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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-g 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.Keywords: leptin; adiponectin; pectus excavatum; scoliosis; Marfan syndrome Chronic lung disease due to nontuberculous mycobacteria (NTM) is a growing public health concern (1-3). Recent studies estimate the incidence in the United States to be five to six cases per 100,000 and as high as 15.5 cases per 100,000 in persons over 50 years of age (3-5). Because the duration of symptomatic NTM lung disease is often years, the prevalence of disease is estimated to be 10 to 40 cases per 100,000 (1).In the United States, the most common NTM species associated with lung disease are Mycobacterium avium complex (MAC), Mycobacterium kansasii, and Mycobacterium abscessus. Although NTM are widespread in water and soil (6, 7), relatively few persons develop disease. Thus, intact immunity is likely pivotal for protection against NTM.Chronic lung disease is the most common form of NTM infection, manifested by two main radiographic patterns: (i) an upper lobe fibrocavitary pattern that occurs mostly in men with underlying lung disease such as chronic obstructive pulmonary disease (COPD) and (ii) a nodular-bronchiectasis pattern that often involves the right middle lobe and lingula and which appears to be more common in women with no clear risk factors (8)...

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-gamma+ 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-gamma 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-gamma 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.

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.

CS suppresses the protective immune response to M. tuberculosis in mice, human THP-1 cells, and primary human alveolar macrophages.

Nuclear factor-kappa B (NFκB) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to Mycobacterium tuberculosis (MTB). However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role of NFκB activation in the immune response of human macrophages to MTB. Targeted pharmacologic inhibition of NFκB activation using BAY 11-7082 (BAY, an inhibitor of IκBα kinase) or an adenovirus construct with a dominant-negative IκBα significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFκB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular MTB. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFκB inhibition decreases viability of intracellular MTB in human macrophages via induction of apoptosis and autophagy.

Rapidly growing mycobacteria (RGM) are ubiquitous in the environment but cause lung disease in only a fraction of exposed individuals. This variable susceptibility to disease implies vulnerability to RGM infection due to weakness in host defense. Since most persons who contract RGM lung disease have no known host defense defect, it is likely that uncharacterized host deficiencies exist that predispose to RGM infection. Alpha-1-antitrypsin (AAT) is a host factor that may protect individuals from respiratory infections. Therefore, we assessed AAT protein anomalies as a risk factor for RGM lung disease. In a cohort of 100 patients with RGM lung disease, Mycobacterium (M.) abscessus was the most prevalent organism, isolated in 64 (64%) subjects. Anomalous AAT proteins were present in 27% of the cohort, which is 1.6 times the estimated prevalence of anomalous AAT proteins in the United States population (p=0.008). In in vitro studies, both AAT and a synthetic inhibitor of serine proteases suppressed M. abscessus infection of monocyte-derived macrophages by up to 65% (p<0.01). AAT may be an anti-RGM host-defense factor, and anomalous AAT phenotypes or AAT deficiency may constitute risk factors for pulmonary disease due to RGM.

The incidence of lung and other diseases due to nontuberculous mycobacteria (NTM) is increasing. NTM sources include potable water, especially in households where NTM populate pipes, taps, and showerheads. NTM share habitats with free-living amoebae (FLA) and can grow in FLA as parasites or as endosymbionts. FLA containing NTM may form cysts that protect mycobacteria from disinfectants and antibiotics. We first assessed the presence of FLA and NTM in water and biofilm samples collected from a hospital, confirming the high prevalence of NTM and FLA in potable water systems, particularly in biofilms. Acanthamoeba spp. (genotype T4) were mainly recovered (8/17), followed by Hartmannella vermiformis (7/17) as well as one isolate closely related to the genus Flamella and one isolate only distantly related to previously described species. Concerning mycobacteria, Mycobacterium gordonae was the most frequently found isolate (9/17), followed by Mycobacterium peregrinum (4/17), Mycobacterium chelonae (2/17), Mycobacterium mucogenicum (1/17), and Mycobacterium avium (1/17). The propensity of Mycobacterium avium hospital isolate H87 and M. avium collection strain 104 to survive and replicate within various FLA was also evaluated, demonstrating survival of both strains in all amoebal species tested but high replication rates only in Acanthamoeba lenticulata. As A. lenticulata was frequently recovered from environmental samples, including drinking water samples, these results could have important consequences for the ecology of M. avium in drinking water networks and the epidemiology of disease due to this species.