CELA1 in Tracheal Lipopolysacharide Model AAT-deficient Emphysema
Borel, et al previously reported that AAT-deficient mice developed more emphysema at 14 days than wildtype mice after tracheal doses of 1 and 0.5 units of lipopolysacharide at days 1 and 10 (8). We intended to test whether Cela1-/-&AAT-deficient mice were protected from emphysema in this model. However, we observed a similar degree of emphysema in wild-type and AAT-deficient mice (Figure 1A), with male and female mice similarly affected. We then performed a dose titration experiment with 1&0.5, 2&1, 5&2.5, and 10&5 units of LPS with evaluation at 21 days instead of 14 and evaluated airspace sizes using a previously described method that generates descriptive statistics of airspace sizes in tile-scanned images of all lobes (6, 10). There was no clear dose-effect and wildtype mice experienced as much or more emphysema as AAT-deficient mice (Supplemental Figure 1). Since in our hands, a previously reported LPS-based model of AAT-deficient emphysema could not be reproduced, we developed a different model of AAT-deficient emphysema.
Low-Dose PPE Model of AAT-deficient Emphysema
Tracheal porcine pancreatic elastase (PPE) is a well-established model that causes significant injury and, in some studies, progressive emphysema (20, 21). In most studies, 1-2 units of PPE is administered tracheally, and emphysema is assessed at or before 21 days. We administered 2 units of PPE to AAT-deficient mice, and 6 of 8 mice died with extensive lung hemorrhage on necropsy. We performed a dose titration experiment and discovered that 0.2 units of tracheal PPE caused substantial emphysema in AAT-deficient mice at 21 days while wild type mice had virtually no emphysema at this dose. Furthermore, emphysema progressed over time (Figure 1B&C, Supplemental Figure 2). We therefore administered 0.2 units of tracheal PPE to AAT-deficient and Cela1-/-&AAT-deficient mice to test for a role for CELA1 in emphysema in the context of AAT-deficiency.
Lung Proteomic Changes in Low-Dose PPE Model
We performed unbiased proteomics of wild type, AAT-deficient, Cela1-/- and Cela1-/-&AAT-deficient lungs (five per genotype) 42 days after low-dose PPE to understand the differential response to injury. In comparing wildtype and AAT-deficient lungs, only one protein had an adjusted p-value<0.1 and at least 2-fold change. This protein was the AAT isoform Serpina1d that is one of the five AAT paralogues deleted in this mouse(8). Seventeen genes had protein levels at least 2-fold lower in AAT-/- compared to wildtype lungs, and 71 genes were at least 2-fold higher (Supplemental Table 1). As expected, many of the most downregulated genes in this analysis were AAT paralogues (e.g., Serpina1a, Serpina1b, and Serpina1d). Genes of many of the upregulated proteins were related to Rho & Rac1 GTPases (Abr and Srgap2), thrombosis and complement (Klkb1, C2 and Mpi), and protein oxidation (Hacd2 and Ermp1) (Figure 2A). Gene ontogeny enrichment for upregulated protein identified neutrophil degranulation, functions related to innate immunity, and nucleotide biosynthesis pathways as the most activated (Figure 2B). The statistical significance of downregulated pathways was greater and largely involved processes related to mRNA translation (Figure 2C). The protein-protein interaction network of both increased and decreased proteins showed many edges related to ribosomes and translation, and that many of the proteins related to neutrophil degranulation were either not adjacent in the network or not within the protein-protein interaction network at all (Figure 2D, Supplemental Figure 3). These data are consistent with the expected pulmonary biology of AAT-deficiency with regards to AAT synthesis and innate immunity but also some unexpected changes with regards to protein synthesis.
CELA1 in Low-Dose PPE Model of AAT-Deficient Emphysema
We tested for a role for CELA1 in progressive emphysema by quantifying mean linear intercepts at 21, 42, and 120 days after low-dose PPE in AAT-deficient and Cela1-/-&AAT-deficient mice. Compared to AAT-deficient mice, Cela1-/-&AAT-deficient mice had less emphysema at 21, 42, and 120 days after low dose PPE administration (Figure 3A&B). Overtime, AAT-deficient but not Cela1-/-&AAT-deficient mice experienced progressive emphysema; between days 42 and 120, AAT-deficient mice had a 31% increase in mean linear intercept (p=0.002) while the increases in this groups between 21 and 42 days or and in the Cela1-/-&AAT-deficient mice over time were smaller and not significant. We observed that at 42 and 120 days, female Cela1-/-&AAT-deficient but not female AAT-deficient mice had more emphysema than male mice (p=0.02 and p=0.01 respectively). However, when analyzing by sex, the 120-day MLI differences remained significant for males (p=0.003) and females (p=0.03). These data indicate that CELA1 plays a role in AAT-deficient emphysema and perhaps this role is more pronounced in males than females.
Impact of CELA1 on Lung Proteome in Low-Dose PPE Model
We then compared the lung proteomes of Cela1-/-&AAT-deficient and AAT-deficient mice 42 days after tracheal administration of low-dose PPE. There were only two proteins with adjusted p-values of less than 0.1 and at least 2-fold change. The genes of these proteins were UBX domain-containing protein 7 and Ig alpha chain C region. There were 19 proteins that were at least 2-fold increased and 28 proteins that were at least 2-fold decreased in Cela1-/-&AAT-deficient compared to AAT-/- mice (Supplemental Table 2). Among the proteins with increased abundance, some of the most significant were from genes related to oxidative stress (Glo1, Msra, and Aldh1b1) and acute inflammation (Saa4 and C8a). Proteins that were most increased were from genes related to ubiquitination (Ubxn7 and Ube2o), elastin fibers (Mmrn2), and neurotransmitters (Nostrin, Palmd, Agap3, and Vps33a) (Figure 4A). Gene ontogeny enrichment for proteins that were increased showed abundance of Rho GTPase proteins, nitric oxide signaling, and muscle-related proteins (Figure 4B). Downregulated processes included elastic fiber formation and neutrophil degranulation (Figure 4C). Protein-protein interaction analysis of increased and decreased proteins identified a number of small but connected networks related to elastin fibers, neutrophil degranulation, and glutathione metabolism (Figure 4D). These data demonstrate that Cela1 mediates protein-level differences in elastin microfibril associated proteins in this AAT-deficient model of emphysema and that there are also potential differences in neutrophil activity and oxidative state.
CELA1 in Cigarette Smoke Induced Emphysema
As cigarette smoke exposure is highly associated with the development of emphysema in AAT-deficient individuals, we performed whole body cigarette smoke exposure to AAT-deficient and Cela1-/-&AAT-deficient mice five days a week for 10 months. Contrary to our expectations, and Cela1-/-&AAT-deficient mice were not protected from CS-induced emphysema and Cela1-/-&AAT-deficient mice had more evidence of airspace destruction than AAT-deficient mice (Figure 5A&B). Given the proteomic findings of altered neutrophil associated and chemotaxis proteins in Cela1-/-&AAT-deficient mice, we hypothesized neutrophil activity might be increased. However, myeloperoxidase activity was reduced by more than half in Cela1-/-&AAT-deficient mice compared to AAT-deficient mice in response to CS and comparable to activity levels seen 42 days after PPE and PBS. (Figure 5C). While the mechanism is unclear, these data indicate the absence of Cela1 enhances airspace destruction in AAT-deficient mice response to sustained cigarette smoke exposure.
CELA1 in Age-Related Alveolar Simplification
In humans and mice, aging is associated with slowly progressive airspace simplification, and AAT-deficient mice were reported to have more rapid simplification that wildtype (8). We validated these findings and discovered that Cela1-/-&AAT-deficient mice experienced a level of age-related airspace simplification similar to that of wildtype mice (Figure 6).