In this study, we used human CpG island microarrays to identify the differentially methylated CpG islands in the lung tissue of IPF patients. Our results indicate that the CpG island methylation profile of the IPF lung samples is very different from that of control samples and greatly overlaps with methylation changes observed in lung adenocarcinoma samples. Despite the observed similarity in CpG methylation between IPF and lung cancer, the lack of LINE-1 methylation in IPF suggests a more specific DNA methylation, which is confined to certain regions of the genome.
One of the most impressive results of our study is the extent of differentially methylated regions in the IPF lungs. Interestingly the majority of the differentially methylated CpG islands rest in promoter-distal sites or intragenic regions and only 8.8% of them are localized in gene promoters. Whereas the methylation status of promoter associated CpG islands can directly affect transcription, the role of the CpG methylation outside the immediate promoter region remains somewhat unclear. It is proposed that methylation of CpG island shores outside the promoter could also control transcription of downstream genes 
or lead to histone modifications 
. Methylation changes that occur in intragenic regions could impact RNA splicing 
. In addition, methylation changes may affect the expression of non-coding RNAs 
and thus indirectly affect global changes in gene expression. The biological impact of modest changes in the degree of CpG methylation is in fact unpredictable. As an example in the case of prostate cancer, a gradual increase in methylation from 12.6% to 19.3% or 21.8% signified a transition from a benign state to a localized or metastatic cancer, respectively 
. However, regardless of the direct downstream effects, the extent of the methylation changes we found, supports previous observations about the degree and profundity of molecular changes in the IPF lung 
Naturally, the motivation to assemble methylation profiles is to find the underlying mechanisms that drive changes in gene expression. The detailed characterization of each one of the differentially methylated CpG islands in IPF patients is beyond the scope of this study. Globally, genes with differentially methylated CpG islands in their promoters were involved in biological processes such as cellular assembly and organization, cellular growth and proliferation, cell morphology, cancer, cell signaling, gene expression and cell death. All of these processes could be implicated in IPF pathogenesis. In our validation we focused on genes with differentially methylated promoters. We selected the Serine/Threonine Kinase 17b (STK17B) and Serine/Threonine Kinase 3 (STK3) because of their role in apoptosis 
and the histone cluster 1 H2ah (HIST1H2AH) because of the recent interest in histone modifications in fibrosis
. STK17B and HIST1H2AH were significantly up-regulated in our IPF samples which is in agreement with the hypo-methylated state of their promoter associated CpG islands. Interestingly, the majority of the differentially methylated islands that were within or close to known genes were outside promoter regions. Some of these methylation changes were in genes that were previously reported to be increased in IPF such as COL18A1 
, genes that are implicated in myofibroblast differentiation such as NOTCH1 
or markers of progressive IPF like SMARCA4 
. In addition, the promoter of CXCL3, a gene which is found to be up-regulated in the lung of bleomycin treated mice 
, was also hypomethylated in our IPF samples. When we looked for the overlap of differentially expressed genes in IPF in our previously published gene expression datasets 
we found that there were 46 genes that had both differentially methylated gene related CpG islands and gene expression changes. While a detailed analysis of methylation and expression changes in the same tissue would be better suited to address the correlation of methylation and gene expression changes our findings suggest that at least some of the methylation changes that we observed do have an effect on lung gene expression and thus may contribute to the lung phenotype in IPF.
A remarkable finding of our study is the similarity in DNA methylation patterns between IPF and lung adenocarcinoma. Recently, Vancheri et al
compared IPF to cancer and described the pathogenic similarities between the two diseases. More specifically, they referred to common genetic and epigenetic alterations, uncontrolled proliferation, tissue invasion and perturbation of signal transduction pathways 
. The similarity between cancer and IPF spreads to microRNA expression such as in the case of let-7d and hsa-miR-21, which are found to be down-regulated or up-regulated respectively in both diseases 
. All of these observations are in accord with published studies reporting high incidence of cancer in IPF patients when compared to healthy individuals 
. DNA hypomethylation is a hallmark of cancer 
and in many types of cancer including lung carcinomas it is accompanied with lower levels of methylation in repetitive DNA elements 
. While the similarity in the differentially methylated CpG islands suggests common epigenetic mechanisms between IPF and cancer, our analysis of LINE-1 methylation indicates that this similarity is limited. LINE-1 repeats comprise about 20% of the human genome 
. LINE-1 elements are usually methylated in somatic tissues but they are often hypomethylated in tumors 
resulting in increased mobility, which in turn leads to gene disruptions 
and chromosomal instability 
. While LINE-1 retrotransposons were hypomethylated in our cancer samples they were not in IPF samples leading to the conclusion that CpG island methylation changes in IPF are somewhat parallel to cancer but are not as extensive and do not involve global changes in LINE-1 methylation. This suggests that despite the similarities between the DNA methylation profiles of IPF and cancer, there are different mechanisms that cause and sustain these changes.
One of the major concerns in our global profiling approach is tissue heterogeneity. The IPF lungs contain mixed areas of normal tissue, myofibroblast foci and honeycombing 
. The IPF lung is also highly variable in its cellular content as it contains normal cells like epithelial, endothelial cells and fibroblasts as well as abnormal ones like hyperplastic type II alveolar epithelial cells, myofibroblasts, potentially altered endothelial cells and varying degrees of inflammatory cells. Thus it is possible that the signal we obtained is only an under-estimation of the real epigenomic changes caused by an admixture of normal and abnormal regions, microenvironments and cell types. Naturally, it is impossible based on our analysis to determine whether the observed DNA methylation changes are cell type specific. In this context, our strategy of averaging signals across an island could also lead to loss of information and underestimation of epigenetic changes. However, we chose this approach because although it is less sensitive, we felt it provided us with global results, reduced the need to deal with probe variability and provided a good approximation of differentially methylated CpG islands. In the future it may make sense to refine both the measurement approach and data analysis to obtain more detailed results. The heterogeneity of our samples as well as the different methodologies used to identify the differences in CpG methylation could also explain the absence of PTGER2 and Thy-1 from our list of significantly methylated genes. The promoters of PTGER2
were found to be hypermethylated in fibrotic lung fibroblasts and fibrotic tissue from IPF patients resulting in low levels of the coded proteins. In fact Thy-1 it is shown that the downregulation occurs only in areas of dense fibrosis and fibrotic foci while the rest of the tissue remains unaffected 
. However, the demonstration of significant global methylation changes despite the limitations of our methods, may be indicative of the importance of epigenomic regulation in IPF and lead to many more detailed discoveries and insights.
To the best of our knowledge our study is the first one to describe global DNA methylation changes in IPF lungs. Taken together with the extensive changes in gene histology, gene expression and microRNA profiles our results highlight the profundity and complexity of events underlying the phenotypic changes in IPF and to some extent suggest that interfering with one pathway may not be sufficient to reverse these changes. The differentially methylated CpG islands we identified should be further studied as their regulation could provide insights about how genotype and the environment interact to determine the lung phenotype in IPF. Based on our results, we believe that epigenetic modifications play a key role in the pathogenesis of IPF and thus could serve as disease biomarkers and therapeutic targets.