3.1 Average body weight gain
There was increase in the average body weight in all the groups after 90 days compared to initial body weight and no mortality was observed throughout the experiment.
3.2 Total leukocyte count and differential leukocyte count analysis.
Blood: Exposure to LPS or high (9.58 mg kg-1) dose of 2,4-D increased TLC of blood along with increase in neutrophils count and decrease in lymphocytes count. Further, exposure to high dose of 2,4-D combined with LPS increased in TLC compared to LPS alone (Table 1). Although treatment with low (5.12 mg kg-1) dose of 2,4-D alone did not alter the TLC of blood, but in combination with LPS increased TLC compared to control and individual low dose group. There was increase (p<0.05) in neutrophils count and decrease in lymphocytes count following exposure to low dose of 2,4-D alone or in combination with LPS compared to control group.
3.3 Bronchoalveolar lavage fluid. LPS increased (p<0.05) the TLC and neutrophils in BAL fluid compared to control group. Similarly, high or low dose of 2,4-D increased (p<0.05) the TLC compared to control and LPS group and neutrophils compared to control (Table 1). Further, high or low dose when combined with LPS increased (p<0.05) TLC and neutrophil count compared to individual high or low group, respectively.
3.4 Histopathological examinations. Hematoxylin and eosin stained lung sections from the control mice showed normal histoarchitecture (Fig. 1 A). Exposure to LPS, high or low doses of 2,4-D individually or the combined with LPS treatments caused lung inflammation characterized by congestion in blood vessels, peribronchial and perivascular accumulation of mononuclear cells and increase in the total histological score (THS) in all the treatment groups compared to the control (Fig. 1 B-F; Suppl Table 1).
3.5 Differentially expressed genes (DEGs) and functional analysis. A total of 5351 genes were differentially expressed (p < 0.05; fold change > ±1.5) following exposure to LPS and high or low dose of 2,4-D alone or a combination of 2,4-D and LPS. LPS treatment alone upregulated 671 genes and down-regulated 655 genes. Treatment with high dose (9.58 mg kg1) and low dose (5.12 mg kg-1) of 2,4-D caused the upregulation of 2178 and 2133 genes and downregulation of 1965 and 1838 genes, respectively. Further 2,4-D in high and low dose in combination with LPS up regulated 2142 and 2054 genes and down regulated 1719 and 1652 genes, respectively as compared to control group (Fig 2A). The gene overlap studies of differentially expressed genes (DEG) in all the groups showed 356 (216 upregulated and 140 down-regulated) commonly expressed genes in all the treatment groups compared to control. The relative expression levels of these genes are illustrated as a Venn diagram (Fig. 2B).
3.6 Biological classification and pathway enrichment analysis of DEGs. Gene ontology enrichment analysis revealed that DEGs were significantly enriched in genes involved in various biological processes including response to oxygen containing compound, tissue development, regulation of protein modification process, regulation of cell population proliferation and nucleic acid metabolic process (Suppl Table 2). KEGG pathway enrichment analysis revealed that SCLC pathway was the topmost dysregulated pathway following exposure to high or low dose of 2,4-D with or without LPS. KEGG pathway enrichment analysis also suggested that p53, Itgb1, Cdk6, NF-kB1 and Apaf1 genes were hub genes primarily associated with SCLC pathway.
3.7 Validation of microarray data by qRT-PCR and immunohistochemistry.
3.7.1 p53: Lung transcriptomic analysis revealed the up regulation of p53 mRNA following exposure to high or low doses of 2,4-D with or without LPS. LPS did not alter the mRNA expression of p53. However, there was 3.28, 3.09, 3.29 and 3.16 folds increase in the mRNA expression of p53, respectively, following exposure to high dose, low dose, high dose in combination with LPS and low dose in combination with LPS. The qRT-PCR data were found to be in concordance with the microarray data (Fig. 3a).
The lung sections incubated without primary antibody did not show any colour development (Fig. 4). Lung tissues from control and LPS-treated mice showed weak staining for p53 in the airways epithelial and alveolar septal cells (Suppl Fig. 1). However, the high or low doses of 2,4-D alone or in combination with LPS showed strong reaction for p53 (Suppl Fig. 1). There was a significant increase in the number of p53-positive cells in lungs of mice exposed to both doses of 2,4-D compared to control and LPS group (Fig. 4a). Further, high or low dose in combination with LPS significantly increased the number of p53 cells compared to LPS group.
3.7.2 Integrin β1: Global view of DEG’s revealed down-regulation of integrin β1 (-0.94 fold) by LPS alone and up regulation following exposure to high (1.76 fold) or low (1.71 fold) doses of 2,4-D alone. There was increase in integrin β1mRNA expression following exposure to, high dose (1.69 fold) and low dose (1.68 fold) in combination with LPS. The qRT-PCR data were found to be in concordance with the microarray data (Fig. 3a).
There was a mild staining for integrin β1 protein in the airway epithelial cells, alveolar septal cells and occasionally in large septal cells/macrophages in lungs from control and LPS group (Suppl Fig. 2). While LPS alone didn't change the expression of the integrin, the high or low doses of 2,4-D alone or in combination with LPS caused an increase in intensity and number of cells positive for integrin β1 (Fig 4b and Suppl Fig. 2).
3.7.3 Cdk6: Lung transcriptomic analysis revealed the up regulation of Cdk6 mRNA following exposure to high or low doses of 2,4-D with or without LPS. Treatment with LPS downregulated (-1.85 folds) Cdk6 mRNA. However, there was increase in the mRNA expression of Cdk6 following exposure to high dose (4.61 folds), low dose (4.64 folds), high dose in combination with LPS (4.62 folds) and low dose in combination with LPS (4.62 folds). The qRT-PCR data were found to be in concordance with the microarray data (Fig. 3c).
Lung sections from the mice in control and LPS groups showed weak to mild reactivity for Cdk6 in the airways epithelial and alveolar septal cells (Suppl Fig. 3). However, the high or the low dose alone or in combination with LPS increased the number of lung cells positive for Cdk6 compared to the control and LPS group (Fig. 4c).
3.7.4 NF-kB1: Microarray analysis revealed the upregulation of NF-kB1 gene following exposure to high and low doses of 2,4-D with or without LPS. However, LPS reduced NF-kB1 mRNA expression by -0.12 folds. There was increase in the expression NF-kB1 following exposure to high dose (4.16 folds), low dose (4.25 folds), high dose in combination with LPS (4.24 folds) and low dose in combination with LPS (4.23 folds). The qRT-PCR data were found to be in concordance with the microarray data (Fig. 3d).
A mild NF-kB1 staining was localized in the airway epithelial cells and alveolar septal cells in lungs of mice from control and LPS group (Suppl Fig. 4). The high or the low dose alone or combined with LPS induced strong staining in alveolar epithelium cells, alveolar septal cells and macrophages compared to the control and LPS group (Suppl Fig. 4). LPS exposure did not cause any change in the number of NF-kB1 positive lung cells compared to control group. There was a significant increase in the number of NF-kB1 positive cells in lungs of mice exposed to high or low dose of 2,4-D compared to control and LPS group (Fig. 4d). Further, high or low dose in combination with LPS significantly increased the number of immunopositive NF-kB1 cells compared to LPS group but did not vary from individual high or low treatment group, respectively (Fig. 4d).
3.7.5 Apaf1: Lung transcriptomic analysis revealed the down regulation of Apaf1 mRNA following exposure to high or low doses of 2,4-D with or without LPS. Treatment with LPS downregulated Apaf1 mRNA by -1.73 folds. Further, the Apaf1 mRNA decreased in lungs of mice treated with high dose (-1.15 fold), low dose (-1.51 fold), high dose combined with LPS (-1.28 fold) and low dose combined with LPS (-1.05 fold). The qRT-PCR data were found to be in concordance with the microarray data (Fig. 3e).
Lungs from control mice showed strong staining for Apaf1 in the alveolar cells (Suppl Fig 5). LPS exposure also showed moderate to strong Apaf1 reactivity in the alveolar cells (Suppl Fig. 5). Further lungs from the mice exposed to the high or the low dose alone or in combination with LPS showed moderate reactivity for Apaf1 protein in alveolar cells (Suppl Fig. 5). There was a significant decrease in the number of Apaf1 positive cells in lungs of mice exposed to high or low dose of 2,4-D compared to control and LPS group (Fig. 4e). Further, high or low dose in combination with LPS significantly decreased the number of immuno-positive Apaf1 cells compared to LPS group but did not vary from individual high or low treatment group, respectively (Fig. 4e).
3.8 Expression of proteins in BAL Fluid. Indirect ELISA was carried out to compare the relative differences in absorbances as a readout of concentrations of p53, Itgb1, Cdk6, NF-kB1 and Apaf1 proteins in the BAL fluid (Fig. 5). LPS treatment did not alter the protein concentration of p53, Itgb1, Cdk6 and NF-kB1 in BAL fluid compared to control group. Exposure to the high or low dose of 2,4-D increased (p<0.05) the BAL fluid concentration of p53 (0.886, 0.898 folds), Itgb1 (0.905, 0.848 folds), Cdk6 (0.833, 0.874 folds) and NF-kB1 (0.833, 0.867 folds) compared to the control and LPS groups, respectively. LPS decreased the concentration of Apaf1 compared to control. Further there was a decrease (p<0.05) in the concentration of Apaf1 (0.176 and 0.150 folds) following exposure to the individual high or low dose as compared to control and LPS group. Furthermore, exposure to the low dose of 2,4-D in combination with LPS decreased (p<0.05) the concentration of Apaf1 (0.077) as compared to individual low dose group (Fig. 5).