PROJECT SUMMARY/ABSTRACT Nontuberculous mycobacteria (NTM) cause an emerging, chronic, and challenging to treat pulmonary disease (PD) whose numbers now undeniably surpass tuberculosis cases in the United States (U.S.) and other geographic regions globally. NTM infections are notoriously difficult and expensive to eradicate, prone to relapse and reinfection, and refractory to current anti-mycobacterial therapies. The environmental niches of NTM also remain largely ill-defined. This lack of knowledge contributes to the on-going frustration to understand where these pulmonary infections are acquired. In the U.S., Hawai’i is a geographic hot spot for NTM PD. Our work in Hawai’i revealed an abundance of respiratory important NTM including Mycobacterium abscessus subsp. abscessus, Mycobacterium avium, and Mycobacterium chimaera in household and non-household water biofilms and soil. A unique environmental factor that may impact NTM PD in Hawai’i is exposure to aerosol pollutants from the Kīlauea volcano, one of the most actively erupting volcanoes on Earth. Fine volcanic ash particles can be breathed deep into the lungs causing irritation and cough. But, is it possible for volcanic ash to also carry infectious NTM? Through our data, we show microbiological culture of viable M. abscessus, M. avium, and M. chimaera from ash recovered during the 2018 eruption of Kīlauea and also from the Kīlauea environment, supporting the possibility of volcanic ash as a newly described fomite for NTM. We demonstrate Kīlauea ash is not directly toxic to Kīlauea-derived NTM and that ash exposure reduces NTM control by human macrophages. Our project’s broad, long-term objective is to elucidate the innate immunological mechanisms used by human lung cells in response to NTM infection and aerosol pollutants such as volcanic ash, providing implications that extend beyond Hawai’i with millions of people worldwide living within kilometers of actively erupting volcanoes. Herein, we leverage our unique collection of volcanic ash and NTM isolates recovered from Kīlauea ash, the Kīlauea environment, and other Hawai’i and non-Hawai’i samples to test the hypothesis that Kīlauea ash exacerbates control of NTM infection by healthy lung cells through antagonizing NLRP3 inflammasome-mediated pyroptosis defenses. In this proposal, we will: (i) Investigate the biological consequences of Kīlauea ash exposures on Kīlauea NTM fitness in the absence of host cells and (ii) Determine the effect of Kīlauea ash on NLRP3 inflammasome-mediated pyroptosis in clearance of NTM using primary human alveolar macrophages and airway epithelial cells cultured at the air-liquid interface from healthy donors, but also introduce a new 3D apical out airway epithelial organoid culture system to study innate immune responses to Kīlauea NTM and Kīlauea ash. The study’s broad application will be a reduction in the biological uncertainties surrounding how exposure to specific environmental respiratory pollutants of global significance such as volcanic ash influences NTM survival inside and outside of healthy human lung cells, while simultaneously informing risk assessments and therapeutic interventions in response to respiratory infections and volcanic ash exposures.

PROJECT NARRATIVE Infectious respiratory diseases such as those caused by nontuberculous mycobacteria (NTM) are acquired through environmental exposures that remain largely under-described. Every day, millions of people inhale hazardous aerosol emissions from erupting volcanoes globally; our evidence supports NTM are hitchhikers on volcanic ash that together overcome innate immune control by lung cells to establish infection. In this proposal, microbiological, molecular, and immunological assays and human lung cells from healthy donors will be studied to reveal the immunological mechanisms of front-line lung cells to NTM and volcanic ash, providing critical new information to improve respiratory health.