Edward D. Chan

Mycobacterium tuberculosis Induces Interleukin-32 Production through a Caspase- 1/IL-18/Interferon-γ-Dependent Mechanism

Background Interleukin (IL)–32 is a newly described proinflammatory cytokine that seems likely to play a role in inflammation and host defense. Little is known about the regulation of IL-32 production by primary cells of the immune system. Methods and Findings In the present study, freshly obtained human peripheral blood mononuclear cells were stimulated with different Toll-like receptor (TLR) agonists, and gene expression and synthesis of IL-32 was determined. We demonstrate that the TLR4 agonist lipopolysaccharide induces moderate (4-fold) production of IL-32, whereas agonists of TLR2, TLR3, TLR5, or TLR9, each of which strongly induced tumor necrosis factor α and IL-6, did not stimulate IL-32 production. However, the greatest amount of IL-32 was induced by the mycobacteria Mycobacterium tuberculosis and M. bovis BCG (20-fold over unstimulated cells). IL-32-induced synthesis by either lipopolysaccharide or mycobacteria remains entirely cell-associated in monocytes; moreover, steady-state mRNA levels are present in unstimulated monocytes without translation into IL-32 protein, similar to other cytokines lacking a signal peptide. IL-32 production induced by M. tuberculosis is dependent on endogenous interferon-γ (IFNγ); endogenous IFNγ is, in turn, dependent on M. tuberculosis–induced IL-18 via caspase-1. Conclusions In conclusion, IL-32 is a cell-associated proinflammatory cytokine, which is specifically stimulated by mycobacteria through a caspase-1- and IL-18-dependent production of IFNγ.

Induction of inducible nitric oxide synthase-NO* by lipoarabinomannan of Mycobacterium tuberculosis is mediated by MEK1-ERK, MKK7-JNK, and NF-kappaB signaling pathways.

ABSTRACT Nitric oxide (NO· ) expression by inducible nitric oxide synthase (iNOS) is an important host defense mechanism againstMycobacterium tuberculosis in mononuclear phagocytes. The objective of this investigation was to examine the role of mitogen-activated protein (MAP) kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways in the regulation of iNOS and NO· by a mycobacterial cell wall lipoglycan known as mannose-capped lipoarabinomannan (ManLAM). Specific pharmacologic inhibition of the extracellular-signal-regulated kinase (ERK) or NF-κB pathway revealed that both these signaling cascades were required in gamma interferon (IFN-γ)-ManLAM-induced iNOS protein and NO2− expression in mouse macrophages. Transient cotransfection of dominant-negative protein mutants of the c-Jun NH2-terminal kinase (JNK) pathway revealed that the MAP kinase kinase 7 (MKK7)-JNK cascade also mediated IFN-γ–ManLAM induction of iNOS promoter activity whereas MKK4 did not. Overexpression of null mutant IκBα, a potent inhibitor of NF-κB activation, confirmed that the IκBα kinase (IKK)–NF-κB signaling pathway enhanced IFN-γ–ManLAM-induced iNOS promoter activity. By contrast, activated p38mapk inhibited iNOS induction. These results indicate that combined IFN-γ and ManLAM stimulation induced iNOS and NO· expression and that MEK1-ERK, MKK7-JNK, IKK–NF-κB, and p38mapksignaling pathways play important regulatory roles.

Drug resistance beyond extensively drug-resistant tuberculosis: individual patient data meta-analysis

The broadest pattern of tuberculosis drug resistance for which a consensus definition exists is extensively drug-resistant tuberculosis (XDR-TB). It is not known if additional drug resistance portends worsened patient outcomes. This study compares treatment outcomes of XDR-TB patients with and without additional resistance to explore the need for a new definition. Individual patient data on XDR-TB outcomes were included in a meta-analysis comparing outcomes between XDR-alone and three non-mutually exclusive XDR-TB patient groups: XDR plus resistance to all the second-line injectables (sli) capreomycin and kanamycin/amikacin (XDR+2sli); XDR plus resistance to second-line injectables and to ≥1 Group 4 drug, i.e. : ethionamide/prothionamide, cycloserine/terizidone or PAS (XDR+sliG4); and XDR+sliG4 plus resistance to ethambutol and/or pyrazinamide (XDR+sliG4EZ). Of 405 XDR-TB cases, 301 were XDR-alone; 68 XDR+2sli; 48 XDR+sliG4; and 42 XDR+sliG4EZ. In multivariate analysis, the odds of cure were significantly lower in XDR+2sli (adjusted Odds Ratio (aOR): 0.4; 95% Confidence Interval: 0.2–0.8) compared to XDR-alone, while odds of failure+death were higher in all XDR patients with additional resistance (aOR range: 2.6–2.8). Patients with additional resistance beyond XDR-TB showed poorer outcomes. Limitations in availability, accuracy and reproducibility of current DST methods preclude the adoption of a useful definition beyond the one currently used for XDR-TB.The broadest pattern of tuberculosis (TB) drug resistance for which a consensus definition exists is extensively drug-resistant (XDR)-TB. It is not known if additional drug resistance portends worsened patient outcomes. This study compares treatment outcomes of XDR-TB patients with and without additional resistance in order to explore the need for a new definition. Individual patient data on XDR-TB outcomes were included in a meta-analysis comparing outcomes between XDR alone and three nonmutually exclusive XDR-TB patient groups: XDR plus resistance to all the second-line injectables (sli) and capreomycin and kanamycin/amikacin (XDR+2sli) XDR plus resistance to second-line injectables and to more than one group 4 drug, i.e. ethionamide/protionamide, cycloserine/terizidone or para-aminosalicylic acid (XDR+sliG4) and XDR+sliG4 plus resistance to ethambutol and/or pyrazinamide (XDR+sliG4EZ). Of 405 XDR-TB cases, 301 were XDR alone, 68 XDR+2sli, 48 XDR+sliG4 and 42 XDR+sliG4EZ. In multivariate analysis, the odds of cure were significantly lower in XDR+2sli (adjusted OR 0.4, 95% CI 0.2–0.8) compared to XDR alone, while odds of failure and death were higher in all XDR patients with additional resistance (adjusted OR 2.6–2.8). Patients with additional resistance beyond XDR-TB showed poorer outcomes. Limitations in availability, accuracy and reproducibility of current drug susceptibility testing methods preclude the adoption of a useful definition beyond the one currently used for XDR-TB.

Current medical treatment for tuberculosis

Instigating effective treatment regimens in a way that improves patient adherence is vital to tackling the global resurgence of tuberculosis About one third of the worlds population has latent tuberculosis, caused by Mycobacterium tuberculosis infection.1 From this pool, roughly 9 million cases of active tuberculosis emerge annually, resulting in 2-3 million deaths. Most new cases occur in the most populated nations—India and China—but the highest rates of disease are seen in sub-Saharan Africa, the Indonesian and Philippine archipelagos, Afghanistan, Bolivia, and Peru. In these regions case rates typically exceed 300 cases per 100 000 per year. 1 2 Although the incidence of tuberculosis declined in North America and western Europe throughout most of the latter half of the 20th century, case rates have increased over the past 10 years mainly because of immigration, HIV/AIDS, and the neglect of tuberculosis control programmes. 3 4 One vital factor in curbing the increase of tuberculosis is the instigation of proper treatment that not only encompasses an effective regimen but also ensures compliance with and response to treatment. This review highlights current treatment recommendations for tuberculosis. #### Summary points Many people worldwide have latent or active tuberculosis, and the number of active cases is expected to increase in the future The most common cause of treatment failure and acquired drug resistance is non-adherence; predicting non-adherence is highly problematic Directly observed therapy is the most effective means of combatting non-adherence; intermittent (less than daily) regimens facilitate the therapy Testing the susceptibility of Mycobacterium tuberculosis to drugs is essential for identifying resistance and tailoring treatment Managing multidrug resistant tuberculosis is complex and should, when possible, be done in specialised programmes We performed a Medline search of the past 10 years using the key words “tuberculosis and treatment or drug therapy” to find pertinent literature. We also searched bibliographies …

Multidrug-resistant and extensively drug-resistant tuberculosis: a review.

Purpose of review The spread of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant TB (XDR-TB) is a major medical and public health concern for the world. These two forms of highly drug-resistant TB threaten to make TB into an untreatable and highly fatal disease, particularly in resource-poor countries with a high prevalence of AIDS. The focus of this review is to highlight the current extent of the problem. Recent findings There is a great variability in clinical outcomes for MDR-TB, in part due to differences in the definitions of outcome measures and retrospective nature of the studies. Outcomes for XDR-TB are uniformly worse than those for MDR-TB. Summary A multifaceted approach is needed to prevent a more widespread epidemic of MDR-TB and XDR-TB. Rapid diagnostic assays to detect highly drug-resistant TB are essential in preventing delays in treatment of MDR-TB and XDR-TB and curbing their spread. Development of new drugs to effectively treat all forms of TB in a shorter period of time is urgently needed.

α-1-Antitrypsin is an endogenous inhibitor of proinflammatory cytokine production in whole blood

Several observations suggest endogenous suppressors of inflammatory mediators are present in human blood. α‐1‐Antitrypsin (AAT) is the most abundant serine protease inhibitor in blood, and AAT possesses anti‐inflammatory activity in vitro and in vivo. Here, we show that in vitro stimulation of whole blood from persons with a genetic AAT deficiency resulted in enhanced cytokine production compared with blood from healthy subjects. Using whole blood from healthy subjects, dilution of blood with RPMI tissue‐culture medium, followed by incubation for 18 h, increased spontaneous production of IL‐8, TNF‐α, IL‐1β, and IL‐1R antagonist (IL‐1Ra) significantly, compared with undiluted blood. Dilution‐induced cytokine production suggested the presence of one or more circulating inhibitors of cytokine synthesis present in blood. Serially diluting blood with tissue‐culture medium in the presence of cytokine stimulation with heat‐killed Staphylococcus epidermidis (S. epi) resulted in 1.2‐ to 55‐fold increases in cytokine production compared with S. epi stimulation alone. Diluting blood with autologous plasma did not increase the production of IL‐8, TNF‐α, IL‐1β, or IL‐1Ra, suggesting that the endogenous, inhibitory activity of blood resided in plasma. In whole blood, diluted and stimulated with S. epi, exogenous AAT inhibited IL‐8, IL‐6, TNF‐α, and IL‐1β significantly but did not suppress induction of the anti‐inflammatory cytokines IL‐1Ra and IL‐10. These ex vivo and in vitro observations suggest that endogenous AAT in blood contributes to the suppression of proinflammatory cytokine synthesis.

Patients with Nontuberculous Mycobacterial Lung Disease Exhibit Unique Body and Immune Phenotypes

RATIONALE Among patients with nontuberculous mycobacterial lung disease is a subset of previously healthy women with a slender body morphotype, often with scoliosis and/or pectus excavatum. We hypothesize that unidentified factors predispose these individuals to pulmonary nontuberculous mycobacterial disease. OBJECTIVES To compare body morphotype, serum adipokine levels, and whole-blood cytokine responses of patients with pulmonary nontuberculous mycobacteria (pNTM) with contemporary control subjects who are well matched demographically. METHODS We enrolled 103 patients with pNTM and 101 uninfected control subjects of similar demographics. Body mass index and body fat were quantified. All patients with pNTM and a subset of control subjects were evaluated for scoliosis and pectus excavatum. Serum leptin and adiponectin were measured. Specific cytokines important to host-defense against mycobacteria were measured in whole blood before and after stimulation. MEASUREMENTS AND MAIN RESULTS Patients with pNTM and control subjects were well matched for age, gender, and race. Patients with pNTM had significantly lower body mass index and body fat and were significantly taller than control subjects. Scoliosis and pectus excavatum were significantly more prevalent in patients with pNTM. The normal relationships between the adipokines and body fat were lost in the patients with pNTM, a novel finding. IFN-γ and IL-10 levels were significantly suppressed in stimulated whole blood of patients with pNTM. CONCLUSIONS This is the first study to comprehensively compare body morphotype, adipokines, and cytokine responses between patients with NTM lung disease and demographically matched controls. Our findings suggest a novel, predisposing immunophenotype that should be mechanistically defined.

Activation of the Mitogen-Activated Protein Kinase Signaling Pathway Is Instrumental in Determining the Ability of Mycobacterium avium to Grow in Murine Macrophages

Of the two common morphotypes of Mycobacterium avium, designated smooth transparent (SmT) or smooth opaque (SmO), the SmO morphotype is avirulent, whereas the SmT morphotype is virulent. The role of the host macrophage in determining these different virulence phenotypes was analyzed using an in vitro model of macrophage infection. Initial studies confirmed previous reports of the increased ability of the SmT bacteria to grow in macrophages; this increased virulence correlated with reduced induction of inflammatory cytokines. Examination of the response of the mitogen-activated protein kinase (MAPK) pathway following infection with either morphotype revealed that all three members of the MAPK pathway were activated. Pharmacologic inhibition of either the extracellular signal-regulated kinase (ERK) or p38MAPK pathways resulted in distinct consequences for the growth of the two morphotypes. In particular, inhibition of the p38MAPK resulted in attenuated growth of the SmT morphotype, which correlated with reduced PGE2 production. Inhibition of cyclooxygenase 2 by indomethacin also inhibited growth of SmT, substantiating the role for PGE2 in promoting the growth of SmT. In contrast, SmO induction of the ERK pathway was increased compared with the SmT morphotype, and inhibition of ERK resulted in decreased TNF-α synthesis and enhanced SmO growth. Pharmacologic inhibitors of the MAPK pathway were present for only the first 4 h of infection and yet had consequences for bacterial growth at 7 days. Therefore, the data suggest that induction of the MAPK pathway during uptake of bacteria is instrumental in determining the eventual fate of the bacteria.

Animal model of Mycobacterium abscessus lung infection

Chronic lung disease as a result of Mycobacterium abscessus is an emerging infection in the United States. We characterized the lung immune responses in mice and guinea pigs infected with M. abscessus. C57BL/6 and leptin‐deficient ob/ob mice challenged with a low‐dose aerosol (LDA) of M. abscessus did not develop an infection. However, when challenged with a high‐dose aerosol (HDA), C57BL/6 and ob/ob mice developed an established infection and a pulmonary immune response consisting of an early influx of IFN‐γ+ CD4+ T cells; this immune response preceded the successful clearance of M. abscessus in both strains of mice, although mycobacterial elimination was delayed in the ob/ob mice. Infected guinea pigs showed an increased influx of lymphocytes into the lungs with bacterial clearance by Day 60. In contrast to the C57BL/6 and ob/ob mice and guinea pigs, IFN‐γ knockout (GKO) mice challenged with a LDA or HDA of M. abscessus showed a progressive lung infection despite a robust influx of T cells, macrophages, and dendritic cells, culminating in extensive lung consolidation. Furthermore, with HDA challenge of the GKO mice, emergence of IL‐4‐ and IL‐10‐producing CD4+ and CD8+ T cells was seen in the lungs. In conclusion, IFN‐γ is critically important in the host defense against M. abscessus. As the number of effective drugs against M. abscessus is limited, the GKO mice provide a model for in vivo testing of novel drugs.

IL-32 Is a Host Protective Cytokine against Mycobacterium tuberculosis in Differentiated THP-1 Human Macrophages

Macrophages provide a first line of defense against Mycobacterium tuberculosis. However, in instances where macrophage activation for killing is suboptimal, M. tuberculosis is capable of surviving intracellularly. IL-32 is a recently described cytokine induced by M. tuberculosis in a variety of cell types including human monocytes and macrophages. In this study, we investigated the biological significance of IL-32 in an in vitro model of M. tuberculosis infection in differentiated THP-1 human macrophages in which IL-32 expression was silenced using stable expression of short hairpin RNA (shRNA). Inhibition of endogenous IL-32 production in THP-1 cells that express one of three distinct shRNA-IL-32 constructs significantly decreased M. tuberculosis induction of TNF-α by ∼60%, IL-1β by 30–60%, and IL-8 by 40–50% and concomitantly increased the number of cell-associated M. tuberculosis bacteria compared with THP-1 cells stably expressing a scrambled shRNA. In THP-1 cells infected with M. tuberculosis and stimulated with rIL-32, a greater level of apoptosis was observed compared with that with M. tuberculosis infection alone. Obversely, there was significant abrogation of apoptosis induced by M. tuberculosis and a concomitant decrease in caspase-3 activation in cells depleted of endogenous IL-32. rIL-32γ significantly reduced the number of viable intracellular M. tuberculosis bacteria, which was modestly but significantly abrogated with a caspase-3 inhibitor. We conclude that IL-32 plays a host defense role against M. tuberculosis in differentiated THP-1 human macrophages.