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Cellular and humoral biomarkers of Bronchopulmonary Dysplasia
EHD-04366; No of Pages 5 Early Human Development xxx (2016) xxx–xxx
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Early Human Development journal homepage: www.elsevier.com/locate/earlhumdev
Cellular and humoral biomarkers of Bronchopulmonary Dysplasia Charitharth Vivek Lal ⁎, Namasivayam Ambalavanan Division of Neonatology, Department of Pediatrics, University of Alabama at Birmingham, 176F Suite 9380, Women and Infants Center, 619 South 19th Street, Birmingham, AL 35249-7335, United States
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Article history: Available online xxxx Keywords: Bronchopulmonary Dysplasia Pulmonary hypertension Microbiome Biomarkers Systems biology
a b s t r a c t The pathogenesis of Bronchopulmonary Dysplasia (BPD) is multifactorial and the clinical phenotype of BPD is extremely variable. Predicting BPD is difficult, as it is a disease with a clinical operational definition but many clinical phenotypes and endotypes. Most biomarkers studied over the years have low predictive accuracy, and none are currently used in routine clinical care or shown to be useful for predicting longer-term respiratory outcome. Targeted cellular and humoral biomarkers and novel systems biology ‘omic’ based approaches including genomic and microbiomic analyses are described in this review. © 2016 Published by Elsevier Ireland Ltd.
Bronchopulmonary Dysplasia (BPD) was described in 1967 by Northway et al. [1] as pulmonary disease resulting from mechanical ventilation in preterm infants with respiratory distress syndrome. Over the next five decades, neonatal intensive care has progressed to a remarkable extent, with therapies such as antenatal glucocorticoids, surfactant, and gentle ventilation strategies, which have improved survival and reduced BPD in more mature preterm infants [2,3] although BPD today continues to be a significant morbidity in very preterm infants born weighing b1 kg at birth [3,4]. The neonatal outcomes of extremely preterm infants from the National Institute of Child Health and Human Development (NICHD) Neonatal Research Network (NRN) show that the increasing survival of infants born earlier in gestation has resulted in the incidence of BPD being relatively stable over the past two decades, although BPD increased between 2009 and 2012 for infants at 26 to 27 weeks' gestation (26 weeks, 50% to 55%; P b 0.001) [3,5]. The National Institute of Health consensus conference on BPD classified BPD as mild, moderate, or severe in very preterm infants based on the magnitude of respiratory support at 28 days age and 36 w postmenstrual age [6]. However, variability in practices of physicians with regards to standards for oxygen requirements and target oxygen saturation ranges influence the incidence and severity of BPD using this definition. To overcome this, Walsh et al. in the NICHD NRN recently developed a physiologic definition of BPD [7]. The physiologic definition reduced the overall rate of BPD and reduced the variation among centers. However, the clinical definition of oxygen requirement even when more precisely defined using the physiologic definition of BPD [7] is only an operational definition, as the magnitude of lung disease ⁎ Corresponding author. E-mail addresses: [email protected] (C.V. Lal), [email protected] (N. Ambalavanan).
or the exact underlying pathology may vary among infants diagnosed with BPD. Recent studies indicate that severe BPD is probably a different entity from mild or moderate BPD in terms of genetic predisposition [8, 9]. This genetic predisposition varies by race/ethnicity, suggesting that biologic pathways contributing to BPD and any biomarkers resulting from this altered genetic background are probably likely to be different [9]. As we have recently stated [10], it is likely that what is now termed “BPD” is not a single entity, nor even a spectrum of disease resulting from a single pathophysiologic process, but a combination of several chronic lung diseases characterized by a common “at-risk population” of infants in the saccular or early alveolar stage of lung development with varying magnitudes of impairment of alveolar septation, lung fibrosis, and abnormal vascular development and remodeling. Infants with BPD may have primarily lung parenchymal disease with low lung compliance and pathology characterized by atelectasis or cystic lesions in their lung parenchyma [1]. Other infants with BPD have more vascular remodeling and impaired lung angiogenesis, with pulmonary hypertension as a major clinical manifestation [11]. Some infants with BPD have airway injury and remodeling, resulting in severe tracheobronchomalacia [12]. There are therefore several phenotypes and endotypes in BPD, each of which may have different pathophysiology mediated directly by cells (cellular mediators) and by macromolecules originating from cells found in extracellular fluids (humoral mediators). Certain cellular and humoral constituents may serve both as mediators of pathophysiology as well as biomarkers of disease, while other molecules may serve as biomarkers but may not be directly involved in pathogenesis. Laboratory-based test markers could be utilized as biomarkers if they characterize disease activity in a manner useful for early diagnosis, prediction of disease severity and monitoring disease processes and response to therapy [13,14]. Earlier diagnosis, before conventional methods of diagnosis are used, may permit the use of therapies that arrest lung injury at an early stage and allow for greater
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Please cite this article as: C.V. Lal, N. Ambalavanan, Cellular and humoral biomarkers of Bronchopulmonary Dysplasia, Early Hum Dev (2016), http://dx.doi.org/10.1016/j.earlhumdev.2016.12.003
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lung development. The biomarkers may also permit quantitation of disease severity, which is useful in following response to therapies. Biomarkers may also help determine prognosis and help plan for additional therapies. In this manuscript, we will review various targeted (i.e., focusing on known targets) cellular and humoral biomarkers of BPD and unbiased systems biology based approaches to identification of biomarkers in BPD.
1. Targeted cellular biomarkers of Bronchopulmonary Dysplasia Abnormalities in the number and function of multiple cell types, primarily those related to inflammation and lung repair, have been shown in human BPD as well as in animal models of BPD (e.g. hyperoxia-exposed newborn rodent models). Early in the course of respiratory distress syndrome in human preterm infants, there is evidence of early systemic activation and transendothelial migration of neutrophils [15], with increased neutrophils in the tracheal aspirate of preterm infants who go on to develop BPD [16]. Anti-neutrophil chemokine therapy [17] or CXCR2 antagonist therapy [18] prevents neutrophil accumulation and preserves alveolar development in hyperoxia-exposed newborn rats. CD18 expression on neutrophils and monocytes and CD62L on neutrophils is decreased in preterm neonates who go on to develop BPD, compared to non-BPD neonates on days 1–28 [19]. Changes in lymphocytes then become evident; Ballabh et al. [20] showed that infants with RDS who went on to develop BPD had fewer lymphocytes, as well as absolute number and % of CD4+ T-cells. The percentage of CD4+ T-cells expressing CD62L was selectively reduced in infants developing BPD, suggesting that reduction of CD4(+) T cells and especially those important in tissue migration and immune surveillance may be relevant to the pathogenesis of BPD [20]. More recently, Mishra et al. [21] evaluated cord blood mononuclear cells from infants (GA b32 weeks), with or without placental histological evidence of chorioamnionitis (CA). Absolute regulatory T-cell (Treg) numbers were not different in CA and non-CA exposed samples. However, the infants who developed BPD had a significant decrease in Treg and nonregulatory T cell numbers, suggesting that the development of BPD is marked by distinct inflammatory changes from those of CA exposed infants [21]. Eosinophil activation has been associated with BPD, as the eosinophil count is higher in BPD compared with RDS and healthy infants, along with increases in the humoral biomarkers eosinophil cationic protein (ECP) and the cellular surface antigen (CD9) [22]. We have also shown recently that the absolute eosinophil count is higher on the first postnatal day in infants who go on to develop BPD as compared to infants who survive without BPD, associated with increases in the humoral biomarker eotaxin-1 (CCL11), a chemokine selective for eosinophils [23]. An increase in pulmonary neuroendocrine cells containing bombesin-like peptide (BLP) has been described in BPD [24,25], and BLP mediates lung injury in a preterm baboon model of BPD [26]. This cellular phenotype of neuroendocrine hyperplasia is accompanied by a humor biomarker, as urinary BLP elevation precedes clinical evidence of BPD [27]. There is some evidence that BLP may mediate premature thymic maturation and thymic nurse-cell depletion leading to autoreactive T-cells in BPD that may injure the lung [28]. Neonatal rat models of hyperoxia exposure also demonstrate an association between hyperoxia, airway remodeling, and pulmonary neuroendocrine hyperplasia [29]. An important characteristic of the histology of BPD is the disrupted capillary development. An initial study by Borghesi et al. demonstrated that circulating endothelial progenitor cells (endothelial colonyforming cells or ECFCs) are fewer at lower gestational ages, and fewer in cord blood in infants who later develop BPD, while endothelial and hematopoietic cell subsets are comparable in infants with and without BPD [30]. However, a subsequent study by the same author indicated
that different subsets of circulating angiogenic cells did not predict BPD [31]. Connective tissue mast cells (chymase expressing) accumulate in BPD lung tissue both in humans and in animal models of BPD, and genes associated with these cells are highly expressed in lung tissue, suggesting a potential role for these cells, not just as biomarkers, but in the development or attempt at resolution of BPD [32]. Mesenchymal stromal cells (MSCs) can be isolated from tracheal aspirates of premature neonates with RDS, primarily in infants who go on to develop BPD and rarely in those who do not develop BPD [33]. These MSCs have a pattern of lung-specific gene expression, are distinct from lung fibroblasts, and secrete pro-inflammatory cytokines [34]. While these MSCs are a cellular biomarker, their role in the development of BPD is uncertain. 2. Targeted humoral biomarkers of Bronchopulmonary Dysplasia Humoral biomarkers refer to biomarkers present in body fluids (called “humors”) which were categorized in ancient literature as blood, yellow bile, black bile, and phlegm. In this review, we will focus primarily on blood and airway secretions (phlegm). Biomarker measurements in blood have been studied due to easy accessibility, but they may not accurately reflect the lung concentrations of the respective mediator. Tracheal aspirates also have limitations, as they are available only in intubated infants and may not necessarily reflect the distal lung parenchymal milieu. As BPD is considered to result from the effect of inflammatory processes upon the substrate of an immature and fragile developing lung, investigators have focused upon changes in inflammatory mediators or changes in growth factors that may be associated with lung injury and alterations in alveolar, airway, or vascular development. As recently reviewed by us [10], increases in blood of multiple biomarkers such as interleukin (IL)-1β, −6, −8, −10, interferon (IFN)-γ, granulocyte colony stimulating factor (GCSF), endostatin, vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) BB, placental growth factor (PGF), Kerbs von Lungren 6 (KL-6), matrix metalloproteinases/tissue inhibitors of metalloproteinases (MMPs/TIMPs), Eselectin, and B-type natriuretic peptide (BNP) have been associated with development of BPD, while concentrations of other biomarkers such as IL-17, chemokine (C-C motif) ligand 5 (CCL5; RANTES or regulated on activation, normal T cell expressed and secreted), tumor necrosis factor (TNF)-β, angiopoietin 1 (ANG-1), VEGF, clara cell proteins (CCP), C-terminal type I procollagen (PICP) have been reduced in infants who developed BPD. Similarly, concentrations of various cytokines and inflammatory molecules (IL-1β, − 6, − 8, TNF-α, monocyte chemoattractant protein (MCP), nuclear factor kappa B (NFkB), neutrophil gelatinase-associated lipocalin (NGAL), MMP-9, transforming growth factor (TGF)-β1, L-selectin, Ang-2, endothelin-1, fibroblast growth factor-2, pepsin, oxyradicals, carbonyls, 3-chlorotyrosine, malondialdehyde) are increased in tracheal aspirates in infants who go on to develop BPD, while concentrations of growth factors and anti-inflammatory substances such as macrophage migration inhibitory factor (MIF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), VEGF, polyunsaturated fatty acids (PUFA), CCP, parathyroid hormone-related protein (PTHrP) are reduced in infants who survive without BPD [10]. As space limitations preclude us from providing a detailed review of changes of each of the biomarkers in blood or tracheal aspirates, we refer the reader to several recent comprehensive reviews on such biomarkers [10,35–37]. A few clinically important issues regarding the use of these biomarkers must be noted. First, most of these biomarkers (either cellular or humoral) have been shown to have poor predictive accuracy (predictive value, sensitivity, specificity) in isolation, and do not add much to the predictive ability of clinical variables (gestational age, birth weight, respiratory illness severity etc) even if they are statistically significant by themselves [38]. In a study of 1067 infants by the NICHD NRN who
Please cite this article as: C.V. Lal, N. Ambalavanan, Cellular and humoral biomarkers of Bronchopulmonary Dysplasia, Early Hum Dev (2016), http://dx.doi.org/10.1016/j.earlhumdev.2016.12.003
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had 25 cytokines measured in the blood at birth and on days 3, 7, 14, and 21, BPD/death was associated with higher peak IL-1β, −6,-8, −10, and IFN-γ, and lower IL-17, RANTES, and TNF-β [38]. However, compared with models with only clinical variables, the addition of cytokine data improved predictive ability by a statistically significant but clinically modest magnitude (for peak cytokines: r-square 0.56 with cytokines vs. 0.53 without cytokines, c-statistic 0.89 vs. 0.88, p b 0.001) [38]. Second, rather than absolute concentrations of a biomarker at one point in time, the pattern of change of multiple biomarkers over time using serial sampling may be informative. Data from infants in the NICHD NRN cytokine study was analyzed in relation to the following lung disease patterns: (1) no lung disease (NLD); (2) respiratory distress syndrome without BPD; (3) classic BPD (persistent exposure to supplemental oxygen until 28 days of age); or (4) atypical BPD (period without supplemental oxygen before 28 days) [39]. Median cytokine levels for infants with BPD were compared with the interquartile range (IQR) of results among infants with NLD [39]. Median levels of 3 cytokines (elevated IL-8, MMP-9; decreased GMCSF) fell outside the IQR for at least 2 time points in both infants with atypical and classic BPD. Profiles of 7 cytokines (IL-6, IL-10, IL-18, macrophage inflammatory protein-1α, C-reactive protein (CRP), brain-derived neurotrophic factor, RANTES) differed between infants with classic and atypical BPD, suggesting that the differences in blood cytokine profiles may be related to variations in pathophysiology of BPD [39]. Third, many of these biomarkers are suitable for analyses in research laboratories using methods such as immunoassays (e.g. ELISA) or multiplex assays (Luminex, Meso Scale Diagnostics), which generally involve batch processing and longer turn-around times, rather than near-patient or point of care testing with rapid results. These assays also demonstrate much variation from laboratory to laboratory, as they are generally done in research laboratories with varying levels of quality control and not College of American Pathologists (CAP) accredited clinical laboratories (with the possible exception of biomarkers such as CRP). Therefore, while the evaluation of the biomarkers provides an improved estimate of risk for BPD or mortality (a competing outcome for death, as infants at high risk of BPD are also at higher risk of death, and infants who die cannot develop BPD), these biomarkers are not yet sufficiently accurate or precise for predicting BPD, or widely available for inclusion into routine clinical practice. Finally, another important issue is that while the focus of most investigations is on prediction of BPD, most infants with BPD eventually improve and are discharged home (some on supplemental oxygen), and it is critically important to be able to predict longer-term abnormalities in airway function (e.g. increased baseline airway resistance or airway reactivity) or impaired gas exchange, and the likelihood of early-onset chronic obstructive pulmonary disease (COPD) in former preterm infants. 3. Unbiased “omic” biomarkers of BPD Advances in molecular genetics and next-generation sequencing (NGS) technology have enabled the examination of BPD at an unbiased molecular ‘omic’ level. In this review, we will focus on biomarkers in the genome, transcriptome, and microbiome. 3.1. The genome in BPD Twin studies indicate that there is a significant genetic contribution to the risk of BPD, ranging from 53% [40] to 82% [41] of the variance in liability for BPD. Many studies to date have focused on targeted analysis of single nucleotide polymorphisms (SNPs), and these studies will not be reviewed further, due to space limitations and the existence of several existing recent comprehensive reviews by us [8] and others [42]. Three unbiased genome-wide association studies (GWAS) have been published to date for BPD, and the first GWAS by Hadchouel et al. [43] identified the SPOCK2 gene in two discovery series, and the most significant polymorphism was confirmed by individual genotyping and in a replication population. The expression pattern of SPOCK2 (increase in
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mRNA levels during alveolar stage, increase with hyperoxia exposure) pointed to a potential role in alveolarization [43]. However, this finding was not replicated in subsequent GWAS by Wang et al. [44] or Ambalavanan et al. [9] which did not identify any SNPs at the genome-wide significance threshold. The gene set pathway analysis by Ambalavanan et al. [9] confirmed involvement of known pathways of lung development and repair (CD44, phosphorus oxygen lyase activity) and indicated novel molecules and pathways (adenosine deaminase, targets of miR-219) involved in genetic predisposition to BPD. Other important findings were that severe BPD or death are associated with pathways distinct from mild/moderate BPD, suggesting that they have a different pathophysiologic basis, and that much variation is present in genetic predisposition to BPD by race/ethnicity [9]. More recently, next generation sequencing (NGS) has been used to gain insight into diseases, utilizing either whole exome sequencing (WES) or whole genome sequencing. In a recent study, Carrera et al. [45] performed exome sequencing and pathway analysis on a cohort of 26 unrelated BPD patients to identify non-common variants. 3369 novel variants were identified, with a median of 400 variations per sample. The top candidate genes highlighted were NOS2, MMP1, CRP, LBP and the toll-like receptor (TLR) family, which were confirmed by Sanger sequencing [45]. Li et al. [46] also performed exome sequencing on 50 BPD-affected and unaffected twin pairs using DNA isolated from neonatal blood spots and identified genes affected by extremely rare nonsynonymous mutations, followed by functional genomic approaches to compare these affected genes. Overall, 258 genes were identified with rare nonsynonymous mutations in patients with BPD. These genes were highly enriched for processes involved in pulmonary structure and function, were significantly up-regulated in expression in lungs, and were increased in a murine model of BPD. It is possible that identification of such mutations may help develop a personalized genomics risk score for BPD.
3.2. The transcriptome in BPD A genome wide transcriptional profiling using microarrays was performed by Bhattacharya et al. [32] on lung tissues obtained at autopsy from 11 BPD cases and 17 age-matched control subjects without BPD, which identified 159 genes differentially expressed in BPD tissues. Three of the five most highly induced genes were mast cell (MC)-specific markers, and an increased accumulation of connective tissue MC(TC) (chymase expressing) mast cells in BPD tissues was identified [32]. However, it is not currently clear if this increase in connective tissue mast cells contributes to the pathogenesis of BPD, or is in response to the tissue injury and damage seen in BPD. As lung tissue is generally not available from living infants with BPD (unless lung biopsy is done), chymase needs to be validated as a biomarker in tracheal aspirates of infants who subsequently go on to develop BPD. Pietrzyk et al. [47] did genome wide transcriptional profiling of RNA extracted from peripheral blood mononuclear cells of BPD subjects and non-BPD controls on the 5th, 14th, and 28th days of life. They found that 2086 genes were differentially expressed on the day 5, 324 on the day 14, and 3498 on the day 28. The cell cycle pathway was up-regulated in the BPD infants, while the most significantly down-regulated pathway was the T cell receptor signaling pathway [47]. In addition to changes in mRNA, changes in microRNA (miRNA) have also been described in BPD. Yang et al. [48] in a recent meta-analysis reported that four up-regulated miRNAs (miRNA-21, miRNA-34a, miRNA-431, and Let-7f) and one down-regulated miRNA (miRNA335) were differentially expressed in BPD compared with normal lung tissue. Eight miRNAs (miRNA-146b, miRNA-29a, miRNA-503, miRNA411, miRNA-214, miRNA-130b, miRNA-382, and miRNA-181a-1) were differentially expressed during both normal lung development and during the progress of BPD [48]. However, while these studies provide some mechanistic insight into BPD, results of studies evaluating
Please cite this article as: C.V. Lal, N. Ambalavanan, Cellular and humoral biomarkers of Bronchopulmonary Dysplasia, Early Hum Dev (2016), http://dx.doi.org/10.1016/j.earlhumdev.2016.12.003
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miRNA profiling in blood and tracheal aspirate for identification and validation as biomarkers of BPD are awaited.
3.3. The airway microbiome in BPD High-throughput sequencing of the bacterial 16S rRNA gene can be subsequently aligned, sorted, and then classified according to publically available taxonomic databases [49,50]. To date, there have been few studies that have used culture-independent methods for detection of bacterial nucleic acids in preterm infant airways. In a small study of 25 preterm infants, Lohmann et al. [51] demonstrated that reduced diversity of the microbiome may be an associated factor in the development of BPD. In another small study including 10 infants, Abman et al. [52] demonstrated by newer techniques that early bacterial colonization with diverse species are present in the airways of intubated preterm infants, and can be characterized by bacterial load and species diversity. In the only validated airway microbiome analysis performed soon after birth, our group recently showed that an early microbial imbalance, or dysbiosis, is predictive for the development of BPD [53]. We also looked at the airway microbiome of ELBW infants with established BPD and found that their microbiome had a decreased diversity of types of microbes, and the pattern was very different from those of ELBW infants shortly after birth or full-term infants at birth. As to specific groups of microbes, the phylum Proteobacteria, which includes bacteria such as E. coli, were involved in BPD pathogenesis, whereas the genus Lactobaccillus, part of the phylum Firmicutes, were associated with reduction in BPD. We found decreased Lactobacillus abundance in the airway microbiome of infants born to mothers who had chorioamnionitis, an independent risk factor for BPD, as well as decreased Lactobacillus abundance at birth in the airways of the BPD-predisposed, ELBW infants, as compared to BPD-resistant infants. Genus Lactobacillus has been known to have strong anti-inflammatory properties [54–56] and has been shown to regulate alveolar development in animal models [57]. Hence we speculate that the early airway microbiome may possibly prime the developing pulmonary immune system, and abnormalities in the establishment of a normal airway microbiome (dysbiosis) may set the stage for subsequent lung disease. At present, however, we do not know the relationship between the airway microbiome and the microbiome in the more distal lung parenchyma, and how this relationship may be disturbed in BPD.
4. Future steps Better phenotyping of BPD and more detailed data collection of clinical variables, in addition to careful determination of unbiased, specific, temporal, systems biology based ‘omic’ biomarkers are needed [58,59]. Strategies that supplement or expand upon genomic, proteomic, metabolomic and microbiomic approaches need to be studied. With the discovery of the presence and role of the neonatal airway microbiome in BPD [53], the study of environmental pathogens and host response has become of increased relevance. The “omic” biomarkers need to be identified and combined with clinical variables to predict BPD. Most importantly, formulation of new phenotypic and endotypic definitions of BPD is warranted, potentially with the aid of the cellular, humoral, and “omic” biomarkers. Additional research is required not just on the short-term outcome of BPD, but on how we can best predict long-term abnormalities in respiratory function, such as impairments of gas exchange and abnormal airway reactivity.
Disclosures The authors have no conflict of interest to disclose.
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Please cite this article as: C.V. Lal, N. Ambalavanan, Cellular and humoral biomarkers of Bronchopulmonary Dysplasia, Early Hum Dev (2016), http://dx.doi.org/10.1016/j.earlhumdev.2016.12.003
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Please cite this article as: C.V. Lal, N. Ambalavanan, Cellular and humoral biomarkers of Bronchopulmonary Dysplasia, Early Hum Dev (2016), http://dx.doi.org/10.1016/j.earlhumdev.2016.12.003
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Cellular and humoral biomarkers of Bronchopulmonary Dysplasia Charitharth Vivek Lal ⁎, Namasivayam Ambalavanan Division of Neonatology, Department of Pediatrics, University of Alabama at Birmingham, 176F Suite 9380, Women and Infants Center, 619 South 19th Street, Birmingham, AL 35249-7335, United States
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Article history: Available online xxxx Keywords: Bronchopulmonary Dysplasia Pulmonary hypertension Microbiome Biomarkers Systems biology
a b s t r a c t The pathogenesis of Bronchopulmonary Dysplasia (BPD) is multifactorial and the clinical phenotype of BPD is extremely variable. Predicting BPD is difficult, as it is a disease with a clinical operational definition but many clinical phenotypes and endotypes. Most biomarkers studied over the years have low predictive accuracy, and none are currently used in routine clinical care or shown to be useful for predicting longer-term respiratory outcome. Targeted cellular and humoral biomarkers and novel systems biology ‘omic’ based approaches including genomic and microbiomic analyses are described in this review. © 2016 Published by Elsevier Ireland Ltd.
Bronchopulmonary Dysplasia (BPD) was described in 1967 by Northway et al. [1] as pulmonary disease resulting from mechanical ventilation in preterm infants with respiratory distress syndrome. Over the next five decades, neonatal intensive care has progressed to a remarkable extent, with therapies such as antenatal glucocorticoids, surfactant, and gentle ventilation strategies, which have improved survival and reduced BPD in more mature preterm infants [2,3] although BPD today continues to be a significant morbidity in very preterm infants born weighing b1 kg at birth [3,4]. The neonatal outcomes of extremely preterm infants from the National Institute of Child Health and Human Development (NICHD) Neonatal Research Network (NRN) show that the increasing survival of infants born earlier in gestation has resulted in the incidence of BPD being relatively stable over the past two decades, although BPD increased between 2009 and 2012 for infants at 26 to 27 weeks' gestation (26 weeks, 50% to 55%; P b 0.001) [3,5]. The National Institute of Health consensus conference on BPD classified BPD as mild, moderate, or severe in very preterm infants based on the magnitude of respiratory support at 28 days age and 36 w postmenstrual age [6]. However, variability in practices of physicians with regards to standards for oxygen requirements and target oxygen saturation ranges influence the incidence and severity of BPD using this definition. To overcome this, Walsh et al. in the NICHD NRN recently developed a physiologic definition of BPD [7]. The physiologic definition reduced the overall rate of BPD and reduced the variation among centers. However, the clinical definition of oxygen requirement even when more precisely defined using the physiologic definition of BPD [7] is only an operational definition, as the magnitude of lung disease ⁎ Corresponding author. E-mail addresses: [email protected] (C.V. Lal), [email protected] (N. Ambalavanan).
or the exact underlying pathology may vary among infants diagnosed with BPD. Recent studies indicate that severe BPD is probably a different entity from mild or moderate BPD in terms of genetic predisposition [8, 9]. This genetic predisposition varies by race/ethnicity, suggesting that biologic pathways contributing to BPD and any biomarkers resulting from this altered genetic background are probably likely to be different [9]. As we have recently stated [10], it is likely that what is now termed “BPD” is not a single entity, nor even a spectrum of disease resulting from a single pathophysiologic process, but a combination of several chronic lung diseases characterized by a common “at-risk population” of infants in the saccular or early alveolar stage of lung development with varying magnitudes of impairment of alveolar septation, lung fibrosis, and abnormal vascular development and remodeling. Infants with BPD may have primarily lung parenchymal disease with low lung compliance and pathology characterized by atelectasis or cystic lesions in their lung parenchyma [1]. Other infants with BPD have more vascular remodeling and impaired lung angiogenesis, with pulmonary hypertension as a major clinical manifestation [11]. Some infants with BPD have airway injury and remodeling, resulting in severe tracheobronchomalacia [12]. There are therefore several phenotypes and endotypes in BPD, each of which may have different pathophysiology mediated directly by cells (cellular mediators) and by macromolecules originating from cells found in extracellular fluids (humoral mediators). Certain cellular and humoral constituents may serve both as mediators of pathophysiology as well as biomarkers of disease, while other molecules may serve as biomarkers but may not be directly involved in pathogenesis. Laboratory-based test markers could be utilized as biomarkers if they characterize disease activity in a manner useful for early diagnosis, prediction of disease severity and monitoring disease processes and response to therapy [13,14]. Earlier diagnosis, before conventional methods of diagnosis are used, may permit the use of therapies that arrest lung injury at an early stage and allow for greater
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Please cite this article as: C.V. Lal, N. Ambalavanan, Cellular and humoral biomarkers of Bronchopulmonary Dysplasia, Early Hum Dev (2016), http://dx.doi.org/10.1016/j.earlhumdev.2016.12.003
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lung development. The biomarkers may also permit quantitation of disease severity, which is useful in following response to therapies. Biomarkers may also help determine prognosis and help plan for additional therapies. In this manuscript, we will review various targeted (i.e., focusing on known targets) cellular and humoral biomarkers of BPD and unbiased systems biology based approaches to identification of biomarkers in BPD.
1. Targeted cellular biomarkers of Bronchopulmonary Dysplasia Abnormalities in the number and function of multiple cell types, primarily those related to inflammation and lung repair, have been shown in human BPD as well as in animal models of BPD (e.g. hyperoxia-exposed newborn rodent models). Early in the course of respiratory distress syndrome in human preterm infants, there is evidence of early systemic activation and transendothelial migration of neutrophils [15], with increased neutrophils in the tracheal aspirate of preterm infants who go on to develop BPD [16]. Anti-neutrophil chemokine therapy [17] or CXCR2 antagonist therapy [18] prevents neutrophil accumulation and preserves alveolar development in hyperoxia-exposed newborn rats. CD18 expression on neutrophils and monocytes and CD62L on neutrophils is decreased in preterm neonates who go on to develop BPD, compared to non-BPD neonates on days 1–28 [19]. Changes in lymphocytes then become evident; Ballabh et al. [20] showed that infants with RDS who went on to develop BPD had fewer lymphocytes, as well as absolute number and % of CD4+ T-cells. The percentage of CD4+ T-cells expressing CD62L was selectively reduced in infants developing BPD, suggesting that reduction of CD4(+) T cells and especially those important in tissue migration and immune surveillance may be relevant to the pathogenesis of BPD [20]. More recently, Mishra et al. [21] evaluated cord blood mononuclear cells from infants (GA b32 weeks), with or without placental histological evidence of chorioamnionitis (CA). Absolute regulatory T-cell (Treg) numbers were not different in CA and non-CA exposed samples. However, the infants who developed BPD had a significant decrease in Treg and nonregulatory T cell numbers, suggesting that the development of BPD is marked by distinct inflammatory changes from those of CA exposed infants [21]. Eosinophil activation has been associated with BPD, as the eosinophil count is higher in BPD compared with RDS and healthy infants, along with increases in the humoral biomarkers eosinophil cationic protein (ECP) and the cellular surface antigen (CD9) [22]. We have also shown recently that the absolute eosinophil count is higher on the first postnatal day in infants who go on to develop BPD as compared to infants who survive without BPD, associated with increases in the humoral biomarker eotaxin-1 (CCL11), a chemokine selective for eosinophils [23]. An increase in pulmonary neuroendocrine cells containing bombesin-like peptide (BLP) has been described in BPD [24,25], and BLP mediates lung injury in a preterm baboon model of BPD [26]. This cellular phenotype of neuroendocrine hyperplasia is accompanied by a humor biomarker, as urinary BLP elevation precedes clinical evidence of BPD [27]. There is some evidence that BLP may mediate premature thymic maturation and thymic nurse-cell depletion leading to autoreactive T-cells in BPD that may injure the lung [28]. Neonatal rat models of hyperoxia exposure also demonstrate an association between hyperoxia, airway remodeling, and pulmonary neuroendocrine hyperplasia [29]. An important characteristic of the histology of BPD is the disrupted capillary development. An initial study by Borghesi et al. demonstrated that circulating endothelial progenitor cells (endothelial colonyforming cells or ECFCs) are fewer at lower gestational ages, and fewer in cord blood in infants who later develop BPD, while endothelial and hematopoietic cell subsets are comparable in infants with and without BPD [30]. However, a subsequent study by the same author indicated
that different subsets of circulating angiogenic cells did not predict BPD [31]. Connective tissue mast cells (chymase expressing) accumulate in BPD lung tissue both in humans and in animal models of BPD, and genes associated with these cells are highly expressed in lung tissue, suggesting a potential role for these cells, not just as biomarkers, but in the development or attempt at resolution of BPD [32]. Mesenchymal stromal cells (MSCs) can be isolated from tracheal aspirates of premature neonates with RDS, primarily in infants who go on to develop BPD and rarely in those who do not develop BPD [33]. These MSCs have a pattern of lung-specific gene expression, are distinct from lung fibroblasts, and secrete pro-inflammatory cytokines [34]. While these MSCs are a cellular biomarker, their role in the development of BPD is uncertain. 2. Targeted humoral biomarkers of Bronchopulmonary Dysplasia Humoral biomarkers refer to biomarkers present in body fluids (called “humors”) which were categorized in ancient literature as blood, yellow bile, black bile, and phlegm. In this review, we will focus primarily on blood and airway secretions (phlegm). Biomarker measurements in blood have been studied due to easy accessibility, but they may not accurately reflect the lung concentrations of the respective mediator. Tracheal aspirates also have limitations, as they are available only in intubated infants and may not necessarily reflect the distal lung parenchymal milieu. As BPD is considered to result from the effect of inflammatory processes upon the substrate of an immature and fragile developing lung, investigators have focused upon changes in inflammatory mediators or changes in growth factors that may be associated with lung injury and alterations in alveolar, airway, or vascular development. As recently reviewed by us [10], increases in blood of multiple biomarkers such as interleukin (IL)-1β, −6, −8, −10, interferon (IFN)-γ, granulocyte colony stimulating factor (GCSF), endostatin, vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) BB, placental growth factor (PGF), Kerbs von Lungren 6 (KL-6), matrix metalloproteinases/tissue inhibitors of metalloproteinases (MMPs/TIMPs), Eselectin, and B-type natriuretic peptide (BNP) have been associated with development of BPD, while concentrations of other biomarkers such as IL-17, chemokine (C-C motif) ligand 5 (CCL5; RANTES or regulated on activation, normal T cell expressed and secreted), tumor necrosis factor (TNF)-β, angiopoietin 1 (ANG-1), VEGF, clara cell proteins (CCP), C-terminal type I procollagen (PICP) have been reduced in infants who developed BPD. Similarly, concentrations of various cytokines and inflammatory molecules (IL-1β, − 6, − 8, TNF-α, monocyte chemoattractant protein (MCP), nuclear factor kappa B (NFkB), neutrophil gelatinase-associated lipocalin (NGAL), MMP-9, transforming growth factor (TGF)-β1, L-selectin, Ang-2, endothelin-1, fibroblast growth factor-2, pepsin, oxyradicals, carbonyls, 3-chlorotyrosine, malondialdehyde) are increased in tracheal aspirates in infants who go on to develop BPD, while concentrations of growth factors and anti-inflammatory substances such as macrophage migration inhibitory factor (MIF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), VEGF, polyunsaturated fatty acids (PUFA), CCP, parathyroid hormone-related protein (PTHrP) are reduced in infants who survive without BPD [10]. As space limitations preclude us from providing a detailed review of changes of each of the biomarkers in blood or tracheal aspirates, we refer the reader to several recent comprehensive reviews on such biomarkers [10,35–37]. A few clinically important issues regarding the use of these biomarkers must be noted. First, most of these biomarkers (either cellular or humoral) have been shown to have poor predictive accuracy (predictive value, sensitivity, specificity) in isolation, and do not add much to the predictive ability of clinical variables (gestational age, birth weight, respiratory illness severity etc) even if they are statistically significant by themselves [38]. In a study of 1067 infants by the NICHD NRN who
Please cite this article as: C.V. Lal, N. Ambalavanan, Cellular and humoral biomarkers of Bronchopulmonary Dysplasia, Early Hum Dev (2016), http://dx.doi.org/10.1016/j.earlhumdev.2016.12.003
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had 25 cytokines measured in the blood at birth and on days 3, 7, 14, and 21, BPD/death was associated with higher peak IL-1β, −6,-8, −10, and IFN-γ, and lower IL-17, RANTES, and TNF-β [38]. However, compared with models with only clinical variables, the addition of cytokine data improved predictive ability by a statistically significant but clinically modest magnitude (for peak cytokines: r-square 0.56 with cytokines vs. 0.53 without cytokines, c-statistic 0.89 vs. 0.88, p b 0.001) [38]. Second, rather than absolute concentrations of a biomarker at one point in time, the pattern of change of multiple biomarkers over time using serial sampling may be informative. Data from infants in the NICHD NRN cytokine study was analyzed in relation to the following lung disease patterns: (1) no lung disease (NLD); (2) respiratory distress syndrome without BPD; (3) classic BPD (persistent exposure to supplemental oxygen until 28 days of age); or (4) atypical BPD (period without supplemental oxygen before 28 days) [39]. Median cytokine levels for infants with BPD were compared with the interquartile range (IQR) of results among infants with NLD [39]. Median levels of 3 cytokines (elevated IL-8, MMP-9; decreased GMCSF) fell outside the IQR for at least 2 time points in both infants with atypical and classic BPD. Profiles of 7 cytokines (IL-6, IL-10, IL-18, macrophage inflammatory protein-1α, C-reactive protein (CRP), brain-derived neurotrophic factor, RANTES) differed between infants with classic and atypical BPD, suggesting that the differences in blood cytokine profiles may be related to variations in pathophysiology of BPD [39]. Third, many of these biomarkers are suitable for analyses in research laboratories using methods such as immunoassays (e.g. ELISA) or multiplex assays (Luminex, Meso Scale Diagnostics), which generally involve batch processing and longer turn-around times, rather than near-patient or point of care testing with rapid results. These assays also demonstrate much variation from laboratory to laboratory, as they are generally done in research laboratories with varying levels of quality control and not College of American Pathologists (CAP) accredited clinical laboratories (with the possible exception of biomarkers such as CRP). Therefore, while the evaluation of the biomarkers provides an improved estimate of risk for BPD or mortality (a competing outcome for death, as infants at high risk of BPD are also at higher risk of death, and infants who die cannot develop BPD), these biomarkers are not yet sufficiently accurate or precise for predicting BPD, or widely available for inclusion into routine clinical practice. Finally, another important issue is that while the focus of most investigations is on prediction of BPD, most infants with BPD eventually improve and are discharged home (some on supplemental oxygen), and it is critically important to be able to predict longer-term abnormalities in airway function (e.g. increased baseline airway resistance or airway reactivity) or impaired gas exchange, and the likelihood of early-onset chronic obstructive pulmonary disease (COPD) in former preterm infants. 3. Unbiased “omic” biomarkers of BPD Advances in molecular genetics and next-generation sequencing (NGS) technology have enabled the examination of BPD at an unbiased molecular ‘omic’ level. In this review, we will focus on biomarkers in the genome, transcriptome, and microbiome. 3.1. The genome in BPD Twin studies indicate that there is a significant genetic contribution to the risk of BPD, ranging from 53% [40] to 82% [41] of the variance in liability for BPD. Many studies to date have focused on targeted analysis of single nucleotide polymorphisms (SNPs), and these studies will not be reviewed further, due to space limitations and the existence of several existing recent comprehensive reviews by us [8] and others [42]. Three unbiased genome-wide association studies (GWAS) have been published to date for BPD, and the first GWAS by Hadchouel et al. [43] identified the SPOCK2 gene in two discovery series, and the most significant polymorphism was confirmed by individual genotyping and in a replication population. The expression pattern of SPOCK2 (increase in
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mRNA levels during alveolar stage, increase with hyperoxia exposure) pointed to a potential role in alveolarization [43]. However, this finding was not replicated in subsequent GWAS by Wang et al. [44] or Ambalavanan et al. [9] which did not identify any SNPs at the genome-wide significance threshold. The gene set pathway analysis by Ambalavanan et al. [9] confirmed involvement of known pathways of lung development and repair (CD44, phosphorus oxygen lyase activity) and indicated novel molecules and pathways (adenosine deaminase, targets of miR-219) involved in genetic predisposition to BPD. Other important findings were that severe BPD or death are associated with pathways distinct from mild/moderate BPD, suggesting that they have a different pathophysiologic basis, and that much variation is present in genetic predisposition to BPD by race/ethnicity [9]. More recently, next generation sequencing (NGS) has been used to gain insight into diseases, utilizing either whole exome sequencing (WES) or whole genome sequencing. In a recent study, Carrera et al. [45] performed exome sequencing and pathway analysis on a cohort of 26 unrelated BPD patients to identify non-common variants. 3369 novel variants were identified, with a median of 400 variations per sample. The top candidate genes highlighted were NOS2, MMP1, CRP, LBP and the toll-like receptor (TLR) family, which were confirmed by Sanger sequencing [45]. Li et al. [46] also performed exome sequencing on 50 BPD-affected and unaffected twin pairs using DNA isolated from neonatal blood spots and identified genes affected by extremely rare nonsynonymous mutations, followed by functional genomic approaches to compare these affected genes. Overall, 258 genes were identified with rare nonsynonymous mutations in patients with BPD. These genes were highly enriched for processes involved in pulmonary structure and function, were significantly up-regulated in expression in lungs, and were increased in a murine model of BPD. It is possible that identification of such mutations may help develop a personalized genomics risk score for BPD.
3.2. The transcriptome in BPD A genome wide transcriptional profiling using microarrays was performed by Bhattacharya et al. [32] on lung tissues obtained at autopsy from 11 BPD cases and 17 age-matched control subjects without BPD, which identified 159 genes differentially expressed in BPD tissues. Three of the five most highly induced genes were mast cell (MC)-specific markers, and an increased accumulation of connective tissue MC(TC) (chymase expressing) mast cells in BPD tissues was identified [32]. However, it is not currently clear if this increase in connective tissue mast cells contributes to the pathogenesis of BPD, or is in response to the tissue injury and damage seen in BPD. As lung tissue is generally not available from living infants with BPD (unless lung biopsy is done), chymase needs to be validated as a biomarker in tracheal aspirates of infants who subsequently go on to develop BPD. Pietrzyk et al. [47] did genome wide transcriptional profiling of RNA extracted from peripheral blood mononuclear cells of BPD subjects and non-BPD controls on the 5th, 14th, and 28th days of life. They found that 2086 genes were differentially expressed on the day 5, 324 on the day 14, and 3498 on the day 28. The cell cycle pathway was up-regulated in the BPD infants, while the most significantly down-regulated pathway was the T cell receptor signaling pathway [47]. In addition to changes in mRNA, changes in microRNA (miRNA) have also been described in BPD. Yang et al. [48] in a recent meta-analysis reported that four up-regulated miRNAs (miRNA-21, miRNA-34a, miRNA-431, and Let-7f) and one down-regulated miRNA (miRNA335) were differentially expressed in BPD compared with normal lung tissue. Eight miRNAs (miRNA-146b, miRNA-29a, miRNA-503, miRNA411, miRNA-214, miRNA-130b, miRNA-382, and miRNA-181a-1) were differentially expressed during both normal lung development and during the progress of BPD [48]. However, while these studies provide some mechanistic insight into BPD, results of studies evaluating
Please cite this article as: C.V. Lal, N. Ambalavanan, Cellular and humoral biomarkers of Bronchopulmonary Dysplasia, Early Hum Dev (2016), http://dx.doi.org/10.1016/j.earlhumdev.2016.12.003
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miRNA profiling in blood and tracheal aspirate for identification and validation as biomarkers of BPD are awaited.
3.3. The airway microbiome in BPD High-throughput sequencing of the bacterial 16S rRNA gene can be subsequently aligned, sorted, and then classified according to publically available taxonomic databases [49,50]. To date, there have been few studies that have used culture-independent methods for detection of bacterial nucleic acids in preterm infant airways. In a small study of 25 preterm infants, Lohmann et al. [51] demonstrated that reduced diversity of the microbiome may be an associated factor in the development of BPD. In another small study including 10 infants, Abman et al. [52] demonstrated by newer techniques that early bacterial colonization with diverse species are present in the airways of intubated preterm infants, and can be characterized by bacterial load and species diversity. In the only validated airway microbiome analysis performed soon after birth, our group recently showed that an early microbial imbalance, or dysbiosis, is predictive for the development of BPD [53]. We also looked at the airway microbiome of ELBW infants with established BPD and found that their microbiome had a decreased diversity of types of microbes, and the pattern was very different from those of ELBW infants shortly after birth or full-term infants at birth. As to specific groups of microbes, the phylum Proteobacteria, which includes bacteria such as E. coli, were involved in BPD pathogenesis, whereas the genus Lactobaccillus, part of the phylum Firmicutes, were associated with reduction in BPD. We found decreased Lactobacillus abundance in the airway microbiome of infants born to mothers who had chorioamnionitis, an independent risk factor for BPD, as well as decreased Lactobacillus abundance at birth in the airways of the BPD-predisposed, ELBW infants, as compared to BPD-resistant infants. Genus Lactobacillus has been known to have strong anti-inflammatory properties [54–56] and has been shown to regulate alveolar development in animal models [57]. Hence we speculate that the early airway microbiome may possibly prime the developing pulmonary immune system, and abnormalities in the establishment of a normal airway microbiome (dysbiosis) may set the stage for subsequent lung disease. At present, however, we do not know the relationship between the airway microbiome and the microbiome in the more distal lung parenchyma, and how this relationship may be disturbed in BPD.
4. Future steps Better phenotyping of BPD and more detailed data collection of clinical variables, in addition to careful determination of unbiased, specific, temporal, systems biology based ‘omic’ biomarkers are needed [58,59]. Strategies that supplement or expand upon genomic, proteomic, metabolomic and microbiomic approaches need to be studied. With the discovery of the presence and role of the neonatal airway microbiome in BPD [53], the study of environmental pathogens and host response has become of increased relevance. The “omic” biomarkers need to be identified and combined with clinical variables to predict BPD. Most importantly, formulation of new phenotypic and endotypic definitions of BPD is warranted, potentially with the aid of the cellular, humoral, and “omic” biomarkers. Additional research is required not just on the short-term outcome of BPD, but on how we can best predict long-term abnormalities in respiratory function, such as impairments of gas exchange and abnormal airway reactivity.
Disclosures The authors have no conflict of interest to disclose.
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