Role of Farnesoid X Receptor in mustard vesicant-induced lung injury, macrophage activation and altered pulmonary functioning
Creators: name: Meshanni, Jaclynn Andres
Publisher: name: No Publisher Supplied
Year: 2023

description: Nitrogen mustard (NM; (bis(2-chloroethyl)methylamine) is a cytotoxic vesicant known to cause pulmonary injury that can progress to fibrosis. This is associated with an accumulation of inflammatory macrophages in the lung, which have been implicated in NM toxicity. Farnesoid X Receptor (FXR) is a nuclear receptor essential for regulating bile acid and lipid homeostasis. Moreover, FXR exerts anti-inflammatory activity. In the current studies, we developed a model to assess pulmonary injury and fibrosis following NM exposure and the role of FXR in regulating macrophage inflammatory activity and lipid homeostasis in the lung. Specifically, male Wistar rats were exposed to phosphate-buffered saline (CTL) or NM (0.125 mg/kg) via i.t. Pencentury Microsrprayer™ aerosolization. Following exposure, rats were treated orally with Obeticholic Acid (OCA), an FXR receptor agonist or vehicle control (peanut butter, 0.13 -0.18 g), 1x/day, 5 days/week. Lungs, bronchoalveolar lavage (BAL), serum, alveolar macrophages (AMs), liver, and ileum were collected 28 days post-exposure. The results showed that NM induced time-related histopathological changes in the lung including epithelial-thickening, alveolar-circularization, and pulmonary edema. Picrosirus Red staining was also increased 28 days following NM exposure, which is associated with increases in hydroxyproline levels in lung tissue, indicative of fibrosis. OCA treatment attenuated NM lung toxicity and fibrosis, as well as reducing the expression of HO-1, a marker of oxidative stress. In addition, NM administration resulted in increased expression of inducible nitric oxide synthase (iNOS), a marker of nitrosative stress. This correlated with an increase in the ratio of nitrates to nitrites in BAL. Oxidative and nitrosative stress were suppressed by OCA treatment. NM-induced structural alterations were associated with reduced pulmonary resistance; these were blunted by OCA administration to rats. We next analyzed the role of FXR in regulating lipid homeostasis following exposure to NM. A marked decrease in serum and lung total bile acid levels was noted in NM treated rats. OCA had no effect on this response. Following NM exposure, enlarged vacuolated macrophages were evident in the lung in areas adjacent to fibrotic lesions; these cells were stained positively for Oil Red O, indicative of profibrotic foamy macrophage formation and dyslipidemia. To explore mechanisms underlying this response, we assessed macrophage mRNA expression of the lipid uptake receptor CD36, the transcription factor regulating cholesterol synthesis protein SREBP-2, and a lipid transporter ACAT-1; NM upregulated expression of the genes encoding these proteins. NM also caused an increase in total and free intracellular cholesterol levels in AMs, and an increase in cholesterol efflux that is associated with increased expression of ABCA-1, a cholesterol efflux transporter. Treatment of rats with OCA mitigated the effects of NM on macrophage foam cell formation; this was correlated with decreases in mRNA expression of Cd36, Srebp-2, Acat-1, and Abca1, normalization of cholesterol levels and cholesterol efflux, and reduced fibrosis in the lung. These findings demonstrate that FXR may play a role in preventing profibrotic foam cell formation following NM exposure by limiting cholesterol accumulation in lung macrophages.The effects of OCA on macrophage inflammatory activity were also evaluated. Expression of Nos2, Arg-1, Il-1β, Il-1RA, Il-12 and Tnfα mRNA was increased following exposure of rats to NM, with no effects on Cd163, or Il-10. Changes in expression were associated with increases in TNFα, ARG-1 and iNOS protein expression, along with increases in nuclear activity of NF-κB p50/p65 subunits; these increases were suppressed by OCA. NM exposure also caused an increased dependence on ATP production in AMs. Treatment with OCA prevented NM-induced increases in proinflammatory and antiinflammatory macrophage activation and blunted changes in bioenergetics. In conclusion, these findings demonstrate that FXR may play a role in limiting NM-induced injury/fibrosis, oxidative stress, and inflammatory macrophage activation, processes critical to the pathogenic response of the lung to mustard vesicants. Furthermore, FXR is important in maintaining lipid homeostasis and limiting macrophage activation following exposure to NM. These studies provide novel data on mechanisms mediating macrophage activity following NM exposure and suggest that activation of FXR may represent an effective therapeutic approach for preventing the development of pulmonary fibrosis after exposure to mustard vesicants.