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.     

Inhibition of IFN-gamma-induced class II transactivator expression by a 19-kDa lipoprotein from Mycobacterium tuberculosis: a potential mechanism for immune evasion

Mycobacterium tuberculosis (MTB) persists inside macrophages despite vigorous immune responses. MTB and MTB 19-kDa lipoprotein inhibit class II MHC (MHC-II) expression and Ag processing by a Toll-like receptor 2-dependent mechanism that is shown in this study to involve a defect in IFN-gamma induction of class II transactivator (CIITA). Exposure of macrophages to MTB or MTB 19-kDa lipoprotein inhibited IFN-gamma-induced MHC-II expression, but not IL-4-induced MHC-II expression, by preventing induction of mRNA for CIITA (total, type I, and type IV), IFN regulatory factor-1, and MHC-II. MTB 19-kDa lipoprotein induced mRNA for suppressor of cytokine signaling (SOCS)1 but did not inhibit IFN-gamma-induced Stat1 phosphorylation. Furthermore, the lipoprotein inhibited MHC-II Ag processing in SOCS1(-/-) macrophages. MTB 19-kDa lipoprotein did not inhibit translocation of phosphorylated Stat1 to the nucleus or Stat1 binding to and transactivation of IFN-gamma-sensitive promoter constructs. Thus, MTB 19-kDa lipoprotein inhibited IFN-gamma signaling independent of SOCS1 and without interfering with the activation of Stat1. Inhibition of IFN-gamma-induced CIITA by MTB 19-kDa lipoprotein may allow MTB to evade detection by CD4(+) T cells.     

Mycobacterium tuberculosis NAD+-dependent DNA ligase is selectively inhibited by glycosylamines compared with human DNA ligase I

DNA ligases are important enzymes which catalyze the joining of nicks between adjacent bases of double-stranded DNA. NAD+-dependent DNA ligases (LigA) are essential in bacteria and are absent in humans. They have therefore been identified as novel, validated and attractive drug targets. Using virtual screening against an in-house database of compounds and our recently determined crystal structure of the NAD+ binding domain of the Mycobacterium tuberculosis LigA, we have identified N1, N(n)-bis-(5-deoxy-alpha-D-xylofuranosylated) diamines as a novel class of inhibitors for this enzyme. Assays involving M.tuberculosis LigA, T4 ligase and human DNA ligase I show that these compounds specifically inhibit LigA from M.tuberculosis. In vitro kinetic and inhibition assays demonstrate that the compounds compete with NAD+ for binding and inhibit enzyme activity with IC50 values in the microM range. Docking studies rationalize the observed specificities and show that among several glycofuranosylated diamines, bis xylofuranosylated diamines with aminoalkyl and 1, 3-phenylene carbamoyl spacers mimic the binding modes of NAD+ with the enzyme. Assays involving LigA-deficient bacterial strains show that in vivo inhibition of ligase by the compounds causes the observed antibacterial activities. They also demonstrate that the compounds exhibit in vivo specificity for LigA over ATP-dependent ligase. This class of inhibitors holds out the promise of rational development of new anti-tubercular agents.     

Cognate B cell signaling via MHC class II: differential regulation of B cell antigen receptor and MHC class II/Ig-alpha beta signaling by CD22

Recent studies demonstrate that MHC class II molecules can signal via associated Ig-alphabeta dimers, signal transducers previously thought to function only in B cell Ag receptor (BCR) signaling. Surprisingly, the biologic outputs of MHC class II and BCR ligation (by thymus-dependent Ags) differ, e.g., MHC class II signaling leads to robust proliferation and extension of pseudopods. It seemed possible that these differences might be due, at least in part, to differential use of inhibitory coreceptors thought to modulate membrane Ig signals. In this study, we demonstrate that CD22, an inhibitory BCR coreceptor, neither associates with nor functions in MHC class II/Ig-alphabeta signaling. Interestingly, CD22 is actively excluded from cell surface MHC class II aggregates.     

Mycobacterium tuberculosis LprG (Rv1411c): a novel TLR-2 ligand that inhibits human macrophage class II MHC antigen processing

MHC class II (MHC-II)-restricted CD4(+) T cells are essential for control of Mycobacterium tuberculosis infection. This report describes the identification and purification of LprG (Rv1411c) as an inhibitor of primary human macrophage MHC-II Ag processing. LprG is a 24-kDa lipoprotein found in the M. tuberculosis cell wall. Prolonged exposure (>16 h) of human macrophages to LprG resulted in marked inhibition of MHC-II Ag processing. Inhibition of MHC-II Ag processing was dependent on TLR-2. Short-term exposure (<6 h) to LprG stimulated TLR-2-dependent TNF-alpha production. Thus, LprG can exploit TLR-2 signaling to inhibit MHC-II Ag processing in human macrophages. Inhibition of MHC-II Ag processing by mycobacterial lipoproteins may allow M. tuberculosis, within infected macrophages, to avoid recognition by CD4(+) T cells.     

Sequential cleavage by metallopeptidases and proteasomes is involved in processing HIV-1 ENV epitope for endogenous MHC class I antigen presentation

Antigenic peptides derived from viral proteins by multiple proteolytic cleavages are bound by MHC class I molecules and recognized by CTL. Processing predominantly takes place in the cytosol of infected cells by the action of proteasomes. To identify other proteases involved in the endogenous generation of viral epitopes, specifically those derived from proteins routed to the secretory pathway, we investigated presentation of the HIV-1 ENV 10-mer epitope 318RGPGRAFVTI327 (p18) to specific CTL in the presence of diverse protease inhibitors. Both metalloproteinase and proteasome inhibitors decreased CTL recognition of the p18 epitope expressed from either native gp160 or from a chimera based on the hepatitis B virus secretory core protein as carrier protein. Processing of this epitope from both native ENV and the hepatitis B virus secretory core chimeric protein appeared to proceed by a TAP-dependent pathway that involved sequential cleavage by proteasomes and metallo-endopeptidases; however, other protease activities could replace the function of the lactacystin-sensitive proteasomes. By contrast, in a second TAP-independent pathway we detected no contribution of metallopeptidases for processing the ENV epitope from the chimeric protein. These results show that, in the classical TAP-dependent MHC class I pathway, endogenous Ag processing of viral proteins to yield the p18 10-mer epitope requires metallo-endopeptidases in addition to proteasomes.     

Hepatocytes express abundant surface class I MHC and efficiently use transporter associated with antigen processing, tapasin, and low molecular weight polypeptide proteasome subunit components of antigen processing and presentation pathway

Hepatic expression levels of class I MHC Ags are generally regarded as very low. Because the status of these Ags and their ability to present peptides are important for the understanding of pathogen clearance and tolerogenic properties of the liver, we set out to identify the factors contributing to the reported phenotype. Unexpectedly, we found that the surface densities of K(b) and D(b) on C57BL/6 mouse hepatocytes are nearly as high as on splenocytes, as are the lysate concentrations of mRNA encoding H chain and beta(2)-microglobulin (beta(2)m). In contrast, the components of the peptide-loading pathway are reduced in hepatocytes. Despite the difference in the stoichiometric ratios of H chain/beta(2)m/peptide-loading machineries, both cell types express predominantly thermostable class I and are critically dependent on TAP and tapasin for display of surface Ags. Minor differences in the expression patterns in tapasin(-/-) background suggest cell specificity in class I assembly. Under immunostimulatory conditions, such as exposure to IFN-gamma or Listeria monocytogenes, hepatocytes respond with a vigorous mRNA synthesis of the components of the Ag presentation pathway (up to 10-fold enhancement) but up-regulate H chain and beta(2)m to a lesser degree (<2-fold). This type of response should promote rapid influx of newly generated peptides into the endoplasmic reticulum and preferential presentation of foreign/induced Ag by hepatic class I.     

Cooperation of Toll-like receptor 2 and 6 for cellular activation by soluble tuberculosis factor and Borrelia burgdorferi outer surface protein A lipoprotein: role of Toll-interacting protein and IL-1 receptor signaling molecules in Toll-like receptor 2 signaling.

Toll-like receptor 2 (TLR2) and TLR4 play important roles in innate immune responses to various microbial agents. We have previously shown that human dermal endothelial cells (HMEC) express TLR4, but very little TLR2, and respond to LPS, but not to Mycobacterium tuberculosis 19-kDa lipoprotein, unless transfected with TLR2. Here we report that HMEC are unresponsive to several additional biologically relevant TLR2 ligands, including, phenol-soluble modulin (PSM), a complex of three small secreted polypeptides from the skin commensal Staphylococcus epidermidis, soluble tuberculosis factor (STF), and Borrelia burgdorferi outer surface protein A lipoprotein (OspA-L). Expression of TLR2 renders HMEC responsive to all these ligands. We further characterized the signaling pathway in response to STF, OspA-L, and PSM in TLR2-transfected HMEC. The TLR2 signaling pathway for NF-kappaB trans-activation shares the IL-1R signaling molecules. Dominant negative constructs of TLR2 or TLR6 inhibit the responses of STF and OspA-L as well as PSM in TLR2-transfected HMEC, supporting the concept of functional cooperation between TLR2 and TLR6 for all these TLR2 ligands. Moreover, we show that Toll-interacting protein (Tollip) coimmunoprecipitates with TLR2 and TLR4 using HEK 293 cells, and overexpression of Tollip inhibits NF-kappaB activation in response to TLR2 and TLR4 signaling. Collectively, these findings suggest that there is functional interaction between TLR2 and TLR6 in the cellular response to STF and OspA-L in addition to S. epidermidis (PSM) Ags, and that engagement of TLR2 triggers a signaling cascade, which shares the IL-1R signaling molecules, similar to the TLR4-LPS signaling cascade. Our data also suggest that Tollip may be an important constituent of both the TLR2 and TLR4 signaling pathways.     

Proteasome, transporter associated with antigen processing, and class I genes in the nurse shark Ginglymostoma cirratum: evidence for a stable class I region and MHC haplotype lineages.

Cartilaginous fish (e.g., sharks) are derived from the oldest vertebrate ancestor having an adaptive immune system, and thus are key models for examining MHC evolution. Previously, family studies in two shark species showed that classical class I (UAA) and class II genes are genetically linked. In this study, we show that proteasome genes LMP2 and LMP7, shark-specific LMP7-like, and the TAP1/2 genes are linked to class I/II. Functional LMP7 and LMP7-like genes, as well as multiple LMP2 genes or gene fragments, are found only in some sharks, suggesting that different sets of peptides might be generated depending upon inherited MHC haplotypes. Cosmid clones bearing the MHC-linked classical class I genes were isolated and shown to contain proteasome gene fragments. A non-MHC-linked LMP7 gene also was identified on another cosmid, but only two exons of this gene were detected, closely linked to a class I pseudogene (UAA-NC2); this region probably resulted from a recent duplication and translocation from the functional MHC. Tight linkage of proteasome and class I genes, in comparison with gene organizations of other vertebrates, suggests a primordial MHC organization. Another nonclassical class I gene (UAA-NC1) was detected that is linked neither to MHC nor to UAA-NC2; its high level of sequence similarity to UAA suggests that UAA-NC1 also was recently derived from UAA and translocated from MHC. These data further support the principle of a primordial class I region with few class I genes. Finally, multiple paternities in one family were demonstrated, with potential segregation distortions.     

402AX teratocarcinoma MHC class I antigen expression is regulated in vivo by Lyt 1, Lyt 2, and L3T4 expressing splenic T cells

The murine 402AX teratocarcinoma is a MHC class I antigen negative tumor of 129 strain origin. Host resistance to the 402AX tumor is genetically controlled. When passed intraperitoneally in genetically resistant mice, the tumor cells are induced to express MHC Class I antigens of the 129 genotype. When passed in genetically susceptible mice, the tumor cells remain MHC class I antigen negative. Earlier studies have demonstrated that resistance to the tumor and regulation of tumor cell MHC class I antigen expression are under the control of the host's immune system. The present studies indicate that splenic Lyt 1-, Lyt 2-, and L3T4-expressing cells regulate tumor cell MHC class I antigen expression, and that these cells require a genetically resistant host environment in which to differentiate. Splenic T cells primed to the 402AX tumor and transferred into genetically susceptible 129 mice give rise to GVHD, suggesting that immunity to the tumor involves reactivity to 129 minor histocompatibility antigens.     

Type I interferon production during herpes simplex virus infection is controlled by cell-type-specific viral recognition through Toll-like receptor 9, the mitochondrial antiviral signaling protein pathway, and novel recognition systems

Recognition of viruses by germ line-encoded pattern recognition receptors of the innate immune system is essential for rapid production of type I interferon (IFN) and early antiviral defense. We investigated the mechanisms of viral recognition governing production of type I IFN during herpes simplex virus (HSV) infection. We show that early production of IFN in vivo is mediated through Toll-like receptor 9 (TLR9) and plasmacytoid dendritic cells, whereas the subsequent alpha/beta IFN (IFN-α/β) response is derived from several cell types and induced independently of TLR9. In conventional DCs, the IFN response occurred independently of viral replication but was dependent on viral entry. Moreover, using a HSV-1 UL15 mutant, which fails to package viral DNA into the virion, we found that entry-dependent IFN induction also required the presence of viral genomic DNA. In macrophages and fibroblasts, where the virus was able to replicate, HSV-induced IFN-α/β production was dependent on both viral entry and replication, and ablated in cells unable to signal through the mitochondrial antiviral signaling protein pathway. Thus, during an HSV infection in vivo, multiple mechanisms of pathogen recognition are active, which operate in cell-type- and time-dependent manners to trigger expression of type I IFN and coordinate the antiviral response.     

High-density lipoprotein-associated 17beta-estradiol fatty acyl ester uptake by Fu5AH hepatoma cells: implications of the roles of scavenger receptor class B, type I and the low-density lipoprotein receptor

17beta-estradiol (E2) fatty acyl esters naturally incorporate into high-density lipoprotein (HDL). The objective was to elucidate mechanisms involved in HDL-associated E2 cellular uptake and to determine the intracellular distribution of E2 and its fatty acyl esters (E2-FAE) after uptake. [3H]E2 or [3H] cholesterol was incubated with human serum for 24 h to allow for fatty acyl esterification. Total-HDL containing [3H]E2-FAE or [3H]cholesterol esters was isolated by sequential density ultracentrifugation and then incubated with Fu5AH rat hepatoma cells for various time points. Cellular uptake was determined by intracellular radioactivity as a percentage of total radioactivity. Chemical inhibition of scavenger receptor class B, type I and low-density lipoprotein (LDL) receptor competition assays were performed to determine cellular uptake mechanisms. Compared to HDL-[3H]cholesterol, cellular uptake of HDL-[3H]E2 occurred at an initially rapid rate. SR-BI inhibition resulted in a decrease in HDL-E2 uptake and LDL impaired this uptake in a concentration-dependent manner. Accordingly, pretreatment of cells with BLT-1 combined with LDL addition significantly attenuated HDL-E2 uptake. HDL-E2-FAE was hydrolyzed into free E2 with the maximum at 24 h. Fu5AH cells facilitate HDL-E2 uptake by at least SR-BI and LDL receptor pathways and intracellular hydrolysis of E2-FAE into free E2 ensues.