Enhanced Th2 cell differentiation and function in the absence of Nox2.

BACKGROUND: Patients with chronic granulomatous disease (CGD), whom inherit abnormal function of NADPH oxidase 2 (Nox2), suffer from hyperinflammatory responses in lung as well as bacterial and fungal infection. There have been studies to reveal the function of Nox2 in hyperinflammatory diseases, especially in asthma, but the exact role of Nox2 in asthma is still unclear and controversial. Therefore, we attempted to clarify the exact role of Nox2 in asthma, using various experimental asthma models. METHODS: Asthma phenotypes were analyzed in response to various allergen-induced experimental asthma using Nox2-deficient mice and recombinase gene-activating-1-deficient mice. To understand the underlying mechanisms of exaggerated Th2 effector functions, we investigated the degree of T-cell activation, levels of activation-induced cell death (AICD), and regulatory T (Treg)-cell differentiation in Nox2-deficient T cells. RESULTS: Asthma phenotypes were increased through enhanced Th2 differentiation and function in Nox2-null mice regardless of dose and route of various allergens. Nox2-deficient T cells also showed hyperactivation, reduced AICD, and diminished Treg-cell differentiation through increased AKT phosphorylation (T308/S473) and enhanced mitochondrial ROS production. CONCLUSION: Our findings indicate that Nox2 deficiency results in exaggerated experimental asthma, which is caused by enhanced Th2 effector function in a T-cell-intrinsic manner.

Role of pancreatic ß-cell death and inflammation in diabetes.

Apoptosis of pancreatic ß-cells is the final step in the development of type 1 diabetes (T1D), leading to critically diminished ß-cell mass and contributing to the onset of hyperglycaemia. The spontaneous apoptosis of pancreatic ß-cells during pancreas ontogeny also induces cell death-associated inflammation, stimulates antigen-presenting cells and sensitizes naïve diabetogenic T cells. The role of pancreatic ß-cell death in type 2 diabetes (T2D) is less clear. In the preclinical period of T2D, hyperinsulinaemia and ß-cell hyperplasia develop to compensate for insulin resistance, which is clearly seen in animal models of T2D. For the development of overt T2D, relative insulin deficiency is critical in addition to insulin resistance. Insulin deficiency could be due to ß-cell dysfunction and/or decreased ß-cell mass. Pancreatic ß-cell apoptosis due to lipid injury (lipoapoptosis), endoplasmic reticulum (ER) stress or JNK activation could contribute to the decreased ß-cell mass in T2D. Activation of inflammasomes by lipid injury, ER stress, human islet amyloid polypeptide, hyperglycaemia or autophagy insufficiency could also lead to ß-cell death or dysfunction. Thus, ß-cell death and cell death-associated inflammation through innate immune receptors could be important in both T1D and T2D.

Induction of cell death in human macrophages by a highly virulent Korean Isolate of Mycobacterium tuberculosis and the virulent strain H37Rv.

Recent studies have suggested that virulent strains of Mycobacterium tuberculosis induce apoptosis in macrophages less often than do attenuated strains. K-strain, which belongs to the Beijing family, is the most frequently isolated clinical strain of M. tuberculosis in Korea. In this study, we investigated the differential induction of cell death in human monocytic THP-1 cells by K-strain and H37Rv, a virulent but laboratory-adapted strain of M. tuberculosis. Although no significant difference in growth rate was observed between the cells exposed to K-strain and those exposed to H37Rv, the levels of protective cytokines such as tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-12p40 were lower in K-strain-infected cells than in H37Rv-infected cells. Cell viability assays showed that both K-strain and H37Rv, but not heat- or streptomycin-killed bacteria, induced THP-1 cell death in a TNF-independent manner. In contrast, double staining with fluorochrome-labelled inhibitors of caspase and propidium iodide and lactate dehydrogenase release assays revealed that K-strain induced significantly higher levels of necrotic cell death, rather than apoptosis, in THP-1 cells than did H37Rv. Anti-apoptotic Bcl-2, Mcl-1, Bfl-1 and Bcl-xL in the cells were significantly upregulated following infection with K-strain compared with H37Rv, whereas Bax was slightly upregulated in response to infection with both H37Rv and K-strain. These results suggest that the highly virulent K-strain keeps cellular apoptosis as a host defense mechanism to a minimum and induces necrosis in macrophages.

Destruction of Bacillus licheniformis spores by microwave irradiation.

AIMS: To investigate the sporicidal mechanisms of microwave irradiation on Bacillus licheniformis spores. METHODS AND RESULTS: We measured spore viability and the release of DNA and proteins, and performed transmission electron microscopy (TEM). A microwave oven (0.5 kW) was modified to output power at 2.0 kW, which allowed a shorter sterilization cycle. A 2.0 kW microwave treatment at the boiling temperature for 1 min did not kill all spores, but killed most spores. The spore inactivation rate was faster than that of boiling and 0.5 kW microwave oven. In contrast to boiling and 0.5 kW microwave treatments, the 2.0 kW microwave resulted in significant leakage of proteins and DNA from spores due to injury to the spore structure. TEM revealed that 2.0 kW microwave irradiation affected spore cortex hydrolysis and swelling, and ruptured the spore coat and inner membrane. CONCLUSIONS: These results suggest that 2.0 kW microwave irradiation ruptures the spore coat and inner membrane, and is significantly different from boiling. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on the sporicidal mechanisms of microwave irradiation on B. licheniformis spores.

Expression and regulation of the CC-chemokine ligand 20 during human tuberculosis.

CC-chemokine ligand 20 (CCL20), a unique chemokine ligand of CC-chemokine receptor 6 (CCR6), play roles in various pathologic conditions. However, the characteristic expression profiles of CCL20 during human tuberculosis (TB) have been largely unknown. The present study analyzed the production and regulatory mechanisms of CCL20 in peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM) from active pulmonary TB patients and healthy controls (HC). The 30-kDa antigen (Ag) of Mycobacterium tuberculosis actively induced the production of CCL20 by human PBMC and MDM. A comparative analysis revealed that the expression of CCL20 protein was prominently up-regulated in PBMC, MDM, bronchoalveolar lavage fluids (not in sera) from TB patients compared with the corresponding cells or body fluids from HC. Blockade of either tumour necrosis factor-alpha or interferon-gamma, but not interleukin-10, significantly attenuated the CCL20 production. In addition, recombinant CCL20 induced CCR6 expression by CD45RO+ T lymphocytes in a dose-dependent manner. Furthermore, the expression of CCR6 was significantly increased in CD45RO+ T lymphocytes from TB patients, as compared with those from HC. Pharmacological inhibition studies showed that the 30-kDa Ag-induced CCL20 mRNA expression involves mitogen-activated protein kinases (MAPK; extracellular signal-regulated kinase 1/2 and p38)- and NF-kappaB-dependent signalling. Collectively, the present study demonstrated that TB patients show the up-regulated expression of CCL20, which is modulated by proinflammatory cytokines, and through MAPK/NF-kappaB-mediated transcriptional mechanisms. The findings suggest important implications of potential roles of CCL20-CCR6 in immunopathogenesis of TB.

Protection of mice against Mycobacterium tuberculosis infection by immunization with aqueous fraction of Triton X-100-soluble cell wall proteins.

The aqueous fraction of Triton X-100-soluble proteins (TSP-Aq) of Mycobacterium tuberculosis cell wall was reported to stimulate T-cell responses in peripheral blood monocytes from tuberculosis (TB) patients and to induce Th1 cytokines, suggesting presence of protective antigens. In this study, therefore, we examined the protective efficacy of TSP-Aq against M. tuberculosis infection in a mouse model. C57BL/6 mice were immunized with TSP-Aq or culture filtrate proteins (CFP) mixed with incomplete Freund's adjuvant or with BCG followed by i.v. challenge with M. tuberculosis H37Rv. TSP-Aq induced strong interferon-gamma production by spleen cells, and mice immunized with TSP-Aq antigens gave a significant reduction in M. tuberculosis CFU counts by 1.17-1.32 log10 CFU in the lungs and 1.31-2.08 log10 CFU in the spleen from 6 to 28 weeks. The degree of protection offered by TSP-Aq was comparable to that of CFP and of the BCG vaccine. The results demonstrated that the TSP-Aq antigens confer a significant level of protection against the growth of the organism in the lungs and spleen in a mouse model of TB and indicate that TSP contains major protective antigens of M. tuberculosis.

The effects of high-power microwaves on the ultrastructure of Bacillus subtilis.

AIMS: To investigate the microbicidal mechanisms of high-power microwave (2.0 kW) irradiation on Bacillus subtilis and to determine the effect of this procedure on the ultrastructure of the cell wall. METHODS AND RESULTS: We performed viability test, examined cells using transmission electron microscopy (TEM), and measured the release of intracellular proteins and nucleic acids. The inactivation rate of B. subtilis by 2.0-kW microwave irradiation was higher than that of a domestic microwave (0.5 kW). Few proteins were released from either microwaved or boiled cells. However, the leakage of nucleic acids from 2.0-kW-microwaved cells was significantly higher than that of 0.5-kW-microwaved or boiled cells. Therefore, we examined ultrastructural alterations of microwaved or boiled cells to analyse the pattern of release of cytoplasmic contents. Although boiled cells did not show any ultrastructural changes on TEM, 2.0-kW-microwaved cells showed disruption of the cell wall. CONCLUSION: The microbicidal mechanisms of 2.0-kW microwave irradiation include damage to the microbial cell wall, breakage of the genomic DNA, and thermal coagulation of cytoplasmic proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: TEM images showed that the cytoplasmic protein aggregation and cell envelope damage by microwave irradiation were different from the ultrastructural changes observed after boiling.

Differential regulation of interleukin-12 and tumour necrosis factor-alpha by phosphatidylinositol 3-kinase and ERK 1/2 pathways during Mycobacterium tuberculosis infection.

Interleukin (IL)-12 and tumour necrosis factor (TNF)-alpha are both thought to be critical factors in the defence against mycobacteria but are known to play different roles. In this study, we investigated the regulatory pathways for IL-12 and TNF-alpha expression in human monocyte-derived macrophages (MDMs) after treatment with Mycobacterium tuberculosis H37Rv or the Triton X-100 solubilized proteins (TSP) purified from M. tuberculosis. We found a rapid phosphorylation of Akt and extracellular signal-regulated kinase (ERK), albeit with differential activation kinetics, in human MDMs treated with M. tuberculosis or TSP. Studies using inhibitors selective for phosphatidylinositol 3-kinase (PI 3-K) and ERK 1/2 show that both pathway plays an essential role in the induction of TNF-alpha at both the transcriptional and translational levels in human MDMs. In contrast, blockade of the PI 3-K/Akt or ERK 1/2 pathways significantly increased M. tuberculosis- or TSP-induced IL-12 p40 and p35 mRNA and bioactive p70 protein. The enhancement of IL-12 levels by inhibition of PI 3-K and ERK 1/2 was not reversed by neutralization of TNF-alpha or addition of rhTNF-alpha, suggesting that the negative regulation of IL-12 is not mediated by concomitant TNF-alpha suppression. Further, PI 3-K activity is required for the M. tuberculosis- or TSP-induced phosphorylation of ERK 1/2 activation. TSP from M. tuberculosis shows a similar dependency on the PI 3-K and ERK 1/2 pathways to those by M. tuberculosis. Collectively, these data suggest that the Th1-driving cytokine IL-12 and proinflammatory cytokine TNF-alpha are differentially regulated by PI 3-K and ERK 1/2 pathways in human MDMs during mycobacterial infection. These results may provide therapeutic targets for precise and specific fine-tuning of cytokine responses.

Ex vivo responses for interferon-gamma and proinflammatory cytokine secretion to low-molecular-weight antigen MTB12 of Mycobacterium tuberculosis during human tuberculosis.

MTB12 protein, also called CFP-2, is a major and early secreted component of Mycobacterium tuberculosis. However, its role during mycobacterial infection has been poorly characterized. In this study, we purified the native MTB12 protein and investigated the profile of MTB12-induced cytokines [interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-6], in early tuberculosis (TB) patients (n = 20) and healthy controls (n = 35). The cytokine profiles were compared with those induced by the 30-kDa antigen (Ag). In healthy controls, MTB12-induced IFN-gamma production was markedly decreased in peripheral blood mononuclear cells compared with 30-kDa Ag-induced IFN-gamma. In TB patients, the mean IFN-gamma level induced by MTB12 was lower than that induced by the 30-kDa Ag, albeit the difference was not significant. After 2 months of anti-TB therapy, both the MTB12- and 30-kDa-induced IFN-gamma levels were significantly increased in TB patients. MTB12-induced TNF-alpha and IL-6 levels were prominently upregulated in monocyte-derived macrophages from TB patients, but they were not significantly different from those induced by the 30-kDa Ag. Further, the activation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase was required for the induction of TNF-alpha and IL-6 by MTB12, as well as by the 30-kDa Ag. Collectively, these data suggest that the MTB12 protein plays an essential role for proinflammatory responses through the MAPK pathway during the early stages of human TB, even though its T-cell immunoreactivity is weaker than that of the 30-kDa Ag.

Dysregulated production of interferon-gamma, interleukin-4 and interleukin-6 in early tuberculosis patients in response to antigen 85B of Mycobacterium tuberculosis.

Both interferon-gamma (IFN-gamma) and interleukin (IL)-4 expression in T cells and IL-6 expression in cells of the monocyte/macrophage lineage were monitored using antigen 85B (Ag85B) protein and purified protein derivative (PPD) antigen in the early stages of tuberculosis (TB). We showed that the levels of cell-associated IFN-gamma and IL-4 (mRNA and intracellular cytokine) in Ag85B-stimulated T cells were significantly depressed in TB patients compared with those in healthy tuberculin reactors. On the other hand, the capacity of peripheral blood mononuclear cells (PBMC) to produce IL-6 spontaneously ex vivo was enhanced in patients (P < 0. 001), but their corresponding capacities to respond to Ag85B were not significantly different from those of normal donors. After 2 months of antituberculosis therapy, the mean blastogenic responses of Ag85B-stimulated PBMC from seven TB patients were increased 6. 1-fold (P = 0.011). Furthermore, the proportions of both IFN-gamma- (P < 0.01) and IL-4- (P = 0.05) producing T cells were significantly increased. However, those of IL-6-producing cells were diminished in response to Ag85B (P = 0.05). Our results suggest that there may be an altered regulation of IFN-gamma, IL-4 and IL-6 to Ag85B in the early stages of TB.

Purification and immunoreactivity of three components from the 30/32-kilodalton antigen 85 complex in Mycobacterium tuberculosis.

The three proteins of the antigen 85 complex (85A, 85B, and 85C), which are major secretory products of Mycobacterium tuberculosis, were purified to homogeneity in large amounts by a combination of chromatography on hydroxylapatite, DEAE-Sepharose, and DEAE-Sephacel and gel filtration from M. tuberculosis culture filtrate. Then we examined the immunological reactivity of the three proteins in tuberculosis patients and healthy controls. Antibody responses to the 85B and 85A proteins in patients were significantly greater than responses to the 85C protein. In contrast, all three antigens induced significant lymphoproliferation and gamma interferon production in peripheral blood mononuclear cells from healthy tuberculin reactors.

Depressed interleukin-12 (IL-12), but not IL-18, production in response to a 30- or 32-kilodalton mycobacterial antigen in patients with active pulmonary tuberculosis.

The secreted 30-kDa antigen (Ag) of Mycobacterium tuberculosis directly stimulates Th1-type protective cytokine responses in healthy tuberculin reactors but not in patients with active tuberculosis (TB). To examine the cytokine profiles attributable to Th1 suppression associated with active TB, interleukin-12 (IL-12), IL-18, and IL-10 production in response to a 30- or 32-kDa Ag in 16 patients with active pulmonary TB and 24 healthy controls was investigated by enzyme-linked immunosorbent assay. In TB patients, production of IL-12 p40, as well as gamma interferon (IFN-gamma), by 30- or 32-kDa Ag-stimulated peripheral blood mononuclear cells (PBMC) was significantly decreased compared with that in healthy tuberculin reactors. There were no significant differences in IL-18 production between patients and controls early during stimulation (16 h). However, PBMC from patients showed significantly enhanced IL-18 proteins after 96 h of stimulation. Similarly, higher IL-10 production was observed in the TB patients than in healthy tuberculin reactors. After 2 months of anti-TB therapy, the mean IFN-gamma and IL-12 p40 production and the mean blastogenic responses were significantly increased in PBMC in the 10 TB patients who were followed up. Our findings provide evidence that depressed IL-12 in response to the 30- or 32-kDa Ag is involved in the immunopathogenesis of human active pulmonary TB.

The production of tumour necrosis factor-alpha is decreased in peripheral blood mononuclear cells from multidrug-resistant tuberculosis patients following stimulation with the 30-kDa antigen of Mycobacterium tuberculosis.

The clearance of intracellular bacteria requires the appropriate induction of proinflammatory cytokines and chemokines to recruit macrophages and T cells to the site of infection. In this study, we investigated the production of tumour necrosis factor (TNF)-alpha, interleukin (IL)-8 and interferon (IFN)-gamma by the peripheral blood mononuclear cells (PBMC) of patients with multidrug-resistant tuberculosis (MDR-TB) in response to in vitro stimulation with the 30-kDa antigen of Mycobacterium tuberculosis. The results were compared with those from cases of newly diagnosed TB (N-TB) and TB with treatment failure (TF-TB), and healthy tuberculin reactors (HTR). The most significantly depressed TNF-alpha levels were found in MDR-TB patients. IFN-gamma production was depressed significantly in all groups of TB patients compared with the HTR group. TNF-alpha secretion in response to the 30-kDa antigen was unchanged by coculturing with recombinant human interferon (rhIFN)-gamma, and was increased dramatically following IL-10 neutralization with an anti-human IL-10 antibody. The IL-8 levels were depressed significantly in MDR-TB patients compared with N-TB patients, but were similar to the IL-8 levels in TF-TB patients. Furthermore, rhTNF-alpha directly increased IL-8 secretion, and neutralizing antibody to TNF-alpha inhibited IL-8 production by the PBMC of MDR-TB patients that were stimulated with the 30-kDa antigen. Taken together, these data suggest that the PBMC of MDR-TB patients typically show TNF-alpha depression in response to the 30-kDa antigen, and this effect is modulated by IL-10. In addition, we highlight the role of TNF-alpha in IL-8 secretion in MDR-TB patients.

Characterization of mutations, including a novel regulatory defect in the first intron, in Bruton's tyrosine kinase gene from seven Korean X-linked agammaglobulinemia families.

In this report, we describe seven mutations, including a novel single base pair substitution in intron 1, of the Bruton's tyrosine kinase (Btk) gene found in 12 Korean patients with X-linked agammaglobulinemia. Various mutations, including three novel genetic alterations, were discovered using single-strand conformation polymorphism analysis and direct DNA sequencing. The effect of the intron 1 point mutation (intron 1 +5G-->A) was further evaluated using reporter constructs. Using luciferase assay experiments, we showed that the transcriptional activity of the mutant was significantly lower than in normal counterparts, indicating that the intronic mutation was functional. In addition, DNase I footprinting analysis showed that a single protected region spanning the position +3 to +15 bp hybridized with a mutant-specific probe, but not with a wild-type probe. EMSA indicated that a distinct nuclear protein has the ability to bind the mutant oligonucleotides to produce a new DNA-protein complex. We also observed decreased expression of Btk proteins in monocytes of patients having the point mutation in intron 1. Taken together with the functional analysis, our results strongly suggest the existence of a novel cis-acting element, which might be involved in the down-regulation of Btk gene transcription. Precise definition of the regulatory defect in the Btk intron 1 may provide valuable clues toward elucidating the pathogenesis of X-linked agammaglobulinemia.

Monocyte chemotactic protein-1 production in patients with active pulmonary tuberculosis and tuberculous pleurisy.

OBJECTIVE: The role of monocyte chemotactic protein (MCP)-1 in human pulmonary and pleural tuberculosis (TB) was assessed by examining its production in clinical samples from patients with active pulmonary TB and tuberculous pleurisy (TBP). METHODS: Serum was obtained from 26 active pulmonary TB patients [14 early TB (E-TB), and 12 chronic refractory TB (CR-TB)] and 15 healthy tuberculin reactors (HTRs). The monocytes and peripheral blood mononuclear cells (PBMCs) were separated and stimulated with purified protein derivatives (PPD) or the 30-kDa antigen of Mycobacterium tuberculosis. Pleural exudates were isolated from 25 patients with TBP and 24 non-TBP patients [malignancy and congestive heart failure (CHF)]. The MCP-1 levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: In sera, the MCP-1 levels of TB patients were similar to those of HTRs. For monocytes, CR-TB patients spontaneously expressed more MCP-1, compared with HTRs and E-TB patients. In addition, MCP-1 production of PPD- or 30-kDa antigen-stimulated monocytes was significantly elevated in CR-TB patients than that from E-TB. Interestingly, the E-TB patients had significantly depressed MCP-1 production by PBMCs in response to PPD or 30-kDa, compared with HTRs and CR-TB patients. In pleural effusions, MCP-1 levels were significantly higher in patients with TBP than in patients with CHF, but lower than in malignant effusions. CONCLUSIONS: The data suggest that MCP-1 production is not uniquely elevated systemically in TB patients, although MCP-1 production might be elevated by monocytes in the chronic phase of TB or with a local pleural infection.

Profiles of IFN-gamma and its regulatory cytokines (IL-12, IL-18 and IL-10) in peripheral blood mononuclear cells from patients with multidrug-resistant tuberculosis.

This study investigated the profiles of IFN-gamma and its regulatory cytokines (IL-12, IL-18 and IL-10) in response to a purified protein derivative (PPD) antigen in peripheral blood mononuclear cells (PBMC) from 18 HIV-negative patients with multidrug-resistant tuberculosis (MDRTB), and compared them with those from 19 healthy tuberculin reactors (HTR). ELISA results showed that following stimulation with PPD, IFN-gamma production was significantly reduced, whereas production of both IL-18 and IL-10 was significantly elevated in MDRTB patients compared with HTR. Three out of 18 patients with MDRTB of greater than 4 years duration showed significantly elevated IL-12 p70 production, induced by in vitro PPD stimulation of their PBMC, when compared with data from HTR. However, when taken as a group, MDRTB patients were similar to HTR in their IL-12 p70-producing capacity. IL-12 p70 protein paralleled IL-12 p40 protein expression. In addition, the production of IL-12 p40 was significantly correlated with IL-10 in all patients, but was not correlated with IFN-gamma. Neutralization of IL-10 increased IL-12 p40 about twofold, but did not significantly alter IFN-gamma induction in MDRTB. IFN-gamma in MDRTB was highly correlated with lymphoproliferation and CD4 counts, but was not correlated with IL-12, IL-18 or IL-10 production. Our findings suggest that patients with MDRTB have dysregulated IL-12, IL-18 and IL-10 production during Mycobacterium tuberculosis infection, and the cytokine profiles are similar to those in patients with drug-sensitive advanced TB previously reported in the literature. In addition, IL-10 may not have a dominant role in defective IFN-gamma production in patients with MDRTB.

IL-18 production in human pulmonary and pleural tuberculosis.

Interleukin-18 (IL-18) has multiple important pro-inflammatory effects, including the induction of interferon-gamma (IFN-gamma) in various diseases. In this study, we investigated the IL-18-producing activities in human pulmonary and pleural tuberculosis (TB) in response to purified protein derivative (PPD) antigen (Ag) from Mycobacterium tuberculosis. The most significant IL-18 production was found in chronic refractory TB (CRTB) patients. However, IFN-gamma production in CRTB patients was significantly less than that in healthy tuberculin reactors or in patients with tuberculous pleurisy (TBP). Elevated levels of both IL-18 and IFN-gamma were found in pleural fluids from TBP patients. In vitro production of IL-18 was dramatically decreased following an 18 h stimulation with PPD. However, IFN-gamma was markedly increased in pleural mononuclear cells from TBP patients after in vitro stimulation with PPD. The mesothelial cell type was the main source of pro-IL-18 in pleural cells from TBP patients, suggesting an important role for these cells in TBP. Taken together, these data indicate that IL-18 is elevated in peripheral blood mononuclear cells from CRTB patients, as well as at the site of TBP, indicating a possible role for IL-18 in both protective immunity and pathologic responses in human TB.