Although a growing body of epidemiologic evidence points to aberrant lung development as a significant contributing factor to chronic respiratory disease, including asthma, little information is available on the molecular determinants of respiratory susceptibility established during pulmonary maturation. One factor that may contribute to asthma susceptibility is the long maturation period of the lung that occurs postnatally, thus limiting human studies. To our knowledge, this is the first study reporting specific developmental differences in lung growth, respiratory function, and gene expression associated with atopic and AHR-specific phenotypes in the rat in the absence of provoked allergen exposures. We report on the interaction of temporal and strain-specific variance in expression of genes important in respiratory growth and development, ECM remodeling, and immune function. Our studies allow us to begin to assemble a genotype–phenotype correlation linking the program of postnatal lung development with specific asthma susceptibility traits.
Mounting evidence suggests that aberrant signals in inflamed airways influence epithelial cell proliferation (17
). Aberrant repair at the mucosal surface can, in turn, trigger abnormal behavior of the underlying mesenchyme, leading to ECM deposition and, eventually, airway remodeling. The developmental program of the lung in BN and Fisher rats model distinct asthma phenotypes that may antedate and relate to asthma susceptibility. Earlier findings demonstrated increased airway smooth muscle and innate AHR to MCh in adult Fisher rats (41
). We now show that newborn Fisher rats also display significantly elevated baseline airway resistance in the absence of allergen challenge. Notable also in the innately hyperresponsive Fisher rat was an increase in neutrophils in BAL. These findings are suggestive of the presence of an early, pre- or perinatal inflammatory process associated with innate AHR. Whether the early inflammation occurs coincident to or precedes the increased airway responsiveness is still unclear, and will require further work. By contrast, the BN rat, with tracheal goblet cell hyperplasia immediately after birth and increased respiratory epithelial proliferation at a time when normal rat lung epithelium has switched from a “proliferation” to a “differentiation” mode, may represent another example of aberrant pulmonary development associated with a proinflammatory phenotype.
That gene expression profiles of developing lungs would help distinguish these rat models was predictable. The highly distinct patterns of gene expression showing interaction of strain with age throughout the period of maximal alveolarization are of great interest. Linear regression identified variant expression of 1,376 genes. The Venn diagrams in highlight the temporal regulation of this variance. Analysis of gene-by-strain interactions also allowed us to identify genes with variant expression restricted to either Fisher or BN rat lung at a given time point. An important objective was to identify genes that contribute to the unique features of the individual models of AHR and atopy, and we therefore focused our validation experiments on these genes.
Subepithelial airway fibrosis, a unique feature of asthma, is a consequence of excessive deposition of collagen I, III, and V in the lamina reticularis (42
). The findings of increased Col3a1 mRNA and collagen III protein in postnatal lung tissue of BN rats is of particular interest in light of the findings of Fedorov and colleagues (43
), demonstrating significant increases in immunoreactive collagen III in the lamina reticularis of bronchial biopsies of children with asthma. Moreover, consistent with a role for mesenchymal epithelial signaling in determining the “phenotype” of atopic risk, is the finding of a parallel increase in Egfr (data not shown) in BN rat lungs.
Members of the Ly6 multigene family encode cell surface antigens with multiple specificities that have not been well characterized. Secreted Ly6/PLAUR (plasminogen activator urokinase receptor) domain containing protein 1 (Slurp1), a Ly6b family member, has been implicated in physiological and structural integrity of the skin (36
). Slurp1 is regulated by Egfr and retinoic acid—key modulators of lung development—as well as by IFN-γ. The dramatic increase in Ly6b mRNA in the BN rat is, to our knowledge, the first association of an Ly6 family member with the asthma-associated phenotype. We were unable to confirm an excess of Ly6b protein, as no rat antibody is currently available.
The combined findings of neutrophilia, increased α1-AT (encoded by Serpina1), and reduced Fcnb in the lungs of Fisher rats define a developmental respiratory phenotype distinct from that of the BN rat. Airway remodeling in asthma involves degradation of the ECM. Critical to the dynamic structure of the normal ECM is the equilibrium between synthesis and degradation of elastin, which is, in turn, dependent on the appropriate balance of elastase and its primary inhibitor, α1-AT. Neutrophils provide the major source of elastase in the lung. In sputum from subjects with asthma, significantly elevated levels of elastase and α1-AT correlate with the percentage of neutrophils (44
). Increased levels of α1-AT appear, however, insufficient to counterbalance the increased levels of elastase in these patients. An imbalance of α1-AT/elastase in the lung has been proposed to contribute to airway inflammation, thereby increasing AHR and impaired lung function in children with asthma (45
). The Fisher rat is innately hyperresponsive, and has increased ASM mass (21
). We suggest that neutrophilia in this model may be associated with an imbalance of α1-AT/elastase that is likely to contribute to AHR in Fisher rats in association with pre-/perinatal inflammation of unknown origin.
This is, to our knowledge, the first report of altered Fcnb expression, a molecule associated with innate immunity, in the developing lung in an animal model of innate airway hyperresponsiveness. The ficolins are pattern-recognition molecules that trigger the innate immune system upon binding to microbial surfaces (37
). Human M-ficolin, the homolog of rodent Fcnb, is exclusively expressed in phagocytic cells. M-ficolin, found in lung and spleen (46
), has been localized to secretory granules in the cytoplasm of peripheral neutrophils and monocytes, and in type II alveolar epithelial cells (47
). Mouse Fcnb is up-regulated in the lysozomes of activated macrophages (48
). Different developmental spatial–temporal expression patterns of Ficolin A (Fcna) and Fcnb suggest that these molecules are likely to play distinct roles in the pre- and postnatal stages (46
). Fcna and Fcnb also appear to have two distinct routes to eliminate the pathogens: one through the lectin pathway (human L-ficolin, Fcna), the other, a more primitive form of opsonophagocytosis performed by Fcnb (46
). Our demonstration of reduced Fcnb in the postnatal lung of Fisher rats suggests that innate AHR in this model may be attributed, at least in part, to deficiency of essential pattern-recognition molecules that serve as a protective interface, protecting the developing lung from pathogenesis and local inflammation.
The GR is the critical mediator of GC effects during lung development. GR also mediates effects of exogenous GC in the control of asthma exacerbations. At PN7, the BN rat lungs had increased GR mRNA and protein. High levels of immunoreactive nuclear GR were observed at PN7 and 14 by immunohistochemistry. These findings are interesting in light of studies showing that steroid-insensitive subjects with asthma have high GR numbers (49
), and that children with asthma express greater quantities of GR mRNA than do healthy children (52
). Increased expression of GR may improve the ability to regulate cytokine responses. Augmented GR may be one mechanism to improve GC responsiveness in a lung that is more vulnerable to inflammation. Notably, we also observed up-regulation of Tgfbr1l1 in the BN rat throughout postnatal development. Tgfb1l1 (also known as hydrogen peroxide-inducible clone 5 protein [Hic-5]) is an important coactivator of several nuclear proteins, including GR in lung epithelium (53
Our data suggest that inflammatory, immune, structural, and physiological (AHR) changes in the lung of the developing rat can be identified and differentiated by using the differing phenotypic susceptibilities of the inbred strains of rat, in conjunction with genomic approaches, in the developing animal. The very early identification of these abnormalities suggests that environmental and genetic processes, operating in utero and in early postnatal life, have a major influence on disease development later in life. Further dissection of these genomic signatures should give important clues that can then be tested in humans. For example, it would be of great interest to relate polymorphism in the genes identified in this investigation to the relevant asthma phenotypes in humans. Such work is currently underway.
We have used both direct assessment of specific genes previously implicated in asthma (GR, IPO13) and global gene expression analysis to identify genes associated with asthma susceptibility phenotypes in our rat models. Our findings affirm the importance of both approaches, and highlight both the power and the limitations of microarray analyses.
In conclusion, through the use of rat models of distinct asthma susceptibility phenotypes, we show unique respiratory gene expression profiles in the perinatal lung. The delineation of specific molecular determinants of respiratory susceptibility has implications for both pediatric and adult pulmonary health. The ultimate goal is to identify how in utero and early life exposures interact with pre-existing respiratory genotypes to initiate asthma pathogenesis to enable the development of early preventive strategies and novel therapeutic protocols targeted to effectively and safely reverse immune dysfunction.