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. 2011;6(7):e21958.
doi: 10.1371/journal.pone.0021958. Epub 2011 Jul 6.

Bacteria-specific neutrophil dysfunction associated with interferon-stimulated gene expression in the acute respiratory distress syndrome

Kenneth C Malcolm  1 , Jennifer E Kret, Robert L Young, Katie R Poch, Silvia M Caceres, Ivor S Douglas, Chris D Coldren, Ellen L Burnham, Marc Moss, Jerry A Nick
Affiliations

Bacteria-specific neutrophil dysfunction associated with interferon-stimulated gene expression in the acute respiratory distress syndrome

Kenneth C Malcolm et al. PLoS One. 2011.

Abstract

Acute respiratory distress syndrome (ARDS) is a poorly understood condition with greater than 30% mortality. Massive recruitment of neutrophils to the lung occurs in the initial stages of the ARDS. Significant variability in the severity and duration of ARDS-associated pulmonary inflammation could be linked to heterogeneity in the inflammatory capacity of neutrophils. Interferon-stimulated genes (ISGs) are a broad gene family induced by Type I interferons. While ISGs are central to anti-viral immunity, the potential exists for these genes to evoke extensive modification in cellular response in other clinical settings. In this prospective study, we sought to determine if ISG expression in circulating neutrophils from ARDS patients is associated with changes in neutrophil function. Circulating neutrophil RNA was isolated, and hierarchical clustering ranked patients' expression of three ISGs. Neutrophil response to pathogenic bacteria was compared between normal and high ISG-expressing neutrophils. High neutrophil ISG expression was found in 25 of 95 (26%) of ARDS patients and was associated with reduced migration toward interleukin-8, and altered responses to Staphylococcus aureus, but not Pseudomonas aeruginosa, which included decreased p38 MAP kinase phosphorylation, superoxide anion release, interleukin-8 release, and a shift from necrotic to apoptotic cell death. These alterations in response were reflected in a decreased capacity to kill S. aureus, but not P. aeruginosa. Therefore, the ISG expression signature is associated with an altered circulating neutrophil response phenotype in ARDS that may predispose a large subgroup of patients to increased risk of specific bacterial infections.

Trial registration: ClinicalTrials.gov NCT00548795.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. ARDS patients include a cohort of individuals with elevated neutrophil ISG expression.
Expression of neutrophil MX1, IFIT1, and ISG15 were determined by real-time PCR and normalized to expression of GAPDH. Log2-transformed, normalized ISG levels were ordered by hierarchical clustering, with the primary clusters designated as either high or normal ISG expression. The dotted line indicates the separation of the normal and high ISG groups. Individual ARDS patients (Pt) are indicated. The difference between the cohorts was highly significant as indicated in the text. (A) The heat-map indicates the range of log2-transformed data. (B) Non-transformed data is aligned to the clustering results for an alternative visualization.
Figure 2
Figure 2. High ISG expression is associated with reduced circulating neutrophil migration in ARDS patients.
(A) Directional migration towards IL8 was measured in isolated neutrophils with a modified Boyden chamber system over 60 minutes and (B) AUC of migration curves shown in (A); ARDS patients with high ISG expression (ISG; red; n = 20) were found to have reduced migration compared to those with normal ISG expression (black; n = 58). Bar and whiskers indicate median, 25th- and 75th-percentile, and range. *, p<0.05.
Figure 3
Figure 3. S. aureus-induced p38 MAP kinase activity is attenuated in circulating neutrophils with high ISG expression.
Time-course of phospho-p38 MAPk phosphorylation induced by (A) S. aureus, or (B) P. aeruginosa was measured by ELISA at 6 time points over 2 hours; stimulated (closed symbols) or unstimulated neutrophils (open symbols) and either normal (black; upper panels) or high ISG expression (red; lower panels). The shaded portion indicates the ΔAUC. Mean ± SEM is shown. Phospho-p38 MAPk ΔAUC values from a phospho-p38 MAPk time-course were determined in neutrophils in response to (C) S. aureus, and (D) P. aeruginosa with normal (black) and high ISG expression (red). Median values are indicated by the like-colored bars; ARDS normal ISG (n = 58 in panels A and C, and 49 in panels B and D), ARDS high ISG (n = 22 in panels A and C, and 18 in panels B and D). *, p<0.05.
Figure 4
Figure 4. Superoxide anion release is suppressed in neutrophils expressing ISG.
Release of superoxide anion (O2 −•) from neutrophils following 60 minutes incubation. (A) Spontaneous; (B) S. aureus-induced; and (C) P. aeruginosa-induced release of O2 −•. ARDS patients demonstrated significant attenuation of O2 −• release when ISG expression was elevated in non-stimulated and S. aureus-stimulated neutrophils. Scatter plot of values in neutrophils with normal (black) and high ISG expression (ISG; red); normal ISG (n = 65 in panels A and B, and 55 in panel C), high ISG (n = 23 in panels A and B, and 17 in panel C). *p<0.05, **, p<0.01.
Figure 5
Figure 5. S. aureus-induced IL8 release is reduced in high ISG-expressing neutrophils from ARDS patients.
Release of IL8 following 2 h incubation with (A) S. aureus, and (B) P. aeruginosa. TNF release following 4 h incubation with( C) S. aureus, and (D) P. aeruginosa. Cytokine release from neutrophils with normal (black) and high ISG expression (ISG; red) are shown; normal ISG (n = 52 in panels A and C and 42 in panels B and D), high ISG (n = 19 in panels A and C and 14 in panels B and D); *p<0.05.
Figure 6
Figure 6. Reciprocal modification of cell death in neutrophils from ARDS patients expressing ISG.
(A–C) The release of LDH to assess necrosis, and (D–F) percent of soluble histone-associated DNA fragments to assess apoptosis, from isolated neutrophils after 4 h treatment. (A) Spontaneous necrosis. (B) S. aureus-induced necrosis. (C) P. aeruginosa-induced necrosis. (D) Spontaneous apoptotic cell death (E) S. aureus-induced apoptotic cell death. (F) P. aeruginosa-induced apoptotic cell death. Scatter plot of values in neutrophils with normal (black) and high ISG expression (ISG; red); median values are indicated by the like-colored bars; normal ISG (n = 50–63), high ISG (n = 17–22). *, p<0.05, **, p<0.01.
Figure 7
Figure 7. High ISG expression is associated with impaired neutrophil killing of S. aureus in ARDS.
Killing of (A) S. aureus and (B) P. aeruginosa. Bacteria were exposed to adherent neutrophils for 60 min, and bacteria remaining was compared to that in the absence of neutrophils to determine percentage of bacteria killed. Values less than zero indicate growth of bacteria during the assay. ISG expression was associated with an impaired bactericidal activity against S. aureus, but not against P. aeruginosa. Scatter plot of values in neutrophils with normal (black) and high ISG expression (ISG; red); median values are indicated by the like-colored bars; normal ISG (n = 54 in panel A, and 49 in panel B), ARDS high ISG (n = 19 in panel A, and 15 in panel B); *, p<0.05.
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