The inflammatory process is usually tightly regulated, involving both signals that initiate and maintain inflammation and signals that shut the process down. An imbalance between the two signals leaves inflammation unchecked, resulting in cellular and tissue damage. Macrophages are a major component of the mononuclear phagocyte system that consists of closely related cells of bone marrow origin, including blood monocytes, and tissue macrophages. From the blood, monocytes migrate into various tissues and transform macrophages. In inflammation, macrophages have three major function; antigen presentation, phagocytosis, and immunomodulation through production of various cytokines and growth factors. Macrophages play a critical role in the initiation, maintenance, and resolution of inflammation. They are activated and deactivated in the inflammatory process. Activation signals include cytokines (interferon gamma, granulocyte-monocyte colony stimulating factor, and tumor necrosis factor alpha), bacterial lipopolysaccharide, extracellular matrix proteins, and other chemical mediators. Inhibition of inflammation by removal or deactivation of mediators and inflammatory effector cells permits the host to repair damages tissues. Activated macrophages are deactivated by anti-inflammatory cytokines (interleukin 10 and transforming growth factor beta) and cytokine antagonists that are mainly produced by macrophages. Macrophages participate in the autoregulatory loop in the inflammatory process. Because macrophages produce a wide range of biologically active molecules participated in both beneficial and detrimental outcomes in inflammation, therapeutic interventions targeted macrophages and their products may open new avenues for controlling inflammatory diseases.

A form of ␣-galactosylceramide, KRN7000, activates CD1d-restricted V␣14-invariant (V␣14i) natural killer (NK) T cells and initiates multiple downstream immune reactions. We report that substituting the C26:0 N-acyl chain of KRN7000 with shorter, unsaturated fatty acids modifies the outcome of V␣14i NKT cell activation. One analogue containing a diunsaturated C20 fatty acid (C20:2) potently induced a T helper type 2-biased cytokine response, with diminished IFN-␥ production and reduced V␣14i NKT cell expansion. C20:2 also exhibited less stringent requirements for loading onto CD1d than KRN7000, suggesting a mechanism for the immunomodulatory properties of this lipid. The differential cellular response elicited by this class of V␣14i NKT cell agonists may prove to be useful in immunotherapeutic applications.cytokines ͉ inflammation ͉ autoimmunity ͉ immunoregulation

Pulmonary surfactant contains homeostatic and antimicrobial hydrolases. When Mycobacterium tuberculosis is initially deposited in the terminal bronchioles and alveoli, as well as following release from lysed macrophages, bacilli are in intimate contact with these lung surfactant hydrolases. We identified and measured several hydrolases in human alveolar lining fluid and lung tissue that, at their physiological concentrations, dramatically modified the M. tuberculosis cell envelope. Independent of their action time (15 min to 12 h), the effects of the hydrolases on the M. tuberculosis cell envelope resulted in a significant decrease (60–80%) in M. tuberculosis association with, and intracellular growth of the bacteria within, human macrophages. The cell envelope-modifying effects of the hydrolases also led to altered M. tuberculosis intracellular trafficking and induced a protective proin-flammatory response to infection. These findings add a new concept to our understanding of M. tuberculosis–macrophage inter-actions (i.e., the impact of lung surfactant hydrolases on M. tuberculosis infection).

Mycobacterium tuberculosis, the causative agent of tuberculosis, has two distinguishing characteristics: its ability to stain acid-fast and its ability to cause long-term latent infections in humans. Although this distinctive staining characteristic has often been attributed to its lipid-rich cell wall, the specific dye-retaining components were not known. Here we report that targeted deletion of kasB, one of two M. tuberculosis genes encoding distinct ␤-ketoacyl-acyl carrier protein synthases involved in mycolic acid synthesis, results in loss of acid-fast staining. Biochemical and structural analyses revealed that the ⌬kasB mutant strain synthesized mycolates with shorter chain lengths. An additional and unexpected outcome of kasB deletion was the loss of ketomycolic acid trans-cyclopropanation and a drastic reduction in methoxymycolic acid trans-cyclopropanation, activities usually associated with the trans-cyclopropane synthase CmaA2. Although deletion of kasB also markedly altered the colony morphology and abolished classic serpentine growth (cording), the most profound effect of kasB deletion was the ability of the mutant strain to persist in infected immunocompetent mice for up to 600 days without causing disease or mortality. This long-term persistence of ⌬kasB represents a model for studying latent M. tuberculosis infections and suggests that this attenuated strain may represent a valuable vaccine candidate against tuberculosis. mycolic acid ͉ Ziehl-Neelsen stain ͉ cording ͉ persistence ͉ FAS-II

Mycobacterium tuberculosis (Mtb) infection remains a global health crisis. Recent genetic evidence implicates specific cell envelope lipids in Mtb pathogenesis, but it is unclear whether these cell envelope compounds affect pathogenesis through a structural role in the cell wall or as pathogenesis effectors that interact directly with host cells. Here we show that cyclopropane modification of the Mtb cell envelope glycolipid trehalose dimycolate (TDM) is critical for Mtb growth during the first week of infection in mice. In addition, TDM modification by the cyclopropane synthase pcaA was both necessary and sufficient for proinflammatory activation of macrophages during early infection. Purified TDM isolated from a cyclopropane-deficient pcaA mutant was hypoinflammatory for macrophages and induced less severe granulomatous inflammation in mice, demonstrating that the fine structure of this glycolipid was critical to its proinflammatory activity. These results established the fine structure of lipids contained in the Mtb cell envelope as direct effectors of pathogenesis and identified temporal control of host immune activation through cyclopropane modification of TDM as a critical pathogenic strategy of Mtb.

We have characterized the gene encoding fatty acid alpha-hydroxylase, a cytochrome P450 (P450) enzyme, from Sphingomonas paucimobilis. A database homology search indicated that the deduced amino acid sequence of this gene product was 44% identical to that of the ybdT gene product that is a 48 kDa protein of unknown function from Bacillus subtilis. In this study, we cloned the ybdT gene and characterized this gene product using a recombinant enzyme to clarify function of the ybdT gene product. The carbon monoxide difference spectrum of the recombinant enzyme showed the characteristic one of P450. In the presence of H2O2, the recombinant ybdT gene product hydroxylated myristic acid to produce beta-hydroxymyristic acid and alpha-hydroxymyristic acid which were determined by high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry. The amount of these products increased with increasing reaction period and amount of H2O2 in the reaction mixture. The amount of beta-hydroxyl product was slightly higher than that of alpha-hydroxyl product at all times during the reaction. However, no reaction products were detected at any time or at any concentration of H2O2 when heat-inactivated enzyme was used. HPLC analysis with a chiral column showed that the beta-hydroxyl product was nearly enantiomerically pure R-form. These results suggest that this P450 enzyme is involved in a novel biosynthesis of beta-hydroxy fatty acid.

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Granulomatous inflammation is characterized morphologically by a compact organized collection of macrophages and their derivatives. It is classified as either a hypersensitivity type or a foreign-body type. Lipid components of the Mycobacterium tuberculosis cell wall participate in the pathogenesis of infection. Strains of M. tuberculosis have cord factor (trehalose 6,6-dimycolate [TDM]) on their surface. To clarify host responses to TDM, including immunogenicity and pathogenicity, we have analyzed the footpad reaction, histopathology, and cytokine profiles of experimental granulomatous lesions in immunized and unimmunized mice challenged with TDM. In the present study, we have demonstrated for the first time that TDM can induce both foreignbody-type (nonimmune) and hypersensitivity-type (immune) granulomas by acting as a nonspecific irritant and T-cell-dependent antigen. Immunized mice challenged with TDM developed more severe lesions than unimmunized mice. At the active lesion, we found monocyte chemotactic, proinflammatory, and immunoregulatory cytokines. The level was enhanced in immunized mice challenged with TDM. This result implies that both nonimmune and immune mechanisms participate in granulomatous inflammation induced by mycobacterial infection. Taken together with a previous report, this study shows that TDM is a pleiotropic molecule against the host and plays an important role in the pathogenesis of tuberculosis.The pathogenesis of tuberculosis is a function of the pathogen, Mycobacterium tuberculosis, and of the immune response of the host to the pathogen (5, 14). Tuberculosis is a chronic infection with M. tuberculosis complex, including M. tuberculosis and Mycobacterium bovis, that is characterized morphologically by granulomatous inflammation, a compact organized collection of macrophages and their derivatives, such as epithelioid and giant cells, at the site of infection (19). The pathogenicity of M. tuberculosis is related to its ability to escape killing by macrophages and induce delayed-type hypersensitivity (DTH) (5,14,19).Granulomatous inflammation can be broadly classified as either a hypersensitivity (immunologic, T-cell-dependent) type or a foreign-body (nonimmunologic, T-cell-independent) type (19,20). There is much known, but we still have a long way to go to understand the mechanism of M. tuberculosis pathogenicity. Mycobacteria are rich in lipids. Lipid components of the M. tuberculosis cell wall participate in pathogenesis. Cord factor (trehalose 6,6Ј-dimycolate [TDM]), a surface glycolipid, causes M. tuberculosis to grow in serpentine cords in vitro. Virulent strains of M. tuberculosis have TDM on their surface (2), and injection of purified TDM into experimental animals induces lesions characterized by chronic granulomatous inflammation (6,29).To clarify host responses to mycobacterial TDM, including immunogenicity and pathogenicity, we have analyzed the footpad reactions, histopathology, and cytokine profiles of experimental granulomatous lesions in immunized and unimmunized mic...