Supplemental Material: Dietary Influence on Lung Injury and Immune Modulation in Cadmium-Exposed Mice

Cadmium (Cd), a toxic heavy metal found in air pollution, poses serious risks to lung health due to its efficient pulmonary absorption and prolonged biological half-life. This study examines how ad-libitum (AL), time-restricted feeding (TRF), and intermittent fasting (IF) influence Cd-induced lung injury and immune responses in mice. Adult male C57BL/6 mice were pre-acclimated to AL, TRF, or IF regimens for three weeks, followed by intratracheal exposure to cadmium chloride (CdCl₂, 0.5 mg/kg). Lung mechanics were assessed using flexiVent, bronchoalveolar lavage (BAL) fluid was analyzed for inflammation, and immune profiling was performed on spleens and mediastinal lymph nodes (MLNs) 14 days post-exposure. Cd exposure increased immune cell infiltration in BAL fluid. IF mice showed significantly elevated inflammatory cytokines, while TRF mice had modest increases. Histological analysis revealed greater lung inflammation in TRF mice, whereas lung mechanics were more impaired in IF mice, suggesting distinct injury profiles. Immune profiling showed that IF reduced activated and effector T-cell populations in the spleen but increased them in MLNs, indicating a shift in immune localization. Furthermore, compared to the AL, Cd-exposed IF mice had minimal changes in T-cell distribution but reduced effector CD4+ and CD8+ T-cells in the spleen and increased in MLNs. In contrast, TRF mice exhibited minimal changes in T-cell distribution. These findings suggest that dietary regimens modulate immune responses and lung injury following Cd exposure. Feeding patterns play a critical role in shaping susceptibility to environmental toxicants and should be considered in future toxicological and immunological studies.