Comprehensive Lung Inflammation Analysis

Bronchoalveolar lavage and measurement of the total protein and cytokines in BALF were performed as previously described in [26 (link),28 (link)]. For the histology, after bronchoalveolar lavage (BAL), the lungs were excised and the left lobe fixed in 4% buffered formalin and embedded in paraffin. Sections of 3 µm thickness were stained with hematoxylin-eosin (H&E) and periodic acid Schiff (PAS). For the real-time PCR, total RNA was extracted from snap-frozen lung tissue as in [25 (link)]. A total of 0.4 μg of RNA was converted to cDNA following manufacturing protocol (Fermentas, ThermoFischer Scientific, Waltham, MA, USA). Real-time PCR was performed with SYBR Green and ViiA™ 7 thermocycler (Applied Biosystems, Foster City, CA, USA). Relative expression levels were calculated using the 2^ddct method, normalized to ACTB. The primer sequences were the following: Arg1 (forward) 5′-AGAGATTATCGGAGCGCCTT-3′ (reverse) 5′-TTTTTCCAGCAGACCAGCTT-3′; Muc5ac (forward) 5′-TGGAGTCAGCACGAAAACAG-3′ (reverse) 5′-GCACTGGGAAGTCAGTGTCA-3′; IL-13 (forward) 5′-TGTGTCTCTCCCTCTGACCC-3′ (reverse) 5′-CACACTCCATACCATGCTGC-3′; KC (forward) 5′-CCACACTCAAGAATGGTCGC-3′ (reverse) 5′-TCTCCGTTACTTGGGGACAC-3′; ACTB (forward) 5′-TTCTTTGCAGCTCCTTCGTT-3′ (reverse) 5′-ATGGAGGGGAATACAGCCC-3′. Data were considered significant with a p value ≤ 0.05 in a confidence interval of 95%.