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Lung (2013) 191:669–675 DOI 10.1007/s00408-013-9504-2
COPD
Functional Characterization of a Porcine Emphysema Model Camilla Sichlau Bruun • Louise Kruse Jensen • Pa´ll Skuli Leifsson Jens Nielsen • Susanna Cirera • Claus Bøttcher Jørgensen • Henrik Elvang Jensen • Merete Fredholm
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Received: 19 June 2013 / Accepted: 17 August 2013 / Published online: 17 September 2013 Ó Springer Science+Business Media New York 2013
Abstract Background Lung emphysema is a central feature of chronic obstructive pulmonary disease (COPD), a frequent human disease worldwide. Cigarette smoking is the major cause of COPD, but genetic predisposition seems to be an important factor. Mutations in surfactant protein genes have been linked to COPD phenotypes in humans. Also, the catalytic activities of metalloproteinases (MMPs) are central in the pathogenesis of emphysema/COPD. Especially MMP9, but also MMP2, MMP7, and MMP12 seem to be involved in human emphysema. MMP12-/- mice are protected from smoke-induced emphysema. ITGB6-/mice spontaneously develop age-related lung emphysema due to lack of ITGB6-TGF-b1 regulation of the MMP12 expression. Methods A mutated pig phenotype characterized by agerelated lung emphysema and resembling the ITGB6-/mouse has been described previously. To investigate the emphysema pathogenesis in this pig model, we examined the expression of MMP2, MMP7, MMP9, MMP12, and TGF-b1 by quantitative PCR (qPCR). In addition,
C. S. Bruun (&) S. Cirera C. B. Jørgensen M. Fredholm Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Grønnega˚rdsvej 3, 1870 Frederiksberg C, Denmark e-mail: cvb@sund.ku.dk L. K. Jensen P. S. Leifsson H. E. Jensen Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Ridebanevej 3, 1870 Frederiksberg C, Denmark J. Nielsen National Veterinary Institute, Technical University of Denmark, Lindholm, 4771 Kalvehave, Denmark
immunohistochemical stainings of the lungs with SP-B, SP-C, MMP9, and MMP12 antibodies were performed. The haematologic/immunologic status of the pigs also was studied. Results The qPCR study showed no difference between pigs with and without emphysema, and no systemic differences were indicated by the haematologic and immunologic studies. However, the immunohistochemical stainings showed an increased expression of MMP9 and MMP12 in older, mutated pigs (with emphysema) compared with normal and young mutated pigs (without emphysema). Conclusions The pig model is comparable to human emphysema patients and the ITGB6-/- mouse model with respect to both morphology and functionality. Keywords Chronic obstructive pulmonary disease Emphysema Matrix metalloproteinases Pig
Lung emphysema is characterized by alveolar enlargement due to degeneration of the alveolar septa. Emphysema is a central hallmark of chronic obstructive pulmonary disease (COPD) in humans, a progressive respiratory condition characterized by inflammation and obstruction of the airways and destruction of the alveoli. COPD has been estimated to become the fifth-leading human disease worldwide in 2020 [1]. Cigarette smoking is the one most important cause of COPD, but because only 10–20 % of all heavy smokers develop COPD, other factors seem to be of importance. Several epidemiologic and genetic studies have provided evidence that genetic predisposition plays an important role [2, 3]. Pulmonary surfactant is a phospholipid-rich film covering the air–liquid interface of the lung reducing the surface tension to prevent the alveoli from collapsing. The
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surfactant proteins (SPs) are the core peptide components of the surfactant [4]. Mutations in SP genes have been associated with emphysema/COPD phenotypes [5, 6], and it has been shown that SP-C -/- mice develop emphysema [7]. Matrix metalloproteinases (MMPs) are other factors known to be central in the pathogenesis of emphysema/ COPD. MMPs are proteolytic enzymes implicated in many physiological and pathological processes. In the lungs, they are produced by several cell types, including pneumocytes, smooth muscle cells, neutrophils, and macrophages, and in healthy individuals their activity is balanced by tissue inhibitors of MMPs [8]. Their catalytic activity has been linked to the tissue destruction observed in emphysema. Especially MMP9 but also MMP2, MMP7, and MMP12 seem central in human emphysema pathogenesis [8–11]. Wild-type mice exposed to cigarette smoke develop emphysema, whereas mice lacking MMP12 (MMP12-/-) are protected against smoke induced emphysema [12]. Conversely, ITGB6-/- mice spontaneously develop age related lung emphysema [13]. Here, the condition is caused by an elevated expression of MMP12. The high level of MMP12 in these mice is caused by a lack of active transforming growth factor beta-1 (TGF-b1), which regulates the expression of MMP12 [14]. ITGB6-/- mice are unable to produce the b-subunit of integrin avb6, which is central in activating latent TGF-b1 [15]. The expression of MMP12 therefore is increased 200-fold in these mice due to a lack of TGF-b1-regulation [14]. Even if single proteases may be considered central determinants in the pathogenesis of emphysema, the human variant of the disease is now considered a complex inflammatory disease involving several types of lymphocytes. The level of alveolar cell apoptosis has been found abnormally elevated in human emphysema patients. The mechanisms underlying the apoptosis are not fully revealed but may involve autoimmune components [16]. A mutated pig phenotype characterized by juvenile hairlessness and age-related lung emphysema has been described previously [17]. The emphysema develops slowly and progresses with age and is thereby comparable to the emphysema component of human COPD. The pig phenotype also resembles that of the ITGB6-/- mice and the candidate region on SSC15 (comprising both ITGAV and ITGB6 encoding the integrin avb6 receptor) has been confirmed but functional and molecular studies have excluded the two candidate genes [17]. Linkage studies in an extended experimental population have now excluded ITGAV and ITGB6 as candidate genes (unpublished). In this study, the mutated pigs are further characterized in a functional comparison to the ITGB6-/- mouse model and to some of the most prominent features of human emphysema patients.
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Materials and Methods Animal Material All samples were collected by veterinarians and in accordance with the institutional guidelines for animal welfare and ethics. In this study, pigs representing the three different genotypes have been used: HH: homozygous for the mutation; Hh: heterozygous for the mutation; and hh: homozygous normal. The pigs used for quantitative PCR (qPCR) and immunohistochemistry are outlined in Table 1. Furthermore, 23 pigs were used for haematologic and immunologic studies. Quantitative PCR qPCR was performed to investigate the expression of MMP2, MMP7, MMP9, MMP12, and TGF-b1 in lung macrophages harvested by lung lavage immediately after euthanization [18] of the following pigs: three HH (198, 204, and 216), three Hh (188, 209, and 218), and one hh (208). RNA was isolated from each sample of lung macrophages using TRI REAGENTÒ, (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions. Three RNA replicates were isolated from the macrophage sample of the hh pig (208). Contaminating genomic DNA was degraded using Qiagen RNeasy mini kit following the manufacturer’s recommendations (Qiagen, GmbH, Hilden, Germany). cDNAs were synthesized from 1 lg of total RNA using ImProm-IITM Reverse Transcription System (Promega, Madison, WI, USA) and a mixture of random hexamers: oligodT in a ratio of 3:1, according to the manufacturer’s recommendations. Before use in qPCR, all cDNAs were diluted 1:8 with H2O.
Table 1 Genotype and age of pigs used for qPCR and IHC Genotype HH
Hh
hh
Pig ID
Age
104
C1 year
149
B2 months
198
C1 year
204
C1 year
216
B2 months
134
B2 months
56
C1 year
188 209
C1 year C1 year
218
B2 months
141
B2 months
So1
C1 year
208
7 months
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qPCR primers were designed to anneal in neighbouring exons separated by large introns in the genomic DNA. Primers were designed using Primer3 software [19].
the gene effect (MMP2, MMP7, MMP9, MMP12, and TGF-B1), genotype(gen) is the effect of genotype (HH, Hh and hh) nested within each gene group and e is the standard error. A significance level of 0.05 was chosen.
Primer Sequences: Immunohistochemistry 0
0
MMP12_qpcr2F: 5 -CATGATGCACAAACCTCGAT-3 MMP12_qpcr2R: 50 -TGGATGGCGTAGTCAACATC-30 MMP7_qpcrF: 50 -GAACAGGCTCAGGGCTAT-30 MMP7_qpcrR: 50 -TGCAACATCTGGTACTCC-30 MMP9_qpcrF: 50 -CGGGAGACCTACGAACCAAT-30 MMP9_qpcrR: 50 -TCCAGGGACTGCTTTCTGTC-30 MMP2_qF: 50 -CTGTGCAATACCTGAACACC-30 MMP2_qR: 50 -TGCATCTTCTTTAGTGTGTCC-30 TGFB1_qpcrF: 50 -CGAGCCCTGGATACCAACT-30 TGFB1_qpcrR: 50 -GCAGAAATTGGCATGGTAG-30 Ribosomal protein L4, hypoxanthine phosphoribosyltransferase, and b-actin were included in this study as reference genes, according to Nyga˚rd et al. [20]. For each target gene, a standard curve was constructed using the purified PCR product (QIAquickÒ PCR Purification Kit (250) Qiagen, GmbH, Hilden, Germany) generated for each specific primer pair. Single reactions were prepared for each cDNA along with each standard curve serial, a genomic DNA sample and a nontemplate control using the BrilliantÒ SYBRÒ Green Master Mix (Stratagene, Santa Clara, CA). Each reaction consisted of 20 ll containing 2 ll of cDNA, 5–20 pmol of each primer, and 10 ll of master mix. The real-time qPCR was run on an Mx3000PTM thermocycler (Stratagene). The cycling conditions were 1 cycle of denaturation at 95 °C/10 min, followed by 40 three-segment cycles of amplification (95 °C/ 30 s, 55 °C/1 min, 72 °C/1 min) where the fluorescence was automatically measured at each cycle and one threesegment cycle of melting curve at the end (95 °C/1 min, 55 °C/30 s, 95 °C/30 s). The baseline adjustment method of the MxPro software (Stratagene) was used to determine the Cq in each reaction. Relative quantities (calculated from each standard curve) for the three reference genes were used in the GeNorm program [21] to calculate a normalization factor (NF). Relative quantities for all target genes were normalized by the NF. Subsequently, fold changes were calculated in relation to the lowest expressed sample. To ensure normal distribution, the fold changes were LOG transformed. To test for statistically significant differences of the mean of the LOG transformed fold changes between the three genotypes, a variance analysis using the GML procedure in SAS was applied: Yijk ¼ l þ litterj þ genek þ genotypeðgenÞl þ eijkl where Yijkl is the LOG transformed fold change for animal i, l is the mean, litter is the effect of litter origin, gene is
Animal Material Blocks of paraffin embedded lung tissue sampled from the diaphragmatic lobes of one young pig and one old pig of each of the three genotypes (HH: 104 and 149; Hh: 56 and 134 and hh: So1 and 141) were used for immunohistochemistry. Antibodies and Staining The production of pig-specific MMP9 and MMP12 antibodies and the immunohistochemical staining procedure have been described recently [22]. The protocol for immunohistochemical staining of SP-C also has been described previously [23] and the same protocol was applied for staining of SP-B (polyclonal rabbit anti-surfactant protein-B antibody, Upstate Biotechnology, Lake Placid, NY) with the following dilution of the primary antibodies 1:1000 (SP-C) and 1:250 (SP-B). Immunolabeling of the lung tissues was evaluated for both intensity and cellular distribution patterns. The intensity was scored semiquantitatively: no immunoreactions (-), minimal immunoreactions (?), and moderate immunoreactions (??). Haematologic and Immunologic Characterization Animal Material Twenty-three weaned pigs from three litters representing all three genotypes, i.e., 6 HH, 11 Hh, and 6 hh, were included. From the age of approximately 6 weeks, peripheral blood samples were collected once a week for a
Table 2 Mean LOG transformed fold changes for each genotype group (HH, Hh, and hh) and p values (t) HH
p value (t)
Hh
p value (t)
hh
MMP2
1.858
0.335
1.387
0.583
1.748
MMP7
1.591
0.484
2.056
0.114
0.614
MMP9
2.771
0.087
2.187
0.227
2.991
MMP12
0.868
0.963
1.117
0.245
0.343
TGF-B1
0.604
0.469
0.307
0.284
0.915
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Table 3 Semiquantitative scoring of labelling for MMP-9 and MMP-12 in old and young pigs within the three genotype groups (HH, Hh, and hh) Lung cell type Alveolar epithelium
Alveolar macrophages
Glandular epithelium
Bronchial/bronchiolar epithelium
MMP9
MMP12
MMP9
MMP12
MMP9
MMP12
MMP9
MMP12
HH old (104)
?
?
??
??
?
-
??
-
HH young (149)
?
?
?
?
?
-
?
-
Hh old (56)
?
?
??
??
-
-
??
-
Hh young (134)
?
?
?
?
?
-
?
-
hh old (So1)
?
?
?
?
-
-
?
-
hh young (141)
?
?
?
?
-
-
?
-
period of 4 weeks in order to study putative divergences between the genotypes. White Blood Cell (WBC), Red Blood Cells (RBC), and Platelets (PTL) Counts, Haemoglobin (HGB) Concentration WBC counts together with determinations of numbers of RBC, PTL, and HGB concentration were performed on EDTA-stabilised blood samples using a semiautomated animal blood cell counter (Vet abcTM, ABX, Montpellier, France). All samples were counted twice, and the mean value was calculated. Flow Cytometry Analyses The phenotyping of leukocytes in peripheral blood was done by flow cytometry, as previously described [24]. Selection of gates was as described elsewhere [25]. This permitted the identification of the T cell subpopulations as CD3?CD4?CD8- naı¨ve Th cells, CD3?CD4?CD8? as memory/activated Th cells, CD3?CD4-CD8? as Tc cells, CD3?CD4-CD8low/- as cd T cells. Natural killer cells (NK) were defined as CD3-CD4-CD8? cells.
but had no significant effect (p [ 0.05). There was no significant effect of genotype (p [ 0.05). In conclusion, there were no significant differences in expression of the studied candidate genes among the three genotypes. Immunohistochemistry Results are presented in Table 3 and Figs. 1, 2. MMP9 was expressed in both alveolar, bronchiolar, bronchial, and glandular epithelial cells and macrophages. Compared with the other animals, a higher expression was observed in the older HH and Hh animals in the alveolar macrophages and bronchial/bronchiolar epithelium. MMP12 was only expressed in the alveolar epithelial cells and the alveolar macrophages. Compared with the other animals, a higher expression also was observed in the older HH and Hh animals in the alveolar macrophages. Positive immunolabeling of SP-B and SP-C was restricted to pulmonary type II cells, but occasionally
Statistical Analysis Data analysis was performed using GraphPad In Stat version 3.00 (GraphPad Software, San Diego, CA). Student’s t test, or alternatively Welch test (W), was used for comparison between means of groups.
Results Quantitative PCR The LOG transformed fold changes and p values are listed in Table 2. Litter was included in the model as a parameter
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Fig. 1 Immunohistochemical staining of MMP12 in lung tissue from an old Hh pig. Multiple alveolar macrophages are positive. Bar 100 lm
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Discussion
Fig. 2 Immunohistochemical staining of MMP9 in lung tissue from an old HH pig. In the emphysematous tissue, positive alveolar epithelial cells are present. Bar 100 lm
positive cells comparable to Clara cells were also seen in the bronchiolar epithelium. In all cases, the positive immunolabeling was localized to granules within the cytoplasm. However, no difference in the level of expression was seen between the three genotypes. Haematologic and Immunologic Characterization During the observation period, no significant differences could be demonstrated between the three genotypes for levels of WBC (Fig. 3a), RBC, PLT, HGB, lymphocytes, monocytes, granulocytes, CD3?, CD4?, CD8?, naı¨ve Th, memory/activated Th, Tc, and NK cells (data not shown). During the observation period, the level of cd T cells in the mutated pigs constantly remained markedly lower compared with the normal pigs (Fig. 3b). However, statistical difference could not be demonstrated.
The haematologic and immunologic investigations proved no significant differences between the normal and mutated pigs. These observations indicate that the development of emphysema in the mutated pigs is unlikely to be linked to systemic immunologic or autoimmunologic processes. However, it remains to be elucidated whether the decreased levels of cd T cells in the mutated pigs at all examined age stages represent a recurrent difference, and, if so, whether low levels of cd T cells may play any role in the development of emphysema in this type of mutated pigs. The study period, however, seems to have been adequate due to the constant difference in quantities of the cd T-cell population. The expression of SP-B and SP-C was evaluated by immunohistochemistry (IHC), which showed no difference between any of the genotypes. SP-B and SP-C are not mapped to the candidate region, but an increased or decreased expression in pigs with emphysema would have indicated that these surfactant proteins were involved in the pathogenesis in the pig model. Although integrin avb6 is functional in these pigs, it is not possible to exclude the integrin avb6 -TGF-b1-MMP12 pathway from being involved in the pathogenesis of the phenotype we observe. To investigate the expression of TGF-b1 and MMP12 qPCR was performed on macrophages lavaged from the lungs of normal and affected pigs. Using lavaged lung macrophages instead of lung tissue ensured that all areas of the lung were represented in the analysis. The qPCR data, however, did not reveal any difference in the expression of MMP12 and TGF-b1 between normal and affected pigs. MMP7 and MMP9, which are known to be upregulated in human emphysema patients, also were expressed at an equal level in the three groups of pigs. Contrasting results were, however, obtained
Fig. 3 Analyses of peripheral blood demonstrated a no significant difference in WBC levels between genotypes and b lower, but statistically not significant, levels of cd T cells in HH (X–X) and Hh (open square) pigs compared to the hh (filled triangle) pigs
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by IHC, which showed an increased expression of both MMP9 and MMP12 in macrophages and the bronchial/ bronchiolar epithelium of the old Hh and HH pigs. The emphysema in these pigs is confined to local areas in the lung, and it is known that the expression and activity of MMPs can be restricted to specific locations in emphysema affected areas [26]. Because the qPCR is performed on lavaged macrophages from the whole lung, an elevated MMP-expression in a subpopulation of macrophages may go unnoticed due to underrepresentation of macrophages with increased expression. The increased expression in bronchial/bronchiolar epithelium is of course not picked up when using lavaged macrophages. This underlines IHC as the assay of choice for expression analysis in this case. Although the number of animals in each genotype group was restricted, the comparability of our results with human expression levels and the agreement with the described functionality of MMP9 and MMP12 strengthens their reliability.
7.
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9. 10.
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Conclusions Using pig-specific MMP9 and MMP12 antibodies [22], we were able to show that these two proteins are overexpressed in pig lung tissue with emphysema. The results are in accordance with that described in human emphysema patients. The fact that both MMP9 and MMP12 are upregulated in this pig model makes it comparable especially to human emphysema patients but also to the ITGB6-/mouse model.
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Acknowledgments Special thanks to Thomas Mark for assistance with the qPCR statistics. 18. Conflict of interest
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