Toll-like receptor 2-dependent inhibition of macrophage class II MHC expression and antigen processing by 19-kDa lipoprotein of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB) induces vigorous immune responses, yet persists inside macrophages, evading host immunity. MTB bacilli or lysate was found to inhibit macrophage expression of class II MHC (MHC-II) molecules and MHC-II Ag processing. This report characterizes and identifies a specific component of MTB that mediates these inhibitory effects. The inhibitor was extracted from MTB lysate with Triton X-114, isolated by gel electroelution, and identified with Abs to be MTB 19-kDa lipoprotein. Electroelution- or immunoaffinity-purified MTB 19-kDa lipoprotein inhibited MHC-II expression and processing of both soluble Ags and Ag 85B from intact MTB bacilli. Inhibition of MHC-II Ag processing by either MTB bacilli or purified MTB 19-kDa lipoprotein was dependent on Toll-like receptor (TLR) 2 and independent of TLR 4. Synthetic analogs of lipopeptides from Treponema pallidum also inhibited Ag processing. Despite the ability of MTB 19-kDa lipoprotein to activate microbicidal and innate immune functions early in infection, TLR 2-dependent inhibition of MHC-II expression and Ag processing by MTB 19-kDa lipoprotein during later phases of macrophage infection may prevent presentation of MTB Ags and decrease recognition by T cells. This mechanism may allow intracellular MTB to evade immune surveillance and maintain chronic infection.

Mycobacterium tuberculosis 19-kDa lipoprotein promotes neutrophil activation.

Certain microbial substances, e.g., LPS, can activate neutrophils or prime them to enhance their response to other activating agents, e.g., fMLP. We investigated the role of the Mycobacterium tuberculosis (MTB) 19-kDa lipoprotein in activation of human neutrophils. MTB 19-kDa lipoprotein initiated phenotypic changes characteristic of neutrophil activation, including down-regulation of CD62 ligand (L-selectin) and up-regulation of CD35 (CR1) and CD11b/CD18 (CR3, Mac-1). In addition, exposure of neutrophils to MTB 19-kDa lipoprotein enhanced the subsequent oxidative burst in response to fMLP as assessed by oxidation of dihydrorhodamine 123 (determined by flow cytometry). LPS also produced these effects with similar kinetics, but an oligodeoxynucleotide containing a CpG motif failed to induce any priming or activation response. Although the effects of LPS required the presence of serum, neutrophil activation by MTB 19-kDa lipoprotein occurred independently of serum factors, suggesting the involvement of different receptors and signaling mechanisms for LPS and MTB 19-kDa lipoprotein. Thus, MTB 19-kDa lipoprotein serves as a pathogen-associated molecular pattern that promotes neutrophil priming and activation.

Mycobacterium tuberculosis inhibits MHC class II antigen processing in murine bone marrow macrophages.

Infection of murine bone-marrow-derived macrophages with viable Mycobacterium tuberculosis (MTB) H37Ra inhibited surface expression of MHC class II (MHC-II) molecules and processing of exogenous antigens for presentation to CD4(+) T hybridoma cells. The inhibition was not dependent on bacterial viability, since it was also produced by exposure to dead bacilli and MTB cytosol preparations, suggesting that it was initiated by a constitutively expressed bacterial component. Northern blot analysis demonstrated that MTB bacilli or cytosol decreased MHC-II mRNA, and immunoprecipitation of biosynthetically labeled molecules confirmed that MHC-II protein synthesis was diminished. Exposure to MTB or MTB cytosol also decreased expression of H2-DM, but H2-DM expression was still sufficient to catalyze conversion of MHC-II to SDS-stable dimers, a measure of MHC-II peptide loading. Thus, infection with MTB decreased both MHC-II and H2-DM expression, but diminished MHC-II synthesis provided the major limitation to antigen processing.

Phagocytic antigen processing and effects of microbial products on antigen processing and T-cell responses.

Processing of exogenous antigens and microbes involves contributions by multiple different endocytic and phagocytic compartments. During the processing of soluble antigens, different endocytic compartments have been demonstrated to use distinct antigen-processing mechanisms and to process distinct sets of antigenic epitopes. Processing of particulate and microbial antigens involves phagocytosis and functions contributed by phagocytic compartments. Recent data from our laboratory demonstrate that phagosomes containing antigen-conjugated latex beads are fully competent class II MHC (MHC-II) antigen-processing organelles, which generate peptide:MHC-II complexes. In addition, phagocytosed antigen enters an alternate class I MHC (MHC-I) processing pathway that results in loading of peptides derived from exogenous antigens onto MHC-I molecules, in contrast to the cytosolic antigen source utilized by the conventional MHC-I antigen-processing pathway. Antigen processing and other immune response mechanisms may be activated or inhibited by microbial components to the benefit of either the host or the pathogen. For example, antigen processing and T-cell responses (e.g. Th1 vs Th2 differentiation) are modulated by multiple distinct microbial components, including lipopolysaccharide, cholera toxin, heat labile enterotoxin of Escherichia coli, DNA containing CpG motifs (found in prokaryotic and invertebrate DNA but not mammalian DNA) and components of Mycobacterium tuberculosis.

CpG oligodeoxynucleotides down-regulate macrophage class II MHC antigen processing.

Unmethylated CpG motifs in bacterial DNA or short oligodeoxynucleotides (ODN) stimulate cells of the immune system and provide adjuvant activity. CpG DNA directly activates macrophages to secrete IL-12 and TNF-alpha and increases transcription of various genes, but its effects on macrophage Ag processing remain uncertain. The effects of CpG ODN on class II MHC (MHC-II) Ag processing and presentation were examined using peritoneal macrophages that were cultured for 18 h with CpG ODN and then pulsed with protein Ags. T cell hybridomas were used to detect presentation of specific peptide:MHC-II complexes. Both CpG ODN and LPS inhibited processing of bovine RNase and hen egg lysozyme. Presentation of exogenous peptides was inhibited to a lesser degree. Treatment of macrophages for 18 h with CpG ODN decreased surface MHC-II expression, as measured by flow cytometry. Furthermore, Northern blot analysis revealed that treatment with CpG ODN decreased I-Ak mRNA. Endocytosis by macrophages, as measured by uptake of fluorescent dextran, was not altered by treatment with CpG ODN. The inhibitory effect of CpG ODN on Ag processing was seen after prolonged (18 h) treatment of macrophages, but not after short treatment (e.g., 2 h) with CpG ODN and protein Ag. Enhancement of macrophage Ag processing was not seen at any time point of CpG ODN exposure, in contrast to data from other studies with dendritic cells. In summary, exposure of macrophages to CpG ODN results in a decrease in macrophage Ag processing and presentation, which is largely mediated by a decrease in synthesis of MHC-II molecules.

Activation of human CD8+ alpha beta TCR+ cells by Mycobacterium tuberculosis via an alternate class I MHC antigen-processing pathway.

Human immune responses to M. tuberculosis are characterized by activation of multiple T cell subsets including CD4+, CD8+, and gammadelta T cells, and the role of CD8+ alphabeta TCR+ T cells in this response is poorly understood. Stimulation of T cells from healthy tuberculin skin test-positive persons with live M. tuberculosis-H37Ra or soluble M. tuberculosis Ags readily up-regulated IL-2Ralpha (CD25) expression on CD8+ T cells. Purified resting and activated CD8+ T cells produced IFN-gamma and proliferated to both M. tuberculosis bacilli and soluble mycobacterial Ags with monocytes as APC. Precursor frequency of mycobacterial Ag-specific CD8+ T cells by IFN-gamma enzyme-linked immunospot was 5-10-fold lower than the precursor frequency of CD4+ T cells, and IFN-gamma secretion by CD8+ T cells was 50-100-fold lower. CD8+ T cells secreted approximately 10-fold less IFN-gamma per cell than CD4+ T cells in response to mycobacterial Ags. CD8+ T cell responses to M. tuberculosis bacilli were blocked by anti-MHC class I antibody and required Ag processing. Processing of M. tuberculosis bacilli by monocytes for presentation to MHC class I-restricted CD8+ T cells was insensitive to brefeldin A treatment, which blocks the conventional MHC class I Ag-processing pathway. These results represent the first demonstration that human cells can process pathogen Ags via an alternate Ag-processing pathway for MHC class I and suggest a mechanism for participation of IFN-gamma-secreting CD8+ T cells in the human immune responses to M. tuberculosis.