Generation of HGPS/hACE2 mouse model
To generate a mouse strain carrying HGPS variants, we utilized CRISPR based base-editing system to introduce a homozygous c.1827 C < T (p.Gly609Gly) mutation in exon 11 of mouse Lmna gene, equivalent to the c.1824 C < T (p.Gly608Gly) mutation found in human LMNA, in mouse embryonic stem cells (mESCs) with humanized ACE218 (Fig. 1A and Supplementary Fig. 1A). Karyotyping and immunofluorescent staining of Lmna mutant mESCs confirmed correct chromosome arrangements and pluripotency (Supplementary Fig. 1C and D). To avoid the breeding issues and ensure an adequate supply of mice for infection studies, we employed tetraploid complementation using our modified mESCs, and generated humanized ACE2 mice carrying homozygous HGPS variants (Fig. 1A and C). These mice exhibit an alternative splicing event observed in HGPS patients (Supplementary Fig. 1B), resulting in the absence of Lamin A protein and the presence of the spliced Lamin protein known as progerin (Fig. B). Similar to HGPS patients, HGPS hACE2 mice display small body size, reduced body weight, and shortened life span (Fig. 1C-E), indicating a premature aging phenotype in these mice.
Tissues such as bone, heart, spleen, and thymus exhibit varying levels of defects in HGPS patients, while not much is known for the lung5. To further investigate the impact of HGPS on the respiratory system in HGPS mice, we carried out transcriptome analysis of lung tissues from 2-month-old wild type and HGPS/hACE2 mice (Fig. 1F). Consistent with previous studies, we observed up-regulation of genes involved in P53-mediated DNA damage pathway including Gadd45a, Gadd45b, Gadd45g, Atf3, Btg2, and Cdkn1a (Supplementary Fig. 1E). Gene Ontology (GO) analysis revealed that immune-related pathways were more enriched in normal hACE2 mice compared to the HGPS ones, indicating a diminished immune response in HGPS mice (Fig. 1G). Conversely, multiple developmental pathways such as epidermis, vasculature, bone marrow, and adipose development were more enriched in HGPS compared to normal hACE2 mice, suggesting a premature developmental stage of lung in HGPS mice (Fig. 1G). Typical marker genes of distinct cell types in lungs show only subtle changes in HGPS/hACE2 compared hACE2 mice, indicating a relatively normal lung function in HGPS/hACE2 mice (Supplementary Fig. 1F). Overall, we have successfully generated a hACE2 mouse model harboring the HGPS mutation, which displays phenotypic similarities to human accelerated aging.
Replication and host response to primary infection with SARS-CoV-2
To investigate infection outcomes of HGPS mice, we examined the pathogenesis of SARS-CoV-2 in 2-month-old HGPS/hACE2 mice infected with the original strain of the coronavirus from Wuhan (WIV04). In parallel, age-matched young hACE2 mice (2 months old) and aged hACE2 mice (over 14 months old) were included for virus challenging. Mice received intranasal inoculation with 1x105 50% tissue culture infective dose (TCID50) of the virus and lung samples were collected at 1, 3, 5, and 7 days post-infection (dpi) for virus replication measurement, clinical response examination, and transcriptome analysis (Fig. 2A). Although young, aged, and HGPS mice exhibited distinct expression levels of hACE2 in lung, transcriptome analysis of SARS-CoV-2 revealed that aged and HGPS mice displayed significantly more expression of SARS-CoV-2 genes in lung including E, M, N, S, and ORF genes in lung at 1 dpi, which then decreased dramatically at 3 dpi (Fig. 2B and Supplementary Fig. 2A-B). In contrast, SARS-CoV-2 genes were detected at 1 dpi in the lungs of young mice, reaching the peaks at 3 dpi (Fig. 2B and Supplementary Fig. 2A). We observed mild decreased body weight in infected young mice compared to the mock-infected ones, while no changes were detected between infected and uninfected aged mice (Fig. 2C). HGPS mice exhibited a slight loss of body weight in both infected and uninfected groups (Fig. 2C). In sum, these findings suggest that SARS-CoV-2 replicates differentially in the lungs of young, aged, and HGPS/hACE2 mice, likely resulting in varied responses upon viral infection among different mouse groups.
Aged hACE2 mice displayed more severe pathological phenotypes induced by SARS-CoV-2 infection
To further evaluate the outcomes of viral infection in different mouse group, we conducted histopathological analysis which illustrated distinct degrees of lung damage among the infected young, aged and HGPS/hACE2 mice. In uninfected control animals, slight inflammation was observed in aged and HGPS mice, characterized by a minor immune cell infiltration, which was not detected in the young mice (Fig. 3A, 3E, and 3I). At 1 dpi, mild changes were observed in the lung tissues of young mice, including the presence of lymphocyte and macrophage infiltration in some alveolar spaces, along with slight thickened alveolar walls (Fig. 3B). Severe pneumonia developed at 3 dpi in the young mice, characterized by multifocal lesions, bleeding, and massive infiltration with increased number of mixed inflammatory cells at peri-vascular regions (Fig. 3C). This phenotype became less severe at 5 dpi, with fewer inflammatory cell infiltration and milder thickened alveolar walls compared to those observed at 3 dpi (Fig. 3D). In consistence with young mice, aged mice displayed hemorrhage and slight thickened alveolar walls at peri-bronchial and peri-vascular regions at 1 dpi, which became more pronounced at 3 dpi (Fig. 3F-G and Supplementary Fig. 2C). By 5 dpi, we found that two out of three aged mice displayed severe bleeding in the bronchus and pulmonary alveoli, along with inflammatory cell infiltration and fibrin exudation, suggesting a severe COVID-19 phenotype (Fig. 3G and Supplementary Fig. 2D). Still, all infected mice survived viral infection till euthanized for sample collection at 7 dpi. Notably, these two mice showing severe bleeding phenotype were in poor condition when being euthanized for sample collection. In contrast, HGPS/hACE2 mice did not display a severe phenotype throughout the infection, showing only a slightly increased number of immune cells at peri-bronchial and peri-vascular areas at 1,3 and 5 dpi (Fig. 3J-L), suggesting a less severe phenotypes in there HGPS mice. Altogether, our histopathological findings demonstrate distinct pathological features induced by SARS-CoV-2 infection between different animal groups, highlighting a severe phenotype in aged mice compared to young hACE2 and HGPS/hACE2 mice.
Gene expression dynamics from different groups with viral infection.
To comprehensively understand how SARS-CoV-2 induces pathological differences among different mouse groups, we conducted transcriptomic analysis to investigate the dynamics gene expression profiles in the lungs of young, aged, and HGPS hACE2 mice throughout the infection. We classified high variable genes into several clusters based on their expression patterns and calculate the averaged expressing trajectories for each cluster. Subsequently, we performed Gene Ontology (GO) analysis to elucidate the affected biological and molecular pathways for each cluster. Although viral gene expression peaked differentially in the lungs of young compared to aged and HGPS/hACE2 mice, we observed a common response to virus and activation of innate immune upon viral infection transiently at 3 dpi in cluster 1 across all the infected groups (Fig. 4A,4D, and Supplementary Fig. 3A). In young and aged groups, mice showed increased B cell-mediated immunity as well as immunoglobulin production in the lungs at 5 dpi, accompanied by upregulation of genes involved in muscle development in cluster 2 (Fig. 4B,4D, and Supplementary Fig. 3B). This indicates an immediate humoral immune response following viral exposure, which further triggers muscle contraction of lung smooth muscle. Interestingly, this activation occurred earlier in HGPS mouse lungs at 1 dpi (Fig. 4B), suggesting a distinct immune response to SARS-CoV-2 in HGPS. Viral infection has been reported to cause cilia loss from ciliated cells, resulting in cilia dysfunction in respiratory epithelium19–21. In agreement with previous findings, we observed a slight downregulation of genes in cluster 3 involved in cilium assembly and movement in young and HGPS mice, which was more pronounced in aged group (Fig. 4C,4D and Supplementary Fig. 3C). This was followed by increased expression of those genes across all groups starting at 3 dpi, suggesting a restoration of function in fluid movement and mucus clearance in the respiratory airway (Fig. 4C, 4D and Supplementary Fig. 3C).
In addition to the common functional enrichment shared by the three groups, we also identified gene clusters with distinct dynamic patterns unique to each mouse group. For instance, cluster 4 in young mice declined linearly throughout the entire infection and was strongly enriched for genes associated with cell junction assembly, extracellular matrix organization, and cell-matrix adhesion (Fig. 4E). Moreover, Notch/vascular genes exhibited a rapid decline rapidly in aged mice at 1 dpi, after which a more gradual decline prevailed, indicating functional defects in vascular and endothelial cells in the lungs of aged mice (Fig. 4E). In infected HGPS mice, genes encoding sulfur, glycoprotein, and liposaccharide metabolic process pathways were featured in cluster 4, exhibiting immediate increase after infection till 3 dpi, followed by a drop towards the end of infection (Fig. 4F). However, cluster 5 contained genes related to lipid transport and exocytosis regulation pathways who reached their lowest expression level at 3 dpi, followed by a pronounced increase till 7 dpi, showing an opposite trend compared to cluster 4 (Fig. 4F). These findings suggest SARS-CoV2-induced metabolic dysfunction in infected HGPS lungs.
Transcriptome comparison reveal strong immune response in young mice upon viral infection.
Next, we focused on infection-induced transcriptomic changes and compared the transcription differences between different mouse groups at each time point post infection. Differential gene expression analysis, together with hierarchical clustering, revealed specifically expressed gene clusters from each group, and GO analysis was applied to these gene sets. Overall, we observed a comparable number of genes between young and aged group at 1, 3, and 5 dpi, while HGPS group displayed subtle changes at 1 and 3 dpi (Fig. 5A-C). Consistent with our histopathological analysis, expression of genes enriched in virus defense, interferon-beta, and innate immune response pathways was significantly higher in the lungs of young mice throughout the infection, suggesting a more active immune response upon viral infection compared to the aged and HGPS mice (Fig. 5D). In the aged mice, immune pathways such as immune cell migration, immunoglobulin production, humoral immune response, and cell chemotaxis represented feature genes in the clusters at 1 and 3 dpi (Fig. 5E). Additionally, we found pathways related to hemorrhage, including wound healing, blood coagulation, and erythrocyte development, were significantly enriched in aged mice, further confirming the bleeding phenotype observed in the lungs of aged mice in the histopathological study (Fig. 5E and Supplementary Fig. 4). We did not obverse a significant pathway enriched in HGPS mice at 1 and 3 dpi, likely due to the low numbers of unique genes (36 genes for 1 dpi and 53 genes for 3 dpi) in this group. Whereas at 5 dpi, 332 genes were more activated in the lungs of HGPS mice. These genes primarily belong to pathways associated with entrainment of circadian clock, regulation of blood circulation, and hormone transport. This suggests a dysregulation of circadian rhythms in HGPS mice, although the enrichment in this pathway was less pronounced (Fig. 5F). Altogether, our results demonstrated that young mice, which have a normal immune system to protect against virus, display the highest immune response activation upon SARS-CoV-2 infection compared to aged and HGPS mice. Conversely, viral infection only causes mild pathological phenomena in HGPS mice, while aged mice exhibit lung hemorrhage when challenged by SARS-CoV-2, which could contribute to severe COVID-19.