Nontuberculous mycobacteria (NTM) infection is common in patients with structural lung damage. To address how NTM infection is established and causes lung damage, we established an NTM mouse model by intranasal inoculation of clinical isolates of M. intracellulare. During the 39-week course of infection, the bacteria persistently grew in the lung and caused progressive granulomatous and fibrotic lung damage with mortality exceeding 50%. Lung neutrophils were significantly increased at 1 week postinfection, reduced at 2 weeks postinfection and increased again at 39 weeks postinfection. IL-17A was increased in the lungs at 1–2 weeks of infection and reduced at 3 weeks postinfection. Depletion of neutrophils during early (0–2 weeks) and late (32–34 weeks) infection had no effect on mortality or lung damage in chronically infected mice. However, neutralization of IL-17A during early infection significantly reduced bacterial burden, fibrotic lung damage, and mortality in chronically infected mice. Since it is known that IL-17A regulates matrix metalloproteinases (MMPs) and that MMPs contribute to the pathogenesis of pulmonary fibrosis, we determined the levels of MMPs in the lungs of M. intracellulare-infected mice. Interestingly, MMP-3 was significantly reduced by anti-IL-17A neutralizing antibody. Moreover, in vitro data showed that exogenous IL-17A exaggerated the production of MMP-3 by lung epithelial cells upon M. intracellulare infection. Collectively, our findings suggest that early IL-17A production precedes and promotes organized pulmonary M. intracellulare infection in mice, at least in part through MMP-3 production.

Neutrophil infiltration of the lungs is associated with granuloma formation and the severity of tuberculosis infection. Although several cytokines and chemokines are known to contribute to lung neutrophil infiltration, the neutrophilic chemotactic factors of Mycobacterium tuberculosis (Mtb) remain unexplored. Therefore, we performed Transwell based chemotactic assays using neutrophils from human peripheral blood and mouse bone marrow to probe the chemotactic activity of the culture filtrates (CF) of Mtb H37Rv. CF of H37Rv induced chemotaxis of both human and mouse neutrophils, and this was also confirmed with CF of 9 clinical isolates and Erdman strain of Mtb with neutrophil chemotactic activity. Sulfasalazine, an N-formyl-Met-Leu-Phe (fMLF) receptor inhibitor, blocked the chemotaxis of neutrophils induced by CF of Mtb, thus indicating the involvement of the fMLF receptor in Mtb CF induced chemotaxis of neutrophils. Mass spectrometry analysis of CF of H37Rv identified three candidate N-formylated heptapeptides. The chemotactic activity of the identified peptides was confirmed with their synthetic mimetics that they induced neutrophil chemotaxis in a manner dependent on N-terminal formylation. For all formylated peptides and CF of Mtb, the induced Ca2+ influx in neutrophils was suppressed by sulfasalazine. Thus, we identified novel formylated Mtb peptides with neutrophil chemotactic activity.

Upon MAC stimulation, the production of IL-1 family cytokines and IL-10 by PBMCs of NTMPD patients was attenuated compared with that of healthy household contacts. Upon MAC stimulation, the expression of TLR2 and TWIK2 (one of the two-pore domain K+ channels) was attenuated in PBMCs of NTMPD patients compared with that of healthy household contacts.

Nontuberculous mycobacteria (NTM) cause pulmonary disease in individuals without obvious immunodeficiency. This study was initiated to gain insight into the immunological factors that predispose persons to NTM pulmonary disease (NTMPD). Blood was obtained from 15 pairs of NTMPD patients and their healthy household contacts. Peripheral blood mononuclear cells (PBMCs) were stimulated with the M. avium complex (MAC). A total of 34 cytokines and chemokines were evaluated in plasma and PBMC culture supernatants using multiplex immunoassays, and gene expression in the PBMCs was determined using real-time PCR. PBMCs from NTMPD patients produced significantly less interleukin (IL)-1β, IL-18, IL-1α and IL-10 than PBMCs from their healthy household contacts in response to MAC. Although plasma RANTES levels were high in NTMPD patients, they had no effect on IL-1β production by macrophages infected with MAC. TLR2 and TWIK2 were impaired in response to MAC in PBMCs of NTMPD patients. A TLR2 inhibitor decreased all 4 cytokines, whereas a TWIK2 inhibitor decreased the production of IL-1β, IL-18, and IL-1α, but not IL-10, by MAC-stimulated PBMCs and monocytes. We suggest that an attenuated TWIK2-mediated IL-1 response may increase susceptibility to NTM pulmonary infection.

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