Arie J. Hoogendijk

A molecular biomarker to diagnose community-acquired pneumonia on intensive care unit admission.

RATIONALE Community-acquired pneumonia (CAP) accounts for a major proportion of intensive care unit (ICU) admissions for respiratory failure and sepsis. Diagnostic uncertainty complicates case management, which may delay appropriate cause-specific treatment. OBJECTIVES To characterize the blood genomic response in patients with suspected CAP and identify a candidate biomarker for the rapid diagnosis of CAP on ICU admission. METHODS The study comprised two cohorts of consecutively enrolled patients treated for suspected CAP on ICU admission. Patients were designated CAP (cases) and no-CAP patients (control subjects) by post hoc assessment. The first (discovery) cohort (101 CAP and 33 no-CAP patients) was enrolled between January 2011 and July 2012; the second (validation) cohort (70 CAP and 30 no-CAP patients) between July 2012 and June 2013. Blood was collected within 24 hours of ICU admission. MEASUREMENTS AND MAIN RESULTS Blood microarray analysis of CAP and no-CAP patients revealed shared and distinct gene expression patterns. A 78-gene signature was defined for CAP, from which a FAIM3:PLAC8 gene expression ratio was derived with area under curve of 0.845 (95% confidence interval, 0.764-0.917) and positive and negative predictive values of 83% and 81%, respectively. Robustness of the FAIM3:PLAC8 ratio was ascertained by quantitative polymerase chain reaction in the validation cohort. The FAIM3:PLAC8 ratio outperformed plasma procalcitonin and IL-8 and IL-6 in discriminating between CAP and no-CAP patients. CONCLUSIONS CAP and no-CAP patients presented shared and distinct blood genomic responses. We propose the FAIM3:PLAC8 ratio as a candidate biomarker to assist in the rapid diagnosis of CAP on ICU admission. Clinical trial registered with www.clinicaltrials.gov (NCT 01905033).

Lipopolysaccharide Inhibits Th2 Lung Inflammation Induced by House Dust Mite Allergens in Mice

The complex biology of asthma compels the use of more relevant human allergens, such as house dust mite (HDM), to improve the translation of animal models into human asthma. LPS exposure is associated with aggravations of asthma, but the mechanisms remain unclear. Here, we studied the effects of increasing LPS doses on HDM-evoked allergic lung inflammation. To this end, mice were intranasally sensitized and challenged with HDM with or without increasing doses of LPS (0.001-10 μg). LPS dose-dependently inhibited HDM-induced eosinophil recruitment into the lungs and mucus production in the airways. LPS attenuated the production of Th2 cytokines (IL-4, IL-5, IL-10, and IL-13) in HDM-challenged lungs, while enhancing the HDM-induced release of IL-17, IL-33, IFN-γ, and TNF-α. The shift toward a Th1 inflammatory response was further illustrated by predominant neutrophilic lung inflammation after LPS administration at higher doses. LPS did not influence HDM-induced plasma IgE concentrations. Although LPS did not significantly affect the activation of coagulation or complement in HDM-challenged lungs, it reduced HDM-initiated endothelial cell activation. This study is the first to provide insights into the effects of LPS in an allergic lung inflammation model making use of a clinically relevant allergen without a systemic adjuvant, revealing that LPS dose-dependently inhibits HDM-induced pulmonary Th2 responses.

Thrombocytopenia is associated with a dysregulated host response in critically ill sepsis patients

Preclinical studies have suggested that platelets influence the host response during sepsis. We sought to assess the association of admission thrombocytopenia with the presentation, outcome, and host response in patients with sepsis. Nine hundred thirty-one consecutive sepsis patients were stratified according to platelet counts (very low <50 × 10(9)/L, intermediate-low 50 × 10(9) to 99 × 10(9)/L, low 100 × 10(9) to 149 × 10(9)/L, or normal 150 × 10(9) to 399 × 10(9)/L) on admission to the intensive care unit. Sepsis patients with platelet counts <50 × 10(9)/L and 50 × 10(9) to 99 × 10(9)/L presented with higher Acute Physiology and Chronic Health Evaluation scores and more shock. Both levels of thrombocytopenia were independently associated with increased 30-day mortality (hazard ratios with 95% confidence intervals 2.00 [1.32-3.05] and 1.72 [1.22-2.44], respectively). To account for baseline differences besides platelet counts, propensity matching was performed, after which the association between thrombocytopenia and the host response was tested, as evaluated by measuring 17 plasma biomarkers indicative of activation and/or dysregulation of pathways implicated in sepsis pathogenesis and by whole genome blood leukocyte expression profiling. In the propensity matched cohort, platelet counts < 50 × 10(9)/L were associated with increased cytokine levels and enhanced endothelial cell activation. All thrombocytopenic groups showed evidence of impaired vascular integrity, whereas coagulation activation was similar between groups. Blood microarray analysis revealed a distinct gene expression pattern in sepsis patients with <50 × 10(9)/L platelets, showing reduced signaling in leukocyte adhesion and diapedesis and increased complement signaling. These data show that admission thrombocytopenia is associated with enhanced mortality and a more disturbed host response during sepsis independent of disease severity, thereby providing clinical validity to animal studies on the role of platelets in severe infection.

Classification of patients with sepsis according to blood genomic endotype: a prospective cohort study

BACKGROUND Host responses during sepsis are highly heterogeneous, which hampers the identification of patients at high risk of mortality and their selection for targeted therapies. In this study, we aimed to identify biologically relevant molecular endotypes in patients with sepsis. METHODS This was a prospective observational cohort study that included consecutive patients admitted for sepsis to two intensive care units (ICUs) in the Netherlands between Jan 1, 2011, and July 20, 2012 (discovery and first validation cohorts) and patients admitted with sepsis due to community-acquired pneumonia to 29 ICUs in the UK (second validation cohort). We generated genome-wide blood gene expression profiles from admission samples and analysed them by unsupervised consensus clustering and machine learning. The primary objective of this study was to establish endotypes for patients with sepsis, and assess the association of these endotypes with clinical traits and survival outcomes. We also established candidate biomarkers for the endotypes to allow identification of patient endotypes in clinical practice. FINDINGS The discovery cohort had 306 patients, the first validation cohort had 216, and the second validation cohort had 265 patients. Four molecular endotypes for sepsis, designated Mars1-4, were identified in the discovery cohort, and were associated with 28-day mortality (log-rank p=0·022). In the discovery cohort, the worst outcome was found for patients classified as having a Mars1 endotype, and at 28 days, 35 (39%) of 90 people with a Mars1 endotype had died (hazard ratio [HR] vs all other endotypes 1·86 [95% CI 1·21-2·86]; p=0·0045), compared with 23 (22%) of 105 people with a Mars2 endotype (HR 0·64 [0·40-1·04]; p=0·061), 16 (23%) of 71 people with a Mars3 endotype (HR 0·71 [0·41-1·22]; p=0·19), and 13 (33%) of 40 patients with a Mars4 endotype (HR 1·13 [0·63-2·04]; p=0·69). Analysis of the net reclassification improvement using a combined clinical and endotype model significantly improved risk prediction to 0·33 (0·09-0·58; p=0·008). A 140-gene expression signature reliably stratified patients with sepsis to the four endotypes in both the first and second validation cohorts. Only Mars1 was consistently significantly associated with 28-day mortality across the cohorts. To facilitate possible clinical use, a biomarker was derived for each endotype; BPGM and TAP2 reliably identified patients with a Mars1 endotype. INTERPRETATION This study provides a method for the molecular classification of patients with sepsis to four different endotypes upon ICU admission. Detection of sepsis endotypes might assist in providing personalised patient management and in selection for trials. FUNDING Center for Translational Molecular Medicine, Netherlands.

Triggering receptor expressed on myeloid cells‐1 (TREM‐1) improves host defence in pneumococcal pneumonia

Streptococcus (S.) pneumoniae is a common Gram‐positive pathogen in community‐acquired pneumonia and sepsis. Triggering receptor expressed on myeloid cells‐1 (TREM‐1) is a receptor on phagocytes known to amplify inflammatory responses. Previous studies showed that TREM‐1 inhibition protects against lethality during experimental Gram‐negative sepsis. We here aimed to investigate the role of TREM‐1 in an experimental model of pneumococcal pneumonia, using TREM‐1/3‐deficient (Trem‐1/3–/–) and wild‐type (Wt) mice. Additionally ex vivo responsiveness of Trem‐1/3–/– neutrophils and macrophages was examined. S. pneumoniae infection resulted in a rapid recruitment of TREM‐1‐positive neutrophils into the bronchoalveolar space, while high constitutive TREM‐1 expression on alveolar macrophages remained unchanged. TREM‐1/3 deficiency led to increased lethality, accompanied by enhanced growth of S. pneumoniae at the primary site of infection and increased dissemination to distant organs. Within the first 3–6 h of infection, Trem‐1/3–/– mice demonstrated a strongly impaired innate immune response in the airways, as reflected by reduced local release of cytokines and chemokines and a delayed influx of neutrophils. Trem‐1/3–/– alveolar macrophages produced fewer cytokines upon exposure to S. pneumoniae in vitro and were less capable of phagocytosing this pathogen. TREM‐1/3 deficiency did not influence neutrophil responsiveness to S. pneumoniae. These results identify TREM‐1 as a key player in protective innate immunity during pneumococcal pneumonia, most likely by enhancing the early immune response of alveolar macrophages. Copyright

AMP-activated Protein Kinase Activation by 5-Aminoimidazole-4-carbox-amide-1-β-d-ribofuranoside (AICAR) Reduces Lipoteichoic Acid-induced Lung Inflammation

Background: AMPK is a highly conserved energy homeostasis-regulating kinase. Results: Activation of AMPK by AICAR in vitro reduced cytokine production in alveolar macrophage cell line and in vivo reduced LTA-induced neutrophil influx, protein leak and cytokine/chemokine levels. Conclusion: AMPK activation inhibits LTA-induced lung inflammation in mice. Significance: AICAR reduces LTA inflammation. Adenosine monophosphate-activated protein (AMP)-activated kinase (AMPK) is a highly conserved kinase that plays a key role in energy homeostasis. Activation of AMPK was shown to reduce inflammation in response to lipolysaccharide in vitro and in vivo. 5-Aminoimidazole-4-carbox-amide-1-β-d-ribofuranoside (AICAR) is intracellularly converted to the AMP analog ZMP, which activates AMPK. Lipoteichoic acid (LTA) is a major component of the cell wall of Gram-positive bacteria that can trigger inflammatory responses. In contrast to lipopolysaccharide, little is known on the effects of AMPK activation in LTA-triggered innate immune responses. Here, we studied the potency of AMPK activation to reduce LTA-induced inflammation in vitro and in lungs in vivo. Activation of AMPK in vitro reduced cytokine production in the alveolar macrophage cell line MH-S. In vivo, AMPK activation reduced LTA-induced neutrophil influx, as well as protein leak and cytokine/chemokine levels in the bronchoalveolar space. In conclusion, AMPK activation inhibits LTA-induced lung inflammation in mice.

Osteopontin Impairs Host Defense During Pneumococcal Pneumonia

BACKGROUND Streptococcus pneumoniae is the most frequently isolated pathogen responsible for community-acquired pneumonia. Osteopontin is involved in inflammation during both innate and adaptive immunity. METHODS To determine the role of osteopontin in the host response during pneumococcal pneumonia, osteopontin knockout (KO) and normal wild-type (WT) mice were intranasally infected with viable S. pneumoniae. RESULTS Pneumonia was associated with a rapid increase in pulmonary osteopontin concentrations in WT mice from 6 h onward. Osteopontin KO mice showed a prolonged survival relative to WT mice, which was accompanied by diminished pulmonary bacterial growth and reduced dissemination to distant body sites. In addition, at 48 h after infection pulmonary inflammation was decreased in osteopontin KO mice as reflected by lower inflammation scores and reduced chemokine concentrations. In contrast to pneumococcal pneumonia, osteopontin deficiency did not influence bacterial growth in primary pneumococcal sepsis induced by direct intravenous infection, suggesting that the detrimental effect of osteopontin on antibacterial defense during pneumonia primarily is exerted in the pulmonary compartment. Moreover, recombinant osteopontin stabilized S. pneumoniae viability in vitro. CONCLUSIONS These results suggest that the pneumococcus misuses osteopontin in the airways for optimal growth and to cause invasive disease after entering the lower airways.

R-roscovitine reduces lung inflammation induced by lipoteichoic acid and Streptococcus pneumoniae.

Bacterial pneumonia remains associated with high morbidity and mortality. The gram-positive pathogen Streptococcus pneumoniae is the most common cause of community-acquired pneumonia. Lipoteichoic acid (LTA) is an important proinflammatory component of the gram-positive bacterial cell wall. R-roscovitine, a purine analog, is a potent cyclin-dependent kinase (CDK)-1, -2, -5 and -7 inhibitor that has the ability to inhibit the cell cycle and to induce polymorphonuclear cell (PMN) apoptosis. We sought to investigate the effect of R-roscovitine on LTA-induced activation of cell lines with relevance for lung inflammation in vitro and on lung inflammation elicited by either LTA or viable S. pneumoniaein vivo. In vitro R-roscovitine enhanced apoptosis in PMNs and reduced tumor necrosis factor (TNF)-α and keratinocyte chemoattractant (KC) production in MH-S (alveolar macrophage) and MLE-12/MLE-15 (respiratory epithelial) cell lines. In vivo R-roscovitine treatment reduced PMN numbers in bronchoalveolar lavage fluid during LTA-induced lung inflammation; this effect was reversed by inhibiting apoptosis. Postponed treatment with R-roscovitine (24 and 72 h) diminished PMN numbers in lung tissue during gram-positive pneumonia; this step was associated with a transient increase in pulmonary bacterial loads. R-roscovitine inhibits proinflammatory responses induced by the gram-positive stimuli LTA and S. pneumoniae. R-roscovitine reduces PMN numbers in lungs upon LTA administration by enhancing apoptosis. The reduction in PMN numbers caused by R-roscovitine during S. pneumoniae pneumonia may hamper antibacterial defense.

Admission Hyperglycemia in Critically Ill Sepsis Patients: Association With Outcome and Host Response

Objectives: To investigate whether admission hyperglycemia is associated with the presentation and/or outcome of sepsis, what the influence of hyperglycemia is on key host responses to sepsis, and whether hyperglycemia differentially affects patients with diabetes mellitus. Design and Setting: A substudy of a prospective observational cohort study was conducted in the intensive care of two tertiary hospitals between January 2011 and July 2013. Patients: Of all consecutive critically ill sepsis patients, admission glucose was used to stratify patients in euglycemia (71–140 mg/dL), mild hyperglycemia (141–199 mg/dL), and severe hyperglycemia (≥ 200 mg/dL), and patients with hypoglycemia were excluded. Fifteen plasma biomarkers providing insight in key host responses implicated in sepsis pathogenesis were measured on admission. Measurements and Main Results: Of 987 sepsis patients with admission glucose levels greater than 70 mg/dL, 519 (52.6%) had normal glucose levels, 267 (27.1%) had mild, and 201 (20.4%) severe hyperglycemia. Admission hyperglycemia was accompanied by mitigated alterations in plasma host response biomarker levels indicative of activation of the cytokine network, the vascular endothelium, and the coagulation system in patients without a history of diabetes. Severe, but not mild, admission hyperglycemia was associated with increased 30-day mortality (adjusted hazard ratio, 1.66 [95% CI, 1.24–2.23]), in both patients without diabetes (adjusted hazard ratio, 1.65 [95% CI, 1.12–2.42]) and with diabetes (adjusted hazard ratio, 1.91 [95% CI, 1.01–3.62]). Conclusion: Admission hyperglycemia is associated with adverse outcome of sepsis irrespective of the presence or absence of preexisting diabetes by a mechanism unrelated to exaggerated inflammation or coagulation.

Interleukin-1 Receptor–Associated Kinase M–Deficient Mice Demonstrate an Improved Host Defense during Gram-negative Pneumonia

Pneumonia is a common cause of morbidity and mortality and the most frequent source of sepsis. Bacteria that try to invade normally sterile body sites are recognized by innate immune cells through pattern recognition receptors, among which toll-like receptors (TLRs) feature prominently. Interleukin-1 receptor (IL-1R)-associated kinase (IRAK)-M is a proximal inhibitor of TLR signaling expressed by epithelial cells and macrophages in the lung. To determine the role of IRAK-M in host defense against bacterial pneumonia, RAK-M-deficient (IRAK-M−/−) and normal wild-type (WT) mice were infected intranasally with Klebsiella pneumoniae. IRAK-M mRNA was upregulated in lungs of WT mice with Klebsiella pneumonia, and the absence of IRAK-M resulted in a strongly improved host defense as reflected by reduced bacterial growth in the lungs, diminished dissemination to distant body sites, less peripheral tissue injury and better survival rates. Although IRAK-M−/− alveolar macrophages displayed enhanced responsiveness toward intact K. pneumoniae and Klebsiella lipopolysaccharide (LPS) in vitro, IRAK-M−/− mice did not show increased cytokine or chemokine levels in their lungs after infection in vivo. The extent of lung inflammation was increased in IRAK-M−/− mice shortly after K. pneumoniae infection, as determined by semiquantitative scoring of specific components of the inflammatory response in lung tissue slides. These data indicate that IRAK-M impairs host defense during pneumonia caused by a common gram-negative respiratory pathogen.