Rationale: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease that is associated with common genetic variants, rare variants, and environmental risk factors. Genome-wide association studies of IPF have included more than 5,000 unique patients with IPF and identified approximately two dozen common genetic variants that are associated with the risk of disease. However, the vast majority of these patients were of European ancestry. Thus, the genetic risk of IPF in ancestries other than European populations is largely unknown. To better understand which genetic variants contribute to IPF in individuals with East Asian ancestry, we conducted a genome-wide association study of IPF in Japanese and Korean populations. Methods: Among individuals of Japanese and Korean ancestry, we genotyped 1,026 patients with IPF and compared them to 1,723 unaffected controls. Genome-wide association analyses were conducted in the Japanese and Korean cohorts separately, which were then combined using a fixed-effects meta-analysis. Ancestry deconvolution was used to infer the local ancestry of each region of chromosome 11 for each subject.Results: We identified loci on chromosomes 4 (FAM13A; rs7690839), 5 (TERT; rs7734992), 6 (DSP; rs2076295), and 11 (MUC5B; rs35705950) that were significantly associated with risk of IPF. Importantly, the sentinel genetic variant indentified in each of these loci are the same as, or in strong linkage disequilibrium with, the common risk variants observed in genetic association studies of European populations. Local ancestry analysis indicates that the presence of the MUC5B promoter variant in the East Asian population is not a result of admixture with European populations.Conclusion: We conclude that the IPF risk loci in East Asian populations are shared with those of European populations. However, some of the frequencies and effect sizes of the associated variants differ. These findings indicate a common etiology of IPF across ancestries that may experience different degrees of genetic selection.

Hypoxic culture conditions have been used to study the impact of oxygen deprivation has on gene expression in a number of disease models. However, hypoxia response elements present in the promoter regions of some commonly used housekeeping genes, such as GAPDH and PGK1, can confound the relative gene expression analysis. Thus, there is ongoing debate as to which housekeeping gene is appropriate for studies investigating hypoxia-induced cell responses. Specifically, there is still contradicting information for which housekeeping genes are stable in hypoxia cultures of mesenchymal stem cells. In this study, candidate housekeeping genes curated from the literature were matched to RNAseq data of normoxic and hypoxic human adipose-derived stem cell cultures to determine if gene expression was modulated by hypoxia or not. Expression levels of selected candidates were used to calculate coefficient of variation. Then, accounting for the mean coefficient of variation, and normalised log twofold change, genes were ranked and shortlisted, before validating with qRT-PCR. Housekeeping gene suitability were then determined using GeNorm, NormFinder, BestKeeper, comparative Δ Ct , RefFinder, and the Livak method. Gene expression levels of 78 candidate genes identified in the literature were analysed in the RNAseq dataset generated from hADSC cultured under Nx and Hx conditions. From the dataset, 15 candidates with coefficient of variation ≤ 0.15 were identified, where differential expression analysis results further shortlisted 8 genes with least variation in expression levels. The top 4 housekeeping gene candidates, ALAS1, RRP1, GUSB, and POLR2B, were chosen for qRT-PCR validation. Additionally, 18S, a ribosomal RNA commonly used as housekeeping gene but not detected in the RNAseq method, was added to the list of housekeeping gene candidates to validate. From qRT-PCR results, 18S and RRP1 were determined to be stably expressed in cells cultured under hypoxic conditions. We have demonstrated that 18S and RRP1 are suitable housekeeping genes for use in hypoxia studies with human adipose-derived stem cell and should be used in combination. Additionally, these data shown that the commonly used GAPDH and PGK1 are not suitable housekeeping genes for investigations into the effect of hypoxia in human adipose-derived stem cell.

The pathogenesis of pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF) and other forms of interstitial lung disease, involves a complex interplay of various factors including host genetics, environmental pollutants, infection, aberrant repair and dysregulated immune responses. Highly variable clinical outcomes of some ILDs, in particular IPF, have made it difficult to identify the precise mechanisms involved in disease pathogenesis and thus the development of a specific cure or treatment to halt and reverse the decline in patient health. With the advent of in-depth molecular diagnostics, it is becoming evident that the pathogenesis of IPF is unlikely to be the same for all patients and therefore will likely require different treatment approaches. Chronic inflammation is a cardinal feature of IPF and is driven by both innate and adaptive immune responses. Inflammatory cells and activated fibroblasts secrete various pro-inflammatory cytokines and chemokines that perpetuate the inflammatory response and contribute to the recruitment and activation of more immune cells and fibroblasts. The balance between pro-inflammatory and regulatory immune cell subsets, as well as the interactions between immune cell types and resident cells within the lung microenvironment, ultimately determines the extent of fibrosis and the potential for resolution. This review examines the role of the innate and adaptive immune responses in pulmonary fibrosis, with an emphasis on IPF. The role of different immune cell types is discussed as well as novel anti-inflammatory and immunotherapy approaches currently in clinical trial or in preclinical development.

Cochlear implants (CIs) allow individuals with profound hearing loss to understand speech and perceive sounds. However, not all patients obtain the full benefits that CIs can provide and the cause of this disparity is not fully understood. One possible factor for the variability in outcomes after cochlear implantation, is the development of fibrotic scar tissue around the implanted electrode. It has been hypothesised that limiting the extent of fibrosis after implantation may improve overall CI function, and longevity of the device. Currently, histology is often used to quantify the extent of intracochlear tissue growth after implantation however this method is labour intensive, time-consuming, often involves significant user bias, and causes physical distortion of the fibrosis. Therefore, this study aimed to evaluate x-ray micro computed tomography (μCT) as a method to measure the amount and distribution of fibrosis in a guinea pig model of cochlear implantation. Adult guinea pigs were implanted with an inactive electrode, and cochleae harvested eight weeks later (n = 7) and analysed using μCT, to quantify the extent of tissue reaction, followed by histological analysis to confirm that the tissue was indeed fibrotic. Cochleae harvested from an additional six animals following implantation were analysed by μCT, before and after contrast staining with osmium tetroxide (OsO4), to enhance the visualisation of soft tissues within the cochlea, including the tissue reaction. Independent analysis by two observers showed that the quantification method was robust and provided additional information on the distribution of the response within the cochlea. Histological analysis revealed that μCT visualised dense collagenous material and new bone formation but did not capture loose, areolar fibrotic tissue. Treatment with OsO4 significantly enhanced the visible tissue reaction detected using μCT. Overall, μCT is an alternative and reliable method that can be used to quantify the extent of the CI-induced intracochlear tissue response and will be a useful tool for the in vivo assessment of novel anti-fibrotic treatments.

RATIONALE Idiopathic pulmonary fibrosis is a rare, irreversible, and progressive disease of the lungs. Common genetic variants, in addition to non-genetic factors, have been consistently associated with IPF. Rare variants identified by candidate gene, family-based, and exome studies have also been reported to associate with IPF. However, the extent to which rare variants genome-wide may contribute to the risk of IPF remains unknown. OBJECTIVES We used whole-genome sequencing to investigate the role of rare variants, genome-wide, on IPF risk. METHODS As part of the Trans-Omics for Precision Medicine Program, we sequenced 2,180 cases of IPF. Association testing focused on the aggregated effect of rare variants (minor allele frequency ≤0.01) within genes or regions. We also identified individual variants that are influential within genes and estimated the heritability of IPF based on rare and common variants. MEASUREMENTS AND MAIN RESULTS Rare variants in both TERT and RTEL1 were significantly associated with IPF. A single rare variant in each of the TERT and RTEL1 genes was found to consistently influence the aggregated test statistics. There was no significant evidence of association with other previously reported rare variants. The SNP-heritability of IPF was estimated to be 32% (s.e. 3%). CONCLUSIONS Rare variants within the TERT and RTEL1 genes and well-established common variants have the largest contribution to IPF risk overall. Efforts in risk profiling or development of therapies for IPF that focus on TERT, RTEL1, common variants, and environmental risk factors are likely to have the largest impact on this complex disease.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease associated with chronic inflammation and tissue remodelling leading to fibrosis, reduced pulmonary function, respiratory failure and death. Bleomycin (Blm)-induced lung fibrosis in mice replicates several clinical features of human IPF, including prominent lymphoid aggregates of predominantly B-cells that accumulate in the lung adjacent to areas of active fibrosis. We have shown previously a requirement for B-cells in the development of Blm-induced lung fibrosis in mice. To determine the therapeutic potential of inhibiting B-cell function in pulmonary fibrosis, we examined the effects of anti-CD20 B-cell ablation therapy to selectively remove mature B-cells from the immune system and inhibit Blm-induced lung fibrosis. Anti-CD20 B-cell ablation did not reduce fibrosis in this model; however, immune phenotyping of peripheral blood and lung resident cells revealed that anti-CD20-treated mice retained a high frequency of CD19+ CD138+ plasma cells. Interestingly, high levels of CD138+ cells were also identified in the lung tissue of patients with IPF, consistent with the mouse model. Treatment of mice with bortezomib, which depletes plasma cells, reduced the level of Blm-induced lung fibrosis, implicating plasma cells as important effector cells in the development and progression of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterised by a dense fibrosing of the lung parenchyma. An association between IPF and cellular senescence is well established and several studies now describe a higher abundance of senescent fibroblasts and epithelial cells in the lungs of IPF patients compared with age-matched controls. The cause of this abnormal accumulation of senescent cells is unknown but evidence suggests that, once established, senescence can be transferred from senescent to non-senescent cells. In this study, we investigated whether senescent human lung fibroblasts (LFs) and alveolar epithelial cells (AECs) could induce a senescent-like phenotype in "naïve" non-senescent LFs in vitro. Primary cultures of LFs from adult control donors (Ctrl-LFs) with a low baseline of senescence were exposed to conditioned medium (CM) from: (i) Ctrl-LFs induced to become senescent using H2O2 or etoposide; (ii) LFs derived from IPF patients (IPF-LFs) with a high baseline of senescence; or (iii) senescence-induced A549 cells, an AEC line. Additionally, ratios of non-senescent Ctrl-LFs and senescence-induced Ctrl-LFs (100:0, 0:100, 50:50, 90:10, 99:1) were co-cultured and their effect on induction of senescence measured. We demonstrated that exposure of naïve non-senescent Ctrl-LFs to CM from senescence-induced Ctrl-LFs and AECs and IPF-LFs increased the markers of senescence including nuclear localisation of phosphorylated-H2A histone family member X (H2AXγ) and expression of p21, IL-6 and IL-8 in Ctrl-LFs. Additionally, co-cultures of non-senescent and senescence-induced Ctrl-LFs induced a senescent-like phenotype in the non-senescent cells. These data suggest that the phenomenon of "senescence-induced senescence" can occur in vitro in primary cultures of human LFs, and provides a possible explanation for the abnormal abundance of senescent cells in the lungs of IPF patients.

The interleukin (IL)-6 family of cytokines and exaggerated signal transducer and activator of transcription (STAT)3 signaling is implicated in idiopathic pulmonary fibrosis (IPF) pathogenesis, but the mechanisms regulating STAT3 expression and function are unknown. Suppressor of cytokine signaling (SOCS)1 and SOCS3 block STAT3, and low SOCS1 levels have been reported in IPF fibroblasts and shown to facilitate collagen production. Fibroblasts and lung tissue from IPF patients and controls were used to examine the mechanisms underlying SOCS1 down-regulation in IPF. A significant reduction in basal SOCS1 mRNA in IPF fibroblasts was confirmed. However, there was no difference in the kinetics of activation, and methylation of SOCS1 in control and IPF lung fibroblasts was low and unaffected by 5′-aza-2′-deoxycytidine’ treatment. SOCS1 is a target of microRNA-155 and although microRNA-155 levels were increased in IPF tissue, they were reduced in IPF fibroblasts. Therefore, SOCS1 is not regulated by SOCS1 gene methylation or microRNA155 in these cells. In conclusion, we confirmed that IPF fibroblasts had lower levels of SOCS1 mRNA compared with control fibroblasts, but we were unable to determine the mechanism. Furthermore, although SOCS1 may be important in the fibrotic process, we were unable to find a significant role for SOCS1 in regulating fibroblast function.

Background: Fibroblasts found in keloid tissues are known to present an altered sensitivity to microenvironmental stimuli. However, the impact of changes in extracellular matrix stiffness on phenotypes of normal fibroblasts (NFs) and keloid fibroblasts (KFs) is poorly understood. Objectives: Investigation the impact of matrix stiffness on NFs and KFs mainly via detecting yesassociated protein (YAP) expression. Methods: We used fibronectin-coated polyacrylamide hydrogel substrates with a range from physiological to pathological stiffness values with or without TGF-beta (fibrogenic inducer). Atomic force microscopy was used to measure the stiffness of fibroblasts. Cellular mechanoresponses were screened by immunocytochemistry, Western blot and Luminex assay. Results: KFs are stiffer than NFs with greater expression of alpha-SMA. In NFs, YAP nuclear translocation was induced by increasing matrix stiffness as well as by stimulation with TGF-beta. In contrast, KFs showed higher baseline levels of nuclear YAP that was not responsive to matrix stiffness or TGF-beta. TGF-beta 1 induced p-SMAD3 in both KFs and NFs, demonstrating the pathway was functional and not hyperactivated in KFs. Moreover, blebbistatin suppressed alpha-SMA expression and cellular stiffness in KFs, linking the elevated YAP signaling to keloid phenotype. Conclusions: These data suggest that whilst normal skin fibroblasts respond to matrix stiffness in vitro, keloid fibroblasts have elevated activation of mechanotransduction signaling insensitive to the microenvironment. This elevated signaling appears linked to the expression of alpha-SMA, suggesting a direct link to disease pathogenesis. These findings suggest changes to keloid fibroblast phenotype related to mechanotransduction contribute to disease and may be a useful therapeutic target. (C) 2021 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Keloid scarring is a fibroproliferative disorder of the skin with unknown pathophysiology, characterised by fibrotic tissue that extends beyond the boundaries of the original wound. Therapeutic options are few and commonly ineffective, with keloids very commonly recurring even after surgery and adjunct treatments. Epigenetics, defined as alterations to the DNA not involving the base-pair sequence, is a key regulator of cell functions, and aberrant epigenetic modifications have been found to contribute to many pathologies. Multiple studies have examined many different epigenetic modifications in keloids, including DNA methylation, histone modification, microRNAs and long non-coding RNAs. These studies have established that epigenetic dysregulation exists in keloid scars, and successful future treatment of keloids may involve reverting these aberrant modifications back to those found in normal skin. Here we summarise the clinical and experimental studies available on the epigenetics of keloids, discuss the major open questions and future perspectives on the treatment of this disease.

Alveolar epithelial cell (AEC) senescence is implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Mitochondrial dysfunction including release of mitochondrial DNA (mtDNA) is a feature of senescence, which led us to investigate the role of the DNA-sensing guanine monophosphate-adenine monophosphate (GMP-AMP) synthase (cGAS) in IPF, with a focus on AEC senescence. cGAS expression in fibrotic tissue from lungs of patients with IPF was detected within cells immunoreactive for epithelial cell adhesion molecule (EpCAM) and p21, epithelial and senescence markers, respectively. Submerged primary cultures of AECs isolated from lung tissue of patients with IPF (IPF-AECs, n = 5) exhibited higher baseline senescence than AECs from control donors (Ctrl-AECs, n = 5–7), as assessed by increased nuclear histone 2AXγ phosphorylation, p21 mRNA, and expression of senescence-associated secretory phenotype (SASP) cytokines. Pharmacological cGAS inhibition using RU.521 diminished IPF-AEC senescence in culture and attenuated induction of Ctrl-AEC senescence following etoposide-induced DNA damage. Short interfering RNA (siRNA) knockdown of cGAS also attenuated etoposide-induced senescence of the AEC line, A549. Higher levels of mtDNA were detected in the cytosol and culture supernatants of primary IPF- and etoposide-treated Ctrl-AECs when compared with Ctrl-AECs at baseline. Furthermore, ectopic mtDNA augmented cGAS-dependent senescence of Ctrl-AECs, whereas DNAse I treatment diminished IPF-AEC senescence. This study provides evidence that a self-DNA-driven, cGAS-dependent response augments AEC senescence, identifying cGAS as a potential therapeutic target for IPF.

Pulmonary fibrosis is characterised by excessive scarring in the lung which leads to compromised lung function, serious breathing problems and in some diseases, death. It includes several lung disorders with idiopathic pulmonary fibrosis (IPF) the most common and most severe. Pulmonary fibrosis is considered to be perpetuated by aberrant wound healing which leads to fibroblast accumulation, differentiation and activation, and deposition of excessive amounts of extracellular matrix (ECM) components, in particular, collagen. Recent studies have identified the importance of changes in the composition and structure of lung ECM during the development of pulmonary fibrosis and the interaction between ECM and lung cells. There is strong evidence that increased matrix stiffness induces changes in cell function including proliferation, migration, differentiation and activation. Understanding how changes in the ECM microenvironment influence cell behaviour during fibrogenesis, and the mechanisms regulating these changes, will provide insight for developing new treatments.

The timely resolution of wound healing is critical for restoring the skin as a protective barrier. The switch from a proinflammatory to a reparative microenvironment must be tightly regulated. Interleukin (IL)-6 is a key modulator of the inflammatory and reparative process: it is involved in the differentiation, activation, and proliferation of leukocytes, endothelial cells, keratinocytes, and fibroblasts. This review examines the role of IL-6 in the healing of cutaneous wounds, and how dysregulation of IL-6 signaling can lead to either fibrosis or a failure to heal. The role of an IL-6/TGF-β feedback loop is discussed in the context of fibrogenesis, while IL-6 expression and responses in advanced age, diabetes, and obesity is outlined regarding the development of chronic wounds. Current research on therapies that modulate IL-6 is explored. Here, we consider IL-6′s diverse impact on cutaneous wound healing.

The lung epithelium is a cellular barrier that protects the lung from environmental exposure to pathogens and chemical insults that could otherwise compromise its physiological role in gas exchange during respiration. In the distal lung, the exchange of oxygen and carbon dioxide occurs within the alveoli, facilitated by type I alveolar epithelial cells (AECs). In addition to their important role in gas exchange, mucus and surfactant secreted by AECs contain a range of host-defence proteins which aid epithelial barrier function and protection from invading pathogens. The lung epithelium is supported by a network of mesenchymal cells including fibroblasts that form the basement membrane that acts as a further layer of protection to prevent access of microbes to the underlying tissue. Many interstitial lung diseases (ILDs) share similar clinical symptoms and findings on radiographic or pathologic assessment often making diagnosis difficult. The accurate diagnosis of an ILD is critical for the long-term management of the disease and can inform the choice of treatment a patient receives and their prognosis. We are beginning to appreciate the importance that immune status has on disease progression and clinical outcome. In this Special Feature of Clinical & Translational Immunology, the reviews include a broad range of topics examining the clinical challenges associated with the diagnosis of ILDs and the nature of the innate and adaptive immune responses elicited following tissue damage in ILDs and in lung regeneration. This series of articles explores how the immune response is modulated during lung cancer and how novel immunotherapeutic approaches are being explored to treat this disease. The use of animal models to study the pathobiology of ILDs is discussed and highlights how these models may inform novel therapeutic strategies to treat ILDs or lung cancer in humans. The review by McLean-Tooke and colleagues examines the challenges associated with the accurate diagnosis of ILDs in patients.1 There are over 200 different entities of ILDs that have been described. Many exhibit low occurrence rates within the human population which can impact on the accuracy of clinical diagnosis and the ability to study disease pathogenesis. The authors focus on two main diseases: idiopathic pulmonary fibrosis, (IPF) which develops late in life and has a poor prognosis of 2–5 years, and interstitial pneumonia with autoimmune features (IPAF), a feature associated with connective tissue disorders (CTD) that include rheumatoid arthritis, systemic lupus erythematosus and systemic sclerosis. Patients who do not meet the specific criteria of CTD are given a diagnosis of IPAF. Both IPF and IPAF share similar disease symptoms, as well as radiologic and pathologic features, making clinical diagnosis difficult. Some IPAF patients can progress to become CTD, while others do not. The review examines the various diagnostic criteria of histopathology and autoimmune serology to screen for various autoantibodies to systemic autoantigens. Although previous genome-wide association studies have identified a number of genes that influence susceptibility to familial IPF and IPAF, including MUC5B, SPC (epithelial cells), TOLLIP (innate immune cells) or PARN, TERT (telomere genes),2-4 genetic screening of IPF or IPAF for diagnostic purposes is not yet routine. The reviews by Warheit-Niemi et al.5 and Denneney et al.6 examine innate immune function in chronic lung disease. The discovery of pattern recognition receptors (PRRs) and their ability to bind both pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) have helped our understanding of the intimate connection between the innate and adaptive immune system.7 Triggering of PRRs on epithelial cells or innate cells such as macrophages and neutrophils stimulates the release of proinflammatory cytokines/chemokines and effector responses (phagocytosis) to help control infection and promote tissue repair mechanisms to limit inflammation. Warheit-Niemi et al.5 provide a comprehensive overview of the innate immune response to lung immunity and fibrosis. They discuss various preclinical models in mice that have been used to dissect the effector response in lung fibrosis. Consideration is also given to the role of microbial infection as a driver of lung fibrosis. As epithelial cells, fibroblasts and innate immune cells share the ability to express PRRs, they play a pivotal role in directing the nature and chronicity of the inflammatory response. Therefore, it is crucial to understand how recognition of common respiratory pathogens or commensal organisms within the lung microbiota may impact on lung fibrosis. Denenney et al.6 focus on the role of mucins and their receptors in chronic lung disease. Mucus secreted by epithelial cells forms a natural barrier to protect the surface of the lung epithelium from microbes. The mucus is composed of a range of mucin proteins, and these can have important immunomodulatory effects on the innate and adaptive immune responses. The functions of various mucins are examined in both health and disease with a focus on pulmonary fibrosis and other chronic lung conditions such as COPD, asthma, bronchiectasis and lung cancer. Lucas et al.8 provide an overview of the cellular and molecular mechanisms that underpin regenerative processes in the lung following acute or chronic damage. They provide insights into the role of innate and adaptive immune responses in this process. The early response to lung damage involves recognition of PAMPS or DAMPs by PRRs on tissue-resident innate or memory T cells. The balance of TH1/TH17 and Tregs appears crucial for tissue repair processes mediated via interleukin (IL)-17, IL-23, IL-10 and IL-22. Dysregulated immune responses are the hallmark of chronic inflammatory diseases such as COPD and IPF. Lucas et al. describe a range of preclinical mouse studies using various anti-cytokine therapy approaches and cytokine gene knockout models. Although some of these approaches have shown promise, this success has not yet been translated to human patients. Collectively, the three reviews by Warheit-Niemi et al., Denneney et al. and Lucas et al. highlight the important role of innate and adaptive immune responses in tissue damage and repair mechanisms. Miles et al.9 investigate a range of animal models that have been used to study the disease pathogenesis of human IPF. Some animals (e.g. dogs and horses) develop a spontaneous form of interstitial lung disease which resembles many of the clinical features of IPF by radiologic criteria. In contrast, mice do not normally develop spontaneous pulmonary fibrosis. Rather, the delivery of various chemical or drug insults can induce acute lung injury that develops into tissue fibrosis. Although the use of some of these animal models and their relevance to human disease has been debated over the years, it is generally accepted that they provide valuable insight into the cellular and molecular mechanisms driving the fibrogenic process. Furthermore, the mouse immune system is highly analogous to that of humans, and the ease of genetic manipulation in mice has made them a common choice to study disease pathogenesis. Genetic studies in congenic mouse strains have helped define genetic loci that predispose to pulmonary fibrosis following bleomycin treatment. Some of the genes are involved in activation of TCRγδ cells10 which are known to play crucial roles in mucosal immune regulation in mouse and humans. The final review by Neeve et al.11 examines the role of T cells in the control of lung cancer. Anti-tumor responses in the immune system require a coordinated response by both innate and adaptive immune cells. The activation of tumor-specific CD8+ cytotoxic T cells is critical for eliminating cancer cells and reducing tumor burden. However, it is now known that tumor cells can subvert the immune system by expressing checkpoint ligands which bind to inhibitory receptors on T cells, rendering them ineffective for tumor surveillance and eradication. A range of checkpoint inhibitory receptors and ligands have been defined and are being evaluated in a range of clinical trials in cancer. Checkpoint therapy has revolutionised cancer therapy for melanoma, and it is being evaluated for the treatment of lung cancer and other cancer types. Early indications are that monotherapies of checkpoint inhibitors in lung cancer may not be effective in all tumors, thus prompting the use of combination therapies.12 Chronic diseases of the lung, such as pulmonary fibrosis or lung cancer, can disrupt the delicate tissue architecture and compromise gas exchange across alveoli. If disease pathology is not arrested, it can severely impact the quality of life and long-term survival of the patient. We need to better understand the complex relationship between host genetics, the environment, and the host immune response, and how this shapes disease pathogenesis within the lung. Improved diagnostics will be developed with integration of new emerging technologies such as immune cell phenotyping, transcriptomics and metabolomics that may help to better stratify patients into appropriate clinical subgroups. This may have the benefit of improving the outcome of clinical trials of novel interventions that may be targeted to a specific response pathway. The authors declare no conflict of interest. Cecilia Prêle: Conceptualization; Writing-original draft; Writing-review & editing. Gerard Hoyne: Conceptualization; Writing-original draft; Writing-review & editing.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease marked by excessive accumulation of lung fibroblasts (LFs) and collagen in the lung parenchyma. The mechanisms that underlie IPF pathophysiology are thought to reflect repeated alveolar epithelial injury leading to an aberrant wound repair response. Recent work has shown that IPF-LFs display increased characteristics of senescence including growth arrest and a senescence-associated secretory phenotype (SASP) suggesting that senescent LFs contribute to dysfunctional wound repair process. Here, we investigated the influence of senescent LFs on alveolar epithelial cell repair responses in a co-culture system. Alveolar epithelial cell proliferation was attenuated when in co-culture with cells or conditioned media from, senescence-induced control LFs or IPF-LFs. Cell-cycle analyses showed that a larger number of epithelial cells were arrested in G2/M phase when co-cultured with IPF-LFs, than in monoculture. Paradoxically, the presence of LFs resulted in increased A549 migration after mechanical injury. Our data suggest that senescent LFs may contribute to aberrant re-epithelialization by inhibiting proliferation in IPF.

The mesothelium when first described was thought to function purely as a non-adhesive surface to facilitate intracoelomic movement of organs. However, the mesothelium is now recognized as a dynamic cellular membrane with many important functions that maintain serosal integrity and homeostasis. For example, mesothelial cells interact with and help regulate the body's inflammatory and immune system following infection, injury, or malignancy. With recent advances in our understanding of checkpoint molecules and the advent of novel immunotherapy approaches, there has been an increase in the number of studies examining mesothelial and immune cell interaction, in particular the role of these interactions in malignant mesothelioma. This review will highlight some of the recent advances in our understanding of how mesothelial cells help regulate serosal immunity and how in a malignant environment, the immune system is hijacked to stimulate tumor growth. Ways to treat mesothelioma using immunotherapy approaches will also be discussed.

Senescence and mitochondrial stress are mutually reinforcing age-related processes that contribute to idiopathic pulmonary fibrosis (IPF); a lethal disease that manifests primarily in the elderly. Whilst evidence is accumulating that GMP-AMP synthase (cGAS) is crucial in perpetuating senescence by binding damaged DNA released into the cytosol, its role in IPF is not known. The present study examines the contributions of cGAS and self DNA to the senescence of lung fibroblasts from IPF patients (IPF-LFs) and age-matched controls (Ctrl-LFs). cGAS immunoreactivity was observed in regions of fibrosis associated with fibroblasts in lung tissue of IPF patients. Pharmacological inhibition of cGAS or its knockdown by silencing RNA (siRNA) diminished the escalation of IPF-LF senescence in culture over 7 days as measured by decreased p21 and p16 expression, histone 2AXγ phosphorylation and/or IL-6 production (P < 0.05, n = 5-8). The targeting of cGAS also attenuated etoposide-induced senescence in Ctrl-LFs (P < 0.05, n = 5-8). Levels of mitochondrial DNA (mDNA) detected by qPCR in the cytosol and medium of IPF-LFs or senescence-induced Ctrl-LFs were higher than Ctrl-LFs at baseline (P < 0.05, n = 5-7). The addition of DNAse I (100 U/ml) deaccelerated IPF-LF senescence (P < 0.05, n = 5), whereas ectopic mDNA or the induction of endogenous mDNA release augmented Ctrl-LF senescence in a cGAS-dependent manner (P < 0.05, n = 5). In conclusion, we provide evidence that cGAS reinforces lung fibroblast senescence involving damaged self DNA. The targeting of cGAS to supress senescent-like responses may have potential important therapeutic implications in the treatment of IPF.

Pulmonary fibrosis occurs in a heterogeneous group of lung disorders and is characterised by an excessive deposition of extracellular matrix proteins within the pulmonary interstitium, leading to impaired gas transfer and a loss of lung function. In the past 10 years, there has been a dramatic increase in our understanding of the immune system and how it contributes to fibrogenic processes within the lung. This review will compare some of the models used to investigate the pathogenesis and treatment of pulmonary fibrosis, in particular those used to study immune cell pathogenicity in idiopathic pulmonary fibrosis, highlighting their advantages and disadvantages in dissecting human disease.

As a part of an abnormal healing process of dermal injuries and irritation, keloid scars arise on the skin as benign fibroproliferative tumors. Although the etiology of keloid scarring remains unsettled, considerable recent evidence suggested that keloidogenesis may be driven by epigenetic changes, particularly, DNA methylation. Therefore, genome-wide scanning of methylated cytosine-phosphoguanine (CpG) sites in extracted DNA from 12 keloid scar fibroblasts (KF) and 12 control skin fibroblasts (CF) (six normal skin fibroblasts and six normotrophic fibroblasts) was conducted using the Illumina Human Methylation 450K BeadChip in two replicates for each sample. Comparing KF and CF used a Linear Models for Microarray Data (Limma) model revealed 100,000 differentially methylated (DM) CpG sites, 20,695 of which were found to be hypomethylated and 79,305 were hypermethylated. The top DM CpG sites were associated with TNKS2, FAM45B, LOC723972, GAS7, RHBDD2 and CAMKK1. Subsequently, the most functionally enriched genes with the top 100 DM CpG sites were significantly (p ≤ 0.05) associated with SH2 domain binding, regulation of transcription, DNA-templated, nucleus, positive regulation of protein targeting to mitochondrion, nucleoplasm, Swr1 complex, histone exchange, and cellular response to organic substance. In addition, NLK, CAMKK1, LPAR2, CASP1, and NHS showed to be the most common regulators in the signaling network analysis. Taken together, these findings shed light on the methylation status of keloids that could be implicated in the underlying mechanism of keloid scars formation and remission.

Malignant mesothelioma is an aggressive fibrous tumor, predominantly of the pleura, with a very poor prognosis. Cell-matrix interactions are recognized important determinants of tumor growth and invasiveness but the role of the extracellular matrix in mesothelioma is unknown. Mesothelioma cells synthesize collagen as well as transforming growth factor-beta (TGF-β), a key regulator of collagen production. This study examined the effect of inhibiting collagen production on mesothelioma cell proliferation in vitro and tumor growth in vivo. Collagen production by mesothelioma cells was inhibited by incubating cells in vitro with the proline analogue thiaproline (thiazolidine-4-carboxylic acid) or by oral administration of thiaproline in a murine tumor model. Cell cytotoxicity was measured using neutral red uptake and lactate dehydrogenase assays. Proliferation was measured by tritiated thymidine incorporation, and inflammatory cell influx, proliferation, apoptosis and angiogenesis in tumors examined by immunohistochemical labelling. Tumor size was determined by tumor weight and collagen production was measured by HPLC. Thiaproline at non-toxic doses significantly reduced basal and TGF-β-induced collagen production by over 50% and cell proliferation by over 65%. In vivo thiaproline administration inhibited tumor growth at 10 days, decreasing the median tumor weight by 80%. The mean concentration of collagen was 50% lower in the thiaproline-treated tumors compared with the controls. There were no significant differences in vasculature or inflammatory cell infiltration but apoptosis was increased in thiaproline treated tumors at day 10. In conclusion, these observations strongly support a role for collagen in mesothelioma growth and establish the potential for inhibitors of collagen synthesis in mesothelioma treatment.