Gliotoxin, an inhibitor of nuclear factor-kappa B, attenuates peptidoglycan-polysaccharide-induced colitis in rats

Gliotoxin is a fungal metabolite that has immunosuppressive properties. First, we determined if gliotoxin could inhibit bacterial peptidoglycan-polysaccharide-stimulated tumor necrosis factor-alpha production, as well as nuclear factor-kappa B (NF-kappaB), in a rat macrophage (NR8383) cell line. Next, the apoptosis-inducing potential of gliotoxin was also evaluated in this cell line. Finally, we evaluated whether gliotoxin could reduce peptidoglycan-polysaccharide-induced colitis in rats. Gliotoxin (2 mg/kg/day) was dosed from day 14 after the initial intramural colonic injection of peptidoglycan-polysaccharide until day 21. A gross colonic injury score, myeloperoxidase activity, and cytokine levels were all evaluated on day 21. Gliotoxin dose dependently inhibited cytokine production, as well as NF-kappaB, and also induced apoptosis in the NR8383 cell line. On day 21, gliotoxin significantly reduced gross colonic injury (adhesions, nodules, mucosal lesions) in rats. Gliotoxin-treated rats also had partially normalized biochemical indices of colitis, such as colonic cytokine levels. The colonic level of NF-kappaB was also partially normalized in gliotoxin treated rats. Gliotoxin also exhibited an antiarthritis effect in peptidoglycan-polysaccharide-treated rats. In summary, gliotoxin effectively attenuated the chronic reactivation phase of peptidoglycan-polysaccharide-induced colitis. This anticolitis effect may be related to the inhibition of NF-kappaB in Lewis rats.     

Immunosuppression in vitro by a metabolite of a human pathogenic fungus.

Gliotoxin , a metabolite of Aspergillus fumigatus, inhibits phagocytosis of macrophages at concentrations of 20-50 ng/ml. Pretreatment of stimulator cells in mixed lymphocyte cultures with gliotoxin (100 ng/ml) abrogates induction of alloreactive cytotoxic T cells. The presence of gliotoxin 48 hr after initiation of cytotoxic T-cell induction has no effect. Inhibition of cytotoxic T-cell induction by gliotoxin at low concentrations, acting on the stimulator cells, can be overridden by concanavalin A-activated cell supernatants. Gliotoxin does not induce immediate cell-surface antigen modification on target cells. The possible role of gliotoxin in the etiology of A. fumigatus-related diseases is discussed.     

Gliotoxin treatment selectively spares M-CSF- plus IL-3-responsive multipotent haemopoietic progenitor cells in bone marrow.

Gliotoxin, an epipolythiodioxopiperazine, is a fungal metabolite that causes genomic DNA degradation preferentially in certain blood cell types including T lymphocytes and macrophages. Gliotoxin has previously been used to treat murine allogeneic bone marrow prior to transplantation into irradiated recipients, and in this situation the drug prevents development of graft-versus-host disease, and permits the establishment of allogeneic bone marrow chimeras. We have examined the nature of the cells that survive gliotoxin treatment and report here that gliotoxin selectively spares a unique class of haemopoietic stem cell that forms large (HPP) colonies in the presence of mixtures of M-CSF and IL-3. We confirm that the cells which survive gliotoxin treatment are capable of reconstituting the haemopoietic system in allogeneic lethally irradiated mice.     

Gliotoxin causes apoptosis and necrosis of rat Kupffer cells in vitro and in vivo in the absence of oxidative stress: exacerbation by caspase and serine protease inhibition

BACKGROUND/AIMS: A potential application of gliotoxin therapy for liver fibrosis was suggested by its apoptotic effect on fibrogenic activated stellate cells. We investigated if gliotoxin exerts similar effects on hepatic macrophage Kupffer cells. METHODS: Effects of gliotoxin on Kupffer cells isolated from the normal liver and in vivo following its administration to CCl(4)-induced cirrhotic rats were studied. RESULTS: Gliotoxin caused apoptosis of cultured Kupffer cells, the effect being apparent at 0.3 microM concentration within 1h; longer incubation caused necrosis. This effect was associated with mitochondrial cytochrome c release, caspase-3 activation and ATP depletion. Interestingly, inhibition of caspase-3 and serine proteases accelerated and augmented gliotoxin-induced cell death via necrosis. Gliotoxin stimulated nuclear translocation of NFkappaB, and phosphorylation of p38, ERK1/2 and JNK MAP kinases, but these signaling molecules were not involved in gliotoxin-induced death of Kupffer cells. In vivo administration of gliotoxin to cirrhotic rats caused apoptosis of Kupffer cells, stellate cells and hepatocytes. In control rats, the effect was minimal on the nonparenchymal cells and not apparent on hepatocytes. CONCLUSIONS: In the fibrotic liver, gliotoxin nonspecifically causes death of hepatic cell types. Modification of gliotoxin molecule may be necessary for selective targeting and elimination of activated stellate cells.     

Gliotoxin reduces the severity of trinitrobenzene sulfonic acid-induced colitis in mice: evidence of the connection between heme oxygenase-1 and the nuclear factor-kappaB pathway in vitro and in vivo

BACKGROUND: Gliotoxin, a fungal metabolite, has been known to show strong immunosuppressive properties, although its mechanisms are not completely understood. In this report, the authors investigated the mechanism whereby gliotoxin has anti-inflammatory properties in vitro and in trinitrobenzene sulfonic acid-induced colitis. MATERIALS AND METHODS: Body weight, histological scores, and myeloperoxidase activity were evaluated in trinitrobenzene sulfonic acid colitis. Nuclear factor-kappaB (NF-kappaB) p65, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-12, and intercellular adhesion molecule-1 were detected by immunohistochemical staining. IL-8 secretion was measured by an enzyme-linked immunosorbent assay. Heme oxygenase-1 (HO-1) expression and I-kappaB degradation were analyzed by Western blot. RESULTS: Pretreatment of human epithelial HT-29 cells with gliotoxin significantly blocked the I-kappaB degradation and NF-kappaB p65 nuclear translocation induced by tumor necrosis factor-alpha or IL-1beta; these were parallel with the inhibition of IL-8 secretion and intercellular adhesion molecule-1 expression in the same cells. Interestingly, gliotoxin induced HO-1 in HT-29 cells and, in turn, inhibition of HO-1 activity by a zinc protoporphyrin IX reversed the effects of gliotoxin in terms of I-kappaB degradation, intercellular adhesion molecule-1 expression, and IL-8 production. In trinitrobenzene sulfonic acid colitis, gliotoxin administration significantly improved the clinical and histopathological symptoms. Notably, gliotoxin also induced HO-1 in the colonic mucosa and zinc protoporphyrin IX reversed the protective effects of gliotoxin in trinitrobenzene sulfonic acid colitis. CONCLUSIONS: These results demonstrate for the first time that the anti-inflammatory actions mediated by gliotoxin include HO-1 induction and the subsequent blockade of NF-kappaB-dependent signaling pathways in vitro and in vivo. The current results also demonstrate that gliotoxin may be an effective agent for the treatment of diseases characterized by mucosal inflammation.     

Gliotoxin ameliorates development of fibrosis and cirrhosis in a thioacetamide rat model

Activation of hepatic stellate cells causes most of the pathological changes in cirrhosis. The fungal metabolite gliotoxin was shown to induce apoptosis of hepatic stellate cells in vitro. We examined whether gliotoxin may prevent or reverse liver fibrosis in a rat model of thioacetamide-inducedcirrhosis, and whether gliotoxin administration in vivo causes apoptosis of activated stellate cells. Gliotoxin treatment resulted in a significant decrease in liver fibrosis in rats, but did not improve liver functions. We observed a significant reduction in the numbers of activated hepatic stellate cells in the gliotoxin-treated rats. Gliotoxin administration also resulted in parenchymal apoptosis of hepatocytes and hepatic stellate cells. In conclusion, gliotoxin reduces hepatic fibrosis, an effect accompanied by reduction of the numbers of activated hepatic stellate cells in the liver.     

In Vitro and In Vivo Effects of Gliotoxin, a Fungal Metabolite:

Gliotoxin is a fungal metabolite that has immunosuppressive properties. First, we determined if gliotoxin could inhibit cytokine production from macrophage and colonic epithelial cell lines, as well as whether it inhibited nuclear factor-kappa B in these same cell types. Second, we evaluated whether gliotoxin could reduce dextran sodium sulfate-induced colitis in rats. A disease activity index, myeloperoxidase activity, and cytokine levels were evaluated on either day 7 or 21. In both cell lines, gliotoxin dose dependently inhibited cytokine production and nuclear factor-kappa B. On day 21, gliotoxin significantly reduced disease activity (diarrhea and bloody stools) in rats. On day 7, gliotoxin treatment significantly improved various indices of colitis, including colonic cytokine levels. Decreased food consumption and weight gain was evident with a larger dose of gliotoxin. In summary, gliotoxin, a nuclear factor-kappa B inhibitor, effectively reduced dextran sodium sulfate-induced colitis in rats. However, gliotoxin exhibited a narrow therapeutic to toxicity ratio in these rats.     

Gliotoxin-mediated apoptosis of activated human hepatic stellate cells

BACKGROUND: Activated hepatic stellate cells (HSCs) play a central role in liver fibrogenesis, and apoptosis of activated HSCs might be essential to clear HSCs from injured liver. Gliotoxin induces apoptosis of activated human and rat HSCs by an unknown mechanism. AIM: This study investigated the role of reactive oxygen species (ROS) and membrane permeability transition (MPT) in gliotoxin-induced apoptosis of activated human HSCs. METHODS: Primary and immortalized human HSCs were analyzed using confocal microscopy for ROS with dichlorodihdrofluorescence diacetate (DCFH-DA) fluorophore and for the mitochondrial membrane potential (MMP) using tetramethylrhodamine methylester (TMRM). RESULTS: Gliotoxin at higher concentrations (> or =7.5 microM) markedly increased ROS formation, and ROS production was also evident at concentrations of gliotoxin causing necrotic cell death (> or =32.5 microM). Gliotoxin rapidly (begins about 20 min at 1.5 microM and 10 min at 7.5 microM) disrupts MMP at a concentration as low as 300nM. MMP disruption was followed by cytochrome c release and caspase-3 activation. The MPT inhibitors, cyclosporine A (5 microM) plus trifluoperazine (12.5 microM), blocked depolarization of the mitochondrial membrane and release of cytochrome c, but did not block apoptosis in HSCs. CONCLUSIONS: Gliotoxin (0.3-7.5 microM) induces apoptosis of activated human HSCs with induction of MPT, cytochrome c release and caspase-3 activation, whereas at higher doses (>32.5 microM), it induces necrosis. However, gliotoxin also activates a mitochondrial independent pathway.     

Cytocidal effect of Streptococcus pyogenes on mouse neutrophils in vivo and the critical role of streptolysin S

We analyzed the in vivo dynamics of peritoneal exudate cells (PECs) in mice injected with group A streptococcus (GAS). A live low-virulence strain, as well as heat-killed low- and high-virulence strains, significantly increased the number of PECs (primarily neutrophils), whereas a live high-virulence strain did not. When coinjected with thioglycollate, the live high-virulence strain, as well as most other GAS strains, suppressed the ability of thioglycollate to induce neutrophil exudation. This suppression was due to a cytocidal effect of GAS on exuded neutrophils rather than an inhibition of neutrophil migration. In addition, GAS enhanced the apoptosis of neutrophils. These cytocidal effects were significantly reduced by the deletion of functional streptolysin S from GAS. Our findings suggest that, in addition to the production of antiphagocytic factors and survival inside phagocytes, GAS uses a more aggressive method--the elimination of neutrophils--to evade the host's innate immune system.     

Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances the resolution of liver fibrosis in rats

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) play a pivotal role in liver fibrosis and stimulating their apoptosis could be an effective treatment for liver fibrosis. METHODS: Activated HSCs, hepatocytes, and rats with liver fibrosis were treated with gliotoxin. RESULTS: Addition of gliotoxin to activated (alpha-smooth muscle actin positive) rat and human HSCs resulted in morphologic alterations typical of apoptosis. Within 2-3 hours of incubation, caspase 3 activity was markedly induced and caspase inhibitor 1 (Z-VAD-FMK)-sensitive oligonucleosome-length DNA fragments were detectable by gel electrophoresis of low molecular weight DNA. Apoptosis was widespread as judged by fluorescence-activated cell sorter analysis and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining in both rat and human HSCs at concentrations that had no effect on the viability of rat hepatocytes. Gliotoxin treatment significantly reduced the number of activated stellate cells and mean thickness of bridging fibrotic septae in livers from rats treated with carbon tetrachloride. CONCLUSIONS: These data demonstrate proof-of-concept that by up-regulating HSC apoptosis, the extent of fibrosis can be decreased in inflammatory liver injury.     

Selective resistance of bone marrow-derived hemopoietic progenitor cells to gliotoxin.

The fungal metabolite gliotoxin at low concentrations prevents mitogen stimulation of mature lymphocytes as a result of gliotoxin-induced genomic DNA degradation. Bone marrow, on the other hand, contains a subpopulation of cells resistant to gliotoxin at similar concentrations. This population includes the hemopoietic progenitor cells that grow in vitro in response to appropriate colony-stimulating factors and cells that form colonies in the spleens of lethally irradiated recipients. Gliotoxin treatment of lymph node cell-enriched bone marrow significantly delayed the onset of graft-versus-host disease in fully allogeneic bone marrow chimeras.     

Mechanism of action of the antifibrogenic compound gliotoxin in rat liver cells

Gliotoxin has been shown to promote a reversal of liver fibrosis in an animal model of the disease although its mechanism of action in the liver is poorly defined. The effects of gliotoxin on activated hepatic stellate cells (HSCs) and hepatocytes have therefore been examined. Addition of gliotoxin (1.5 microM) to culture-activated HSCs resulted in its rapid accumulation, resulting in increased levels of glutathione and apoptosis without any evidence of oxidative stress. In contrast, although hepatocytes also rapidly sequestered gliotoxin, cell death only occurred at high (50-microM) concentrations of gliotoxin and by necrosis. At high concentrations, gliotoxin was metabolized by hepatocytes to a reduced (dithiol) metabolite and glutathione was rapidly oxidized. Fluorescent dye loading experiments showed that gliotoxin caused oxidative stress in hepatocytes. Antioxidants--but not thiol redox active compounds--inhibited both oxidative stress and necrosis in hepatocytes. In contrast, HSC apoptosis was not affected by antioxidants but was potently abrogated by thiol redox active compounds. The adenine nucleotide transporter (ANT) is implicated in mitochondrial-dependent apoptosis. HSCs expressed predominantly nonliver ANT isoform 1, and gliotoxin treatment resulted in a thiol redox-dependent alteration in ANT mobility in HSC extracts, but not hepatocyte extracts. In conclusion, these data suggest that gliotoxin stimulates the apoptosis of HSCs through a specific thiol redox-dependent interaction with the ANT. Further understanding of this mechanism of cell death will aid in finding therapeutics that specifically stimulate HSC apoptosis in the liver, a promising approach to antifibrotic therapy.     

Aspergillus fumigatus suppresses the human cellular immune response via gliotoxin-mediated apoptosis of monocytes

Aspergillus fumigatus (AF) is a ubiquitous mold and is the most common cause of invasive aspergillosis, an important source of morbidity and mortality in immunocompromised hosts. Using cytokine flow cytometry, we assessed the magnitude of functional CD4+ and CD8+ T-cell responses following stimulation with Aspergillus antigens. Relative to those seen with cytomegalovirus (CMV) or superantigen stimulation, responses to Aspergillus antigens were near background levels. Subsequently, we confirmed that gliotoxin, the most abundant mycotoxin produced by AF, was able to suppress functional T-cell responses following CMV or staphylococcal enterotoxin B (SEB) stimulation. Additional studies demonstrated that crude AF filtrates and purified gliotoxin inhibited antigen-presenting cell function and induced the preferential death of monocytes, leading to a marked decrease in the monocyte-lymphocyte ratio. Analysis of caspase-3 activation confirmed that gliotoxin preferentially induced apoptosis of monocytes; similar effects were observed in CD83+ monocyte-derived dendritic cells. Importantly, the physiologic effects of gliotoxin in vitro were observed below concentrations recently observed in the serum of patients with invasive aspergillosis. These studies suggest that the production of gliotoxin by AF may constitute an important immunoevasive mechanism that is mediated by direct effects on antigen-presenting cells and both direct and indirect effects on T cells.     

我国小肠结肠炎耶氏菌毒力株的特征

本文对我国各地分离的小肠结肠炎耶氏菌的毒力特征进行测定,所有毒力株除个别外,均有VW抗原、Vi抗原和毒力因子,它们的检测结果一致。有毒株还有一条40～50MDa的质粒带,即所谓毒力质粒,但有此毒力质粒的菌株并非都是有毒株,视其有否特异性外膜蛋白(约200KDa)而定。自凝性也是检测耶氏菌毒力的一种方法,但其敏感性与特异性均不及VW抗原测定。因此测定菌株毒力,以VW抗原、Vi抗原和毒力因子测定最特异而敏感,特别后两种方法简单、易行、可靠,可在基层单位推广使用。     

Immunological studies of experimental tsutsugamushi disease in congenitally athymic (nude) mice

Athymic mice were taken ill and died from infection with the high virulence as well as the low virulence strains of Rickettsia tsutsugamushi, and they did not improve in spite of tetracycline therapy. Moreover, neither 7S nor IgM antibody was detected by immunofluorescent antibody method in serum samples of athymic mice infected with the high virulence strain. Although immune serum-transfer exhibited some protective effect in athymic mice infected with the high virulence strain, it was far lower than in euthymic mice. Although both athymic and euthymic mice having received non-immune T-lymphocytes were taken ill and died, the mice having received immune T-lymphocytes survived infection with the high virulence strain. This protective capacity of T-lymphocytes was weak by 10 days after immunization of donor mice, became firm after a month and lasted as long as 12 months without decay. For athymic mice infected with the low virulence strain, not only immune but also non-immune T-lymphocytes from euthymic mice exhibited significant protective effect. By treatment of immune T-lymphocytes with anti-Thy-1.2 or anti-Lyt-1.2 alloserum, the protective capacity was lost entirely, and considerably diminished by treatment with anti-Lyt-2.2 alloserum in a homologous system using the high virulence strain. The results show that the inhibition of progress of tsutsugamushi disease is principally dependent on cellular immune mechanism(s) and that the production of antibody against R. tsutsugamushi is thymus-dependent.     

Genetic characterization of Mycobacterium tuberculosis clinical isolates with deletions in the plcA-plcB-plcC locus

SETTING: The basis for Mycobacterium tuberculosis virulence is not completely understood. Analysis of the genomic structure of clinical isolates will give information that can be related to biological activities involved in virulence. OBJECTIVE: To determine the extension of the deletion in the plcA-plcB-plcC locus of selected M. tuberculosis isolates, as well as other changes in the chromosome. DESIGN: In the present work we characterized a group of M. tuberculosis isolates devoid of the plcA-plcB-plcC locus by PCR, sequencing and microarrays. RESULTS: PCR amplification of this region demonstrated a complete lack of plcA and plcB ORF's in all of the isolates. The plcC gene was completely deleted in one of the strains (DR-689) and the other three isolates still conserved part of this ORF. The loss of lateral DNA sequences ranged from 3723 to 7646bp. An IS6110 element was present in all tested strains cases, and some isolates presented the insertion of ORF's coding for proteins homologous to the ESAT-6 and QILSS families. Genomic DNA of all the strains was extracted and analyzed with an in-house microarray system to observe loss of other genes possibly implicated in attenuated virulence. Two of the strains presented novel deletions; the rest of the isolates showed deletions already reported for other M. tuberculosis strains. DR-689, a Beijing type M. tuberculosis strain isolated in Canada, showed an IS6110 RFLP and a genomic deletion pattern similar to a San Francisco family of strains, although completely unrelated epidemiologically. CONCLUSION: Genomic changes in M. tuberculosis seem to occur in a controlled manner and they are possibly related to changes in its pathogenic properties.     

Polymorphic family of injected pseudokinases is paramount in Toxoplasma virulence

Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa, has the unusual ability to infect virtually any warm-blooded animal. It is an extraordinarily successful parasite, infecting an estimated 30% of humans worldwide. The outcome of Toxoplasma infection is highly dependent on allelic differences in the large number of effectors that the parasite secretes into the host cell. Here, we show that the largest determinant of the virulence difference between two of the most common strains of Toxoplasma is the ROP5 locus. This is an unusual segment of the Toxoplasma genome consisting of a family of 4-10 tandem, highly divergent genes encoding pseudokinases that are injected directly into host cells. Given their hypothesized catalytic inactivity, it is striking that deletion of the ROP5 cluster in a highly virulent strain caused a complete loss of virulence, showing that ROP5 proteins are, in fact, indispensable for Toxoplasma to cause disease in mice. We find that copy number at this locus varies among the three major Toxoplasma lineages and that extensive polymorphism is clustered into hotspots within the ROP5 pseudokinase domain. We propose that the ROP5 locus represents an unusual evolutionary strategy for sampling of sequence space in which the gene encoding an important enzyme has been (i) catalytically inactivated, (ii) expanded in number, and (iii) subject to strong positive selection. Such a strategy likely contributes to Toxoplasma's successful adaptation to a wide host range and has resulted in dramatic differences in virulence.