scholarly journals Ex vivo mass cytometry analysis reveals a profound myeloid proinflammatory signature in psoriatic arthritis synovial fluid

2021 ◽  
pp. annrheumdis-2021-220280
Author(s):  
Nicole Yager ◽  
Suzanne Cole ◽  
Alicia Lledo Lara ◽  
Ash Maroof ◽  
Frank Penkava ◽  
...  
Keyword(s):  
Psoriatic Arthritis ◽  
Synovial Fluid ◽  
Cytokine Production ◽  
Ex Vivo ◽  
Gene Array ◽  
Transcriptomic Analysis ◽  
Cell Populations ◽  
Mass Cytometry ◽  
Proinflammatory Mediators ◽  
Intermediate Monocytes

ObjectivesA number of immune populations have been implicated in psoriatic arthritis (PsA) pathogenesis. This study used mass cytometry (CyTOF) combined with transcriptomic analysis to generate a high-dimensional dataset of matched PsA synovial fluid (SF) and blood leucocytes, with the aim of identifying cytokine production ex vivo in unstimulated lymphoid and myeloid cells.MethodsFresh SF and paired blood were either fixed or incubated with protein transport inhibitors for 6 hours. Samples were stained with two CyTOF panels: a phenotyping panel and an intracellular panel, including antibodies to both T cell and myeloid cell secreted proteins. Transcriptomic analysis by gene array of key expanded cell populations, single-cell RNA-seq, ELISA and LEGENDplex analysis of PsA SF were also performed.ResultsWe observed marked changes in the myeloid compartment of PsA SF relative to blood, with expansion of intermediate monocytes, macrophages and dendritic cell populations. Classical monocytes, intermediate monocytes and macrophages spontaneously produced significant levels of the proinflammatory mediators osteopontin and CCL2 in the absence of any in vitro stimulation. By contrast minimal spontaneous cytokine production by T cells was detected. Gene expression analysis showed the genes for osteopontin and CCL2 to be among those most highly upregulated by PsA monocytes/macrophages in SF; and both proteins were elevated in PsA SF.ConclusionsUsing multiomic analyses, we have generated a comprehensive cellular map of PsA SF and blood to reveal key expanded myeloid proinflammatory modules in PsA of potential pathogenic and therapeutic importance.

scholarly journals Ex vivo mass cytometry analysis reveals a profound myeloid proinflammatory signature in psoriatic arthritis synovial fluid

2021 ◽  
Author(s):  
Nicole Yager ◽  
Suzanne Cole ◽  
Alicia Lledo Lara ◽  
Ash Maroof ◽  
Frank Penkava ◽  
...  
Keyword(s):  
Psoriatic Arthritis ◽  
Synovial Fluid ◽  
Cytokine Production ◽  
Ex Vivo ◽  
Gene Array ◽  
Transcriptomic Analysis ◽  
Cell Populations ◽  
Mass Cytometry ◽  
Proinflammatory Mediators ◽  
Intermediate Monocytes

Objectives: A number of immune populations have been implicated in psoriatic arthritis (PsA) pathogenesis. This study used mass cytometry (CyTOF) combined with transcriptomic analysis to generate a high-dimensional dataset of matched PsA synovial fluid (SF) and blood leucocytes, with the aim of identifying cytokine production ex vivo in unstimulated lymphoid and myeloid cells. Methods: Fresh SF and paired blood were either fixed or incubated with protein transport inhibitors for 6 h. Samples were stained with two CyTOF panels: a phenotyping panel and an intracellular panel, including antibodies to both T cell and myeloid cell secreted proteins. Transcriptomic analysis by gene array of key expanded cell populations and single-cell RNA-seq, and ELISA and LEGENDplex analysis of PsA SF were also performed. Results: We observed marked changes in the myeloid compartment of PsA SF relative to blood, with expansion of intermediate monocytes, macrophages and dendritic cell populations. Classical monocytes, intermediate monocytes and macrophages spontaneously produced significant levels of the proinflammatory mediators osteopontin and CCL2 in the absence of any in vitro stimulation. By contrast minimal spontaneous cytokine production by T cells was detected. Gene expression analysis showed the genes for osteopontin and CCL2 to be amongst those most highly upregulated by PsA monocytes/macrophages; and both proteins were elevated in PsA SF. Conclusions: Using multiomic analyses we have generated a comprehensive cellular map of PsA SF and blood to reveal key expanded myeloid proinflammatory modules in PsA of potential pathogenic and therapeutic importance.

scholarly journals Mass Cytometry Analysis of Dysregulated Cytokine Production and Intracellular Signaling in Myelofibrosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4277-4277 ◽  
Author(s):  
Daniel A.C. Fisher ◽  
Cathrine A. Miner ◽  
Elizabeth K. Engle ◽  
Taylor M. Brost ◽  
Olga Malkova ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Cytokine Production ◽  
Jak2 V617f ◽  
Cell Populations ◽  
Mass Cytometry ◽  
Advisory Committees ◽  
Downstream Signaling ◽  
Cd34 Cells ◽  
Multiple Cell

Abstract Myeloproliferative neoplasms including myelofibrosis (MF) are characterized by a malignant clone containing JAK2 V617F or other mutations leading to unregulated JAK2 kinase activity. MF is characterized by anemia, splenomegaly, bone marrow fibrosis, inflammatory cytokine production, and a propensity for transformation to secondary acute myeloid leukemia. Inhibition of JAK2 with ruxolitinib improves constitutional symptoms and splenomegaly and lowers circulating plasma cytokine levels. However, improvements in anemia, fibrosis, and malignant clonal burden are infrequent. These observations illustrate the need for improved therapy for MF. The objective of this study is to better understand the relationship between dysregulated cytokines and downstream signaling in MF, with the goal of determining how these pathways can be more effectively manipulated for therapeutic benefit. To interrogate altered signaling in MF, we have employed mass cytometry (CyTOF), a method which enables the quantitative analysis of signaling throughout hematopoiesis. In our survey of signaling in MF, hyperactivation of the NFκB signaling pathway was found to be widespread. This finding was corroborated by gene set enrichment analysis (GSEA) of a published gene expression dataset of CD34+ cells from MF versus normal controls (Norfo et al. 2014 Blood). Evidence for NFκB hyperactivation, both by mass cytometry and GSEA, was strongest in JAK2V617F-mutant MF patients. This supports a hypothesis that pronounced NFκB signaling hyperactivation is a consequence of mutant JAK2. In MF, hyperactivated NFκB signaling was widespread among hematopoietic cell populations, including T cells. This distribution suggests that NFκB activation may be partly driven by non-cell-autonomous mechanisms. Consistent with previous studies, plasma TNFα levels were found to be elevated in these patients, suggesting that excessive production of TNFα could result in downstream activation of NFκB across multiple cell populations in a non-cell-autonomous fashion. To further elucidate the etiology of systemic NFκB hyperactivation and understand the interplay of inflammatory cytokines and downstream signaling, we extended our mass cytometry approach to study the cellular distribution of cytokine production in MF. A panel of 22 surface marker antibodies and 12 cytokine antibodies was developed for these experiments. Examination of peripheral blood from two JAK2V617F-mutant MF patients revealed that intracellular levels of several cytokines were constitutively elevated in both MF patients compared to healthy controls. Monocytes produced the highest levels of TNFα among hematopoietic populations, and these were higher in MF versus control monocytes. Supranormal cytokine expression was accentuated by stimulation with PMA/ionomycin or TLR ligands R848 or PAM3CSK4. Incubation with TNFα led to supranormal levels of the cytokines MIP1β and IL-6, in monocytes from one or both patients. Therefore, abnormal production of TNFα by MF patient monocytes could result in overproduction of IL-6, and MIP1β in the same cells. PMA/ionomycin led to above normal production of TNFα from MF Lin-CD34+ cells and CD33+CD34- immature myeloid cells, suggestive that these cells could be hypersensitive to pathophysiologic signaling stimulations in a manner resulting in elevated cytokine production. MF patient T cells also showed hypersensitivity to PMA/ionomycin stimulation, compared with controls, in their production of IFNγ and MIP1β. These cytokines, along with the MF monocyte-overexpressed cytokines TNFα, IL-6, and MIP1β, were found to be elevated in MF patient plasma, consistent with prior studies. These findings imply that multiple cell populations in JAK2 V617F-mutant MF patients overexpress inflammatory cytokines and are hypersensitive to inflammatory insults. The upregulation of cytokines is likely to underlie the systemic hyperactivation of NFκB signaling observed in MF, and could generate non-cell-autonomous effects on the malignant myeloid clone. While NFκB phosphorylation responses to TNFα appear strongest in Lin-CD34+ cells, other cytokines may mediate signaling abnormalities across a variety of cell types. Future experiments will attempt to identify signaling effects of multiple elevated cytokines, which may underlie features of MF that persist despite JAK2 inhibitor therapy. Disclosures Oh: CTI: Research Funding; Janssen: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding.

MO074TILVESTAMAB, A FUNCTION-BLOCKING MONOCLONAL ANTIBODY INHIBITOR OF AXL RTK SIGNALLING, LIMITS THE ONSET OF RENAL FIBROTIC CHANGES IN HUMAN KIDNEYS EX VIVO

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Linn Hodneland Nilsson ◽  
Sturla Magnus Grøndal ◽  
Magnus Blø ◽  
Anna Boniecka ◽  
Barbara VanderHoeven ◽  
...  
Keyword(s):  
Ace Inhibitor ◽  
Ex Vivo ◽  
Kidney Tissue ◽  
Human Kidney ◽  
Receptor Activation ◽  
Cell Populations ◽  
Mass Cytometry ◽  
Kidney Fibrosis ◽  
Mesenchymal Transition ◽  
Antibody Panel

Abstract Background and Aims Interstitial fibrosis, characterised by the accumulation of extracellular matrix in the cortical interstitium, is directly correlated with progressive chronic kidney disease secondary to inflammatory, immunologic, obstructive or metabolic causes. An invariant histologic marker of this progression is the accumulation of fibroblasts, with the phenotypic appearance of activated myofibroblasts expressing alpha smooth muscle actin (αSMA) within intracellular contractile stress fibres. Once present, these myofibroblasts are prognostic indicators of expansion of fibrotic matrix and progressive tubular atrophy, leading towards end-stage disease. The Receptor Tyrosine Kinase AXL is involved in a range of kidney pathologies, with increased activity associated with Epithelial to Mesenchymal Transition (EMT) and tubular proliferation following podocyte loss. In mice treated with an angiotensin-converting enzyme (ACE) inhibitor, enhancement of AXL expression is localised to tubular segments within the medulla and there is evidence of parallel regulatory control of ACE and AXL. We have demonstrated enhanced expression of AXL and the mesenchymal marker, vimentin in diseased human kidney tissue secondary to diabetes or hypertension. Targeting AXL with a small-molecule inhibitor has previously been reported to attenuate fibrosis and reduce inflammation in the unilateral ureteric-outflow obstruction (UUO) model of kidney fibrosis in mice (Landolt et al., 2019). Tilvestamab is a novel function blocking humanized anti-AXL antibody. Tilvestamab blocks GAS6-mediated AXL receptor activation in fibroblasts and renal tubule epithelial cells and mediates AXL receptor internalization and degradation. In this study we aimed to further characterise AXL as a target in CKD and to investigate anti-fibrotic efficacy of tilvestamab. Method Eight weeks old male C57BL/6 mice underwent UUO operation. After 15 days, kidneys were dissociated and stained with a high dimensional single cell mass cytometry 33 markers antibody panel. Data were analysed using JMP Genomics (v.8.2). Precision Cut Kidney Slices (PCKSs) from explanted human kidney tissue were propagated in a bioreactor (Paish et al., 2019, FibroFind, UK). PCKS were incubated for 72hrs in the presence of investigational drugs. Secreted collagen1a1 were quantified by ELISA. RNA was reverse transcribed to cDNA and used in qPCRs to measure Col1a1 and αSMA. FFPE sections were stained for αSMA. High magnification images were taken of each slide and analysed for surface area covered by the stain. Results Expression pattern of AXL during development of kidney fibrosis in the UUO model was investigated using a mass cytometry antibody panel designed for identifying subpopulations of immune cells as well as cell populations of the fibrotic stroma. Two predominant cell populations were affected by ligation; the mesenchymal and the immune island. AXL was a marker characterising several of the key populations that expanded upon ligation supporting a role for AXL in kidney fibrosis pathogenesis. In an ex vivo model of human PCKS, tilvestamab dose-dependently reduced the levels of αSMA. When combined with the lower of two doses of the ACE inhibitor enalapril, the lowest dose of tilvestamab synergized to reduce αSMA levels further as well as reducing secreted Collagen 1a1. Conclusion AXL expression is induced in key cell populations during development of kidney fibrosis supporting AXL as a novel target in CKD. Tilvestamab represents a promising strategy for the pharmacologic intervention of kidney fibrosis, and the potential synergy with current reno-protective therapies warrants further exploration.

scholarly journals Aging and interferon gamma response drive the phenotype of neutrophils in the inflamed joint

2021 ◽  
Author(s):  
Ricardo Grieshaber-Bouyer ◽  
Tarik Exner ◽  
Nicolaj S Hackert ◽  
Felix A Radtke ◽  
Scott A Jelinsky ◽  
...  
Keyword(s):  
Synovial Fluid ◽  
Interferon Gamma ◽  
Ex Vivo ◽  
Joint Fluid ◽  
Mass Cytometry ◽  
Healthy Human ◽  
Healthy Donors ◽  
Active Arthritis ◽  
Blood Neutrophils ◽  
Prominent Response

Objectives: Neutrophils are typically the most abundant leukocyte in arthritic synovial fluid. We sought to understand changes that occur in neutrophils as they migrate from blood to joint. Methods: We performed RNA sequencing of neutrophils from healthy human blood, arthritic blood, and arthritic synovial fluid, comparing transcriptional signatures with those from murine K/BxN serum transfer arthritis. We employed mass cytometry to quantify protein expression and sought to reproduce the synovial fluid phenotype ex vivo in cultured healthy blood neutrophils. Results: Blood neutrophils from healthy donors and patients with active arthritis exhibited largely similar transcriptional signatures. By contrast, synovial fluid neutrophils exhibited more than 1,600 differentially expressed genes. Gene signatures identified a prominent response to interferon gamma (IFNγ), as well as to tumor necrosis factor, interleukin 6, and hypoxia, in both humans and mice. Mass cytometry also found healthy and arthritic donor blood neutrophils largely indistinguishable but revealed a range of neutrophil phenotypes in synovial fluid defined by downregulation of CXCR1 and upregulation of FcγRI, HLA-DR, PD-L1, ICAM-1 and CXCR4. Reproduction of key elements of this signature in cultured blood neutrophils required both IFNγ and prolonged culture. Conclusions: Circulating neutrophils from arthritis patients resemble those from healthy controls, but joint fluid cells exhibit a network of changes, conserved across species, that implicate IFNγ response and aging as complementary drivers of the synovial neutrophil phenotype.

scholarly journals Chronic low-level cadmium exposure in rats affects cytokine production by activated T cells

2019 ◽  
Vol 8 (2) ◽  
pp. 227-237 ◽  
Author(s):  
Alexandra E. Turley ◽  
Joseph W. Zagorski ◽  
Rebekah C. Kennedy ◽  
Robert A. Freeborn ◽  
Jenna K. Bursley ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
Low Dose ◽  
Cytokine Production ◽  
Ex Vivo ◽  
Cadmium Exposure ◽  
Activated T Cells ◽  
Low Level

The purpose of this study was to determine the effect of subchronic, oral, low-dose cadmium exposure (32 ppm over 10 weeks) on the rat immune system. We found that cadmium exposure increased the induction of IFNγ and IL-10 in T cells activated ex vivo after cadmium exposure.

scholarly journals AB0108 IRAK4 INHIBITION SUPPRESSES PROINFLAMMATORY CYTOKINE PRODUCTION FROM HUMAN MACROPHAGES STIMULATED WITH SYNOVIAL FLUID FROM RHEUMATOID ARTHRITIS PATIENTS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1353.2-1353
Author(s):  
A. Yadon ◽  
D. Ruelas ◽  
G. Min-Oo ◽  
J. Taylor ◽  
M. R. Warr
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Signaling Pathways ◽  
Proinflammatory Cytokines ◽  
Proinflammatory Cytokine ◽  
Cytokine Production ◽  
Interleukin 1 ◽  
Proinflammatory Cytokine Production ◽  
Human Macrophages ◽  
Percent Suppression

Background:Rheumatoid arthritis (RA) is characterized by chronic, uncontrolled joint inflammation and tissue destruction. Macrophages are thought to be key mediators in both the initiation and perpetuation of this pathology.1,2The RA synovium contains a complex inflammatory milieu that can stimulate macrophage-dependent production of proinflammatory cytokines through multiple signaling pathways.1,2Existing evidence indicates that toll-like receptors (TLRs) and interleukin-1 receptors (IL-1R) along with their agonists, damage-associated molecular patterns (DAMPs) and IL-1β, are highly expressed in RA joints and are important mediators of synovial macrophage activation and proinflammatory cytokine production.1-9IRAK4 (interleukin-1 receptor-associated kinase 4) is a serine/threonine kinase that facilitates TLR and IL-1R signaling in many cell types, including macrophages.10IRAK4 inhibition represents an opportunity to reduce proinflammatory cytokine production in the joints of patients with RA.Objectives:To investigate the effect of a highly selective IRAK4 inhibitor on proinflammatory cytokine production from human macrophages stimulated with synovial fluid from patients with RA.Methods:Primary human monocytes from 2 independent donors were differentiated for 6 days with granulocyte-macrophage colony-stimulating factor (GM-CSF) to generate human monocyte-derived macrophages (hMDMs). hMDMs were then pretreated with an IRAK4 inhibitor for 1 hour and subsequently stimulated for 24 hours with RA synovial fluid from 5 patients. Culture supernatants were then assessed for secretion of proinflammatory cytokines by MesoScale Discovery.Results:RA synovial fluid stimulation of hMDMs resulted in the production of several proinflammatory cytokines, including IL-6, IL-8, and TNFα. Pretreatment of hMDMs with an IRAK4 inhibitor resulted in the dose-dependent inhibition of IL-6, IL-8, and TNFα production, with an average EC50± SD of 27 ± 31, 26 ± 41, and 28 ± 22 nM, respectively. Maximal percent suppression ± SD of IL-6, IL-8, and TNFα were 76 ± 8.8, 73 ± 15, and 77 ± 13, respectively. To evaluate the specific IRAK4-dependent signaling pathways mediating this response, hMDMs were pretreated with inhibitors of TLR4 (TAK242) and IL-1R (IL-1RA) prior to stimulation with RA synovial fluid. Both TAK242 and IL-1RA inhibited proinflammatory cytokine production. For TAK242, maximal percent suppression ± SD of IL-6, IL-8, and TNFα were 39 ± 25, 48 ± 24, and 50 ± 21, respectively. For IL-1RA maximal percent suppression ± SD of IL-6, IL-8, and TNFα were 18 ± 18, 20 ± 23, and 16 ± 18, respectively. The broad range of inhibition across each stimulation highlights the complexity and variability in the signaling pathways mediating proinflammatory cytokine production from hMDMs stimulated with RA synovial fluid, but demonstrates that RA synovial fluid can stimulate proinflammatory cytokine production in hMDMs, at least partly, through IRAK4-dependent pathways.Conclusion:This work demonstrates that IRAK4 inhibition can suppress proinflammatory cytokine production from macrophages stimulated with synovial fluid from patients with RA and supports a potential pathophysiological role for IRAK4 in perpetuating chronic inflammation in RA.References:[1]Smolen JS, et al.Nat Rev Dis Primers.2018;4:18001.[2]Udalova IA, et al.Nat Rev Rheumatol.2016;12(8):472-485.[3]Joosten LAB, et al.Nat Rev Rheumatol.2016;12(6):344-357.[4]Huang QQ, Pope RM.Curr Rheumatol Rep.2009;11(5):357-364.[5]Roh JS, Sohn DH.Immune Netw.2018;18(4):e27.[6]Sacre SM, et al.Am J Pathol.2007;170(2):518-525.[7]Ultaigh SNA, et al.Arthritis Res Ther.2011;13(1):R33.[8]Bottini N, Firestein GS.Nat Rev Rheumatol.2013;9(1):24-33.[9]Firestein GS, McInnes IB.Immunity.2017;46(2):183-196.[10]Janssens S, Beyaert R.Mol Cell.2003;11(2):293-302.Disclosure of Interests:Adam Yadon Employee of: Gilead, Debbie Ruelas Employee of: Gilead, Gundula Min-Oo Employee of: Gilead, James Taylor Employee of: Gilead, Matthew R. Warr Employee of: Gilead

scholarly journals An Evidence-Based Systematic Review of Human Knee Post-Traumatic Osteoarthritis (PTOA): Timeline of Clinical Presentation and Disease Markers, Comparison of Knee Joint PTOA Models and Early Disease Implications

2021 ◽  
Vol 22 (4) ◽  
pp. 1996 ◽  
Author(s):  
Christine M. Khella ◽  
Rojiar Asgarian ◽  
Judith M. Horvath ◽  
Bernd Rolauffs ◽  
Melanie L. Hart
Keyword(s):  
Synovial Fluid ◽  
Knee Joint ◽  
Ex Vivo ◽  
Disease Process ◽  
Early Disease ◽  
Knee Trauma ◽  
Post Traumatic ◽  
Acute Knee

Understanding the causality of the post-traumatic osteoarthritis (PTOA) disease process of the knee joint is important for diagnosing early disease and developing new and effective preventions or treatments. The aim of this review was to provide detailed clinical data on inflammatory and other biomarkers obtained from patients after acute knee trauma in order to (i) present a timeline of events that occur in the acute, subacute, and chronic post-traumatic phases and in PTOA, and (ii) to identify key factors present in the synovial fluid, serum/plasma and urine, leading to PTOA of the knee in 23–50% of individuals who had acute knee trauma. In this context, we additionally discuss methods of simulating knee trauma and inflammation in in vivo, ex vivo articular cartilage explant and in vitro chondrocyte models, and answer whether these models are representative of the clinical inflammatory stages following knee trauma. Moreover, we compare the pro-inflammatory cytokine concentrations used in such models and demonstrate that, compared to concentrations in the synovial fluid after knee trauma, they are exceedingly high. We then used the Bradford Hill Framework to present evidence that TNF-α and IL-6 cytokines are causal factors, while IL-1β and IL-17 are credible factors in inducing knee PTOA disease progresssion. Lastly, we discuss beneficial infrastructure for future studies to dissect the role of local vs. systemic inflammation in PTOA progression with an emphasis on early disease.

scholarly journals The endogenous capacity to produce proinflammatory mediators by the ex vivo human perfused lung

2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Aleksandra Leligdowicz ◽  
James T. Ross ◽  
Nicolas Nesseler ◽  
Michael A. Matthay
Keyword(s):  
Ex Vivo ◽  
Cold Ischemia Time ◽  
Cold Ischemia ◽  
Experimental Conditions ◽  
Ischemia Time ◽  
Proinflammatory Mediators ◽  
Donor Lung ◽  
Perfused Lung ◽  
Lung Response ◽  
Percent Weight Gain

Abstract Background The ex vivo human perfused lung model has enabled optimizing donor lungs for transplantation and delineating mechanisms of lung injury. Perfusate and airspace biomarkers are a proxy of the lung response to experimental conditions. However, there is a lack of studies evaluating biomarker kinetics during perfusion and after exposure to stimuli. In this study, we analyzed the ex vivo-perfused lung response to three key perturbations: exposure to the perfusion circuit, exogenous fresh whole blood, and bacteria. Results Ninety-nine lungs rejected for transplantation underwent ex vivo perfusion. One hour after reaching experimental conditions, fresh whole blood was added to the perfusate (n = 55). Two hours after reaching target temperature, Streptococcus pneumoniae was added to the perfusate (n = 42) or to the airspaces (n = 17). Perfusate and airspace samples were collected at baseline (once lungs were equilibrated for 1 h, but before blood or bacteria were added) and 4 h later. Interleukin (IL)-6, IL-8, angiopoietin (Ang)-2, and soluble tumor necrosis factor receptor (sTNFR)-1 were quantified. Baseline perfusate and airspace biomarker levels varied significantly, and this was not related to pre-procurement PaO2:FiO2 ratio, cold ischemia time, and baseline alveolar fluid clearance (AFC). After 4 h of ex vivo perfusion, the lung demonstrated a sustained production of proinflammatory mediators. The change in biomarker levels was not influenced by baseline donor lung characteristics (cold ischemia time, baseline AFC) nor was it associated with measures of experimental epithelial (final AFC) or endothelial (percent weight gain) injury. In the presence of exogenous blood, the rise in biomarkers was attenuated. Lungs exposed to intravenous (IV) bacteria relative to control lungs demonstrated a significantly higher rise in perfusate IL-6. Conclusions The ex vivo-perfused lung has a marked endogenous capacity to produce inflammatory mediators over the course of short-term perfusion that is not significantly influenced by donor lung characteristics or the presence of exogenous blood, and only minimally affected by the introduction of systemic bacteremia. The lack of association between biomarker change and donor lung cold ischemia time, final alveolar fluid clearance, and experimental percent weight gain suggests that the maintained ability of the human lung to produce biomarkers is not merely a marker of lung epithelial or endothelial injury, but may support the function of the lung as an immune cell reservoir.

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