Microarray Analysis Identifies IL-1 Receptor Type 2 as a Novel Candidate Biomarker in Patients With Acute Respiratory Distress Syndrome

Melissa A Kovach; Kathleen A Stringer; Rachel Bunting; Xiaoying Wu; Lani San Mateo; Michael W Newstead; Robert Paine III; Theodore J Standiford


Respiratory Research. 2015;16(29) 

In This Article

Abstract and Introduction


Background Acute respiratory distress syndrome (ARDS) is a disease associated with a high mortality rate. The initial phase is characterized by induction of inflammatory cytokines and chemokines and influx of circulating inflammatory cells, including macrophages which play a pivotal role in the innate and adaptive immune responses to injury. Growing evidence points to phenotypic heterogeneity and plasticity between various macrophage activation states.

Methods In this study, gene expression in alveolar macrophages and circulating leukocytes from healthy control subjects and patients with ARDS was assessed by mRNA microarray analysis.

Results Both alveolar macrophages and circulating leukocytes demonstrated up-regulation of genes encoding chemotactic factors, antimicrobial peptides, chemokine receptors, and matrix metalloproteinases. Two genes, the pro-inflammatory S100A12 and the anti-inflammatory IL-1 decoy receptor IL-1R2 were significantly induced in both cell populations in ARDS patients, which was confirmed by protein quantification. Although S100A12 levels did not correlate with disease severity, there was a significant association between early plasma levels of IL-1R2 and APACHE III scores at presentation. Moreover, higher levels of IL-1R2 in plasma were observed in non-survivors as compared to survivors at later stages of ARDS.

Conclusions These results suggest a hybrid state of alveolar macrophage activation in ARDS, with features of both alternative activation and immune tolerance/deactivation.. Furthermore, we have identified a novel plasma biomarker candidate in ARDS that correlates with the severity of systemic illness and mortality.


Acute respiratory distress syndrome (ARDS) is a deadly disease, with an incidence of roughly 200,000 cases per year in the United States and a mortality rate of approximately 40%.[1] ARDS is characterized by marked hypoxemia (PaO2/FiO2 < 300), diffuse bilateral infiltrates, and decreased lung compliance either due to direct injury (e.g., pneumonia, aspiration) or indirect injury (e.g., sepsis, pancreatitis, transfusion-related acute lung injury).[2–4] Following the initial injury, a stereotyped process of tissue injury, inflammation, and alveolar capillary damage evolves to a fibroproliferative phase.[5] During the inflammatory phase, resident immune cells in the lung express pro-inflammatory chemokines and cytokines, which in turn stimulates the influx of circulating inflammatory cells into the interstitium and alveolar spaces.[4]

Macrophages play a pivotal role in the innate and adaptive immune responses during host defense and response to injury. Recent research has uncovered distinctly heterogeneous populations of macrophages, as well as plasticity between different macrophage phenotypes.[6] This phenotypic polarization of macrophages is believed to be a consequence of factors present within the cell's microenvironment.[7,8] Several broad categories of distinct macrophage phenotypes have been described. Classically-activated macrophages (M1) are generally associated with an inflammatory environment and mediate microbial phagocytosis, pro-inflammatory cytokine expression, and cellular immune responses.[6,9,10] Alternatively-activated macrophages (M2) are anti-inflammatory in nature, and are associated with tissue repair and humoral immune responses.[9–11] A third macrophage phenotype, referred to deactivation or immunoparalysis, can be induced by repeated exposure to various pathogen-associated molecular patterns (PAMPs) in-vitro (referred to as immune tolerance) or during the systemic inflammatory response in-vivo.[12–14] Macrophage deactivation is characterized by suppression of pro-inflammatory cytokines (e.g., TNF-α, IL-6, nitric oxide synthase-2) and enhanced expression of anti-inflammatory molecules (IL-10), and this phenotype has been described in circulating blood monocytes isolated from critically ill patients, including patients with sepsis.[12,15–19] Recent evidence also points to the presence of phenotypic heterogeneity, with a spectrum between classically activated, alternatively activated, and deactivated macrophages, particularly in the setting of chronic inflammatory states.[10,20–22]

The identification of biomarkers correlating to disease activity in ARDS is an important goal, potentially allowing for earlier diagnosis and treatment, as well as providing prognostic information.[23] Nevertheless, successful association of biomarkers to disease severity remains elusive. S100 calcium binding protein A12 (S100A12) is a known chemoattractant, and is a pro-inflammatory ligand for the RAGE receptor.[24] Previous studies have found that S100A12 protein is upregulated in both the bronchoalveolar lavage (BAL) fluid and plasma of patients with ARDS.[25–27] However, these studies did not assess whether S100A12 levels correlated with indices of ARDS severity. Conversely, the anti-inflammatory IL-1 decoy receptor, IL-1R2, has recently been shown to be elevated in plasma during sepsis,[28] as well as a mouse model of chemically-induced lung injury.[29] However, there have been no studies examining the association of IL-1R2 and human lung disease, specifically ARDS.

In this pilot study, we analyzed gene expression profiles in isolated alveolar macrophages (AM) obtained from BAL samples and buffy coat leukocytes isolated from patients with ARDS. As compared to cells from healthy subjects, there was considerable over-representation of immune activation genes, including genes involved in leukocyte trafficking, phagocytosis, microbial killing, apoptosis and lymphocyte activation. We also found a gene expression profile in AM with features of both alternative macrophage activation and immune tolerance/deactivation, suggesting the presence of unique macrophage phenotypes present early in the course of ARDS that may have important functional significance in the pathogenesis and course of disease in this patient population. Furthermore, we assessed plasma and BAL levels of two of the most highly induced genes identified in our microarray analysis, S100A12 and IL-1R2. While both analytes were significantly up-regulated in both the plasma and BAL fluid of ARDS patients as compared with healthy control subjects, only plasma levels of IL-1R2 correlated significantly with APACHE III scores and mortality. We have therefore identified a novel plasma biomarker candidate for ARDS, which may aid in early diagnosis and which carries prognostic implications for disease severity in ARDS.