Abstract Rationale People with cystic fibrosis (pwCF) are at high risk for developing nontuberculous mycobacteria pulmonary disease (NTM-PD), which is difficult to treat and increases morbidity and mortality. Yet only a fraction of pwCF develop NTM-PD (10-15%). To better understand heterogeneity in NTM-PD susceptibility, the role of granulocyte-macrophage colony stimulating factor (GM-CSF) in host resistance to NTM-PD caused by Mycobacterium abscessus (Mabsc) was investigated. GM-CSF is essential for murine defense against Mabsc acute pneumonia. Therapeutic nebulized GM-CSF is being tested for recalcitrant NTM-PD in pwCF and non-CF bronchiectasis, with some successes. GM-CSF is constitutively produced by alveolar pneumocytes; however, epithelia in the airways (where bronchiectasis occurs) rarely transcribe CSF2 (GM-CSF), but Pseudomonas aeruginosa (Pa) can induce GM-CSF production by epithelial cell lines in vitro. Whether Pa can stimulate GM-CSF production by primary airway epithelia in vitro or in vivo has not been investigated. Moreover, which immune cells respond to GM-CSF to protect against Mabsc remain unknown. We hypothesized that specific Pa isolates can stimulate airway epithelial GM-CSF production, and that focal airway GM-CSF programs macrophages for effective killing of Mabsc. Methods Human monocyte-derived macrophages (MDMs) from healthy donors were matured with or without GM-CSF, infected with Mabsc, and colony forming units were determined after 96 hours. GM-CSF reporter mice were inoculated oropharyngeally with 100-200 micron diameter agar beads (which reliably lodge in small airways) containing heat killed Pa, live Mabsc, or no infection. Primary human bronchial epithelia differentiated to model small airways as air-liquid interface (ALI) cultures were exposed to Pa or Mabsc; RNA abundance and secreted cytokines were quantitated. Results GM-CSF matured MDMs showed enhanced ability to kill Mabsc compared to those matured in M-CSF, which demonstrated minimal Mabsc killing (p 0.001). Immunofluorescence microscopy of GM-CSF reporter mice lungs revealed that heat killed Pa beads but not sterile beads nor Mabsc beads induced GM-CSF production by airways epithelial cells. ALI cultures exposed to Pa demonstrated increased Csf2 mRNA (p 0.0073) and apical GM-CSF protein expression (p 0.019) at 24 hours compared to unstimulated cells and cells exposed to Mabsc. Conclusions Pa-induced GM-CSF production by airway epithelia may create focal environments where recruited MDMs become programmed to develop anti-Mabsc phenotypes. Ongoing studies aim to (a) create murine co-infection models to test whether Pa co-infection decreases Mabsc bacterial burden in mice inoculated with Mabsc agar beads, and (b) determine if in-host evolution drives development of Pa isolates that lose ability to induce airway GM-CSF. This abstract is funded by: CFF, NIH
Hisert et al. (Fri,) studied this question.
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