Total cell count confirmed no differences between sham and PM10sum-treated mice, 24 h after the last PM10sum intratracheal instillation (, A). On the contrary, differential cell count disclosed a significant decrease of AMs percentage and a significant increase of PMNs percentage (99% neutrophils) in treated mice (, A). A significant increase of IL-1β and MIP-2 concentrations were evident in the BALf of PM10sum-treated mice in comparison to sham, though in presence of unchanged concentration of TNF-α (, A). Lactate Dehydrogenase (LDH) and Alkaline Phosphatase (ALP) activities were analyzed in the BALf as markers of cytotoxicity. LDH activity was significantly increased in PM10sum-treated mice (, A), while no difference was detected in ALP activity (, A). Finally, PM10sum-treatment induced a significant increase in both MPO and Hsp70 levels (, A and , B).
BALf, lung, heart, brain and plasma immunoblotings.
Lung Genes Expression Analysis
PM10sum-treatment induced a significant MIP-2 up-regulation while no significant changes were found for HMOX1, Cyp1B1, IL-1β and MPO; an induction of miR-21 in the lung tissues of PM10sum-treated mice was evidenced during miRNA gene expression analysis ().
Lung, Heart and Brain Protein Analyses
ET-1, a potent regulator of the vascular tone, was significantly higher in the lungs of PM10sum-treated mice in comparison to sham (, B and , A). Moreover a significant increase in HO-1 and Hsp70 levels (, B and , A), both involved in the protection against protein unfolding, as well as in the inflammation and oxidative stress, were observed in the lungs of PM10sum-treated mice. The level of Cyp1B1, a cytochrome of the P450 superfamily involved in the activation of many xenobiotics and in PAHs metabolism, significantly decreased 24 h after the last PM10sum intratracheal instillation (, B and , A). iNOS, MPO, Caspase8-p18 and Caspase3-p17 showed no differences between sham and PM10sum-treated mice (, A). TLR4 pathway was not activated as no increases of the IRAK1, TAK1 and IKBα phosphorylation were detected (, A).
Heart tissues of PM10sum-treated mice disclosed significant increases of ET-1, Cyp1B1 and Hsp70 levels (, C and , B). MPO, HO-1, Caspase8-p18 and Caspase3-p17 shown no differences between sham and PM10sum treated mice (, B).
In the brain of PM10sum-treated mice, ET-1 and HO-1 levels were significantly increased (, D and , C); Cyp1B1, CD68, soluble and membrane bound TNF-α, Hsp-70, GFAP, Caspase8-p18, Caspase3-p17 were basically unchanged comparing sham and PM10sum-treated mice (, C).
Blood and Plasma Analysis
Gene expression analysis evidenced no up-regulation of HMOX1 or IL-1β in blood RNA of PM10sum-treated mice, compared to sham, together with no induction of miR-21 ().
Blood and plasma of sham and PM10sum-treated mice were analysed for pro-inflammatory and pro-trombogenic markers. Total cell count, neutrophils percentage and sP-selectin concentration, a marker of the activated platelet/endothelium interface, were significantly increased 24 h after the last intratracheal instillation of PM10sum (, A). The cytokines analyses in the plasma of sham and PM10sum-trated mice, performed by ELISA assay, were under the kit detection limits.
Finally, both MPO and ET-1 increased in PM10sum-treated mice (, E and , B).
Despite the relatively high cumulative dose of PM10sum instilled, there were no signs of pulmonary overloading with particles; moreover, only few free particles and no large particle aggregates were seen in the BALf or lung tissue of treated mice.
As evidenced by the histological lung analysis, when compared to sham (, A–B) PM10sum has induced inflammatory cell recruitment in the connective surrounding the terminal bronchioles and the proximal alveolar sacs (, C–D). Lungs instilled with PM10sum showed extended inflammatory infiltrate and particles engulfed by alveolar macrophages (, C–D). These histological evaluations have supported the cytological and biochemical assays on BALf. Immunohistochemical detection of HO-1 in the lungs of PM10sum-treated mice has confirmed the biochemical analyses, (, A–D). HO-1 induction appeared to be distributed along both airways and the respiratory tract, particularly well distinguishable within the epithelium of distal bronchioles (, C–D).
Lung immunohistochemical analysis.