Bovine monocyte TLR2 receptors differentially regulate the intracellular fate of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium

Pathogenic mycobacterial organisms have the capacity to inhibit macrophage activation and phagosome maturation. Although the mechanism is complex, several studies have incriminated signaling through TLR2 receptors with subsequent activation of the MAPK pathway p38 (MAPKp38) and overproduction of IL-10 in the survival of pathogenic mycobacterial organisms. In the present study, we compared the response of bovine monocytes with infection by Mycobacterium avium subspecies paratuberculosis (MAP), the cause of paratuberculosis in ruminants, with the closely related organism M. avium subspecies avium (Maa), which usually does not cause disease in ruminants. Both MAP and Maa induced phosphorylation of MAPKp38 by bovine monocytes; however, addition of a blocking anti-TLR2 antibody partially prevented MAPKp38 phosphorylation of MAP-infected monocytes but not Maa-infected monocytes. Addition of anti-TLR2 antibody enhanced phagosome acidification and phagosome-lysosome fusion in MAP-containing phagosomes and enabled monocytes to kill MAP organisms. These changes were not observed in Maa-infected monocytes. The effect on phagosome maturation appears to occur independently from the previously described inhibitory effects of IL-10 on phagosome acidification and organism killing, as IL-10 production was not affected by addition of anti-TLR2 antibody to monocyte cultures. Therefore, signaling through the TLR2 receptor appears to play a role in phagosome trafficking and antimicrobial responses in MAP-infected bovine mononuclear phagocytes.     

Glycopeptidolipids from Mycobacterium avium promote macrophage activation in a TLR2- and MyD88-dependent manner

The Toll-like receptors (TLRs) are key components in the immune response against numerous pathogens. Previous studies have indicated that TLR2 plays an essential role in promoting immune responses against mycobacterial infections. Prior work has also shown that mice deficient in TLR2 are more susceptible to infection by Mycobacterium tuberculosis, Mycobacterium bovis bacillus Calmette-Guerin, and Mycobacterium avium. Therefore, it is important to define the molecules expressed by pathogenic mycobacteria, which bind the various TLRs. Although a number of TLR agonists have been characterized for M. tuberculosis, no specific TLR ligand has been identified in M. avium. We have found that glycopeptidolipids (GPLs), which are highly expressed surface molecules on M. avium, can stimulate the nuclear factor-kappaB pathway as well as mitogen-activated protein kinase p38 and Jun N-terminal kinase activation and production of proinflammatory cytokines when added to murine bone marrow-derived macrophages. This stimulation was dependent on TLR2 and myeloid differentiation primary-response protein 88 (MyD88) but not TLR4. M. avium express apolar and serovar-specific (ss)GPLs, and it is the expression of the latter that determines the serotype of a particular M. avium strain. It is interesting that the ssGPLs activated macrophages in a TLR2- and MyD88-dependent manner, and no macrophage activation was observed when using apolar GPLs. ssGPLs also differed in their ability to activate macrophages with Serovars 1 and 2 stimulating inhibitor of kappaB p38 and phosphorylation and tumor necrosis factor alpha (TNF-alpha) secretion, while Serovar 4 failed to stimulate p38 activation and TNF-alpha production. Our studies indicate that ssGPLs can function as TLR2 agonists and promote macrophage activation in a MyD88-dependent pathway.     

Role of gamma interferon and tumor necrosis factor alpha during T-cell-independent and -dependent phases of Mycobacterium avium infection.

To design an effective immunotherapy for Mycobacterium avium infections, the protective host response to the infection must be known. Here we analyzed the role of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in the innate and acquired responses to M. avium infections in mice. T-cell depletion studies showed that CD4+ T cells were required for control of the infection. CD(4+)-depleted mice showed enhanced bacterial proliferation and at the same time showed a reduction in the level of expression of both IFN-gamma and TNF-alpha mRNAs in spleen cells. In contrast, M. bovis BCG immunization restricted M. avium proliferation and at the same time promoted expression of the mRNAs for the two cytokines. In vivo depletion studies using specific monoclonal antibodies showed that both IFN-gamma and TNF-alpha are involved in an early protection possibly involving NK cells, and furthermore, IFN-gamma is involved in the later T-cell-protective response to infection. In vivo neutralization of IFN-gamma during M. avium infection also blocked the priming for enhanced TNF-alpha secretion triggered by endotoxin. Both cytokines were found to be involved in the resistance expressed in BCG-immunized animals and exhibited additive bacteriostatic effects in vitro on bone marrow-derived macrophages infected with different strains of M. avium. These data suggest that both cytokines act in an additive or synergistic fashion in the induction of bacteriostasis and that IFN-gamma is also involved in priming TNF-alpha secretion.     

Regulation of expression of major histocompatibility antigens by bovine macrophages infected with Mycobacterium avium subsp. paratuberculosis or Mycobacterium avium subsp. avium

Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium are antigenically and genetically very similar organisms; however, they differ markedly in their virulence for cattle. We evaluated the capacity of bovine macrophages infected with M. avium subsp. paratuberculosis or M. avium subsp. avium to express major histocompatibility complex (MHC) class I and class II antigens on their surface and to interact with primed autologous lymphocytes. Our results indicate that infection of bovine macrophages with M. avium subsp. paratuberculosis promoted the downregulation of MHC class I and class II molecules on the macrophage surface within 24 and 12 h, respectively. Alternatively, MHC class II expression by M. avium subsp. avium-infected macrophages was not detected until 24 h after infection, and the magnitude of the decrease was smaller. Decreased MHC class I expression by M. avium subsp. avium-infected macrophages was not detected. Unlike M. avium subsp. paratuberculosis-infected macrophages, M. avium subsp. avium-infected macrophages upregulated MHC class I and class II expression after activation by gamma interferon or tumor necrosis factor alpha. Further, M. avium subsp. avium-infected macrophages were lysed by primed autologous lymphocytes, whereas M. avium subsp. paratuberculosis-infected macrophages were not. Overall, the results support the hypothesis that the difference in the virulence of M. avium subsp. paratuberculosis and M. avium subsp. avium for cattle is dependent on a difference in the capacity of the organisms to suppress mycobacterial antigen presentation to T lymphocytes.     

Beta-chemokines are induced by Mycobacterium tuberculosis and inhibit its growth

Chemokines (CK) are potent leukocyte activators and chemoattractants and aid in granuloma formation, functions critical for the immune response to Mycobacterium tuberculosis. We hypothesized that infection of alveolar macrophages (AM) with different strains of M. tuberculosis elicits distinct profiles of CK, which could be altered by human immunodeficiency virus (HIV) infection. RANTES, macrophage inflammatory protein-1 alpha (MIP-1 alpha), and MIP-1 beta were the major beta-CK produced in response to M. tuberculosis infection. Virulent M. tuberculosis (H37Rv) induced significantly less MIP-1 alpha than did the avirulent strain (H37Ra), while MIP-1 beta and RANTES production was comparable for both strains. MIP-1 alpha and MIP-1 beta were induced by the membrane, but not cytosolic, fraction of M. tuberculosis. M. tuberculosis-induced CK secretion was partly dependent on tumor necrosis factor alpha (TNF-alpha). AM from HIV-infected individuals produced less TNF-alpha and MIP-1 beta than did normal AM in response to either M. tuberculosis strain. We tested the functional significance of decreased beta-CK secretion by examining the ability of beta-CK to suppress intracellular growth of M. tuberculosis. MIP-1 beta and RANTES suppressed intracellular growth of M. tuberculosis two- to threefold, a novel finding. Thus, beta-CK contribute to the innate immune response to M. tuberculosis infection, and their diminution may promote the intracellular survival of M. tuberculosis.     

TSA通过组蛋白乙酰化影响TLR2基因表达

目的组蛋白去乙酰化酶抑制剂(Trichostatin A,TSA)处理人THP-1细胞,干预组蛋白H3乙酰化水平,探讨组蛋白H3乙酰化对人THP-1细胞中TLR2基因表达水平的影响。方法构建THP-1巨噬细胞模型,分别利用不同浓度TSA处理细胞,Real-time PCR和Western blot方法检测m RNA和蛋白的表达;染色质免疫共沉淀技术(Chromatin immunoprecipitation,Ch IP)比较TSA处理前后启动子区H3乙酰化水平。结果TSA以浓度依赖方式上调表达水平。TSA上调组蛋白H3乙酰化水平,使启动子区H3乙酰化水平明显上升。结论 TSA通过组蛋白H3乙酰化影响基因表达,这是乙酰化调控TLR2基因表达的一种可能机制。     

Whole chromosomal DNA probes for rapid identification of Mycobacterium tuberculosis and Mycobacterium avium complex.

Whole chromosomal DNA probes were used to identify clinical isolates of Mycobacterium tuberculosis, Mycobacterium avium complex, and Mycobacterium gordonae. The probe for M. tuberculosis was prepared from Mycobacterium bovis BCG, which has been shown to be closely related to M. tuberculosis. A probe for the M. avium complex was prepared from three strains representing each of the three DNA homology groups in the M. avium complex. The probes were used in dot blot assays to identify clinical isolates of mycobacteria. The dot blot test correctly identified 57 of the 61 (93%) cultures grown on solid media, and 100% of antibiotic-treated broth-grown cells were correctly identified. Identification by dot blot required a maximum of 48 h. When the probes were tested against 63 positive BACTEC (Johnston Laboratories, Inc., Towson, Md.) cultures of clinical specimens, 59% were correctly identified. However, of the 14 BACTEC cultures that had been treated with antibiotics before being lysed, 13 (93%) were correctly identified.     

Isolation and partial characterization of glycolipid fractions from Mycobacterium avium serovar 2 (Mycobacterium paratuberculosis 18) that inhibit activated macrophages.

Glycolipid fractions from Mycobacterium avium serovar 2 (Mycobacterium paratuberculosis 18) inhibited the killing of Candida albicans by activated bovine peripheral-blood-derived macrophages. Fractions were derived by using the matrix solid-phase dispersion technique, which is a new method of simultaneous lysis and partial fractionation of components of bacterial cells. Further purification of active fractions was performed by concanavalin A affinity chromatography, centrifugal filtration, and differing solvent solubility. Three different fractions were isolated and partially characterized. Two of these fractions have characteristics typical of glycolipids, and the third fraction has characteristics compatible with a peptidoglycolipid. This peptidoglycolipid fraction has been purified and named MIF-A3.     

Improved detection times for Mycobacterium avium complex and Mycobacterium tuberculosis with the BACTEC radiometric system.

A total of 2,559 routine clinical specimens were cultured for mycobacteria by using BACTEC Middlebrook 7H12 medium (BACTEC), Lowenstein-Jensen slants (LJ), and Mycobactosel selective Middlebrook 7H11 slants (M7H11). Thirty-three isolates (1.3%) of M. avium complex and 82 isolates (3.2%) of M. tuberculosis were recovered. The BACTEC mean detection time of M. avium complex from 27 smear-negative specimens was earlier than that of conventional media for both decontaminated respiratory specimens (BACTEC, 12 days; LJ, 32 days; and M7H11, 38 days) and untreated tissue and fluid specimens (BACTEC, 8 days; LJ, 30 days; and M7H11, 31 days). The sensitivity for smear-negative M. avium complex with BACTEC (74%) was comparable to that with LJ (74%) and M7H11 (63%). The mean detection times of M. tuberculosis from 56 smear-positive respiratory specimens were 8 days for BACTEC, 16 days for LJ, and 17 days for M7H11, and sensitivities for the detection of positive cultures were 98% for BACTEC, 76% for LJ, and 79% for M7H11. The BACTEC mean detection time of M. tuberculosis in smear-negative specimens was better for tissues and fluids (14 days) than for respiratory specimens (24 days). BACTEC yielded substantially earlier detection of M. avium complex from all specimen types and of M. tuberculosis from smear-positive respiratory specimens. The rapid identification and susceptibility testing of M. tuberculosis in BACTEC agreed completely with conventional tests and provided a 3-week reduction in median time to final reports.     

TLR2 deficiency by compromising p19 (IL-23) expression limits Th 17 cell responses to Mycobacterium tuberculosis

CD4(+) T(h)1 cells producing IFN-γ are of extreme importance in controlling infections by Mycobacterium tuberculosis both in mice and in men. In addition to IFN-γ-producing T cells, IL-17-producing T cells (T(h)17) have been observed during mycobacterial infections. Nevertheless, their contribution for the host immune response to mycobacteria as well as the signals triggering M. tuberculosis -specific T(h)17 cell differentiation and maintenance are not fully understood. We show that signaling via Toll-like receptor (TLR) 2 has a major impact on the regulation of p19 (IL-23) expression in response to M. tuberculosis and therefore on the establishment of T(h)17 cell responses to M. tuberculosis infection. Diminished T(h)17 responses in the lung of M. tuberculosis -infected TLR2-deficient animals were not caused by defective cell differentiation in the draining lymph node (LN) but rather by reduced maintenance at the site of infection. Consistent with the decreased numbers of T(h)17 cells in the lungs of infected TLR2-deficient animals, we observed reduced expression of CXCL9, CXCL10 and CXCL11, chemokines involved in recall responses to M. tuberculosis. Our data provides insights into the TLR2 role in infection with M. tuberculosis, with implications in pathophysiology of the disease and vaccine design.     

Induction of macrophage-derived SLPI by Mycobacterium tuberculosis depends on TLR2 but not MyD88

Macrophages respond to Mycobacterium tuberculosis by regulating expression of gene products that initiate a host innate response to this micro-organism. In this study, we report that exposure of murine peritoneal macrophages to heat-killed Mycobacterium tuberculosis (HK-Mtb) led to an increase in secretory leucocyte protease inhibitor (SLPI) gene expression and protein secretion in a time- and dose-dependent manner. HK-Mtb-induced SLPI mRNA expression was sensitive neither to a protein synthesis inhibitor, cycloheximide, nor to an actin polymerization blocker, cytochalasin D. Treatment of macrophages with interferon (IFN)-gamma inhibited HK-Mtb-induced SLPI expression. RAW264.7 cells stably expressing SLPI produced a reduced level of tumour necrosis factor (TNF) in response to HK-Mtb as compared with mock transfectants. Aerosol infection of mice with live M. tuberculosis resulted in an induction of SLPI gene expression in infected lungs. Macrophages from Toll-like receptor 4 (TLR4)-/- or MyD88-/- mice responded to M. tuberculosis similarly to wild-type macrophages by exhibiting increased SLPI expression. In contrast, macrophages from TLR2-/- mice were incapable of inducing SLPI in response to M. tuberculosis. This induction signifies the presence of a TLR2-dependent but MyD88-independent M. tuberculosis signalling pathway, suggesting involvement of adaptor protein(s) other than MyD88 in M. tuberculosis-mediated induction of SLPI.     

Identification of mycobacteria from culture by using the Gen-Probe Rapid Diagnostic System for Mycobacterium avium complex and Mycobacterium tuberculosis complex.

Commercially available kits (Mycobacterium avium Complex Rapid Diagnostic System and Mycobacterium tuberculosis Complex Rapid Diagnostic System; Gen-Probe, Inc., San Diego, Calif.) utilizing nucleic acid hybridization for the rapid identification of members of the M. avium-M. intracellulare complex and M. tuberculosis complex were evaluated by using 339 clinical and American Type Culture Collection (Rockville, Md.) isolates. The tests, which can be performed in approximately 2 h, use specific [125I]DNA probes complementary to the rRNAs of M. avium, M. intracellulare, and M. tuberculosis complex, the latter of which includes M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, and M. microti. The M. avium-M. intracellulare probes correctly identified 99 of 114 M. avium-M. intracellulare isolates, with 7 false-negatives and 8 false-positives, for a sensitivity of 93.4% and a specificity of 96.6%. After repeat testing, 110 of 114 were correctly identified, with 4 false-negatives and no false-positives, for a sensitivity of 96.5% and a specificity of 100%. The M. tuberculosis complex probe correctly identified 99 of 102 M. tuberculosis isolates, with 1 false-negative and 2 false-positives, for a sensitivity of 99% and a specificity of 99.2%. After repeat testing, 100 of 102 isolates were correctly identified, with no false-negatives and 2 false-positives, for a sensitivity of 100% and a specificity of 99.2%. Overall, there were 15 discrepant M. avium-M. intracellulare results, with 4 such results after repeat testing, and 3 discrepant M. tuberculosis complex results, with 2 such results after repeat testing. The Gen-Probe kits are highly sensitive and specific for use in identifying M. avium-M. intracellulare complex and M. tuberculosis complex isolates and will be useful in the clinical laboratory which can use the present radionuclide-containing kits cost effectively.     

Cytokine secretion and adhesion molecule expression by granuloma T lymphocytes in Mycobacterium avium infection.

Mice experimentally infected with Mycobacterium avium develop a chronic disease characterized by widespread noncaseating granulomas. In this report, we describe the phenotype and cytokine secretion profile of these granuloma-infiltrating effector T lymphocytes. In response to specific antigen, granuloma T cells and, to a lesser extent, spleen cells secrete interferon-gamma, but no interleukin-4 or -5. The importance of this Th1-like response to the host was demonstrated by the massively increased bacterial load and lethal disease in interferon-gamma knockout mice. One function of localized cytokine secretion is to recruit inflammatory T cells bearing surface adhesion molecules complementary to counter-receptors on vascular endothelial cells. Granuloma T cells express high levels of these pro-inflammatory adhesion molecules but have down-regulated their expression of L-selectin (CD62L). The expression of these adhesion molecules on granuloma-infiltrating T lymphocytes would alter the migration pathway of these cells and is likely to be important in facilitating the traffic of effector T cells to the granulomatous inflammatory site. In addition, T cells from Schistosoma mansoni granulomas express the same set of adhesion molecules, showing that this phenotype is not specifically dependent upon the Th1 pattern of cytokine secretion.     

Molecular cloning and expression of an alpha-mannosidase gene in Mycobacterium tuberculosis

Mannose is a major component of glycolipids and glycoproteins of the cell envelope of M. tuberculosis (Mtb). However, the enzymes involved in the biosynthesis and catabolism of mannosylated glycans are largely unknown. We demonstrate alpha-mannosidase activity towards the fluorescent substrate 4-methylumberlliferyl-alpha-D-mannopyranoside (4MU-Man) in cell lysates of attenuated and virulent Mtb bacilli, with two-fold higher activity in the virulent strain Erdman. Mannosidase activity was optimal at pH 6.5, was not inhibited by deoxymannojirimycin (dMNJ), was mildly inhibited by swainsonine (SW) and stimulated two-fold by EDTA. GenBank BLAST analysis for sequences homologous to eukaryotic alpha-mannosidases revealed a 3.6 kb putative gene (Rv0648) in Mtb cosmid SCY20H10 (Acc# z92772), with strong homology (48%) to the rat ER/cytosolic alpha-mannosidase and containing signature sequences of class 2 mannosidases. By RT-PCR, gene Rv0648 was found differentially expressed, with lower expression during growth in A549 pneumocyte cultures. Gene Rv0648 was cloned, expressed in E. coli, and alpha-mannosidase activity in cell lysates determined. Expression of alphaMan-pET in E. coli cells resulted in an eight-fold increase in mannosidase activity toward 4-MU-Man, upon IPTG induction. Partial purification of the histidine-tagged Mtb mannosidase by metal chelation affinity chromatography, and analysis by SDS-PAGE, showed a protein with the predicted m.w. of 137.5 kDa. Enzyme assays of the column fractions showed alpha-mannosidase activity toward synthetic aryl-mannose substrates, in fractions enriched in the recombinant Mtb mannosidase. These results demonstrate that gene Rv0648 encodes an active alpha-mannosidase in Mtb.     

Reduction of TLR2 gene expression in allergic and nonallergic rhinitis.

BACKGROUND: Immunomodulators, including toll-like receptors (TLRs) and defensins, produced in response to pathogenic stimuli, can direct the developing immune system toward a T(H)1 nonallergic phenotype. Increased human beta-defensin (HBD) 4 expression is associated with infection. OBJECTIVE: To determine whether reduced mucosal levels of TLRs and defensins contribute to the inflammation seen in chronic allergic and nonallergic rhinitis. METHODS: Real-time polymerase chain reaction was used to determine gene expression levels of HBDs 1 through 4 and TLRs 2 and 4. Immunohistochemical analysis was used to study the localization and distribution of protein for alpha-defensins 1 through 3, HBD2, neutrophil elastase, and TLR2 in sections of nasal turbinate tissue from adults with persistent allergic and idiopathic rhinitis, healthy nasal mucosa, and tonsil tissue. RESULTS: Allergic mucosa showed a significant (P = .02) reduction in TLR2 messenger RNA expression compared with control mucosa and generally reduced expression for TLR4 and HBDs. Although not significant, the nonallergic group also showed reduced expression for TLRs and HBDs. With the exception of HBD4, increased target gene levels were seen in tonsil tissue. Protein expression of HBD2 and TLR2 was localized in lining and submucosal glandular epithelium but insignificant differences were seen for HBDs, TLRs, neutrophils, and a-defensin between the rhinitic and control patient groups. CONCLUSIONS: Subjects with allergic and nonallergic rhinitis show reduced TLR and HBD gene expression. The significant reduction in TLR2 gene expression in allergic adults supports the concept that increased TLR2 protects against the development of allergy. The low levels of HBD4 detected in both rhinitis groups suggest lack of an underlying infection pathophysiological feature.     

腺病毒介导的mPPARγ1基因转染抑制IFN-γ诱导ECV304细胞galectin-9基因和蛋白表达

目的：观察腺病毒介导的mPPARγ1转染抑制IFN-γ诱导ECV304细胞galectin-9基因和蛋白表达。方法：构建表达小鼠PPARγ1基因的复制缺陷型腺病毒表达载体；将融合80%的ECV304细胞给予不同刺激量（1×104 U/L、5×104 U/L、1×105 U/L和2×105 U/L）的IFN-γ干预；将IFN-γ（1×105 U/L）预刺激并孵育 24 h 的ECV304细胞分成对照组（C）、PPARγ基因过度表达组(P)、PPARγ活化剂曲格列酮干预组(T)以及PPARγ基因过度表达和曲格列酮共刺激组(PT)进行干预，观察不同剂量IFN-γ对ECV304细胞galectin-9基因和蛋白表达的作用，以及PPARγ基因过度表达和/或活化对上述作用的影响。结果：正常ECV304细胞galectin-9基因表达弱。IFNγ孵育24 h后， ECV304细胞galectin-9基因和蛋白表达增加，且galectin-9表达与IFN-γ具有量效关系。PPARγ1基因转染抑制IFN-γ诱导galectin-9基因/蛋白表达，曲格列酮对上述作用无影响；PPARγ1基因转染和曲格列酮共刺激抑制IFN-γ诱导galectin-9基因/蛋白表达与单一PPARγ1基因转染效应相似。正常ECV304细胞PPARγ表达量低，而PPARγ基因过表达和活化不影响内源性PPARγ基因表达。结论：PPARγ1基因转染抑制IFN-γ诱导ECV304细胞galectin-9基因/蛋白表达可能是PPARγ基因发挥免疫调控作用的一个重要机制。