Previous studies have shown that fibroblasts isolated from SSc-ILD  and IPF  lungs, while displaying substantial heterogeneity, are generally more proliferative, migratory, resistant to apoptosis, and ECM producing, than control lung fibroblasts . A greater proportion display elevated αSMA expression, also enhancing their contractility. These features are all consistent with the fibroblast accumulation and scar tissue formation observed in fibrotic lung. The so called myofibroblast phenotype is maintained over several passages in culture , suggesting an underlying epigenetic regulation, whether established and maintained in local cells within the chronic disease setting, or supporting the phenotype of a specialised infiltrating wound healing cell type . Regardless of cell origin, this feature enables in vitro studies of mechanisms underlying the fibrotic fibroblast phenotype.
In this study we observed a high number of differentially expressed genes between SSc-ILD and/or IPF derived fibroblasts, compared with controls. There was a large overlap between expression profiles of SSc-ILD and IPF fibroblasts, suggesting several common pathways at this stage of the two diseases. Indeed, a direct comparison demonstrated only seven genes with significantly different expression between the two disease groups. Caution should be applied, however, when interpreting data from this direct comparison since only three IPF samples were included here. It is possible that more genes with differential expression between the two disease groups would be identified with a larger numbers of IPF fibroblasts samples. Such studies would also be required to verify the sporadic observations made in this study, such as the elevated expression levels of desmoplakin in SSc-ILD, but not in IPF fibroblasts. Therefore, we stress that the main objective of the study presented here was to gain an overview of potential SSc-ILD target genes, and the study was not designed to detect differences between different fibrotic entities.
Among significantly upregulated genes in both SSc-ILD and IPF/UIP fibroblasts, we identify a recognised fibrosis signature, including smooth muscle actin (ACTA2), CTGF, and PAI1 (SERPINE1), along with genes more recently associated with lung fibrogenesis, including ID1, ID3, IL11, and NOX4. Inhibitor of DNA binding 1 and 3 (ID1 and ID3), are target genes of bone morphogenetic proteins, and control cell differentiation by dominant negative inhibition of helix-loop-helix transcription factors . Chambers et al. described upregulation of ID1 in lung fibroblasts in response to TGF-β, and linkage to the smooth muscle phenotypic switch . Increased ID1 and ID3 gene expression was also observed in lung tissue and fibroblasts from patients with SSc-ILD by Hsu and colleagues, . The ID proteins are overexpressed in many cancers, controlling cell growth and apoptosis, and have been suggested as a therapeutic target [39, 40]. In fibroproliferative stages of fibrosis, high ID1/ID3 expression could maintain fibroblasts in a dedifferentiated, hyperproliferative, and apoptosis resistant state. Another significantly upregulated gene, though somewhat variably in SSc-ILD fibroblasts, is NOX4, encoding a member of the NADPH oxidase (NOX) proteins which generate superoxide by electron transfer to oxygen . Through its involvement in TGF-β-induced fibroblast differentiation into myofibroblasts, two recent studies have suggested a role for NOX4 in IPF, where it is found to be overexpressed . Specific NOX4 inhibitors are now being developed as possible antifibrotic agents . Another potential novel target gene, GADD45B, highly induced and coexpressed with the fibrosis related genes in this study, is a pro-survival factor associated with stress-resistant tumours , and has been found to be upregulated in SSc skin biopsies .
The most striking of our observations is the strongly and uniformly repressed ISG profile in fibrotic fibroblasts. Among these are genes coding for: antiviral protein myxovirus (influenza virus) resistance 1 (MX1), interferon gamma inducible protein p16 (IFI16), 2’,5’-oligoadenylate synthetase 1 (OAS1), the chemokines chemokine (C-X-C motif) ligand 10 (CXCL10/IP10) and chemokine (C-C motif) ligand 11 (CCL11), and antigen presenting MHC I molecules. Key transcriptional regulators of this program, including IRF1, IRF7, and STAT1 are also suppressed. To our knowledge, the reduction in expression of a large set of immune response/interferon related genes has not previously been described in either SSc-ILD or IPF derived fibroblasts, or other fibrotic lung diseases. Global gene expression was recently evaluated in scleroderma whole skin biopsies and matched dermal fibroblasts. While 26 genes were differentially expressed in both scleroderma whole skin and fibroblasts compared to controls, nine were found to be discordant . Interestingly, the majority of the discordant genes were upregulated in skin biopsies but downregulated in fibroblasts, including the ISG genes we identify as repressed in lung fibroblasts, such as MX1, IFI16, intercellular adhesion molecule 1 (ICAM1), and OAS1. While the authors argue that the discordant genes are an indication of a scarce representativeness of skin fibroblast gene expression in vivo, in light of our data, it is likely that they were in fact observing the same phenomenon reported in the current study. As these are mainly interferon regulated genes, the discordance between fibroblasts and whole tissue gene expression could be explained by the known increase of immune cell populations in SSc skin, likely to be overexpressing immunoregulatory genes. The upregulation of MX1, IRF7 and STAT1 in PBMC from SSc patients would support this notion [46, 47]. The downregulation of the ISG program both in SSc-ILD and in IPF fibroblasts suggests that this phenomenon relates to a local fibroblast specific, rather than systemic, profibrotic process, perhaps underpinned by a general susceptibility for tissue fibrosis, common to both diseases.
Additional support for our finding of an aberrantly regulated ISG program in fibrotic lung fibroblasts, comes from work on two of the signature genes from this group, CXCL10 (IP10) and STAT1. IP10 levels were found to be downregulated in IPF lung fibroblasts by Keane et al. . More recently, Coward et al. have shown, again in IPF fibroblasts, that epigenetic dysregulation involving both histone deacetylation and hypermethylation is responsible for targeted repression of IP10. By contrast, Hsu et al. did not report a significant difference in expression in isolated lung fibroblasts , a difference which may relate to experimental design, as discussed below. Further support of a role of suppressed ISGs in pulmonary fibrosis comes from two animal models. Mice deficient in IP10, and in STAT1, displayed enhanced susceptibility to pulmonary fibrosis. Based on these studies, the suppressed ISG program indentified in fibroblasts isolated from SSc-ILD and IPF lung, as presented here, would support enhanced lung fibrosis progression through promoting fibroblast proliferation, migration, and apoptosis resistance. Interestingly, IFN-γ treatment in IPF has failed to show a benefit , and there was a suggestion of worse pulmonary outcomes in a study investigating treatment of SSc patients with IFN-α compared with placebo , with the latter providing indirect support for IFN-related mechanisms involved in organ-specific SSc complications. There could be several possible explanations for these disappointing results, including unexpected adverse effects through circulating cell populations. Activation of pathways downstream of systemically administered interferons is likely to have different direct and indirect effects depending on the cell type and tissue location. The findings shown here add important information to this complexity, and need to be investigated in future detailed mechanistic cell and animal studies.
The signatures observed in our study are remarkably strong, both in terms of fold difference and statistical significance. One possible reason for this is that lung tissue samples were obtained from biopsies at the time of diagnosis, when the disease may be at a relatively early or active wound healing stage, and when perhaps fibroblast proliferation (accumulation) and elastin synthesis, rather than contraction and collagen remodelling, dominate. This is in contrast to the study by Hsu et al., in which gene expression profiles were investigated in SSc lung tissue and fibroblasts from transplant, and therefore possible end-stage, material, also noted by the authors as a potential limitation . Another possibility relates to differences in in vitro culture condition as we employed serum free media before harvest, similarly to others, including Coward et al. , as opposed to in low serum (0.5%) as applied by e.g. Hsu et al. . Serum withdrawal, a form of cellular stress, may evoke the clear differential expression profiles observed in our study. Whereas in the fibrotic fibroblasts an anti-apoptotic survival gene program is maintained, which may be a result of the suppressed ISGs, this gene program may not be subject to repression in the normal fibroblasts.
While an accepted source of control tissue in studies of ILD [10, 12], the use of control fibroblasts from cancer resected specimens, rather than from healthy control subjects (not available for this study), represents a potential limitation. Although obtained from areas of lung with normal histological appearance, differential gene expression in fibroblasts derived from lungs in which cancer has developed cannot be excluded. However, significant gene expression differences were observed in lung cancer associated fibroblasts compared to matched fibroblasts from areas of normal lung from the same patient, suggesting that the cancer associated phenotype of lung fibroblasts is regionally limited to the cancer stroma . A further possible source of bias in gene expression is smoking history. Smoking has been shown to be associated with interstitial fibrotic changes , and is itself likely to cause changes in the expression of certain genes. We observed no significant differences among differentially expressed genes according to smoking status in the pooled fibrotic samples, suggesting that the observed changes were related to the fibrotic lung disease itself rather than to smoking. However, as subgroup numbers were small, and it was not possible to separately analyse the two fibrotic lung diseases, further studies are needed to carefully assess the contribution of smoking to gene expression changes in the context of fibrotic lung disease.
While the general hypothesis, that a repressed interferon stimulated gene program at least in part underpins the fibrotic fibroblast phenotype, will be tested in future studies, it is interesting to note the same phenomenon in several other clinical settings where hyperplasia and apoptosis resistance are key features; certain viruses, including high-risk human papillomaviruses (HPV), have evolved a mechanism to down-regulate ISGs in host cells as an immunoevasive strategy , and persistent HPV infection may lead to cervical cancer development; breast cancer metastasis is promoted by IRF7 silencing ; fibroblasts from patients with Li-Fraumeni syndrome become spontaneously immortalised through the downregulation of interferon pathway genes . Conversely, IRF1 expression reverts the phenotype of oncogenically transformed fibroblasts , and IRF-1 enhancing drugs with tumour suppressing properties are currently being developed . Many similar examples in the literature lead to questions about whether fibrosis is a pre-cancerous state . The repressed, or aberrantly regulated, fibroblast specific interferon response network may therefore be a common necessary determinant allowing lung fibrosis progression to occur.
In summary, in this study comparing gene expression profiles of fibroblasts explanted from fibrotic lung tissue (SSc-ILD and IPF), with control fibroblasts from areas of normal lung, we observe: an overall elevated expression of previously reported fibrosis associated genes, with marked heterogeneity across samples; differentially regulated myofibroblast markers which correlate with the expression heterogeneity between samples; and a strongly suppressed interferon stimulated gene program, uniformly present across fibrotic samples. This suppressed gene program displays both the greatest significance and largest fold differences in expression in our data set. Similarly to functional findings in parallel fields, particularly cancer, this group of genes, and the suppression of their expression, could explain essential aspects of the profibrotic fibroblast phenotype. This hypothesis will need to be tested by future studies, with particular focus on epigenetic silencing as a potential underlying mechanism.