induction, whereas HIF1α controls Nos2 expression. C/EBPß also connects TLR signals to cord factor responsiveness through MYD88-dependent upregulation of Mincle.

C-type lectin receptor dectin-3 mediates trehalose 6,6'-dimycolate (TDM)-induced Mincle expression through CARD9/Bcl10/MALT1-dependent nuclear factor (NF)-κB activation.

Previous studies indicate that both Dectin-3 (also called MCL or Clec4d) and Mincle (also called Clec4e), two C-type lectin receptors, can recognize trehalose 6,6'-dimycolate (TDM), a cell wall component from mycobacteria, and induce potent innate immune responses. Interestingly, stimulation of Dectin-3 by TDM can also induce Mincle expression, which may enhance the host innate immune system to sense Mycobacterium infection. However, the mechanism by which Dectin-3 induces Mincle expression is not fully defined. Here, we show that TDM-induced Mincle expression is dependent on Dectin-3-mediated NF-κB, but not nuclear factor of activated T-cells (NFAT), activation, and Dectin-3 induces NF-κB activation through the CARD9-BCL10-MALT1 complex. We found that bone marrow-derived macrophages from Dectin-3-deficient mice were severely defective in the induction of Mincle expression in response to TDM stimulation. This defect is correlated with the failure of TDM-induced NF-κB activation in Dectin-3-deficient bone marrow-derived macrophages. Consistently, inhibition of NF-κB, but not NFAT, impaired TDM-induced Mincle expression, whereas NF-κB, but not NFAT, binds to the Mincle promoter. Dectin-3-mediated NF-κB activation is dependent on the CARD9-Bcl10-MALT1 complex. Finally, mice deficient for Dectin-3 or CARD9 produced much less proinflammatory cytokines and keyhole limpet hemocyanin (KLH)-specific antibodies after immunization with an adjuvant containing TDM. Overall, this study provides the mechanism by which Dectin-3 induces Mincle expression in response to Mycobacterium infection, which will have significant impact to improve adjuvant and design vaccine for antimicrobial infection.

Neutrophils Promote Mycobacterial Trehalose Dimycolate-Induced Lung Inflammation via the Mincle Pathway.

Trehalose 6,6'-dimycolate (TDM), a cord factor of Mycobacterium tuberculosis (Mtb), is an important regulator of immune responses during Mtb infections. Macrophages recognize TDM through the Mincle receptor and initiate TDM-induced inflammatory responses, leading to lung granuloma formation. Although various immune cells are recruited to lung granulomas, the roles of other immune cells, especially during the initial process of TDM-induced inflammation, are not clear. In this study, Mincle signaling on neutrophils played an important role in TDM-induced lung inflammation by promoting adhesion and innate immune responses. Neutrophils were recruited during the early stage of lung inflammation following TDM-induced granuloma formation. Mincle expression on neutrophils was required for infiltration of TDM-challenged sites in a granuloma model induced by TDM-coated-beads. TDM-induced Mincle signaling on neutrophils increased cell adherence by enhancing F-actin polymerization and CD11b/CD18 surface expression. The TDM-induced effects were dependent on Src, Syk, and MAPK/ERK kinases (MEK). Moreover, coactivation of the Mincle and TLR2 pathways by TDM and Pam3CSK4 treatment synergistically induced CD11b/CD18 surface expression, reactive oxygen species, and TNFα production by neutrophils. These synergistically-enhanced immune responses correlated with the degree of Mincle expression on neutrophil surfaces. The physiological relevance of the Mincle-mediated anti-TDM immune response was confirmed by defective immune responses in Mincle⁻/⁻ mice upon aerosol infections with Mtb. Mincle-mutant mice had higher inflammation levels and mycobacterial loads than WT mice. Neutrophil depletion with anti-Ly6G antibody caused a reduction in IL-6 and monocyte chemotactic protein-1 expression upon TDM treatment, and reduced levels of immune cell recruitment during the initial stage of infection. These findings suggest a new role of Mincle signaling on neutrophils during anti-mycobacterial responses.

A mycobacteriophage-derived trehalose-6,6'-dimycolate-binding peptide containing both antimycobacterial and anti-inflammatory abilities.

Bacteriophages, the viruses of eubacteria, have developed unique mechanisms to interact with their host bacteria. They have been viewed as potential antibacterial therapeutics. Mycobacteriophage-derived compounds may interact with Mycobacterium tuberculosis (MTB) and/or its components, such as the cord factor, trehalose-6,6'-dimycolate (TDM), which is the most abundant glycolipid produced on the surface of MTB. TDM emulsion injected intravenously into mice induces lung immunopathology that mimics many aspects of MTB infection. Thus, TDM is an important target for anti-MTB agent development. On the basis of genomics information of mycobacteriophages, 200 peptides were synthesized. Their effects on MTB, their interactions with TDM, and anti-inflammatory activities were tested. One of them (PK34) showed MTB-killing activity with a minimal inhibitory concentration of 50 µg/ml and TDM-binding ability. In a mouse model, PK34 showed comparable ability to clear MTB as rifampin did in vivo. It also exerted strong activity to inhibit MTB or TDM-induced inflammation in vivo. PK34 significantly inhibited inflammatory cytokines secretions by inactivating MAPK and PKB signals while it maintained certain proinflammatory cytokine production. It is possible to prospect for TDM-binding and/or anti-MTB peptides by mining the mycobacteriophages genome. In addition to its direct MTB-killing ability, PK34 might be a useful adjunct in the treatment of granulomatous inflammation occurring during mycobacterial infection or a template for developing antituberculosis (TB) agents because of its immunoregulative effects. As a TDM-binding peptide, PK34 may be a promising tool to study TDM's interactions with corresponding receptors and signal pathways.

Mycobacterial trehalose dimycolate reprograms macrophage global gene expression and activates matrix metalloproteinases.

Trehalose 6,6'-dimycolate (TDM) is a cell wall glycolipid and an important virulence factor of mycobacteria. In order to study the role of TDM in the innate immune response to Mycobacterium tuberculosis, microarray analysis was used to examine gene regulation in murine bone marrow-derived macrophages in response to 90-µm-diameter polystyrene microspheres coated with TDM. A large number of genes, particularly those involved in the immune response and macrophage function, were up- or downregulated in response to these TDM-coated beads compared to control beads. Genes involved in the immune response were specifically upregulated in a myeloid differentiation primary response gene 88 (MyD88)-dependent manner. The complexity of the transcriptional response also increased greatly between 2 and 24 h. Matrix metalloproteinases (MMPs) were significantly upregulated at both time points, and this was confirmed by quantitative real-time reverse transcription-PCR (RT-PCR). Using an in vivo Matrigel granuloma model, the presence and activity of MMP-9 were examined by immunohistochemistry and in situ zymography (ISZ), respectively. We found that TDM-coated beads induced MMP-9 expression and activity in Matrigel granulomas. Macrophages were primarily responsible for MMP-9 expression, as granulomas from neutrophil-depleted mice showed staining patterns similar to that for wild-type mice. The relevance of these observations to human disease is supported by the similar induction of MMP-9 in human caseous tuberculosis (TB) granulomas. Given that MMPs likely play an important role in both the construction and breakdown of tuberculous granulomas, our results suggest that TDM may drive MMP expression during TB pathogenesis.

Prospective Subunit Nanovaccine against Mycobacterium tuberculosis Infection─Cubosome Lipid Nanocarriers of Cord Factor, Trehalose 6,6' Dimycolate.

An improved vaccine is urgently needed to replace the now more than 100-year-old Bacillus Calmette-Guérin (BCG) vaccine against tuberculosis (TB) disease, which represents a significant burden on global public health. Mycolic acid, or cord factor trehalose 6,6' dimycolate (TDM), a lipid component abundant in the cell wall of the pathogen Mycobacterium tuberculosis (MTB), has been shown to have strong immunostimulatory activity but remains underexplored due to its high toxicity and poor solubility. Herein, we employed a novel strategy to encapsulate TDM within a cubosome lipid nanocarrier as a potential subunit nanovaccine candidate against TB. This strategy not only increased the solubility and reduced the toxicity of TDM but also elicited a protective immune response to control MTB growth in macrophages. Both pre-treatment and concurrent treatment of the TDM encapsulated in lipid monoolein (MO) cubosomes (MO-TDM) (1 mol %) induced a strong proinflammatory cytokine response in MTB-infected macrophages, due to epigenetic changes at the promoters of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) in comparison to the untreated control. Furthermore, treatment with MO-TDM (1 mol %) cubosomes significantly improved antigen processing and presentation capabilities of MTB-infected macrophages to CD4 T cells. The ability of MO-TDM (1 mol %) cubosomes to induce a robust innate and adaptive response in vitro was further supported by a mathematical modeling study predicting the vaccine efficacy in vivo. Overall, these results indicate a strong immunostimulatory effect of TDM when delivered through the lipid nanocarrier, suggesting its potential as a novel TB vaccine.

Cord factors from atypical mycobacteria (Mycobacterium alvei, Mycobacterium brumae) stimulate the secretion of some pro-inflammatory cytokines of relevance in tuberculosis.

The ability to induce several cytokines relevant to tuberculosis (TNF-α, IL-1ß, IL-6, IL-12p40 and IL-23) by cord factor (trehalose dimycolate) from Mycobacterium alvei CR-21(T) and Mycobacterium brumae CR-270(T) was studied in the cell lines RAW 264.7 and THP-1, and compared to the ability of cord factor from Mycobacterium tuberculosis H37Rv, where this glycolipid appears to be implicated in the pathogenesis of tuberculosis. Details of the fine structure of these molecules were obtained by NMR and MS. The mycoloyl residues were identified as α and (ω-1)-methoxy in M. alvei CR-21(T) and α in M. brumae CR-270(T); in both cases they were di-unsaturated instead of cyclopropanated as found in M. tuberculosis. In RAW 264.7 cells, cord factors from M. alvei CR-21(T), M. brumae CR-270(T) and M. tuberculosis differed in their ability to stimulate IL-6, the higher levels corresponding to the cord factor from M. tuberculosis. In THP-1 cells, a similar overall profile of cytokines was found for M. alvei CR-21(T) and M. brumae CR-270(T), with high proportions of IL-1ß and TNF-α, and different from M. tuberculosis, where IL-6 and IL-12p40 prevailed. The data obtained indicate that cord factors from the atypical mycobacteria M. alvei CR-21(T) and M. brumae CR-270(T) stimulated the secretion of several pro-inflammatory cytokines, although there were some differences with those of M. tuberculosis H37Rv. This finding seems to be due to their particular mycoloyl substituents and could be of interest when considering the potential adjuvanticity of these molecules.

Trehalose Recycling Promotes Energy-Efficient Biosynthesis of the Mycobacterial Cell Envelope.

The mycomembrane layer of the mycobacterial cell envelope is a barrier to environmental, immune, and antibiotic insults. There is considerable evidence of mycomembrane plasticity during infection and in response to host-mimicking stresses. Since mycobacteria are resource and energy limited under these conditions, it is likely that remodeling has distinct requirements from those of the well-characterized biosynthetic program that operates during unrestricted growth. Unexpectedly, we found that mycomembrane remodeling in nutrient-starved, nonreplicating mycobacteria includes synthesis in addition to turnover. Mycomembrane synthesis under these conditions occurs along the cell periphery, in contrast to the polar assembly of actively growing cells, and both liberates and relies on the nonmammalian disaccharide trehalose. In the absence of trehalose recycling, de novo trehalose synthesis fuels mycomembrane remodeling. However, mycobacteria experience ATP depletion, enhanced respiration, and redox stress, hallmarks of futile cycling and the collateral dysfunction elicited by some bactericidal antibiotics. Inefficient energy metabolism compromises the survival of trehalose recycling mutants in macrophages. Our data suggest that trehalose recycling alleviates the energetic burden of mycomembrane remodeling under stress. Cell envelope recycling pathways are emerging targets for sensitizing resource-limited bacterial pathogens to host and antibiotic pressure.IMPORTANCE The glucose-based disaccharide trehalose is a stress protectant and carbon source in many nonmammalian cells. Mycobacteria are relatively unique in that they use trehalose for an additional, extracytoplasmic purpose: to build their outer "myco" membrane. In these organisms, trehalose connects mycomembrane biosynthesis and turnover to central carbon metabolism. Key to this connection is the retrograde transporter LpqY-SugABC. Unexpectedly, we found that nongrowing mycobacteria synthesize mycomembrane under carbon limitation but do not require LpqY-SugABC. In the absence of trehalose recycling, compensatory anabolism allows mycomembrane biosynthesis to continue. However, this workaround comes at a cost, namely, ATP consumption, increased respiration, and oxidative stress. Strikingly, these phenotypes resemble those elicited by futile cycles and some bactericidal antibiotics. We demonstrate that inefficient energy metabolism attenuates trehalose recycling mutant Mycobacterium tuberculosis in macrophages. Energy-expensive macromolecule biosynthesis triggered in the absence of recycling may be a new paradigm for boosting host activity against bacterial pathogens.

Adjuvant-dependent immune response to malarial transmission-blocking vaccine candidate antigens.

Immune responses in major histocompatibility complex (MHC)-disparate congenic mouse strains immunized with sexual stage malaria parasites or purified recombinant protein were adjuvant dependent. Whereas mice exhibited a limited antibody response to immunization with newly emerged Plasmodium falciparum gametes in Freund's adjuvant, all five congenic mouse strains responded to several transmission-blocking vaccine candidate antigens, when parasites were emulsified in a monophosphoryl lipid A (MPL) and trehalose dimycolate (TDM) adjuvant. The humoral response in those animals immunized with the antigen in a MPL/TDM adjuvant was helper T cell dependent, as evident by boosting of the antibody response after a second immunization. If the immunogen consisted of purified recombinant protein, then the immune response was not MHC class II limited in mice immunized with either complete Freund's adjuvant or TDM/MPL. The potential role of adjuvants in overcoming apparent immune nonresponsiveness and the implications for development of a malaria transmission-blocking vaccine are discussed.

Comparison of BCG, MPL and cationic liposome adjuvant systems in leishmanial antigen vaccine formulations against murine visceral leishmaniasis.

BACKGROUND: The development of an effective vaccine against visceral leishmaniasis (VL) caused by Leishmania donovani is an essential aim for controlling the disease. Use of the right adjuvant is of fundamental importance in vaccine formulations for generation of effective cell-mediated immune response. Earlier we reported the protective efficacy of cationic liposome-associated L. donovani promastigote antigens (LAg) against experimental VL. The aim of the present study was to compare the effectiveness of two very promising adjuvants, Bacille Calmette-Guerin (BCG) and Monophosphoryl lipid A (MPL) plus trehalose dicorynomycolate (TDM) with cationic liposomes, in combination with LAg, to confer protection against murine VL. RESULTS: All the three formulations afforded significant protection against L. donovani in both the visceral organs, liver and spleen. Although comparable level of protection was observed in BCG+LAg and MPL-TDM+LAg immunized mice, highest level of protection was exhibited by the liposomal LAg immunized group. Significant increase in anti-LAg IgG levels were detected in both MPL-TDM+LAg and liposomal LAg immunized animals with higher levels of IgG2a than IgG1. But BCG+LAg failed to induce any antibody response. As an index of cell-mediated immunity DTH responses were measured and significant response was observed in mice vaccinated with all the three different formulations. However, highest responses were observed with liposomal vaccine immunization. Comparative evaluation of IFN-gamma and IL-4 responses in immunized mice revealed that MPL-TDM+LAg group produced the highest level of IFN-gamma but lowest IL-4 level, while BCG+LAg demonstrated generation of suboptimum levels of both IFN-gamma and IL-4 response. Elicitation of moderate levels of prechallenge IFN-gamma along with optimum IL-4 corresponds with successful vaccination with liposomal LAg. CONCLUSION: This comparative study reveals greater effectiveness of the liposomal vaccine for protection against progressive VL in BALB/c. Again, evaluation of the immune responses by vaccination emphasizes the need of stimulation of potent cellular immunity based on both Th1 and Th2 cell responses to confer protection against VL.

Type I IFN, Ly6C+ cells, and Phagocytes Support Suppression of Peritoneal Carcinomatosis Elicited by a TLR and CLR Agonist Combination.

Metastatic cancer involving spread to the peritoneal cavity is referred to as peritoneal carcinomatosis and has a very poor prognosis. Our previous study demonstrated a Toll-like receptor and C-type lectin receptor agonist pairing of monophosphoryl lipid A (MPL) and trehalose-6,6'-dicorynomycolate (TDCM) effectively inhibits tumor growth and ascites development following TA3-Ha and EL4 challenge through a mechanism dependent on B-1a cell-produced natural IgM and complement. In this study, we investigated additional players in the MPL/TDCM-elicited response. MPL/TDCM treatment rapidly increased type I IFN levels in the peritoneal cavity along with myeloid cell numbers, including macrophages and Ly6Chi monocytes. Type I IFN receptor (IFNAR1-/-) mice produced tumor-reactive IgM following MPL/TDCM treatment, but failed to recruit Ly6C+ monocytes and were not afforded protection during tumor challenges. Clodronate liposome depletion of phagocytic cells, as well as targeted depletion of Ly6C+ cells, also ablated MPL/TDCM-induced protection. Cytotoxic mediators known to be produced by these cells were required for effects. TNFα was required for effective TA3-Ha killing and nitric oxide was required for EL4 killing. Collectively, these data reveal a model whereby MPL/TDCM-elicited antitumor effects strongly depend on innate cell responses, with B-1a cell-produced tumor-reactive IgM and complement pairing with myeloid cell-produced cytotoxic mediators to effectively eradicate tumors in the peritoneal cavity.

Trehalose 6,6'-dimycolate on the surface of Mycobacterium tuberculosis modulates surface marker expression for antigen presentation and costimulation in murine macrophages.

Trehalose 6,6'-dimycolate (TDM) is the most abundant lipid extracted from Mycobacterium tuberculosis (MTB). TDM promotes MTB survival by decreasing phagosomal acidification and phagolysosomal fusion in macrophages. Delipidation of MTB using petroleum ether removes TDM and decreases MTB survival within host cells. TDM reconstituted onto MTB restores its virulent wild-type characteristics. We investigated the role of TDM in regulating surface marker expression in MTB-infected macrophages. Macrophages were infected with wild-type, delipidated, and TDM-reconstituted MTB for 24h and measured for changes in surface marker expression. TDM on MTB was found to specifically target MHCII, CD1d, CD40, CD80 and CD86. Both wild-type and TDM-reconstituted MTB suppressed or induced no change in expression of these surface markers, whereas delipidated MTB increased expression of the same markers. MTB-infected macrophages were also overlaid with MHCII-restricted T cell hybridomas which recognize Antigen 85B. Macrophages infected by wild-type and TDM-reconstituted MTB did not present antigen as well as delipidated MTB-infected macrophages. The evidence shown furthers supports the notion that TDM present on MTB promotes its survival and persistence in host macrophages.

Activation of B-1 Cells Promotes Tumor Cell Killing in the Peritoneal Cavity.

Metastatic cancer involving spread to the peritoneal cavity is referred to as peritoneal carcinomatosis and has a very poor prognosis. Activating the antitumor immune response in the characteristically immune-suppressive peritoneal environment presents a potential strategy to treat this disease. In this study, we show that a toll-like receptor (TLR) and C-type lectin receptor (CLR) agonist pairing of monophosphoryl lipid A (MPL) and trehalose-6,6'-dicorynomycolate (TDCM) effectively inhibits tumor growth and ascites development in a mouse model of aggressive mammary cancer-induced peritoneal carcinomatosis. MPL/TDCM treatment similarly inhibited peritoneal EL4 tumor growth and ascites development. These effects were not observed in mice lacking B cells or mice lacking CD19, which are deficient in B-1a cells, an innate-like B-cell population enriched in the peritoneal cavity. Remarkably, adoptive transfer of B-1a cells, but not splenic B cells from WT mice, restored MPL/TDCM-induced protection in mice with B-cell defects. Treatment induced B-1 cells to rapidly produce high levels of natural IgM reactive against tumor-associated carbohydrate antigens. Consistent with this, we found significant deposition of IgM and C3 on peritoneal tumor cells as early as 5 days post-treatment. Mice unable to secrete IgM or complement component C4 were not protected by MPL/TDCM treatment, indicating tumor killing was mediated by activation of the classical complement pathway. Collectively, our findings reveal an unsuspected role for B-1 cell-produced natural IgM in providing protection against tumor growth in the peritoneal cavity, thereby highlighting potential opportunities to develop novel therapeutic strategies for the prevention and treatment of peritoneal metastases. SIGNIFICANCE: This work identifies a critical antitumor role for innate-like B cells localized within the peritoneal cavity and demonstrates a novel strategy to activate their tumor-killing potential.See related commentary by Tripodo, p. 5.

Effect of mycobacterial lipids on surface properties of Curosurf: implications for lung surfactant dysfunction in tuberculosis.

In this study, the effect of mycobacterial lipids on the surface activity of lung surfactant was evaluated. Mycolic acid and cord factor, the most abundant surface active lipids of the mycobacterial cell wall when combined with Curosurf led to alteration of its surface properties. Addition of graded amounts of mycolic acid increased the minimum surface tension of Curosurf monolayers from <10 mN/m to approximately 20-27 mN/m. Presence of mycolic acid also slowed the rate of Curosurf adsorption. Similarly, presence of cord factor increased the minimum surface tension achieved by Curosurf to approximately 16-27 mN/m. AFM imaging revealed presence of aggregates on addition of mycobacterial lipids to Curosurf monolayers. Results of this study show that mycolic acid and cord factor can biophysically inactivate porcine lung surfactant extract (Curosurf). This suggests that biophysical inhibition of lung surfactant is possible in vivo in pulmonary tuberculosis which could aggravate areas of alveolar atelectasis.

Characterization of cationic liposomes based on dimethyldioctadecylammonium and synthetic cord factor from M. tuberculosis (trehalose 6,6'-dibehenate)-a novel adjuvant inducing both strong CMI and antibody responses.

Incorporation of the glycolipid trehalose 6,6'-dibehenate (TDB) into cationic liposomes composed of the quaternary ammonium compound dimethyldioctadecylammonium (DDA) produce an adjuvant system which induces a powerful cell-mediated immune response and a strong antibody response, desirable for a high number of disease targets. We have used differential scanning calorimetry (DSC) to investigate the effect of TDB on the gel-fluid phase transition of DDA liposomes and to demonstrate that TDB is incorporated into DDA liposome bilayers. Transmission Electron Microscopy (TEM) and cryo-TEM confirmed that liposomes were formed when a lipid film of DDA containing small amounts of TDB was hydrated in an aqueous buffer solution at physiological pH. Furthermore, time development of particle size and zeta potential of DDA liposomes incorporating TDB during storage at 4 degrees C and 25 degrees C, indicates that TDB effectively stabilizes the DDA liposomes. Immunization of mice with the mycobacterial fusion protein Ag85B-ESAT-6 in DDA-TDB liposomes induced a strong, specific Th1 type immune response characterized by substantial production of the interferon-gamma cytokine and high levels of IgG2b isotype antibodies. The lymphocyte subset releasing the interferon-gamma was identified as CD4 T cells.

Chemotactic response of leukocytes to cord factor (trehalose-6,6'-dimycolate).

Cord factor (trehalose-6,6'-dimycolate), a glycolipid extractable from myocobacteria, was chemotactic for peritoneal cells of mice at concentrations from 5 to 25 mug/ml medium, as well as for peripheral white blood cells of mice and humans. At higher concentrations, presumably only macrophages were attracted.

Mincle-mediated translational regulation is required for strong nitric oxide production and inflammation resolution.

In response to persistent mycobacteria infection, the host induces a granuloma, which often fails to eradicate bacteria and results in tissue damage. Diverse host receptors are required to control the formation and resolution of granuloma, but little is known concerning their regulatory interactions. Here we show that Mincle, the inducible receptor for mycobacterial cord factor, is the key switch for the transition of macrophages from cytokine expression to high nitric oxide production. In addition to its stimulatory role on TLR-mediated transcription, Mincle enhanced the translation of key genes required for nitric oxide synthesis through p38 and eIF5A hypusination, leading to granuloma resolution. Thus, Mincle has dual functions in the promotion and subsequent resolution of inflammation during anti-mycobacterial defence using both transcriptional and translational controls.

Interaction of cord factor (alpha, alpha'-trehalose-6,6'-dimycolate) with phospholipids.

We previously reported that cord factor (alpha,alpha'-trehalose-6,6'-dimycolate) isolated from Nocardia asteroides strain GUH-2 strongly inhibits fusion between unilamellar vesicles containing acidic phospholipid. We chose to study the effects of this molecule on liposome fusion since the presence of N. asteroides GUH-2 in the phagosomes of mouse macrophages had been shown to prevent phagosomal acidification and inhibit phagosome-lysosome fusion. A virtually non-virulent strain, N. asteroides 10905, does not prevent acidification or phagosome-lysosome fusion and, further, contains only trace amounts of cord factor. In the present paper, we have investigated the effects of cord factor on phospholipid bilayers that could be responsible for the inhibition of fusion. We show that cord factor increases molecular area, measured by isothermal compression of a monolayer film, in a mixed monolayer more than would be expected based in its individual contribution to molecular area. Cord factor, as well as other glycolipids investigated, increased the overall hydration of bilayers of dipalmitoylphosphatidylcholine by 50%, as estimated from the unfrozen water fraction measured by differential scanning calorimetry. The effect of calcium on this increased molecular area and headgroup hydration was measured by fluorescence anisotropy and FTIR spectroscopy of phosphatidylserine liposomes. Both techniques showed that cord factor, incorporated at 10 mol%, increased acyl chain disorder over controls in the presence of Ca2+. However, FTIR showed that cord factor did not prevent headgroup dehydration by the Ca2+. The other glycolipids tested did not prevent either the Ca(2+)-induced chain crystallization or headgroup dehydration of phosphatidylserine bilayers. These data point to a possible role of the bulky mycolic acids of cord factor in preventing Ca(2+)-induced fusion of liposomes containing acidic phospholipids.

Modifications of glycosylation patterns in macrophages upon activation.

Activated macrophages, in contrast to inflammatory and resident macrophages, are able to inhibit the growth of intracellular pathogens and tumor cells. In order to understand the adaptative changes which allow macrophages to express antitumor activity, we compared, among several parameters, the glycoproteins of cytotoxic and non-cytotoxic macrophages. After activation of mouse peritoneal macrophages by two stimuli applied in a sequence (trehalose dimycolate in vivo, lipopolysaccharide in vitro), we observed that: (1) surface sialic acid residues (labeled by tritiated borohydride after treatment of intact cells in culture by periodate) were reduced by 37%; (2) total sialic acid, as measured by an adaptation to HPLC of the thiobarbituric assay, was reduced by 30%. Variations in the intensity of the labeling after periodate/borohydride treatment were especially pronounced for a few high-molecular-weight glycoproteins. Analysis of glycopeptides indicated that the reduction of sialylation was accompanied by a slight increase in the relative importance of high mannose-type oligosaccharides (glycopeptides sensitive to endoglycosidase H or retained on concanavalin A-Sepharose) but did not affect the ratio of the various anionic species separated on QAE-Sephadex. A reduced sialylation of glycans after activation may facilitate interactions of macrophages with microbes and tumor cells.