Selective p38α mitogen-activated protein kinase inhibitor attenuates lung inflammation and fibrosis in IL-13 transgenic mouse model of asthma

Figures & data

Figure 3 Effect of SD-282 on the hyperplasia of airway wall epithelial cells induced in the CC10:IL-13 transgenic asthma model and analyzed following PASH stain. [a] SD-282 at a high-dose significantly reduces the thickness of epithelial cells (p < 0.05). The noted values represent the MEAN ± SD on a minimum of eight animals. [b] PASH staining of lung sections, x400 from Tg (−) naïve (A), Tg (+) baseline (B), Tg (+) vehicle(C), and Tg (+) SD-282H (D). Increased thickness of epithelial cells is seen in both baseline (B) and vehicle-treated Tg (+) mice (C) compared with Tg (−) mice (A), and decreased thickness of epithelial cells is seen in the SD-282 high-dose treated Tg (+) mice (D).

Figure 4 Effect of SD-282 on goblet cell metaplasia and mucus hypersecretion induced in the CC10:IL-13 transgenic asthma model, analyzed after PASH stain. [a] SD-282 at a high-dose significantly inhibited the goblet cell metaplasia and mucus hypersecretion (p < 0.001). The noted values represent the MEAN ± SD on a minimum of eight animals. [b] Neutral and acidic mucus are overproduced in goblet cells of segmental bronchus. PASH and Alcian blue stain, x200 showing lung sections from Tg (−) naïve (A&B) and Tg (+) baseline (C&D). PASH showing the red-purple cytoplasmic inclusion (neutral mucus) and Alcian blue showing the blue cytoplasmic inclusion (acid mucus). There is almost no neutral and acid mucus production in the epithelial cell in Tg (−) mice (A&B). In contrast, there is hypersecretion of both neutral and acid mucus from goblet cells in Tg (+) mice from baseline (C&D). [c] PASH, x200 (in green box) and x400 showing lung sections from Tg (−) naïve (A), Tg (+) baseline (B), Tg (+) vehicle (C) and Tg (+) SD-282H (D). Mucus overproduction and goblet cell metaplasia are seen in both baseline (B) and vehicle-treated Tg (+) mice (C) compared with Tg (−) mice (A), and decreased mucus production and fewer goblet cells are seen in the SD-282 high-dose treated Tg (+) mice (D).

Figure 5 Effect of SD-282 on fibrosis induced in the CC10:IL-13 transgenic asthma model analyzed after Masson’s trichrome stain. [a] SD-282 at high-dose significantly reduces fibrosis of lung parenchyma compared with the vehicle-treated group (p < 0.01). The noted values represent the MEAN ± SD on a minimum of eight animals. [b] Thickness of basement membrane following Masson’s trichrome stain. SD-282 at high-dose significantly reduces subepithelial fibrosis compared with the vehicle-treated group (p < 0.05).The noted values represent the MEAN ± SD on a minimum of eight animals. [c] Masson’s trichrome stain, x200 showing lung sections from Tg (+) baseline (A), Tg (+) vehicle (B) and Tg (+) SD-282H (C). Very mild collagen deposition stained in blue (black arrow) around the basement is seen in Tg (+) mice in baseline (A). Enhanced accumulation of collagen stained in blue (black arrow) is seen in the vehicle-treated Tg (+) mice (B). Less collagen accumulation and reduced thickening in the basement membrane (black arrow) are seen in the SD-282 high-dose treated Tg (+) mice (C). Masson’s trichrome stain, x100 and x400 (in large green box with arrow) showing lung sections from Tg (+) baseline (D), Tg (+) vehicle (E), and Tg (+) SD-282H (F). Mild fibrotic change is seen in the Tg (+) mice in baseline (D). Numerous fibrotic beads with collagen deposition stained in blue (green box) and elongated fibroblasts are seen in the vehicle-treated Tg (+) mice (E). Reduced fibrotic beads are seen in SD-282 high-dose treated Tg (+) mice (F).

Figure 1 p38 MAPK and SMAD 2/3 phosphorylation in lungs of CC10:IL-13 transgenic mice during development of asthma-like phenotype (8–16 weeks of age). The noted values represent the MEAN±SD, n = 3. [a] Score of p-p38 MAPK (IHC) activation in the epithelial cells of airway and infiltrated lymphocytes. [b] Percentage of p-SMAD2/3 (IHC) positively labeled epithelial cells in the airway. [c] Percentage of p-SMAD2/3 (IHC) positively labeled lung parenchyma cells.

Figure 2 Effect of SD-282 on inflammation induced in the CC10:IL-13 transgenic asthma model analyzed following H&E stain. [a] SD-282 at high-dose significantly reduces the inflammation (p < 0.05). The noted values represent the MEAN ± SD on a minimum of eight animals. [b] SD-282 reduces the inflammatory response. H&E, x40 showing lung sections from Tg (−) naïve (A), Tg (+) baseline (B), Tg (+) vehicle (C) and Tg (+) SD-282H (D). C showing lungs extremely enlarged and consolidated; alveoli, small and large airway filled with inflammatory cells in the Tg (+) mice treated with vehicle as compared with mild inflammatory response at onset of the asthma (B). D showing lung with less inflammatory response in SD-282 high-dose treated Tg (+) mice. [c] SD-282 reduces crystal formation and prevents eosinophil, lymphocyte, and macrophage infiltration. H&E, x400 showing lung sections from Tg (+) baseline (A), Tg (+) vehicle (B and C) and Tg (+) SD-282H (D). Mild inflammatory cell infiltration is seen at onset of the asthma/baseline (A). Long, thin, rectangular, and needle-like crystals (thin arrow), stained with eosin are seen in the vehicle-treated Tg (+) mice (B). Collections of lymphocytes (arrow), eosinophils (arrowhead), and enlarged macrophages (thin arrow) are seen in the vehicle-treated Tg (+) mice (C). Reduced crystal formation and fewer eosinophil, lymphocyte, and macrophage are seen in the SD-282 high-dose treated Tg (+) mice (D).

Figure 6 Effect of p38 MAPK inhibition on immune cell infiltration and activation in CC10:IL-13 mice [1] p-p38 MAPK IHC stain, x400 showing no p-p38 MAPK activation in Tg (−) mice (1-A), enhanced p-p38 MAPK activation in the epithelial cells of airway (1-B) and infiltrated lymphocytes(1-C) in the vehicle-treated Tg (+) mice and completed inhibition of p-p38 MAPK activation in the epithelial cells in the SD-282 high-dose treated Tg (+) mice (1-D). [2] IL-1β IHC stain, x400 showing no IL-1β expression in Tg (−) mice (2-A), mild IL-1β expression in the PMN cells in Tg (+) mice in baseline (2-B), increased IL-1β expression in the vehicle-treated Tg (+) mice (2-C) and less IL-1β expression in the PMN cells in the SD-282 high-dose treated Tg (+) mice (2-D). [3] CD3 IHC stain, x400 showing few CD3⁺ T-lymphocytes in the lung of Tg (−) mice (3-A), mild CD3⁺ T-lymphocyte infiltration in the lung of Tg (+) mice in baseline (3-B), markedly increased number of CD3⁺ T-lymphocyte in the lung of Tg (+) mice treated with vehicle (3-C) and significantly decreased number of CD3⁺ T-lymphocytes in the lung of Tg (+) mice treated with the high-dose of SD-282 (3-D). [4] F4/80 IHC, x400 showing no activated macrophages in the lung of Tg (−) mice (4-A), few activated macrophages in the lung of Tg (+) mice in baseline (4-B), markedly increased number of activated macrophages in the lung of Tg (+) mice treated with vehicle (4-C), and decreased number of activated macrophages in the lung of Tg (+) mice treated with high-dose of SD-282 (4-D).

Table 1 Effect of SD-282 on p38 MAPK activation in the airway epithelial cells and infiltrated lymphocytes (analyzed after p-p38 MAPK IHC staining); on IL-1β expression in the inflammatory PMN cells (analyzed after IL-1β IHC staining); on number of infiltrated CD3⁺ T-lymphocytes (analyzed after CD3 IHC staining), and on activated macrophages in the parenchyma of the lung (analyzed after F4/80 IHC staining) in the CC10:IL13 transgenic mouse asthma model. SD-282 at a high-dose significantly inhibits the level of phosphorylated p38 MAPK in the epithelial cells of airway and infiltrated lymphocytes compared with the vehicle-treated group (p < 0.001). SD-282 at a high-dose significantly inhibits IL-1β expression compared with the vehicle-treated group (p < 0.05). SD-282 at a high-dose significantly decreases the number of CD3⁺ T-lymphocytes compared with the vehicle-treated group (p < 0.05). SD-282 at a high-dose significantly decreases the number of activated macrophages in the parenchyma of the lung compared with the vehicle group (p < 0.001). The noted values represent the MEAN ± SD on a minimum of eight animals.

Genotype	Treatment	*p-p38 MAPK	*IL-1β	#CD3	#F4/80
Tg (−)	naive	0 ± 0	0 ± 0	64 ± 17	29 ± 8
Tg (+)	baseline	2.13 ± 0.99	1.5 ± 0.53	142 ± 54	110 ± 29
Tg (+)	vehicle	3.3 ± 0.67	2.6 ± 1.07	382 ± 210	183 ± 42
Tg (+)	SD-282	0.13 ± 0.35	0.75 ± 1.04	196 ± 117	92 ± 18
p value		p < 0.001	p < 0.05	p < 0.05	p < 0.001

* Values represent scores on the intensity of the IHC staining and

# values represent the number of IHC positively labeled cells

Table 2 Effect of TGFβRI kinase inhibitor, SD-208 and p38α-selective MAPK inhibitor, SD-282 both at a high-dose significantly reduce fibrosis of lung parenchyma compared with the vehicle-treated group (p < 0.05 and p < 0.01). The noted values represent the MEAN ± SD on a minimum of eight animals

Genotype	Treatment	Fibrosis of lung parenchyma mm^2/per section	p value
Tg (−)	naive	0 ± 0
Tg (+)	baseline	0.00098 ± 0.00059
Tg (+)	vehicle	0.02631 ± 0.01210
Tg (+)	SD-208	0.01629 ± 0.0038	p < 0.05
Tg (+)	SD-282	0.01213 ± 0.0077	p < 0.01