Defining Interstitial Macrophages Populations in the Lungs and How They Change Based on Nanomaterials

Nanoparticles are increasingly utilized in multiple fields such as but not limited to, medicine, clothing, agriculture, and industry. Carbon nanotubes have important uses in many of these fields so understanding their potential for adverse effects is vital. Macrophages are a key component of the immune system and are known to separate into functional subsets that generate distinct responses in the body. Pulmonary macrophages, key regulators of immune responses to respiratory exposures, have not yet fully been separated in subsets. Alveolar macrophages have been broken in subsets such as M1 and M2a while interstitial macrophage subsets have yet to be fully described. Using the alveolar macrophage model as a basis we hoped to distinguish subsets of pulmonary macrophages using Flow Cytometry. Methods: Our group has generated a vast array of data while still being preliminary. Balb/c mice were exposed to FA21, A highly inflammatory multiwall carbon nanotube, or silica and harvested a week later. The lungs were lavaged to obtain alveolar macrophages and interstitial macrophages were obtained through a collagenase treatment and percoll gradient centrifugation. The cells were then analyzed using Flow Cytometry. Alveolar macrophages were also cultured and polarized in vitro into either M1 or M2a subsets, to distinguish differences between them. Results: The data showed slight trends in gated pulmonary macrophage subsets. Following in vivo particle exposures alveolar macrophages showed a decrease in MHC II expression and an increase in Ly6C expression in both the FA21 and silica groups; while the interstitial macrophages, which were separated into inflammatory interstitial macrophages, showed an increase in MHC II expression in the FA21 and silica groups. It was also found that the M1 subset of alveolar macrophages from the plated cell experiment had a very large expression of MHC II compared to the M0 and M2a subsets. Following in vitro polarization of alveolar macrophages, we detected increased levels of YM1/YM2 in the M2a polarized cells compared to the controls.  We were also able to find marker differences in subsets of pulmonary macrophages following the seven-day exposure. Using Flow Cytometry and in vitro polarization of alveolar macrophages we could conclude that macrophages were expressing predicted markers of polarization. The data collected suggests that subsets within the interstitial macrophages do exist and further research is needed to separate and classify them. The varying levels of MHC II expression could be due to different subsets having varying levels of activation. Being able to separate the macrophages from the seven-day exposure into two groups, interstitial and inflammatory interstitial, demonstrates differences between interstitial macrophages in the lungs which needs to be studied further.