Lung injury caused by an imbalance in the release of proinflammatory and anti-inflammatory cytokines, occurring as a result of volutrauma or barotrauma, sepsis, pulmonary edema, or hyperoxia, damages the immature lung of the NB. This is followed either by healing (resolution of injury) or by repair of the lung (BPD) (2
). The pathology of the “new” BPD involves dysregulated angiogenesis and impaired alveolarization (1
). Limited information is available regarding the mechanisms that cause structural remodeling in BPD. Although the infiltration of lung tissue by inflammatory cells was demonstrated in hyperoxia-induced lung injury in adult mice, the key finding in early stages before neutrophil infiltration was an increased expression of IFNγ in lungs (10
Hence, we postulated that increased IFNγ could constitute a critical early response molecule in the pathogenesis of BPD. Using developmentally appropriate animal models, we showed that IFNγ-induced impaired alveolarization is MMP9-dependent. Specifically, we confirmed that IFNγ increases in response to hyperoxia exposure in the developing NB lung. Next, using a unique, lung-targeted, externally regulatable TTG murine model of IFNγ overexpression, we show impaired alveolarization. The IFNγ-induced NB murine lung phenotype mirrors human BPD, as exemplified by alveolar simplification. Our study showed that IFNγ causes structural remodeling along with the activation of enzymes involved in cell death, angiogenesis, and protein degradation. In addition, significant rescue of the pulmonary phenotype occurs secondary to a partial deficiency of MMP9, and is associated with a reversal of changes evident in cell death pathways and angiogenic markers. The clinical relevance of our work is indicated by the increased mortality of NB mice exposed to hyperoxia, in the presence of increased IFNγ, and with the increased mortality being rescued by a partial deficiency of MMP9. Furthermore, the clinical significance of our findings was supported by data regarding the enhanced expression of downstream targets of IFNγ in murine and baboon models and in the human neonate with BPD.
A significant increase of IFNγ in BALF within 48–72 hours of exposure to hyperoxia was reported in adult mice (11
). Hyperoxia-induced lung injury is believed to result from the direct toxic effects of reactive oxygen species and the indirect effects of inflammatory cell activation, with a resultant synthesis of cytokines. A previous study in adult mice (24
) showed that the depletion of IFNγ resulted in attenuated neutrophil accumulation in the lung airspace and in an inhibition of hyperoxia-induced increases in pulmonary alveolar permeability in the early phase of hyperoxic injury. Whereas earlier reports in adult mice also noted that increased caspases, cathepsins, and MMPs (23
) followed increased IFNγ in the lung, this finding had not been established in the developing NB lung. This is important, because the developmental regulation of molecules and their targets is well-described. In fact, significant differences occur in the response to the same stimulus and molecule by the mature versus the developing lung, as shown elsewhere (41
). Hence, independent testing in developmentally appropriate in vivo
modeling systems is important, as opposed to interpolating the results of in vitro
experiments or data derived from adults.
We emphasize that we used a neonatal murine model of hyperoxia-induced alveolar development to mimic human BPD. Differences secondary to additional volutrauma/barotrauma may be attributable to invasive ventilation in preterm human lungs. However, the baboon model attempts to mimic closely what is practiced in most neonatal units, and our data support a role for IFNγ-induced effects.
Angiogenic factors play a significant role in remodeling and tissue repair (36
). We found a significant elevation of Ang2 in lung tissue, secondary to IFNγ induction. We previously reported on the important role of Ang2 in hyperoxia-induced cell death and lung injury (36
) and human BPD (36
). IFNγ-induced Ang2 may affect caspase-dependent cell death pathways, and contribute to alveolar simplification. It may also contribute to the increased mortality in NB IFNγ TTG mice exposed to hyperoxia.
Although we could not find any reports of a role for IFNγ in lung tissue of animal models or human BPD, elevated serum concentrations of IFNγ were associated in extremely preterm infants with BPD or death (45
). We are not aware of any studies of IFNγ concentrations in the TAs of premature infants, to help us predict the kinetics of IFNγ production and release. IFNγ produced in the lung in preterm neonates may dissipate quickly (before our collection of the TA sample), and hence we could only detect it in a few samples (albeit all with BPD or death). However, after IFNγ is released, it triggers a cascade of specific events, including the production of downstream markers such as IP9 and IP10. Therefore, we focused on detecting those markers in human and baboon lungs. Elevated MMP9 concentrations were detected in BALF collected from preterm infants who subsequently developed BPD (46
), in alveolar Type II epithelial cells in adult rats after 85% hyperoxia exposure (47
), and in the lungs of NB rat pups after hyperoxic exposure (48
). Increased MMP concentrations were evident when IFNγ was overexpressed in our animals. MMP9 caused damage by degrading the extracellular matrix (ECM). In the lung, the ECM is composed of Type I collagen, and Type III collagen is also present in the alveolar walls. Both are substrates for MMP9. The destruction of the ECM may result in impaired alveolar and vascular development (50
). In NB IFNγ TTG MMP9HZ mice, we observed a reversal of the damage noted in the NB IFNγ TTG mice, despite their similarly elevated IFNγ lung concentrations. Our data are in accordance with studies of NB MMP9 null mutant mice exposed to hyperoxia (27
), but not with the IL-1β–induced murine model of BPD, where a deficiency of MMP9 worsened alveolar architecture (52
). Variations in the types, timing, and amounts of cytokines under study could account for the different results. Importantly, the baboon model (53
) and human BPD (28
) studies consistently showed increased lung MMP9 concentrations. We also detected the elevated expression of other MMPs (i.e., MMPs 2, 12, and 14 in our NB IFNγ TTG murine lungs) (). The contributions of these other MMPs to the “BPD” phenotype call for further experimentation, which was beyond the scope of the present study.
sepsis (chorioamnionitis) also contributes to the pathogenesis of BPD (1
). In the endotoxin-exposed lamb model of chorioamnionitis, increased IP10 was evident in the bronchiolar epithelium of preterm lambs, and was speculated to contribute to lung injury (54
). Our data of increased IP10 and IP9 in murine, baboon, and human BPD lungs suggest that IFNγ may be contribute to lung injury and repair responses.
The present study enhances our understanding of the mechanism of IFNγ-induced tissue remodeling and destruction. We demonstrate that IFNγ, a potent inducer of multiple caspases, proteases, and cathepsins, causes injury in the developing lung, and that these responses are partly mediated by an MMP9-dependent mechanism. By demonstrating an amelioration of lung injury after reducing the activity of MMP9 in NB IFNγ TTG MMP9HZ mice, we show that MMP9-induced ECM damage is a critical event in IFNγ-induced lung pathology. In a cause-and-effect fashion, states of inflammation, enhanced protease activity, cellular DNA injury, cell death, and tissue destruction define a novel pathway of IFNγ-induced tissue remodeling that may be operative in diverse biologic settings. In neonatal lung disease, these observations provide the first pathogenetic construct that can unify the seemingly disparate inflammatory, protease/antiprotease, and cell death theories of disease pathogenesis.
BPD of the premature neonate and emphysema of the adult lung are characterized by increased airspace size and respiratory insufficiency. BPD and emphysema exhibit striking similarities in their pathophysiology, including the effects of oxidative stress, sustained inflammation, enhanced apoptosis, protease–antiprotease imbalance, elastic fiber deterioration, and altered microvascularization (55
). Hence, our findings may also be relevant to pulmonary emphysema and chronic obstructive lung disease (COPD). In fact, emerging data indicate that BPD may be a precursor to COPD as infants become older (1
In conclusion, our study highlights an MMP9-dependent lung injury pathway, and demonstrates that MMP9 plays a key role in IFNγ-induced alveolar remodeling and chronic lung disease. These findings validate IFNγ, its downstream targets (including MMP9), and cell injury response regulators as potential targets for therapies directed at the treatment of neonatal lung disease, COPD, and other IFNγ-mediated diseases as diverse as atherosclerosis, rheumatoid arthritis, and Crohn's disease. Additional investigations of the potential therapeutic utility of anti-IFNγ–based and anti-MMP9–based therapies for lung disease are warranted.