Farnesoid X receptor in mustard lung toxicity; new approaches for assessing lung structure and function

The overall objective of these studies was to characterize the role of farnesoid X receptor (FXR) in nitrogen mustard (NM)-induced lung injury, assess NM-induced lung injury and fibrosis using imaging techniques (i.e., MRI and CT), and predict alterations in lung functioning using mathematical modeling. We hypothesized that NM exposure results in upregulation of FXR in the lung leading to reduced M1 macrophage activation and increased M2 macrophage activation, increased lipid uptake and foam cell formation, and pulmonary fibrosis. In the first specific aim, we utilized male and female FXR knockout (FXR-/-) mice to test this hypothesis. Interestingly, female WT and FXR-/- mice were less sensitive to NM toxicity; this was assessed by histopathological analysis and bronchoalveolar lavage (BAL) cell number and protein content. Based on these findings, we continued our analysis of lung responsiveness to NM in male WT and FXR-/- mice. We found that FXR regulates inflammatory macrophage activation in the lung following NM exposure. We utilized flow cytometry to assess the phenotype of pro and anti-inflammatory macrophages in BAL in response to NM. Loss of FXR resulted in a significant increase in proinflammatory macrophages at 3 d post NM exposure, as expected. This increase in proinflammatory macrophages correlated with increased expression of COX-2 and ARL11, markers of M1 activation in lung macrophages demonstrating that these proinflammatory macrophages are activated. The miRNAs related to NF-κB pathway activation were analyzed and were found to be also upregulated in FXR-/- mice, which may exacerbate proinflammatory macrophage activity. We also found that gene expression of Nur77, a nuclear receptor associated with anti-inflammatory macrophage activation, was significantly reduced in WT mice treated with NM at 14 d post exposure, suggesting an impairment of M2 macrophage activity. As FXR is a nuclear receptor involved in lipid homeostasis, we also analyzed the expression of genes related to lipid metabolism and transport. Both Apoe and Abca1 mRNA levels were significantly increased in FXR-/- mice at 14 d post NM exposure. Conversely, at 28 d post NM, Abcg1 mRNA levels were significantly reduced, which correlates with accumulation of foamy macrophages in lung tissue. In the second specific aim, we refined MRI and CT imaging methodology to track the progression of NM-induced lung injury and response to the therapeutic, anti-TNFα antibody in the same animal over time. Rats were imaged prior to exposure and then 1-28 days thereafter. High-signal lung volume present in MR images corresponded to edema, inflammation and tissue remodeling; pathologies that were found to persist for 28 d following NM exposure. CT scans were used to assess structural components of the lung and register alterations in tissue radiodensities. Changes in lung volume relative to pre-exposure lung volumes were reduced in rats exposed to NM at 28 d (-1.3 x 10^⁵ vs. 3.9 x 10^⁵ Hounsfield units); this was mitigated by anti-TNFα antibody. Loss of respiratory area caused by NM was restored by the treatment of rats with anti-TNFα antibody. Following imaging, at 30 d post exposure, rats were anesthetized, and lung function assessed at a positive end expiratory pressure (PEEP) of 3 cm H₂O using a SciReq flexiVent® small animal ventilator. Structural changes generated from MRI and CT imaging analyses (e.g., % of lung injury, AUC of the air portion of the lung, and AUC of the wet/consolidated portion of the lung) were then used to predict alterations in pulmonary mechanics, which was the focus of the third specific aim. Forward modeling was used to measure (using flexiVent® generated data) and calculate (using imaging derived parameters) respiratory system impedance (Zrs), a sensitive indicator of lung function. Strong correlations (r²≥0.96) were observed between measured and calculated Zrs confirming that the model is highly prognostic. We found that measured and calculated real and imaginary Zrs spectra were increased by 30 d post exposure. Using the real and imaginary components of Zrs, we calculated resistance (RL) and elastance (EL) for measured and calculated data. RL and EL were elevated indicating increased lung stiffness, a finding that is typically associated with mustard exposure. As imaging data were collected for earlier time points (e.g., 1, 3, 7, 14, and 21 d post NM exposure), simulations of real and imaginary Zrs spectra were performed using our model. We observed a shift from reduced to increased real Zrs spectra relative to control animals over time, supporting that pathologies progress and alter lung function following NM exposure. In conclusion, the current studies suggest that FXR plays a key role in mediating pro and anti-inflammatory macrophage activation and foam cell formation associated with NM-induced lung injury. The refinement of MRI and CT imaging methodologies allowed us to quantify NM-induced pathological alterations over time in the same animal and confirm the ability of anti-TNFα to abrogate lung toxicity. We further implemented the structural data derived from these imaging methods into a mathematical model to predict lung function. Overall, these data provide new insights into mechanisms that promote the pathogenesis of NM lung toxicity and strategies to assess lung injury and response to therapeutics.Ph.D.Includes bibliographical reference