Overall the study consisted of 24 lung tissue samples from different sources as mentioned above. The collected tissues were classified into six different groups based on age, the smoking and disease status. Further, comparative
gene analysis was also done based on the smoking and disease status irrespective of the age. There were no genes in common that were changed in any of the comparisons involving all the three groups i.e. non-smokers, smokers and COPD. However, the individual group wise comparisons were reported here.
Differentially expressed genes in young non-smokers versus young smokers versus young COPD groups
First, we analyzed differentially expressed transcript levels among young non-smokers vs. young smokers, young smokers vs. young COPD and young non-smokers vs. young COPD (Figure 1). We found 5 genes were differentially expressed in young non-smokers vs. young smokers’ pairwise comparison. Out of 5 genes, the transcript levels of 4 genes (NFATC1, NFATC2, GADD45A, and CDKN1A) were decreased and 1 gene (PARP1) was increased in the young smokers as compared to young non-smokers group (Figures 2 and 3 A). Next, we compared genes differentially expressed in young smokers vs. young COPD pairwise comparison. Out of 5 genes, transcript levels of 1 gene (SIRT6) was decreased and the remaining 4 genes (RAD17, CDKN1C, COX10 and KLOTHO) were significantly increased in young smokers as compared to young COPD group (Figures 2A and 3B). Finally, we found 6 genes differentially expressed among young non-smokers vs. young COPD pairwise comparison. Out of 6 genes, the transcript levels of 2 genes CDKN1C and KLOTHO that belong to cellular senescence panel were decreased in the young COPD as compared to young non-smokers group. While the transcript levels of remaining 4 genes PARP1, SIRT6, TERT and SLX4 were increased in young COPD as compared to young non-smokers group (Figures 2A and 3C). Overall, 4 genes PARP1, SIRT6, KLOTHO and CDKN1C were among the common target genes that were differentially expressed in young COPD as compared to young non-smokers and young smokers groups.
Differentially expressed genes in old non-smokers versus old smokers versus old COPD groups
Here we analyzed differentially expressed transcript levels among old non-smokers vs. old smokers, old smokers vs. old COPD and old non-smokers vs. old COPD groups. Out of 7 genes, we found 3 genes IGF1, COX18 and RIF1 were decreased and remaining 4 genes NFATC1, NFATC2, RAD17 and PCNA were increased in old smokers as compared to old non-smokers group (Figures 2B and 4A). The transcript levels of IGF1, PARP1, PTEN, NBN, HSPD1 and RIF1 were decreased and GAR1 was increased in old smokers as compared to old COPD group (Figures 2B and 4B). Only 2 genes were affected among old non-smokers and old COPD group; RPA2 and PCNA were increased in old COPD as compared to old non-smokers group (Figures 2B and 4C). Overall, a total of 3 genes as mentioned above (IGF1, RIF1 and PCNA) were among the common targets that was found differentially expressed in old smokers as compared to old non-smokers and old COPD groups.
Altered gene expression levels in young and old non-smokers versus smokers versus COPD groups
We then analyzed differentially expressed genes among young non-smokers vs. old non-smokers, young smokers vs. old smokers and young COPD vs. old COPD pairwise comparisons. Transcript levels across different age groups were performed to better understand, whether age factor influences the measured outcomes in the current study. Accordingly, we found that 9 genes were significantly elevated in young non-smokers as compared to old non-smokers group. The following are the genes that were increased among young non-smokers (PCNA, NFATC2, ACD, GSK3β, HAT1, UCP2, CDKN1A, CDKN1C and SIRT1) as compared to old non-smokers group (Figures 2C and 5A). Although, young smokers show increased transcript levels of PARP1, UCP3 and E2F1 genes but decreased levels of NFATC1, NFATC2, MYC and GADD45A as compared to old smokers group as seen in Figures 2C and 5B. Additionally, 2 genes TNSK2 and PTEN were decreased in young COPD as compared to old COPD group. The transcript levels of GAR1, TERT, H2AX and FEN1 tend to increase in younger COPD as compared to old COPD group (Figures 2C and 5C). Interestingly, we noted that transcript levels of NFATC2 was decreased in lungs of old non-smokers, but increased in lungs of old smokers.
Combined analysis of differentially expressed genes among non-smokers, smokers and COPD groups
We performed grouped analysis of differentially expressed transcripts (comparisons without considering the age factor (young or old) among non-smokers, smokers and patients with COPD groups. Data from young and old that belong to same experimental groups were combined (young nonsmokers with old non-smokers group, young smokers with old smokers group and young COPD and old COPD group; n=8/group, as given in Figures 6 and 7A). Results indicated that smokers show decreased FOXO1 and increased RAD17 levels as compared to non- smokers group (Figure 8A). While decreased PARP1 and increased RAD17 levels were observed in smokers as compared to COPD group (Figure 8B). In patients with COPD KLOTHO gene was decreased and PARP1 and SLX4 genes were increased as compared to non-smokers group (Figure 8C). Furthermore, these comparisons revealed that smokers have significantly higher levels of RAD17 expression compared to both non-smokers and COPD groups. Whereas, COPD patients showed significantly higher levels of PARP1 as compared to both non- smokers and smokers groups.
Altered gene expression levels in young and old non-smokers versus IPF groups
Pairwise analysis of young non-smokers vs young IPF showed 25 significantly altered genes, which were given in Table 2. Comparisons between old non-smokers and old IPF showed 13 significantly altered genes, as listed in table 3 along with their observed level of significance. The gene comparisons among these groups were given in Figure 7B.
Altered gene expression levels in young and old COPD versus IPF groups
As detailed above both COPD and IPF are chronic age related diseases that severely alter lung function and share certain common features for the disease occurrence and progression. Here, we compared the altered gene levels related to the same pathways among COPD and IPF subjects as detailed above. There was no change in any of the genes analyzed in comparisons between young IPF (n=3) and old IPF (n=13). While, 16 genes were found to be altered in the comparisons between young (COPD vs IPF), as indicated in Table 4. A total of 6 genes were altered in comparisons between old COPD vs old IPF) as shown in Table 5.
Gene expression analysis of differentially expressed targets
Some of the differentially expressed mRNA targets predicted using Nanostring were selected for qPCR study. As given in the Figure 9 the expression trends of these selected genes matches with the trend observed using the NanoString mRNA analysis with varied levels of fold changes. Combined gene analysis for PARP1 was also given, and matches with the trend are given in Figure 8. The results clearly indicate that the genes validated using qPCR are in agreement and significant across all the pairwise comparisons made.
Protein expression levels of crucial SARS-CoV-2 targets in the lung homogenates
Western blots analysis (Figures 10 and 11) revealed a significant increase in the protein levels of TMPRSS2 protease (which plays a crucial role in the processing of the SARS-CoV-2 proteins) in COPD subjects as compared to smokers and non-smokers. Similarly, the levels of another important protease furin a spike protein, was also increased in smokers and COPD subjects, with a significant expression in COPD subjects. ACE2, which is considered crucial for the SARS-CoV2 binding as a receptor, was found to increase in smokers as compared to the rest of the groups. The samples were also further probed for the expression of DPP4, another crucial protein which plays an important role in MERS‐CoV binding. The DPP4 expression was found to be significantly higher in smokers as compared to COPD and non-smokers. These results indicate that the levels of proteases, which aid in the processing and binding of the viral spike proteins were highly expressed in COPD and smokers. The expression results showed varied protein intensities in smokers and COPD for TMPRSS2 and DPP4 in the lungs, which may suggest a varied effect of virus entry/susceptibility based on specific cells in smokers and COPD/IPF subjects.a