Carmen Mikacenic

Variation in the TLR10/TLR1/TLR6 locus is the major genetic determinant of interindividual difference in TLR1/2-mediated responses

Toll-like receptor (TLR)-mediated innate immune responses are important in early host defense. Using a candidate gene approach, we previously identified genetic variation within TLR1 that is associated with hyper-responsiveness to a TLR1/2 agonist in vitro and with death and organ dysfunction in patients with sepsis. Here we report a genome-wide association study (GWAS) designed to identify genetic loci controlling whole blood cytokine responses to the TLR1/2 lipopeptide agonist, Pam3CSK4 (N-palmitoyl-S-dipalmitoylglyceryl Cys-Ser-(Lys)4) ex vivo. We identified a very strong association (P<1 × 10−27) between genetic variation within the TLR10/1/6 locus on chromosome 4, and Pam3CSK4-induced cytokine responses. This was the predominant association explaining over 35% of the population variance for this phenotype. Notably, strong associations were observed within TLR10, suggesting that genetic variation in TLR10 may influence bacterial lipoprotein-induced responses. These findings establish the TLR10/1/6 locus as the dominant common genetic factor controlling interindividual variability in Pam3CSK4-induced whole blood responses in the healthy population.

Biomarkers of Endothelial Activation Are Associated with Poor Outcome in Critical Illness

Background Endothelial activation plays a role in organ dysfunction in the systemic inflammatory response syndrome (SIRS). Angiopoietin-1 (Ang-1) promotes vascular quiescence while angiopoietin-2 (Ang-2) mediates microvascular leak. Circulating levels of Ang-1 and Ang-2 in patients with SIRS could provide insight on risks for organ dysfunction and death distinct from inflammatory proteins. In this study, we determined if biomarkers of endothelial activation and inflammation exhibit independent associations with poor outcomes in SIRS. Methods We studied 943 critically ill patients with SIRS admitted to an Intensive Care Unit (ICU) of an academic medical center. We measured plasma levels of endothelial markers (Ang-1, Ang-2, soluble vascular cell adhesion molecule-1 (sVCAM-1)) and inflammatory markers (interleukin-6 (IL-6), interleukin-8 (IL-8), granulocyte-colony stimulating factor (G-CSF), soluble tumor necrosis factor receptor-1 (sTNFR-1)) within 24 hours of enrollment. We tested for associations between each marker and 28 day mortality, shock, and day 3 sequential organ failure assessment (SOFA) score. For 28 day mortality, we performed sensitivity analysis for those subjects with sepsis and those with sterile inflammation. We used multivariate models to adjust for clinical covariates and determine if associations identified with endothelial activation markers were independent of those observed with inflammatory markers. Results Higher levels of all biomarkers were associated with increased 28 day mortality except levels of Ang-1 which were associated with lower mortality. After adjustment for comorbidities and sTNFR-1 concentration, a doubling of Ang-1 concentration was associated with lower 28 day mortality (Odds ratio (OR) = 0.81; p<0.01), shock (OR = 0.82; p<0.001), and SOFA score (β = -0.50; p<0.001), while Ang-2 concentration was associated with increased mortality (OR = 1.55; p<0.001), shock (OR = 1.51; p<0.001), and SOFA score (β = +0.63; p<0.001). sVCAM-1 was not independently associated with SIRS outcomes. Conclusions In critically ill patients with SIRS, early measurements of Ang-1 and Ang-2 are associated with death and organ dysfunction independently of simultaneously-measured markers of inflammation.

Host derived biomarkers of inflammation, apoptosis, and endothelial activation are associated with clinical outcomes in patients with bacteremia and sepsis regardless of microbial etiology

William O. Hahn, Carmen Mikacenic, Brenda L. Price, Susanna Harju-Baker, Ronit Katz, Jonathan Himmelfarb, Mark M. Wurfelb,y, and W. Conrad Lilesa,y Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, WA, USA; Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Washington, Seattle, WA, USA; Department of Biostatistics, University of Washington, Seattle, WA, USA; Kidney Research Institute, University of Washington, Seattle, WA, USA

Interleukin-17A Is Associated with Alveolar Inflammation and Poor Outcomes in Acute Respiratory Distress Syndrome

Objective:Interleukin-17A is a proinflammatory cytokine known to play a role in host defense and pathologic inflammation in murine models of lung injury. The relationship between interleukin-17A and inflammation in human lung injury is unknown. Our primary objective was to determine whether interleukin-17A levels are associated with alveolar measures of inflammation and injury in patients with acute respiratory distress syndrome. Our secondary objective was to test whether interleukin-17A levels are associated with acute respiratory distress syndrome–related outcomes. Design:Observational study. Setting:Six North American medical centers. Patients:We studied two groups of patients with acute respiratory distress syndrome: 1) patients previously enrolled in a placebo-controlled clinical trial of omega-3 fatty acids performed at five North American medical centers (n = 86, acute respiratory distress syndrome 1), and 2) patients with systemic inflammatory response syndrome admitted to an ICU who developed acute respiratory distress syndrome (n = 140, acute respiratory distress syndrome 2). In acute respiratory distress syndrome 1, we used paired serum and bronchoalveolar lavage fluid samples obtained within 48 hours of acute respiratory distress syndrome onset, whereas in acute respiratory distress syndrome 2, we used plasma obtained within the first 24 hours of ICU admission. Interventions:None. Measurements and Main Results:We measured circulating interleukin-17A in acute respiratory distress syndrome 1 and acute respiratory distress syndrome 2. We also measured interleukin-17A, neutrophil counts, and total protein in bronchoalveolar lavage fluid from acute respiratory distress syndrome 1. We found that bronchoalveolar lavage interleukin-17A was strongly associated with higher bronchoalveolar lavage percent neutrophils (p < 0.001) and bronchoalveolar lavage total protein (p < 0.01) in acute respiratory distress syndrome1. In both acute respiratory distress syndrome 1 and acute respiratory distress syndrome 2, elevated interleukin-17A was associated with higher Sequential Organ Failure Assessment scores (p < 0.05). Conclusions:Elevated circulating and alveolar levels of interleukin-17A are associated with increased percentage of alveolar neutrophils, alveolar permeability, and organ dysfunction in acute respiratory distress syndrome.

Cutting Edge: Genetic Variation in TLR1 Is Associated with Pam3CSK4-Induced Effector T Cell Resistance to Regulatory T Cell Suppression

TLR play essential roles in the initiation and modulation of immune responses. TLR1/TLR2 heterodimers recognize triacylated bacterial lipopeptides, including the synthetic TLR1/2 lipopeptide Pam3CSK4. Genetic variation in TLR1 is associated with outcomes in diseases in which regulatory T cells (Treg) play a role, including asthma and allergy. To determine whether genetic polymorphisms in TLR1 are associated with alterations in Treg suppression of effector T cells (Teff), we performed in vitro suppression assays in healthy individuals with various haplotypes in TLR1. We show that functional genetic polymorphisms in TLR1 modify surface expression of TLR1 on T lymphocytes and confer enhanced Teff resistance to Treg suppression in the presence of Pam3CSK4. These effects are mediated, in part, by IL-6 and inhibited by blocking IL-6 signaling through STAT3. These findings suggest that TLR1 polymorphisms could influence immune-related disease through Teff resistance to Treg suppression.

Pentraxin-3 and the right ventricle: the Multi-Ethnic Study of Atherosclerosis-Right Ventricle Study.

Pentraxin-3 (PTX3) is a protein mediator of innate immunity that is elevated in the setting of left heart disease and pulmonary arterial hypertension. The relationship between PTX3 and right ventricular (RV) structure and function is not known. We included men and women with magnetic resonance imaging assessment of RV structure and function and measurement of PTX3 from the Multi-Ethnic Study of Atherosclerosis, a study of individuals free of clinical cardiovascular disease. Multivariable linear regression estimated associations between PTX3 protein levels and RV measures after adjusting for demographic characteristics, anthropometrics, smoking status, diabetes mellitus, hypertension, and corresponding left ventricular (LV) parameters. Instrumental variable analysis exploiting Mendelian randomization was attempted using two-stage least squares regression. The study sample included 1,779 participants with available PTX3 levels, RV measures, and all covariables. Mean PTX3 level was 2.1 ng/mL. Higher PTX3 was independently associated with greater RV mass and larger RV end-diastolic volume with and without adjustment for the corresponding LV parameters or C-reactive protein (all P < .05). There was no association between PTX3 and RV ejection fraction or stroke volume. Single-nucleotide polymorphisms were not associated with PTX3 protein levels or RV measures after accounting for race. Instrumental variable analysis could not be reliably performed. Higher PTX3 protein levels were associated with greater RV mass and larger RV end-diastolic volume. These associations were independent of common cardiovascular risk factors and LV morphologic changes. Inflammation is associated with differences in the pulmonary circulation-RV axis in adults without clinical cardiovascular disease.

A Two-Biomarker Model Predicts Mortality in the Critically Ill with Sepsis.

Rationale: Improving the prospective identification of patients with systemic inflammatory response syndrome (SIRS) and sepsis at low risk for organ dysfunction and death is a major clinical challenge. Objectives: To develop and validate a multibiomarker‐based prediction model for 28‐day mortality in critically ill patients with SIRS and sepsis. Methods: A derivation cohort (n = 888) and internal test cohort (n = 278) were taken from a prospective study of critically ill intensive care unit (ICU) patients meeting two of four SIRS criteria at an academic medical center for whom plasma was obtained within 24 hours. The validation cohort (n = 759) was taken from a prospective cohort enrolled at another academic medical center ICU for whom plasma was obtained within 48 hours. We measured concentrations of angiopoietin‐1, angiopoietin‐2, IL‐6, IL‐8, soluble tumor necrosis factor receptor‐1, soluble vascular cell adhesion molecule‐1, granulocyte colony‐stimulating factor, and soluble Fas. Measurements and Main Results: We identified a two‐biomarker model in the derivation cohort that predicted mortality (area under the receiver operator characteristic curve [AUC], 0.79; 95% confidence interval [CI], 0.74‐0.83). It performed well in the internal test cohort (AUC, 0.75; 95% CI, 0.65‐0.85) and the external validation cohort (AUC, 0.77; 95% CI, 0.72‐0.83). We determined a model score threshold demonstrating high negative predictive value (0.95) for death. In addition to a low risk of death, patients below this threshold had shorter ICU length of stay, lower incidence of acute kidney injury, acute respiratory distress syndrome, and need for vasopressors. Conclusions: We have developed a simple, robust biomarker‐based model that identifies patients with SIRS/sepsis at low risk for death and organ dysfunction.

Peripheral and Alveolar Cell Transcriptional Programs Are Distinct in Acute Respiratory Distress Syndrome

1. Center for International Blood and Marrow Transplant Research (CIBMTR), National Marrow Donor Program (NMDP), European Blood and Marrow Transplant Group (EBMT), American Society of Blood and Marrow Transplantation (ASBMT), Canadian Blood and Marrow Transplant Group (CBMTG), Infectious Disease Society of America (IDSA), Society for Healthcare Epidemiology of America (SHEA), Association of Medical Microbiology and Infectious Diseases Canada (AMMI), Centers for Disease Control and Prevention (CDC). Guidelines for preventing infectious complications among hematopoietic cell transplant recipients: a global perspective. Bone Marrow Transplant 2009;44:453–558. 2. Wilson MR, Naccache SN, Samayoa E, Biagtan M, Bashir H, Yu G, et al. Actionable diagnosis of neuroleptospirosis by next-generation sequencing. N Engl J Med 2014;370:2408–2417. 3. Doan T, Wilson MR, Crawford ED, Chow ED, Khan LM, Knopp KA, et al. Illuminating uveitis: metagenomic deep sequencing identifies common and rare pathogens. Genome Med 2016;8:90. 4. Langelier C, Zinter MS, Kalantar KK, Yanik GA, Christenson S, O’Donovan B, et al. Data from: Metagenomic sequencing detects respiratory pathogens in hematopoietic cellular transplant patients. Dryad Digital Repository. 2017. Available from: https://doi.org/10.5061/ dryad.800tj. 5. Jain S, Self WH, Wunderink RG, Fakhran S, Balk R, Bramley AM, et al. CDC EPIC Study Team. Community-acquired pneumonia requiring hospitalization among U.S. adults. N Engl J Med 2015;373:415–427. 6. Tunkel AR, Sepkowitz KA. Infections caused by viridans streptococci in patients with neutropenia. Clin Infect Dis 2002;34:1524–1529. 7. Dı́ez-Aguilar M, Ruiz-Garbajosa P, Fernández-Olmos A, Guisado P, Del Campo R, Quereda C, et al. Non-diphtheriae Corynebacterium species: an emerging respiratory pathogen. Eur J Clin Microbiol Infect Dis 2013;32:769–772. 8. Heinonen S, Jartti T, Garcia C, Oliva S, Smitherman C, Anguiano E, et al. Rhinovirus detection in symptomatic and asymptomatic children: value of host transcriptome analysis. Am J Respir Crit Care Med 2016; 193:772–782. 9. Abreu NA, Nagalingam NA, Song Y, Roediger FC, Pletcher SD, Goldberg AN, et al. Sinus microbiome diversity depletion and Corynebacterium tuberculostearicum enrichment mediates rhinosinusitis. Sci Transl Med 2012;4:151ra124. 10. Liberzon A, Birger C, Thorvaldsdóttir H, Ghandi M, Mesirov JP, Tamayo P. The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst 2015;1:417–425.

A novel and rapid method to quantify Treg mediated suppression of CD4 T cells

Measuring regulatory T cell suppression provides important insight into T cell dysfunction in autoimmune disease. However, to date, suppression assays are limited by the requirement for freshly isolated cells, and significant cell numbers. Here, we present a novel and rapid in vitro assay using effector T cell surface expression of both CD25 and CD134 as a surrogate marker of regulatory T cell-mediated suppression. This surface marker-based suppression assay works for frozen samples and for samples with limited cell numbers. It is also shorter taking two days to complete compared to the four days required for proliferation-based assays. Furthermore, this assay works with both in vitro expanded and natural Tregs, as well as anti-CD3/anti-CD28 bead-based and APC stimulation conditions. In conclusion, we have developed and validated a new suppression assay for cryopreserved samples with limited cell numbers that may be helpful to investigate T cell regulation in the context of infection or autoimmune diseases.

Cytometry TOF identifies alveolar macrophage subtypes in acute respiratory distress syndrome

Studies in human peripheral blood monocyte-derived macrophages in vitro have shown clear evidence that multiple macrophage polarization states exist. The extent to which different alveolar macrophage (AM) polarization states exist in homeostasis or in the setting of severe injury such as acute respiratory distress syndrome (ARDS) is largely unknown. We applied single-cell cytometry TOF (CyTOF) to simultaneously measure 36 cell-surface markers on CD45+ cells present in bronchoalveolar lavage from healthy volunteers, as well as mechanically ventilated subjects with and without ARDS. Visualization of the high-dimensional data with the t-distributed stochastic neighbor embedding algorithm demonstrated wide diversity of cell-surface marker profiles among CD33+CD71+CD163+ AMs. We then used a κ-nearest neighbor density estimation algorithm to statistically identify distinct alveolar myeloid subtypes, and we discerned 3 AM subtypes defined by CD169 and PD-L1 surface expression. The percentage of AMs that were classified into one of the 3 AM subtypes was significantly different between healthy and mechanically ventilated subjects. In an independent cohort of subjects with ARDS, PD-L1 gene expression and PD-L1/PD-1 pathway-associated gene sets were significantly decreased in AMs from patients who experienced prolonged mechanical ventilation or death. Unsupervised CyTOF analysis of alveolar leukocytes from human subjects has potential to identify expected and potentially novel myeloid populations that may be linked with clinical outcomes.