Figure 1.
Generation of Retnla-Tg mice, and the expression pattern of Retnla in lung.
(a) DNA construct for generating Retnla-Tg mice. Mouse Retnla cDNA was cloned into the EcoRI site of the pCAGGS vector. The transgene was excised as a SpeI and PstI fragment for microinjection. (b) Northern blot analysis of total RNA extracted from lungs of Retnla-Tg and non-Tg mice. 28S and 18S rRNA were used as controls for RNA amount and integrity. (c) Immunoblot analysis of mouse Retnla from lung and bronchoalveolar lavage (BAL) fluid of Retnla-Tg and non-Tg mice. β-actin was also analyzed as a loading control. (d) Control staining using non-immunized rabbit serum. Representative Retnla staining in lungs of OVA sensitized/challenged mice (upper right) and Retnla-Tg mice (bottom row). Nuclei were stained with DAPI (blue). Inset: original magnification, 200×. (e) Quantitative RT-PCR analysis showing mRNA expression of Retnla in peritoneal macrophages of non-Tg and Retnla-Tg mice (n = 5 per group). Data are represented as mean ±SD. Mann-Whitney U-test; ***P<0.005.
Figure 2.
Retnla overexpression did not alter lung histology or function under normal conditions.
(a) Representative lung sections from non-Tg (top row) and Retnla-Tg (bottom row) mice stained with hematoxylin and eosin (left panel) or trichrome (right panel) (collagen is stained blue). Inset: original magnification, 200×. There was no significant inflammatory change in hematoxylin and eosin staining, and collagen content (blue in trichrome staining) was not changed in Retnla-Tg mice. (b) Fibrosis was assessed by the measurement of hydroxyproline in the lungs from non-Tg and Retnla-Tg mice (n = 8 for non-Tg, n = 5 for Retnla-Tg). (c–g) Analysis of pulmonary functional parameters including (c) respiratory frequency (fR, BPM indicates breaths per minute), (d) minute ventilation (VE), (e) tidal volume (VT), (f) peak expiratory flow (PEF), and (g) peak inspiratory flow (PIF) were measured using whole-body plethysmography (n = 7 to 8 per group). There were no significant changes in pulmonary function of Retnla-Tg mice compared to those of non-Tg mice. Data are presented as mean ±SD. Statistical analysis was performed with the Mann-Whitney U-test.
Figure 3.
Retnla overexpression did not influence immune cell composition of lung under normal conditions.
(a) Gating strategy for identification of immune cell populations from lung of Retnla-Tg and non-Tg mice. (b) Immune cell populations in lungs from Retnla-Tg and non-Tg mice were quantified by flow cytometry (n = 7 to 8 per group). Data are presented as mean ±SD. Statistical analysis was performed using the Mann-Whitney U-test.
Figure 4.
Retnla overexpression reduced immune cells in BAL fluid from OVA-sensitized/challenged mice.
(a) Inflammatory cell infiltration was examined in BAL fluid from OVA-challenged Retnla-Tg and non-Tg mice (n = 7 to 8 per group). (b) The level of Retnla protein in BAL fluid was analyzed by immunoblotting. Each lane represents an individual mouse. Band intensities were quantitated by computerized densitometry. (c) Immune cell populations in BAL fluid of Retnla-Tg and non-Tg mice were quantified by flow cytometry (n = 4 for non-Tg, n = 6 for Retnla-Tg). Data are presented as mean ±SD. Statistical analysis was performed with the Mann-Whitney U-test; *P<0.05; # P = 0.06.
Figure 5.
Retnla overexpression attenuated pulmonary inflammation and mucus production in OVA-sensitized/challenged mice.
(a) Representative H&E-stained tissue sections demonstrating peribronchial inflammatory infiltrates (arrowheads) in lung. The average histological inflammation score was significantly decreased in Retnla-Tg mice. Each dot represents an individual mouse. (b) Representative Alcian blue-stained sections demonstrating mucus production (mucus is stained blue) are shown on the left. The numbers of mucus-producing bronchial epithelial cells per mouse are shown in right. Each dot represents an average number of Alcian blue-positive bronchial epithelial cells from each individual mouse (five sections analyzed/animal, per 200× field). Inset: original magnification, 200×. (c) The mRNA level of Muc5ac was determined by qPCR analysis of RNA from lungs. (d) Airway responsiveness measured in Retnla-Tg and non-Tg mice after stimulation with increasing doses of inhaled methacholine (n = 6 per group). (e) Fibrosis was measured by quantifying hydroxyproline level in the lungs. Data are presented as mean ±SD. Statistical analysis was performed using the Mann-Whitney U-test; *P<0.05; **P<0.01.
Figure 6.
Retnla overexpression decreased the levels of IL-4, IL-5, and IL-13 in OVA-induced asthmatic lung.
(a–c) The mRNA levels of IL-4 (a), IL-5 (b), and IL-13 (c) were determined by qPCR analysis of RNA from lungs of OVA-induced Retnla-Tg and non-Tg mice (n = 7 to 8 per group). (d–k) Levels of IL-4 (d), IL-5 (e), IL-10 (f), IL-13 (g), IgG1 (h), IgG2a (i), IgG2c (j) and IgE (k) in the BAL fluid were measured by ELISA. Data are presented as mean ±SD. Statistical analysis was performed with the Mann-Whitney U-test; *P<0.05.
Figure 7.
Retnla overexpression reduced ERK activation in OVA-induced asthmatic lung.
(a,c) The phosphorylation of MAPKs (ERK, p38, and JNK) (a) and STAT6 (c) in lung tissue was analyzed by immunoblotting. Each lane represents an individual mouse. (b,d) Band intensities in the immunoblot were quantitated by computerized densitometry. The phosphorylation of ERK was markedly reduced in Retnla-Tg mice. Data are presented as mean ±SD. Statistical analysis was performed with the Mann-Whitney U-test; **P<0.01.