Differential responses of bovine macrophages to Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium

Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium are antigenically and genetically similar organisms; however, they differ in their virulence for cattle. M. avium subsp. paratuberculosis causes a chronic intestinal infection leading to a chronic wasting disease termed paratuberculosis or Johne's disease, whereas M. avium subsp. avium causes only a transient infection. We compared the response of bovine monocyte-derived macrophages to ingestion of M. avium subsp. paratuberculosis and M. avium subsp. avium organisms by determining organism survival, superoxide and nitric oxide production, and expression of the cytokines tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), interleukin-8 (IL-8), IL-10, IL-12, and granulocyte-monocyte colony-stimulating factor (GM-CSF). Unlike M. avium subsp. paratuberculosis, macrophages were able to kill approximately half of the M. avium subsp. avium organisms after 96 h of incubation. This difference in killing efficiency was not related to differences in nitric oxide or superoxide production. Compared to macrophages activated with IFN-gamma and lipopolysaccharide, macrophages incubated with M. avium subsp. paratuberculosis showed greater expression of IL-10 and GM-CSF (all time points) and IL-8 (72 h) and less expression of IL-12 (72 h), IFN-gamma (6 h), and TNF-alpha (6 h). When cytokine expression by macrophages incubated with M. avium subsp. paratuberculosis was compared to those of macrophages incubated with M. avium subsp. avium, M. avium subsp. paratuberculosis-infected cells showed greater expression of IL-10 (6 and 24 h) and less expression of TNF-alpha (6 h). Therefore, the combination of inherent resistance to intracellular degradation and suppression of macrophage activation through oversecretion of IL-10 may contribute to the virulence of M. avium subsp. paratuberculosis in cattle.     

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.     

Apoptosis of human monocytes and macrophages by Mycobacterium avium sonicate.

Mycobacterium avium is an intracellular organism which multiplies predominantly within human macrophages. This organism has previously been shown to induce apoptosis in human macrophages. With a view to identifying M. avium components that induce cell death in infected host cells, sonicated extracts of M. avium as well as individual components isolated from the M. avium sonicate were tested in various assays with a human monocytic cell line (THP-1). THP-1 cells incubated with M. avium sonicate showed significantly reduced viability after a 2-day exposure compared to control cells incubated with media alone. This effect was dose dependent, with only 6.6% +/- 5.2% and 48.8% +/- 10.3% of the cells being viable by trypan blue exclusion at 600 and 300 microg/ml, respectively. Control cells, on the other hand, exhibited a viability of 98.8% +/- 1.0%. In addition, an 80% ammonium sulfate fraction of the M. avium sonicate and the previously characterized 68-kDa protein were found to have similar effects on THP-1 cells. In both cases, the reduction in viability was due to apoptosis characterized by chromatin condensation, DNA fragmentation by agarose gel electrophoresis, or terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling (TUNEL) and release of nuclear matrix protein (NMP) into the culture medium. M. avium sonicate-induced apoptosis of THP-1 cells was completely inhibited by the commonly used antioxidants pyrrolidinedithiocarbamate (PDTC) and butylated hydroxyanisole (BHA), indicating that the generation of free oxygen radicals may be responsible for inducing cell death. M. avium sonicate was found to induce apoptosis of monocyte-derived macrophages (MDMs) as well. This effect was not reversed in the presence of PDTC and was not accompanied with DNA fragmentation when determined by agarose gel electrophoresis, as seen in the case of THP-1 cells. However, these MDMs were found to contain fragmented DNA by TUNEL. These findings suggest that the mechanism of cell death in MDMs may be different from that observed with THP-1 cells. Furthermore, these results provide new insight into the effect of M. avium components on host cell responses during M. avium infection.     

Inhibition by normal human serum of Mycobacterium avium multiplication in cultured human macrophages.

Normal human serum used as a protein supplement in RPMI 1640 medium inhibited growth in blood-derived human macrophages (MP) of virulent Mycobacterium avium serovars 4 and 8, derived from patients with acquired immunodeficiency syndrome, but not virulent Mycobacterium tuberculosis. A defined serum substitute (SS) promoted the intramacrophage growth of M. avium but not of M. tuberculosis. The effects of serum or SS were measured by counting viable bacteria in lysates of the MP at 0, 4, and 7 days after their infection by the bacteria. Neither serum nor SS inhibited or enhanced M. avium growth in the absence of MP. The results suggest that a nutrient essential for intracellular replication of M. avium is made by MP from a pronutrient present in both SS and serum and that something in serum inhibits MP conversion of the pronutrient to nutrient. This inhibition may be an important mechanism of native resistance against M. avium in normal people.     

Characterization of inhibition of Mycobacterium avium replication in macrophages by normal human serum.

Serum from some AIDS patients permits the rapid multiplication of Mycobacterium avium in cultured human macrophages. Serum from human immunodeficiency virus-negative individuals inhibits replication. The characteristics of the serum-induced inhibition were examined here. M. avium 7497 serovar 4 grew exponentially in macrophages when they were cultured in serumless medium. Growth was measured by determining the CFU after infected macrophages were lysed at 0 to 7 days after infection. Normal AB serum (5 to 10%) added to infected macrophages resulted in an initial 4-day lag of bacterial growth followed by rapid replication from 4 to 7 days. Serum also inhibited bacterial replication in medium without macrophages. This inhibition was not biphasic but was sustained over 7 days. Macrophage-associated M. avium became less responsive to serum inhibitor within 24 h after infection of macrophages. Within 2 days of culture, M. avium no longer responded to inhibitor. Replication of macrophage-derived M. avium (Vi) was in some instances serum inhibitable and at other times was enhanced by serum, when it was used to infect fresh macrophages. The Vi phenotype remained serum inhibitable without macrophages. Preinfection of macrophages with heat-killed M. avium did not alter serum-induced bacterial inhibition or escape from inhibition.     

Activation of macrophages and interference with CD4+ T-cell stimulation by Mycobacterium avium subspecies paratuberculosis and Mycobacterium avium subspecies avium

Mycobacterium avium subspecies paratuberculosis (M. ptb) and M. avium subspecies avium (M. avium) are closely related but exhibit significant differences in their interaction with the host immune system. The macrophage line, J774, was infected with M. ptb and M. avium and analysed for cytokine production and stimulatory capacity towards antigen-specific CD4+ T cells. Under all conditions J774 cells were activated to produce proinflammatory cytokines. No influence on the expression of major histocompatibility complex (MHC) class II, intracellular adhesion molecule-1 (ICAM-1), B7.1, B7.2 or CD40 was found. However, the antigen-specific stimulatory capacity of J774 cells for a CD4+ T-cell line was significantly inhibited after infection with M. ptb, but not with M. avium. When a T-cell hybridoma expressing a T-cell receptor identical to that of the T-cell line was used, this inhibition was not observed, suggesting that costimulation which is essential for the CD4+ T-cell line is influenced by the pathogenic bacterium M. ptb.     

Recombinant tumour necrosis factor-alpha decreases whereas recombinant interleukin-6 increases growth of a virulent strain of Mycobacterium avium in human macrophages.

The ability of a virulent strain of Mycobacterium avium to infect and replicate within human monocyte-derived macrophages of normal donors was assessed. Moreover, the ability of selected cytokines to modulate the intracellular growth of M. avium was investigated. Our virulent strain of M. avium grew progressively in human macrophages. Treatment of macrophage monolayers with interferon-gamma (IFN-gamma) did not lead to any significant change in the infection pattern. Conversely, treatment with tumour necrosis factor-alpha (TNF-alpha) led to a significant reduction in the growth of M. avium in the macrophages. In contrast, treatment of macrophages with interleukin-6 (IL-6) enhanced their susceptibility to M. avium significantly. This finding was substantiated by other results which showed that IL-6 increased the growth of M. avium in tissue culture medium. These results suggest that cytokines may influence the M. avium-macrophage interaction, in a positive or negative manner.     

A pre-existing infection by Mycobacterium avium subsp. avium modulates anti-Cryptococcus neoformans and anti-Candida albicans activities in human macrophages

Mycobacterium avium is a facultative intracellular microorganism, able to survive and multiply within mammalian macrophages by circumventing antimicrobial mechanisms. In this study we hypothesize that pre-existing M. avium infection could result in macrophage superinfections by other microorganisms. We found that 24 h after ingestion of M. avium at a low multiplicity of infection, macrophages are unable to efficiently produce superoxide anions when over-stimulated with phorbol esters, and that the generation of oxidative burst is only partially restored 72 h after bacteria ingestion. We also demonstrate that intracellular killing of Cryptococcus neoformans is markedly impaired in human macrophages that have previously ingested M. avium (but not other bacteria such as Escherichia coli). This inhibitory effect is observed with live mycobacteria, but not when heat-inactivated bacteria are ingested. In contrast, when Candida albicans is given to macrophages instead of C. neoformans, an enhancement of intracellular killing is observed, suggesting that cytocidal mechanisms other than respiratory burst are involved in the anti- Candidacidal activity of macrophages.     

Growth inhibition of Mycobacterium avium complex in human alveolar macrophages by the combination of recombinant macrophage colony-stimulating factor and interferon-gamma

The reservoir of Mycobacterium avium complex (MAC) during human infection is the mononuclear phagocyte. In these studies, the ability of certain macrophage-active cytokines to affect MAC growth in human alveolar macrophages was evaluated. Neither recombinant interferon-gamma (2 x 10(2) to 10(3) U/well of 5 x 10(5) cells) nor recombinant macrophage colony-stimulating factor (20 to 50 ng/well), when tested alone, exhibited a consistent ability to induce macrophage targets to inhibit the growth of a clinical strain of MAC serovar 4. However, the combination of these cytokines (1 to 50 ng macrophage colony-stimulating factor + 10(3) U interferon per well) was remarkably effective in diminishing replication of MAC in all experiments. These cytokines were also able to induce alveolar macrophages to restrict MAC growth even though cells were obtained from several individuals with acquired immunodeficiency syndrome (AIDS) or from normal donors and infected in vitro with the human immunodeficiency virus type 1. The effect of this cytokine combination was not abrogated by 10(4) neutralizing U/ml of anti-tumor necrosis factor-alpha antibody. Rather, the combination of interferon-gamma and macrophage colony-stimulating factor appeared to activate intrinsic macrophage mechanisms for restricting MAC growth and deserves further study to determine the potential value of this cytokine combination in the treatment of human infection.     

Growth of Mycobacterium avium in activated macrophages harvested from inbred mice with differing innate susceptibilities to mycobacterial infection.

The growth of Mycobacterium avium in macrophages obtained from Mycobacterium bovis BCG-infected mice was compared with that in macrophages from uninfected mice. BCG vaccination resulted in substantial macrophage activation, measured as increased acid phosphatase and superoxide anion production, as well as enhanced leishmanicidal activity. However, the activated macrophages were only able to reduce the rate of intracellular growth by Listeria monocytogenes and M. avium in vivo and did not express detectable levels of mycobactericidal activity in vitro. Exposure of the macrophage monolayers to concanavalin A-stimulated spleen cell supernatant fluid and lipopolysaccharide did not further enhance the ability of the BCG-activated macrophages to control the intracellular replication of the M. avium. Macrophages from BCG-infected C57BL/6 (BCGs) mice were quantitatively better able to control the intracellular replication of the M. avium challenge than were similar phagocytes obtained from BCGr (A/J) mice. These findings have important implications with respect to the expression of acquired resistance to these atypical mycobacterial infections.     

Effector mechanisms involved in cytokine-mediated bacteriostasis of Mycobacterium avium infections in murine macrophages.

In this study we found that addition of a range of doses of interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), or granulocyte-macrophage colony stimulating factor (GM-CSF) to cultures of bone marrow-derived murine macrophages infected with the 25291 strain of Mycobacterium avium gave rise to varying degrees of bacteriostasis. In contrast, similar treatment with interleukin-4 (IL-4) or IL-6 had no effect. However, when similar experiments with the former set of cytokines were performed using a panel of M. avium isolates, substantial isolate-to-isolate variation was observed. In cultures containing IFN-gamma, synthesis of substantial levels of reactive nitrogen intermediates was observed; however, neither these materials, nor reactive oxygen intermediates, were found to be responsible for observed bacteriostasis. In further experiments, in which the culture medium was supplemented with various concentrations of a weak acid or a weak base in order to influence the pH of macrophage intracellular compartments, it was found that the presence of the weak acid augmented the activity of IFN-gamma, whilst the weak base counteracted this effect. These data support the hypothesis, therefore, that the bacteriostatic effect of IFN-gamma against the growth of M. avium, rather than depending on reactive radical production, is mediated through acidification of the infected phagosome, perhaps through activation of proton pumps in the phagosomal membrane.     

Multilocus enzyme electrophoresis analysis of the Mycobacterium avium complex and other mycobacteria.

Multilocus enzyme electrophoresis analysis was used to evaluate the Mycobacterium avium complex (MAC), M. paratuberculosis, and nine other mycobacterial species. The average number of alleles per locus was 2.8 for the 35 MAC and 2 M. paratuberculosis strains which represented 24 electrophoretic types (ETs) and two distinct groups. The M. avium group was resolved into 17 ETs and contained the M. paratuberculosis ET. The M. intracellulare group consisted of six ETs. There was complete agreement between Gen-Probe identification and group placement by multilocus enzyme electrophoresis. The mean genetic diversity per locus for the 24 MAC ETs was 0.38. This procedure subdivided some serovars and, if implemented, should prove to be a powerful epidemiologic tool for the MAC. Eleven additional ETs were formed after the data for the other mycobacterial species were pooled with those for the MAC.     

Resistance of macrophages to Mycobacterium avium is induced by alpha2-adrenergic stimulation

The ability of macrophages to control the growth of microorganisms is increased by macrophage activation. Previously, it was shown that epinephrine activated mouse macrophages to resist the growth of Mycobacterium avium via alpha(2)-adrenergic stimulation. In the present study, we show that the alpha(2)-adrenergic agonist (alpha(2)-agonist) clonidine induced resistance to M. avium growth in the RAW264.7 mouse macrophage cell line. The ability of catecholamines to induce resistance to mycobacteria was specific to alpha(2)-adrenergic stimulation, as alpha(1)-, beta(1)-, and beta(2)-agonists had no effect. Receptor signaling through Gi proteins was required. A G-protein antagonist specific for the alpha subunits of the Go/Gi family blocked the increased resistance induced by clonidine, while a Gs-protein antagonist was without effect. Both nitric oxide (NO) production and superoxide (O(2)(-)) production were required for the increased resistance to M. avium growth induced by clonidine. Although NO production was required, clonidine did not increase the level of NO in M. avium-infected cells. Since NO and O(2)(-) interact to produce peroxynitrite (ONOO(-)), we examined whether ONOO(-) mediates the increased resistance to M. avium induced by clonidine. 5,10,15,20-Tetrakis(4-sulfonatophenyl)prophyrinato iron (III) chloride (FeTPPS), a specific scavenger of ONOO(-), inhibited the effect of clonidine on M. avium growth. Clonidine also increased the production of ONOO(-) in M. avium-infected RAW264.7 cells, as measured by the oxidation of 123-dihydrorhodamine and the production of nitrated tyrosine residues. We therefore conclude that alpha(2)-adrenergic stimulation activates macrophages to resist the growth of M. avium by enhancing the production of ONOO(-).     

鸟结核分枝杆菌感染巨噬细胞后的外泌体刺激巨噬细胞产生的细胞因子及其蛋白质表达改变

目的 探讨鸟结核分枝杆菌(M.avium)感染巨噬细胞后分泌的外泌体[(+)外泌体]刺激巨噬细胞后产生的分子免疫机制及其差异蛋白质成分研究.方法 冷冻高速离心法收集经鸟分枝结核杆菌感染及未感染的巨噬细胞上清中的外泌体,然后利用外泌体刺激巨噬细胞;ELISA方法检测经外泌体刺激巨噬细胞后细胞上清中IFN-γ、TNF-α的含量;流式细胞术从蛋白水平检测巨噬细胞受外泌体刺激后CD80、CD86蛋白表达情况;2-DE MALDI-TOF/TOF MS技术分析鉴定巨噬细胞受M.avium感染后分泌的外泌体中的差异蛋白.结果 IFN-γ、TNF-α在(+)外泌体刺激巨噬细胞后培养上清中含量增加;CD80、CD86蛋白在巨噬细胞受(+)外泌体刺激后表达上调.外泌体经2-DE MALDI TOF/TOF MS分析获得18个差异蛋白,且质谱成功鉴定12个.结论 (+)外泌体促进巨噬细胞TNF-α、IFN-γ的分泌从而增强巨噬细胞的炎症反应,并且可促进巨噬细胞CD80、CD86蛋白表达增强;筛选出差异蛋白的功能与细胞骨架结构、蛋白质合成与加工,炎症反应密切相关.     

Typing of Mycobacterium avium isolates by PCR.

A Mycobacterium avium typing method based on PCR amplification of genomic sequences located between the recently described repetitive elements IS1245 and IS1311 was developed. This method was applied to a set of epidemiologically related and unrelated strains and compared with restriction fragment length polymorphism analysis with IS1245 as the probe. This PCR typing consists of a rapid and simple technique, providing a reproducible M. avium characterization as discriminant as restriction fragment length polymorphism analysis.     

Multiplication of Mycobacterium marinum within phagolysosomes of murine macrophages.

Both in vivo and in vitro, Mycobacterium marinum organisms were found to multiply within phagolysosomes of murine macrophages. It thus appears that M. marinum are neither killed nor inhibited from multiplying by lysosomal enzymes.     

Characterization of IS1110-like sequences found in Mycobacterium species other than Mycobacterium avium

During study of a polycyclic aromatic hydrocarbon-degrading gene cluster in Mycobacterium sp. SNP11, which is a fast-growing strain related to Mycobacterium gilvum, a DNA segment with very high similarities to the IS1110 element of Mycobacterium avium was identified. Insertion sequence IS1110 was discovered for the first time in 1994 during a study of plasmid incidence in AIDS-derived M. avium strains. This element had thus far been detected in most human, veterinary, and environmental M. avium isolates, but not in other Mycobacterium species such as M. bovis, M. tuberculosis, M. xenopi, M. kansasii or M. gordonae. In the present paper, we describe the isolation and characterization of ISMysp1, an IS1110-like element present in several copies in the genome of Mycobacterium sp. strain SNP11. PCR and hybridization experiments revealed that this element is commonly found in fast-growing Mycobacterium strains. Moreover, Blast searches against the recently sequenced genome of M. smegmatis mc(2)155 revealed that this strain contains four IS1110-like elements. Analysis and sequence comparison of the whole of the IS1110-like elements revealed several common features not found in the most closely related mycobacterial members, IS900, IS901, IS902, IS1626 and IS1547.     

Human immunodeficiency virus type 1 enhances intracellular growth of Mycobacterium avium in human macrophages.

Disseminated Mycobacterium avium infections are common in patients with AIDS and result in a reduced life expectancy. Human monocytes/macrophages are important target cells for both human immunodeficiency virus (HIV) and M. avium. We have studied the interaction in vitro of M. avium and HIV type 1 (HIV-1) in human macrophages. Human monocytes isolated from the peripheral blood of healthy individuals were infected with HIV-1, M. avium, or both. The intracellular growth of M. avium and the replication of HIV-1 were monitored for up to 5 weeks. Intracellular mycobacterial growth was seen in all M. avium infected cell cultures and was paralleled by increased production of interleukin 1 alpha and beta. Preinfection of the macrophages with HIV-1 reduced the interleukin 1 production and accelerated the intracellular growth of M. avium. These findings may explain in part the impaired control of mycobacterial infections seen with patients with AIDS.