Cluster analysis of blood biomarkers to identify molecular patterns in pulmonary fibrosis: assessment of a multicentre, prospective, observational cohort with independent validation.

BACKGROUND: Pulmonary fibrosis results from alveolar injury, leading to extracellular matrix remodelling and impaired lung function. This study aimed to classify patients with pulmonary fibrosis according to blood biomarkers to differentiate distinct disease patterns, known as endotypes. METHODS: In this cluster analysis, we first classified patients from the PROFILE study, a multicentre, prospective, observational cohort of individuals with incident idiopathic pulmonary fibrosis or non-specific interstitial pneumonia in the UK (Nottingham University Hospitals, Nottingham; and Royal Brompton Hospital, London). 13 blood biomarkers representing extracellular matrix remodelling, epithelial stress, and thrombosis were measured by ELISA in the PROFILE study. We classified patients by unsupervised consensus clustering. To evaluate generalisability, a machine learning classifier trained on biomarker signatures derived from consensus clustering was applied to a replication dataset from the Australian Idiopathic Pulmonary Fibrosis Registry (AIPFR). Biomarker associations with mortality and change in percentage of predicted forced vital capacity (FVC%) were assessed, adjusting for age, gender, baseline FVC%, and antifibrotic treatment and steroid treatment before and after baseline. Mortality risk associated with the clusters in the PROFILE cohort was evaluated with Cox proportional hazards models, and mixed-effects models were used to analyse how clustering was associated with longitudinal FVC% in the PROFILE and AIPFR cohorts. FINDINGS: 455 of 580 participants from the PROFILE study (348 [76%] men and 107 [24%] women; mean age 72·4 years [SD 8·3]) were included in the analysis. Within this group, three clusters were identified based on blood biomarkers. A basement membrane collagen (BM) cluster (n=248 [55%]) showed high concentrations of PRO-C4, PRO-C28, C3M, and C6M, whereas an epithelial injury (EI) cluster (n=109 [24%]) showed high concentrations of MMP-7, SP-D, CYFRA211, CA19-9, and CA-125. The third cluster (crosslinked fibrin [XF] cluster; n=98 [22%]) had high concentrations of X-FIB. In the replication dataset (117 of 833 patients from AIPFR; 87 [74%] men and 30 [26%] women; mean age 72·9 years [SD 7·9]), we identified the same three clusters (BM cluster, n=93 [79%]; EI cluster, n=8 [7%]; XF cluster, n=16 [14%]). These clusters showed similarities with clusters in the PROFILE dataset regarding blood biomarkers and phenotypic signatures. In the PROFILE dataset, the EI and XF clusters were associated with increased mortality risk compared with the BM cluster (EI vs BM: adjusted hazard ratio [HR] 1·88 [95% CI 1·42-2·49], p<0·0001; XF vs BM: adjusted HR 1·53 [1·13-2·06], p=0·0058). The EI cluster showed the greatest annual FVC% decline, followed by the BM and XF clusters. A similar FVC% decline pattern was observed in these clusters in the AIPFR replication dataset. INTERPRETATION: Blood biomarker clustering in pulmonary fibrosis identified three distinct blood biomarker signatures associated with lung function and prognosis, suggesting unique pulmonary fibrosis biomarker patterns. These findings support the presence of pulmonary fibrosis endotypes with the potential to guide targeted therapy development. FUNDING: None.

Forced vital capacity trajectories in patients with idiopathic pulmonary fibrosis: a secondary analysis of a multicentre, prospective, observational cohort.

BACKGROUND: Idiopathic pulmonary fibrosis is a progressive fibrotic lung disease with a variable clinical trajectory. Decline in forced vital capacity (FVC) is the main indicator of progression; however, missingness prevents long-term analysis of patterns in lung function. We aimed to identify distinct clusters of lung function trajectory among patients with idiopathic pulmonary fibrosis using machine learning techniques. METHODS: We did a secondary analysis of longitudinal data on FVC collected from a cohort of patients with idiopathic pulmonary fibrosis from the PROFILE study; a multicentre, prospective, observational cohort study. We evaluated the imputation performance of conventional and machine learning techniques to impute missing data and then analysed the fully imputed dataset by unsupervised clustering using self-organising maps. We compared anthropometric features, genomic associations, serum biomarkers, and clinical outcomes between clusters. We also performed a replication of the analysis on data from a cohort of patients with idiopathic pulmonary fibrosis from an independent dataset, obtained from the Chicago Consortium. FINDINGS: 415 (71%) of 581 participants recruited into the PROFILE study were eligible for further analysis. An unsupervised machine learning algorithm had the lowest imputation error among tested methods, and self-organising maps identified four distinct clusters (1-4), which was confirmed by sensitivity analysis. Cluster 1 comprised 140 (34%) participants and was associated with a disease trajectory showing a linear decline in FVC over 3 years. Cluster 2 comprised 100 (24%) participants and was associated with a trajectory showing an initial improvement in FVC before subsequently decreasing. Cluster 3 comprised 113 (27%) participants and was associated with a trajectory showing an initial decline in FVC before subsequent stabilisation. Cluster 4 comprised 62 (15%) participants and was associated with a trajectory showing stable lung function. Median survival was shortest in cluster 1 (2·87 years [IQR 2·29-3·40]) and cluster 3 (2·23 years [1·75-3·84]), followed by cluster 2 (4·74 years [3·96-5·73]), and was longest in cluster 4 (5·56 years [5·18-6·62]). Baseline FEV1 to FVC ratio and concentrations of the biomarker SP-D were significantly higher in clusters 1 and 3. Similar lung function clusters with some shared anthropometric features were identified in the replication cohort. INTERPRETATION: Using a data-driven unsupervised approach, we identified four clusters of lung function trajectory with distinct clinical and biochemical features. Enriching or stratifying longitudinal spirometric data into clusters might optimise evaluation of intervention efficacy during clinical trials and patient management. FUNDING: National Institute for Health and Care Research, Medical Research Council, and GlaxoSmithKline.

Transcriptional analysis of adipose tissue during development reveals depot-specific responsiveness to maternal dietary supplementation.

Brown adipose tissue (BAT) undergoes pronounced changes after birth coincident with the loss of the BAT-specific uncoupling protein (UCP)1 and rapid fat growth. The extent to which this adaptation may vary between anatomical locations remains unknown, or whether the process is sensitive to maternal dietary supplementation. We, therefore, conducted a data mining based study on the major fat depots (i.e. epicardial, perirenal, sternal (which possess UCP1 at 7 days), subcutaneous and omental) (that do not possess UCP1) of young sheep during the first month of life. Initially we determined what effect adding 3% canola oil to the maternal diet has on mitochondrial protein abundance in those depots which possessed UCP1. This demonstrated that maternal dietary supplementation delayed the loss of mitochondrial proteins, with the amount of cytochrome C actually being increased. Using machine learning algorithms followed by weighted gene co-expression network analysis, we demonstrated that each depot could be segregated into a unique and concise set of modules containing co-expressed genes involved in adipose function. Finally using lipidomic analysis following the maternal dietary intervention, we confirmed the perirenal depot to be most responsive. These insights point at new research avenues for examining interventions to modulate fat development in early life.

Brown adipose tissue development and function and its impact on reproduction.

Although brown adipose tissue (BAT) is one of the smallest organs in the body, it has the potential to have a substantial impact on both heat production as well as fat and carbohydrate metabolism. This is most apparent at birth, which is characterised with the rapid appearance and activation of the BAT specific mitochondrial uncoupling protein (UCP)1 in many large mammals. The amount of brown fat then gradually declines with age, an adaptation that can be modulated by the thermal environment. Given the increased incidence of maternal obesity and its potential transmission to the mother's offspring, increasing BAT activity in the mother could be one mechanism to prevent this cycle. To date, however, all rodent studies investigating maternal obesity have been conducted at standard laboratory temperature (21°C), which represents an appreciable cold challenge. This could also explain why offspring weight is rarely increased, suggesting that future studies would benefit from being conducted at thermoneutrality (~28°C). It is also becoming apparent that each fat depot has a unique transcriptome and show different developmental pattern, which is not readily apparent macroscopically. These differences could contribute to the retention of UCP1 within the supraclavicular fat depot, the most active depot in adult humans, increasing heat production following a meal. Despite the rapid increase in publications on BAT over the past decade, the extent to which modifications in diet and/or environment can be utilised to promote its activity in the mother and/or her offspring remains to be established.

Gene pathway development in human epicardial adipose tissue during early life.

Studies in rodents and newborn humans demonstrate the influence of brown adipose tissue (BAT) in temperature control and energy balance and a critical role in the regulation of body weight. Here, we obtained samples of epicardial adipose tissue (EAT) from neonates, infants, and children in order to evaluate changes in their transcriptional landscape by applying a systems biology approach. Surprisingly, these analyses revealed that the transition to infancy is a critical stage for changes in the morphology of EAT and is reflected in unique gene expression patterns of a substantial proportion of thermogenic gene transcripts (~10%). Our results also indicated that the pattern of gene expression represents a distinct developmental stage, even after the rebound in abundance of thermogenic genes in later childhood. Using weighted gene coexpression network analyses, we found precise anthropometric-specific correlations with changes in gene expression and the decline of thermogenic capacity within EAT. In addition, these results indicate a sequential order of transcriptional events affecting cellular pathways, which could potentially explain the variation in the amount, or activity, of BAT in adulthood. Together, these results provide a resource to elucidate gene regulatory mechanisms underlying the progressive development of BAT during early life.

Impact of maternal dietary fat supplementation during gestation upon skeletal muscle in neonatal pigs.

BACKGROUND: Maternal diet during pregnancy can modulate skeletal muscle development of the offspring. Previous studies in pigs have indicated that a fat supplemented diet during pregnancy can improve piglet outcome, however, this is in contrast to human studies suggesting adverse effects of saturated fats during pregnancy. This study aimed to investigate the impact of a fat supplemented (palm oil) "high fat" diet on skeletal muscle development in a porcine model. Histological and metabolic features of the biceps femoris muscle obtained from 7-day-old piglets born to sows assigned to either a commercial (C, n = 7) or to an isocaloric fat supplementation diet ("high fat" HF, n = 7) during pregnancy were assessed. RESULTS: Offspring exposed to a maternal HF diet demonstrated enhanced muscular development, reflected by an increase in fractional growth rate, rise in myofibre cross-sectional area, increased storage of glycogen and reduction in lipid staining of myofibres. Although both groups had similar intramuscular protein and triglyceride concentrations, the offspring born to HF mothers had a higher proportion of arachidonic acid (C20:4n6) and a reduction in α-linolenic acid (C18:3n3) compared to C group offspring. The HF group muscle also exhibited a higher ratio of C20:3n6 to C20:4n6 and total n-6 to n-3 in conjunction with up-regulation of genes associated with free fatty acid uptake and biogenesis. CONCLUSION: In conclusion, a HF gestational diet accelerates the maturation of offspring biceps femoris muscle, reflected in increased glycolytic metabolism and fibre cross sectional area, differences accompanied with a potential resetting of myofibre nutrient uptake.

Molecular characterization of adipose tissue in the African elephant (Loxodonta africana).

Adipose tissue (AT) is a dynamic and flexible organ with regulatory roles in physiological functions including metabolism, reproduction and inflammation; secreted adipokines, including leptin, and fatty acids facilitate many of these roles. The African elephant (Loxodonta africana) is experiencing serious challenges to optimal reproduction in captivity. The physiological and molecular basis of this impaired fertility remains unknown. AT production of leptin is a crucial molecular link between nutritional status, adiposity and fertility in many species. We propose that leptin has a similar function in the African elephant. African elephant visceral and subcutaneous adipose tissue (AT) was obtained from both sexes and a range of ages including females with known pregnancy status. RNA was extracted and histological sections created and analyzed by microarray, PCR and immunohistochemistry respectively. Gas-chromatography was used to determine the fatty acid composition of AT. Microarray expression profiling was used to compare gene expression profiles of AT from pre-pubertal versus reproductively competent adult African elephants. This study demonstrates, for the first time, leptin mRNA and protein expression in African elephant AT. The derived protein sequence of the elephant leptin protein was exploited to determine its relationship within the class I helical cytokine superfamily, which indicates that elephant leptin is most closely related to the leptin orthologs of Oryctolagus cuniculus (European rabbit), Lepus oiostolus (woolly hare), and members of the Ochotonidae (Pika). Immunohistological analysis identified considerable leptin staining within the cytoplasm of adipocytes. Significant differences in fatty acid profiles between pregnant and non-pregnant animals were revealed, most notably a reduction in both linoleic and α linoleic acid in pregnant animals. This report forms the basis for future studies to address the effect of nutrient composition and body condition on reproduction in captive and wild elephants.

Reduced neonatal mortality in Meishan piglets: a role for hepatic fatty acids?

The Meishan pig breed exhibits increased prolificacy and reduced neonatal mortality compared to commercial breeds, such as the Large White, prompting breeders to introduce the Meishan genotype into commercial herds. Commercial piglets are highly susceptible to hypoglycemia, hypothermia, and death, potentially due to limited lipid stores and/or delayed hepatic metabolic ability. We therefore hypothesized that variation in hepatic development and lipid metabolism could contribute to the differences in neonatal mortality between breeds. Liver samples were obtained from piglets of each breed on days 0, 7, and 21 of postnatal age and subjected to molecular and biochemical analysis. At birth, both breeds exhibited similar hepatic glycogen contents, despite Meishan piglets having significantly lower body weight. The livers from newborn Meishan piglets exhibited increased C18∶1n9C and C20∶1n9 but lower C18∶0, C20∶4n6, and C22∶6n3 fatty acid content. Furthermore, by using an unsupervised machine learning approach, we detected an interaction between C18∶1n9C and glycogen content in newborn Meishan piglets. Bioinformatic analysis could identify unique age-based clusters from the lipid profiles in Meishan piglets that were not apparent in the commercial offspring. Examination of the fatty acid signature during the neonatal period provides novel insights into the body composition of Meishan piglets that may facilitate liver responses that prevent hypoglycaemia and reduce offspring mortality.

Impact of early onset obesity and hypertension on the unfolded protein response in renal tissues of juvenile sheep.

Childhood obesity has reached epidemic proportions. Obesity is an independent risk factor for the development of end-stage renal disease. Endoplasmic reticulum stress and subsequent activation of the unfolded protein response (UPR) are implicated in the development of adipose tissue dysregulation and type 2 diabetes mellitus in obesity. The present study explored the impact of adolescent-onset obesity on the UPR after obesity-related hypertension and nephropathy, using an ovine model in which obesity was induced by increased food intake and reduced activity. Obese young adults had a higher mean arterial pressure (lean, 89.6+/-1.7 mm Hg versus obese, 101+/-3.0 mm Hg; P<0.01) and greater sensitivity to low physiological doses of angiotensin II. Obesity increased the glomerular area and was associated with activation of the UPR in renal cells with a greater abundance of glucose-regulated protein 78, C/EBP homologous protein, Bax, phosphorylated c-Jun amino-terminal kinase, and activating transcription factor 6 (all P<0.05). In addition, there was a marked upregulation of proinflammatory genes, most notably those involved in macrophage signaling. Reactive oxygen species production and handling were also perturbed in obese adults. Renal endoplasmic reticulum stress was positively correlated with macrophage content (r=0.83; P<0.001), phosphorylated c-Jun amino-terminal kinase (r=0.73; P<0.01), and adiposity (r=0.71; P<0.01). In conclusion, adolescent-onset, obesity-related renal endoplasmic reticulum stress was associated with activation of the UPR, apoptosis, and inflammation, potentially increasing the associated renal damage observed in young adults. The UPR may prove to be a useful therapeutic target for the treatment and prevention of obesity-related nephropathy and associated hypertension, thereby reducing the burden of end-stage renal disease.

Maternal nutrient restriction during pregnancy differentially alters the unfolded protein response in adipose and renal tissue of obese juvenile offspring.

Maternal diet during pregnancy can program an offspring's risk of disease in later life. Obesity adversely alters renal and adipose tissue function, resulting in chronic kidney disease and insulin resistance, respectively, the latter associated with dysregulation of the unfolded protein response (UPR). In view of the current obesity epidemic, we explored the combined effects of in utero early- to midgestational nutrient restriction and postnatal obesity on the UPR in ovine juvenile offspring. Nutrient restriction was coincident with fetal kidney development but prior to exponential adipose tissue deposition. Nutrient restricted (NR) and normal diet (control) offspring were exposed to an obesogenic environment throughout adolescence, resulting in similar degrees of juvenile obesity. NR offspring showed enhanced adipose tissue dysregulation characterized by activation of the UPR, perturbed insulin signaling, and marked inflammation, as demonstrated by increased abundance of crownlike structures and proinflammatory genes. Conversely, in renal tissue NR offspring had marked attenuation of cellular stress and inflammation evident as reduced activation of the UPR, down-regulation of proinflammatory genes, and less histological damage. In conclusion, obesity-related activation of the UPR can be determined by the in utero nutritional environment, demonstrating organ-specific effects dependent on the developmental phase targeted within the fetus.

Global regulation of virulence and the stress response by CsrA in the highly adapted human gastric pathogen Helicobacter pylori.

Although successful and persistent colonization of the gastric mucosa depends on the ability to respond to changing environmental conditions and co-ordinate the expression of virulence factors during the course of infection, Helicobacter pylori possesses relatively few transcriptional regulators. We therefore investigated the contribution of the regulatory protein CsrA to global gene regulation in this important human pathogen. CsrA was necessary for full motility and survival of H. pylori under conditions of oxidative stress. Loss of csrA expression deregulated the oxidant-induced transcriptional responses of napA and ahpC, the acid induction of napA, cagA, vacA, the urease operon, and fur, as well as the heat shock responses of napA, groESL and hspR. Although the level of napA transcript was higher in the csrA mutant, its stability was similar in the wild-type and mutant strains, and less NapA protein was produced in the mutant strain. Finally, H. pylori strains deficient in the production of CsrA were significantly attenuated for virulence in a mouse model of infection. This work provides evidence that CsrA has a broad role in regulating the physiology of H. pylori in response to environmental stimuli, and may be important in facilitating adaptation to the different environments encountered during colonization of the gastric mucosa. Furthermore, CsrA appears to mediate its effects in H. pylori at the post-transcriptional level by influencing the processing and translation of target transcripts, with minimal effect on the stability of the target mRNAs.