Background
Methods
Mice
Design-based stereological analysis of lung structures and lung function measurements
Lung function measurements
RNA and Protein extraction
Transcript and protein analyses
Immunohistochemistry
Statistics
Results
Design-based stereological analysis of lung architecture
Morphometric Parameters | C3H/HeJ | JF1/MsJ |
P value | C3H/HeJ | JF1/MsJ |
p value |
---|---|---|---|---|---|---|
Number of mice | MALE: n = 5 | FEMALE: n = 3–5 | ||||
Age (weeks) | 13 | 12–14 | 13 | 12–14 | ||
Fixed lung volume (μl) | 1092 ± 123 | 735 ± 71 | 0.001 | 987 ± 54 | 667 ± 23 | 0.001 |
Mean chord length (Lcm) (μm) | 74.3 ± 9.9 | 48.5 ± 5.5 | 0.002 | 69.2 ± 14.8 | 47.3 ± 2.0 | 0.011 |
Mean alveolar volume (103/μm3) | 61.1 ± 7.9 | 40.8 ± 5.9 | 0.01 | 71.5 ± 11.4 | 42.3 ± 7.0 | 0.007 |
Alveolar number density (106/cm3 parenchyma) | 10.2 ± 1.1 | 14.7 ± 2.0 | 0.005 | 8.8 ± 1.5 | 14.5 ± 2.2 | 0.001 |
Alveolar septum width (μm) | 0.72 ± 0.07 | 0.66 ± 0.08 | ns | 0.82 ± 0.14 | 0.63 ± 0.11 | 0.005 |
Total number of alveoli (106/both lungs) | 9.2 ± 1.1 | 8.9 ± 1.7 | ns | 6.9 ± 1.4 | 8.2 ± 1.6 | ns |
Total alveolar surface area (cm2) | 952 ± 88 | 957 ± 125 | ns | 847 ± 70 | 906 ± 102 | ns |
Elastin - collagen ratio | 1.40 ± 0.3 | 1.95 ± 0.3 | 0.004 | 1.83 ± 0.3 | 2.12 ± 0.2 | 0.035 |
Absolute volumes | ||||||
Parenchymal volume (mm3) | 933 ± 112 | 629 ± 72 | 0.001 | 849 ± 71 | 571 ± 28 | 0.001 |
Non-parenchymal volume (mm3) | 145 ± 33 | 98 ± 23 | 0.002 | 126 ± 22 | 78 ± 11 | 0.003 |
Alveolar airspace volume (mm3) | 574 ± 73 | 377 ± 53 | 0.001 | 514 ± 41 | 340 ± 21 | 0.001 |
Alveolar duct airspace volume (mm3) | 261 ± 32 | 167 ± 26 | 0.041 | 237 ± 28 | 155 ± 15 | ns |
Alveolar septal volume (mm3) | 69 ± 12 | 63 ± 5 | ns | 69 ± 11 | 58 ± 5 | ns |
Total alveolar surface density (μm2/μm3) | 0.10 ± 0.01 | 0.15 ± 0.01 | ns | 0.11 ± 0.01 | 0.16 ± 0.01 | ns |
Alveolar epithelial type I cells (mm3) | 19.5 ± 3.3 | 19.2 ± 3.2 | ns | 20,8 ± 4.02 | 15.5 ± 3.45 | ns |
Alveolar epithelial type II cells (mm3) | 6.0 ± 2.8 | 7.8 ± 4.6 | ns | 4.3 ± 1.5 | 6.5 ± 2.7 | ns |
Capillary endothelial cells (mm3) | 16.3 ± 3.7 | 13.6 ± 1.4 | ns | 15.8 ± 1.9 | 11.8 ± 2.5 | ns |
Interstitial tissue (mm3) | 22.7 ± 5.3 | 17.5 ± 2.1 | ns | 22.5 ± 4.5 | 19.3 ± 6.1 | ns |
Total alveolar surface area (cm2) | 952 ± 88 | 957 ± 125 | ns | 847 ± 70 | 906 ± 102 | ns |
Volume densities | ||||||
Alveolar epithelial type I cells (mm3/mm3) | 28.2 ± 1.5 | 30.5 ± 4.2 | ns | 30.2 ± 2.1 | 27.23 ± 2.4 | ns |
Alveolar epithelial type II cells (mm3/mm3) | 9.0 ± 4.2 | 12.5 ± 6.7 | ns | 6.3 ± 3.4 | 10.9 ± 4.0 | ns |
Capillary endothelial cells (mm3/mm3) | 23.3 ± 2.3 | 21.7 ± 3.1 | ns | 22.8 ± 1.9 | 21.2 ± 2.5 | ns |
Interstitial tissue (mm3/mm3) | 32.9 ± 3.7 | 28.2 ± 2.6 | ns | 32.5 ± 3.0 | 33.2 ± 3.1 | ns |
Collagen (mm3/mm3) | 2.78 ± 0.49 | 2.63 ± 0.51 | ns | 3.02 ± 0.62 | 2.42 ± 0.41 | ns |
Elastin (mm3/mm3) | 3.87 ± 1.23 | 5.08 ± 1.06 | ns | 5.3 ± 1.55 | 5.08 ± 0.72 | ns |
Lung function |
n = 4 |
n = 3 | n-3 | n-3 | ||
Total Lung capacity (TLC) (μl) | 1425 ± 164 | 750 ± 50 | <0.05 | 1467 ± 125 | 800 ± 82 | <0.05 |
Compliance air (μl/cm H20) | 143 ± 5 | 36 ± 7 | <0.05 | 130 ± 6 | 34 ± 6 | <0.05 |
Compliance saline (μl/cm H20) | 306 ± 46 | 96 ± 13 | <0.05 | 296 ± 37 | 107 ± 20 | <0.05 |
Specific compliance saline (μl/cm H20) | 214 ± 15 | 128 ± 5 | <0.05 | 202 ± 9 | 134 ± 23 | <0.05 |
Compliance saline/Compliance air
| 2.2 ± 0.4 | 2.7 ± 0.1 | <0.05 | 2.3 ± 0.2 | 3.2 ± 0.9 | <0.05 |
Transcript analysis in the lung (E18, P28 and P70)
Gene | Gene symbol | Entrez ID | Fold Difference |
---|---|---|---|
E18 decreased transcripts of GO term extracellular region; Enrichment Score = 2.83; p = 0.0005 | |||
Chemokine (C-X-C motif) ligand 15 |
Cxcl15
| 20,309 | −14.6 |
Hemolytic complement |
Hc
| 15,139 | −8.61 |
Kallikrein 1-related peptidase b21 |
Klk1b21
| 16,616 | −8.25 |
Surfactant associated protein A1 |
Sftpa1
| 20,387 | −3.80 |
Collagen, type XXVIII, alpha 1 |
Col28a1
| 213,945 | −3.15 |
Paraoxonase 3 |
Pon3
| 269,823 | −2.96 |
Ceruloplasmin |
Cp
| 12,870 | −2.95 |
Neuron-derived neurotrophic factor |
Ndnf
| 68,169 | −2.41 |
Fibroblast growth factor 1 |
Fgf1
| 14,164 | −2.21 |
EGF-like, fibronectin type III and laminin G domains |
Egflam
| 268,780 | −2.13 |
Transferrin |
Trf
| 22,041 | −2.12 |
Interleukin 33 |
Il33
| 77,125 | −2.04 |
P70 decreased transcripts of GO term extracellular region; Enrichment Score = 4.18; p = 0.04 | |||
Hemolytic complement |
Hc
| 15,139 | −27.88 |
Chitinase 3-like 3 |
Chi3l3
| 12,655 | −15.93 |
Kallikrein 1-related peptidase b21 |
Klk1b21
| 16,616 | −11.92 |
Hepcidin antimicrobial peptide |
Hamp
| 84,506 | −4.87 |
Chemokine (C-C motif) ligand 6 |
Ccl6
| 20,305 | −3.92 |
Matrix metallopeptidase 3 |
Mmp3
| 17,392 | −3.62 |
Insulin-like growth factor binding protein 6 |
Igfbp6
| 16,012 | −2.83 |
Collagen, type XXVIII, alpha 1 |
Col28a1
| 213,945 | −2.76 |
Angiopoietin-like 7 |
Angptl7
| 654,812 | −2.72 |
Leptin receptor |
Lepr
| 16,847 | −2.28 |
Chitinase, acidic |
Chia
| 81,600 | −2.26 |
Oxidized low density lipoprotein (lectin-like) receptor 1 |
Olr1
| 108,078 | −2.21 |
Coagulation factor VII |
F7
| 14,068 | −2.18 |
Paraoxonase 3 |
Pon3
| 269,823 | −2.18 |
Plasminogen activator, tissue |
Plat
| 18,791 | −2.10 |
Secreted Ly6/Plaur domain containing 1 |
Slurp1
| 57,277 | −2.02 |
Gene Name | Gene symbol | Entrez ID | Probeset | Fold Difference | ||
---|---|---|---|---|---|---|
E18 | P28 | P70 | ||||
Glutaredoxin 3 |
Glrx3
| 30,926 | 6,923,313 | 12.22 | 6.08 | 3.27 |
RIKEN cDNA G730007D18 gene |
G730007D18Rik
| 100,038,502 | 7,006,004 | 3.66 | 2.87 | 2.41 |
Holliday junction recognition protein |
Hjurp
| 381,280 | 6,760,490 | 3.58 | 2.09 | 2.20 |
Synaptonemal complex central element protein 1 |
Syce1
| 74,075 | 6,972,143 | 3.00 | 2.06 | 2.13 |
Predicted gene 14,403 |
Gm14403
| 433,520 | 6,883,977 | 2.98 | 2.79 | 2.68 |
RIKEN cDNA 2,610,028 J07 gene |
2610028J07Rik
| 71,813 | 6,859,453 | 2.94 | 2.74 | 2.82 |
Microspherule protein 1 |
Mcrs1
| 51,812 | 6,918,337 | 2.62 | 2.03 | 2.30 |
RIKEN cDNA 2610507I01 gene |
2610507I01Rik
| 72,203 | 6,788,659 | 2.60 | 5.35 | 3.74 |
Olfactory receptor 1061 |
Olfr1061
| 259,022 | 6,888,451 | 2.51 | 2.81 | 3.77 |
Olfactory receptor 170 |
Olfr170
| 258,959 | 6,844,403 | 2.47 | 4.67 | 3.43 |
Uncharacterized protein C130090J04 |
C130090J04
| 328,049 | 6,860,959 | 2.36 | 2.12 | 2.20 |
RIKEN cDNA 6,330,403 K07 gene |
6330403K07Rik
| 103,712 | 6,789,483 | 2.32 | 7.15 | 6.56 |
Glucagon-like peptide 1 receptor |
Glp1r
| 14,652 | 6,849,762 | −2.02 | −2.33 | −2.06 |
Solute carrier family 39 (metal ion transporter), member 8 |
Slc39a8
| 67,547 | 6,901,634 | −2.07 | −2.43 | −2.51 |
Sodium channel, voltage-gated, type III, alpha |
Scn3a
| 20,269 | 6,887,324 | −2.08 | −5.31 | −4.49 |
Sterile alpha motif domain containing 9-like |
Samd9l
| 209,086 | 6,951,281 | −2.08 | −2.74 | −3.05 |
Interferon-induced protein 44 |
Ifi44
| 99,899 | 6,910,592 | −2.10 | −2.86 | −2.19 |
Transmembrane protein 181A |
Tmem181a
| 77,106 | 6,848,520 | −2.13 | −2.60 | −2.40 |
NME/NM23 family member 7 |
Nme7
| 171,567 | 6,754,701 | −2.13 | −2.49 | −2.70 |
Protein phosphatase 1, regulatory (inhibitor) subunit 3A |
Ppp1r3a
| 140,491 | 6,951,783 | −2.19 | −2.10 | −3.06 |
Megalencephalic leukoencephalopathy with subcortical cysts 1 homolog (human) |
Mlc1
| 170,790 | 6,837,773 | −2.24 | −2.78 | −3.13 |
Cytochrome P450, family 2, subfamily J, polypeptide 6 |
Cyp2j6
| 13,110 | 6,923,520 | −2.29 | −2.27 | −2.39 |
RIKEN cDNA A330076H08 gene |
A330076H08Rik
| 320,026 | 6,967,799 | −2.35 | −2.53 | −2.06 |
Aminolevulinate, delta-, dehydratase |
Alad
| 17,025 | 6,922,241 | −2.82 | −2.54 | −2.67 |
RIKEN cDNA D230046O15 gene |
D230046O15Rik
| 106,824 | 6,856,766 | −3.00 | −3.63 | −2.54 |
Nuclear paraspeckle assembly transcript 1 (non-protein coding) |
Neat1
| 66,961 | 6,867,774 | −3.54 | −3.45 | −2.70 |
Surfactant associated 2 |
Sfta2
| 433,102 | 6,850,183 | −4.02 | −2.19 | −2.23 |
Transmembrane protein 181A |
Tmem181a
| 77,106 | 6,812,918 | −4.26 | −4.08 | −3.40 |
RIKEN cDNA B930063I24 gene |
B930063I24Rik
| 319,330 | 6,756,745 | −5.06 | −4.33 | −6.19 |
G protein-coupled receptor 137B |
Gpr137b
| 83,924 | 7,003,081 | −5.39 | −5.38 | −3.06 |
Zinc finger, X-linked, duplicated B |
Zxdb
| 668,166 | 7,021,109 | −6.18 | −7.68 | −7.23 |
Kallikrein 1-related peptidase B21 |
Klk1b21
| 16,616 | 6,960,239 | −8.25 | −19.87 | −11.92 |
Hemolytic complement |
Hc
| 15,139 | 6,886,022 | −8.61 | −32.16 | −27.88 |
RIKEN cDNA A730017L22 gene |
A730017L22Rik
| 613,258 | 6,890,967 | −13.13 | −15.62 | −14.60 |
Evaluation of selected transcripts
Expression quantitative trait loci (eQTL) analysis
Trait | QTL Region | QTL Location (Basepair) | Gene Description | Gene symbol/location (basepair) | EntrezID | Fold Difference | ||
---|---|---|---|---|---|---|---|---|
E18 | P28 | P70 | ||||||
Lfnq3 Chromosome: 15 | ||||||||
TLC, TLC/Bw |
D15Mit85 to D15Mit105
| 40,313,626–72,500,731 | Leucine rich repeat containing 6 (testis) |
Lrrc6
(66379858–66,500,910) | 54,562 | −2.03 | ns | ns |
Lfnq4 Chromosome: 17 | ||||||||
TLC |
D17Mit156 to D17Mit234
| 10,987,597–39,053,168 | Radial spoke head 1 homolog (Chlamydomonas) |
Rsph1
(31255019–31,277,356) | 22,092 | ns | 2.81 | 2.63 |
Formyl peptide receptor 1 |
Fpr1
(17876471–17,883,939) | 14,293 | −2.05 | −1.75 | −2.26 | |||
ATP-binding cassette, sub-family G (WHITE), member 1 |
Abcg1
(31057694–31,117,984) | 11,307 | ns | −1.76 | −2.44 | |||
Surfactant associated 2 |
Sfta2
(35649708–35,650,569) | 433,102 | −4.02 | −2.19 | −2.23 | |||
Histocompatibility 2, class II antigen E beta |
H2-Eb1
(34305867–34,316,674) | 14,969 | ns | −2.23 | −2.21 | |||
Histocompatibility 2, D region locus 1 |
H2-D1
(35263094–35,267,497) | 14,964 | ns | 2.21 | 2.11 | |||
Histocompatibility 2, Q region locus 4 |
H2-Q4
(35379555–35,384,674) | 15,015 | −1.52 | −3.19 | −2.86 | |||
Gamma-aminobutyric acid (GABA) B receptor, 1 |
Gabbr1
(37045966–37,074,305) | 54,393 | −2.20 | −1.68 | −1.62 |