Abstract
BACKGROUND: Metals in particulate matter (PM), like iron (Fe), were associated with lung injury. Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) was suggested to inhibit lung inflammation. However, the effect of metals in PM, particularly Fe, on lung inflammation involving ITIH4 remained unclear. METHODS: We investigated the effects of recombinant ITIH4 (rITIH4) against acute lung injury in C57BL/6JNarl and B6.Sftpc-CreER(T2);Ai14(RCL-tdT)-D mice exposed to Fe-containing PM. Mice were exposed to diesel exhaust particles (DEP) or soluble iron (FeCl₃) via intratracheal instillation, while rITIH4 treatment was administered intranasally after exposure. Lung function, Fe levels (both bulk and single-cell by inductively-coupled plasma mass spectrometry (ICP-MS) and single-cell ICP-MS, respectively), inflammatory cell infiltration, and Hippo pathway regulation in type II alveolar epithelial cells (AECII) were assessed. RESULTS: We observed correlation between lung function changes and Fe levels, both in bulk and single-cell Fe in peripheral blood mononuclear cells. Single-cell RNA sequencing of the control group identified AECII-related cells characterized by high Sftpc, Sftpa1, Mzb1, B3 gnt5, Cacna1e, and Agbl1 expression. rITIH4 treatment in DEP-exposed mice restored Hippo pathway Cdh1, Itih4, Pdpn, Wwtr1, and Yap1 in AECII. rITIH4 reversed DEP- and Fe-induced increases in neutrophil infiltration, neutrophil-to-lymphocyte ratio, and monocyte depletion in bronchoalveolar lavage fluid (BALF). rITIH4 reduced BALF CXCL1/KC levels by DEP and serum 8-isoprostane levels by Fe. rITIH4 also reduced DEP-induced lung damage, increased ⍺-catenin and p-YAP in Fe-exposed mice, and pTAZ/TAZ ratio in both DEP- and Fe-exposed mice. rITIH4 increased pYAP/YAP ratio in DEP-exposed mice while decreasing LC3BII/I ratio in Fe-exposed mice. CONCLUSION: ITIH4 attenuated acute lung injury in mice exposed to PM, specifically Fe, by modulating the Hippo pathway in AECII.