Whole blood transcriptomics reveals granulocyte colony-stimulating factor as a mediator of cardiopulmonary bypass-induced systemic inflammatory response syndrome.

Objectives: Systemic inflammatory response syndrome (SIRS) is a frequent complication of cardiopulmonary bypass (CPB). SIRS is associated with significant morbidity and mortality, but its pathogenesis remains incompletely understood, and as a result, biomarkers are lacking and treatment remains expectant and supportive. This study aimed to understand the pathophysiological mechanisms driving SIRS induced by CPB and identify novel therapeutic targets that might reduce systemic inflammation and improve patient outcomes. Methods: Twenty-one patients undergoing cardiac surgery and CPB were recruited, and blood was sampled before, during and after surgery. SIRS was defined using the American College of Chest Physicians/Society of Critical Care Medicine criteria. We performed immune cell profiling and whole blood transcriptomics and measured individual mediators in plasma/serum to characterise SIRS induced by CPB. Results: Nineteen patients fulfilled criteria for SIRS, with a mean duration of 2.7 days. Neutrophil numbers rose rapidly with CPB and remained elevated for at least 48 h afterwards. Transcriptional signatures associated with neutrophil activation and degranulation were enriched during CPB. We identified a network of cytokines governing these transcriptional changes, including granulocyte colony-stimulating factor (G-CSF), a regulator of neutrophil production and function. Conclusions: We identified neutrophils and G-CSF as major regulators of CPB-induced systemic inflammation. Short-term targeting of G-CSF could provide a novel therapeutic strategy to limit neutrophil-mediated inflammation and tissue damage in SIRS induced by CPB.

CD98 defines a metabolically flexible, proinflammatory subset of low-density neutrophils in systemic lupus erythematosus.

BACKGROUND: Low-density neutrophils (LDN) are a distinct subset of neutrophils rarely detected in healthy people but appear in the blood of patients with autoimmune diseases, including systemic lupus erythematosus (SLE), and are mobilised in response to granulocyte colony-stimulating factor (G-CSF). The aim of this study was to identify novel mechanisms responsible for the pathogenic capacity of LDN in SLE. METHODS: Neutrophils were isolated from donors treated with G-CSF, and whole-cell proteomic analysis was performed on LDN and normal-density neutrophils. RESULTS: CD98 is significantly upregulated in LDN from G-CSF donors and defines a subset of LDN within the blood of SLE patients. CD98 is a transmembrane protein that dimerises with L-type amino acid transporters. We show that CD98 is responsible for the increased bioenergetic capacity of LDN. CD98 on LDN mediates the uptake of essential amino acids that are used by mitochondria to produce adenosine triphosphate, especially in the absence of glucose. Inhibition of CD98 reduces the metabolic flexibility of this population, which may limit their pathogenic capacity. CD98+ LDN produce more proinflammatory cytokines and chemokines than their normal density counterparts and are resistant to apoptosis, which may also contribute to tissue inflammation and end organ damage in SLE. CONCLUSIONS: CD98 provides a phenotypic marker for LDN that facilitates identification of this population without density-gradient separation and represents a novel therapeutic target to limit its pathogenic capacity.

Mechanistic Target of Rapamycin Inhibition Prevents Coronary Artery Remodeling in a Murine Model of Kawasaki Disease.

OBJECTIVE: Remodeling of the coronary arteries is a common feature in severe cases of Kawasaki disease (KD). This pathology is driven by the dysregulated proliferation of vascular fibroblasts, which can lead to coronary artery aneurysms, stenosis, and myocardial ischemia. We undertook this study to investigate whether inhibiting fibroblast proliferation might be an effective therapeutic strategy to prevent coronary artery remodeling in KD. METHOD: We used a murine model of KD (induced by the injection of the Candida albicans water-soluble complex [CAWS]) and analyzed patient samples to evaluate potential antifibrotic therapies for KD. RESULTS: We identified the mechanistic target of rapamycin (mTOR) pathway as a potential therapeutic target in KD. The mTOR inhibitor rapamycin potently inhibited cardiac fibroblast proliferation in vitro, and vascular fibroblasts up-regulated mTOR kinase signaling in vivo in the CAWS mouse model of KD. We evaluated the in vivo efficacy of mTOR inhibition and found that the therapeutic administration of rapamycin reduced vascular fibrosis and intimal hyperplasia of the coronary arteries in CAWS-injected mice. Furthermore, the analysis of cardiac tissue from KD fatalities revealed that vascular fibroblasts localizing with inflamed coronary arteries up-regulate mTOR signaling, confirming that the mTOR pathway is active in human KD. CONCLUSION: Our findings demonstrate that mTOR signaling contributes to coronary artery remodeling in KD, and that targeting this pathway offers a potential therapeutic strategy to prevent or restrict this pathology in high-risk KD patients.

BCL-XL antagonism selectively reduces neutrophil life span within inflamed tissues without causing neutropenia.

Neutrophils help to clear pathogens and cellular debris, but can also cause collateral damage within inflamed tissues. Prolonged neutrophil residency within an inflammatory niche can exacerbate tissue pathology. Using both genetic and pharmacological approaches, we show that BCL-XL is required for the persistence of neutrophils within inflammatory sites in mice. We demonstrate that a selective BCL-XL inhibitor (A-1331852) has therapeutic potential by causing apoptosis in inflammatory human neutrophils ex vivo. Moreover, in murine models of acute and chronic inflammatory disease, it reduced inflammatory neutrophil numbers and ameliorated tissue pathology. In contrast, there was minimal effect on circulating neutrophils. Thus, we show a differential survival requirement in activated neutrophils for BCL-XL and reveal a new therapeutic approach to neutrophil-mediated diseases.

Inhibition of interleukin-1ß signalling promotes atherosclerotic lesion remodelling in mice with inflammatory arthritis.

OBJECTIVES: Rheumatoid arthritis (RA), an inflammatory joint disorder, independently increases the risk of cardiovascular disease (CVD). IL-1ß contributes to both RA and CVD. We hypothesised that inhibiting IL-1 signalling with the IL-1R antagonist, anakinra, would dampen inflammation and promote resolution of atherosclerosis in arthritic mice. METHODS: Low-density lipoprotein receptor (Ldlr)-deficient mice were fed a Western-type diet for 14 weeks to develop atherosclerotic plaques. Mice were then switched to a chow diet, promoting lesion regression, and randomised to a control group or into groups where arthritis was induced by passive transfer of K/BxN arthritogenic serum. The arthritic mice were further randomised to vehicle or anakinra. RESULTS: Arthritis impaired atherosclerotic lesion regression when cholesterol was lowered. This was associated with a higher burden of plaque macrophages, likely due to monocytosis, driven by myelopoiesis in the bone marrow and spleen. Interestingly, delayed intervention with anakinra had no effect on arthritis in these mice. However, a significant improvement in atherosclerotic plaque remodelling to a more stable phenotype was observed. This was associated with fewer circulating monocytes, caused by a reduction in splenic extramedullary myelopoiesis. CONCLUSION: We show that inhibiting IL-1 signalling in arthritic mice with pre-existing atherosclerosis promotes lesion remodelling to a more stable phenotype, that is less likely to rupture and cause ischemic events such as myocardial infarction. This suggests that IL-1R antagonism may suppress CVD complications in patients with RA. Furthermore, inhibiting IL-1ß signalling in other patients with inflammatory diseases that also predispose to CVD may also benefit from anti-IL-1 therapy.

TNF and IL-1 Play Essential but Temporally Distinct Roles in Driving Cardiac Inflammation in a Murine Model of Kawasaki Disease.

Kawasaki disease (KD) is a leading cause of pediatric heart disease, characterized by the emergence of life-threatening coronary vasculitis. Identifying which cytokines drive KD has been a major research goal, and both TNF and IL-1 have been identified as potential candidates. Using a murine model of KD induced by the injection of the water-soluble component of Candida albicans, we therefore undertook a mechanistic study to determine how and when these two cytokines mediate cardiac inflammation. In this study, we show that TNF signaling is active in the acute phase of cardiac inflammation, which is characterized by a diffuse myocarditis that precedes the development of coronary vasculitis. Mechanistically, TNF is produced by the myeloid cells and triggers acute cardiac inflammation by stimulating both stromal and immune compartments of the heart. In contrast to this early involvement for TNF, IL-1 signaling is dispensable for the development of acute myocarditis. Critically, although mice deficient in IL-1 signaling have extensive acute inflammation following C. albicans water-soluble complex challenge, they do not develop coronary vasculitis. Thus, TNF and IL-1 appear to play temporally distinct roles in KD, with TNF being active in acute cardiac inflammation and IL-1 in the subsequent development of coronary vasculitis. These observations have important implications for understanding the progression of cardiac pathology in KD and the relative therapeutic use of targeting these cytokines.

The Selective Expansion and Targeted Accumulation of Bone Marrow-Derived Macrophages Drive Cardiac Vasculitis.

The adult heart contains macrophages derived from both embryonic and adult bone marrow (BM)-derived precursors. This population diversity prompted us to explore how distinct macrophage subsets localize within the heart, and their relative contributions in cardiac disease. In this study, using the reciprocal expression of Lyve-1 and Ccr2 to distinguish macrophages with distinct origins, we show that, in the steady state, both embryonic (Lyvepos) and BM-derived (Ccr2pos) macrophages populate the major vessels of the heart in mice and humans. However, cardiac macrophage populations are markedly perturbed by inflammation. In a mouse model of Kawasaki disease, BM-derived macrophages preferentially increase during acute cardiac inflammation and selectively accumulate around major cardiac vessels. The accumulation of BM-derived macrophages coincides with the loss of their embryonic counterparts and is an initiating, essential step in the emergence of subsequent cardiac vasculitis in this experimental model. Finally, we demonstrate that the accumulation of Ccr2pos macrophages (and the development of vasculitis) occurs in close proximity to a population of Ccr2 chemokine ligand-producing epicardial cells, suggesting that the epicardium may be involved in localizing inflammation to cardiac vessels. Collectively, our findings identify the perivascular accumulation of BM-derived macrophages as pivotal in the pathogenesis of cardiac vasculitis and provide evidence about the mechanisms governing their recruitment to the heart.

Therapeutic Effects of a TANK-Binding Kinase 1 Inhibitor in Germinal Center-Driven Collagen-Induced Arthritis.

OBJECTIVE: The production of class-switched high-affinity autoantibodies derived from organized germinal centers (GCs) is a hallmark of many autoimmune inflammatory diseases, including rheumatoid arthritis (RA). TANK-binding kinase 1 (TBK-1) is a serine/threonine kinase involved in the maturation of GC follicular helper T (Tfh) cells downstream of inducible costimulator signaling. We undertook this study to assess the therapeutic potential of TBK-1 inhibition using the small-molecule inhibitor WEHI-112 in antibody-dependent models of inflammatory arthritis. METHODS: Using the models of collagen-induced arthritis (CIA), antigen-induced arthritis (AIA), and K/BxN serum-transfer-induced arthritis (STIA), we determined the effectiveness of WEHI-112 at inhibiting clinical and histologic features of arthritis in C57BL/6 and DBA/1 mice. We used immunohistochemistry to characterize GC populations during CIA development, and we used enzyme-linked immunosorbent assays to determine levels of Ig autoantibodies in WEHI-112-treated mice compared to vehicle-treated mice. RESULTS: WEHI-112, a tool compound that is semiselective for TBK-1 but that also has activity against IKKε and JAK2, abolished TBK-1-dependent activation of interferon (IFN) regulatory factor 3 and inhibited type I IFN responses in vitro. In vivo, treatment with WEHI-112 selectively abrogated clinical and histologic features of established, antibody-dependent CIA, but had minimal effects on an antibody-independent model of AIA or on K/BxN STIA. In keeping with these findings, WEHI-112 reduced arthritogenic type II collagen-specific IgG1 and IgG2b antibody production. Furthermore, WEHI-112 altered the GC Tfh cell phenotype and GC B cell function in CIA. CONCLUSION: We report that TBK-1 inhibition using WEHI-112 abrogated antibody-dependent CIA. As WEHI-112 failed to inhibit non-antibody-driven joint inflammation, we conclude that the major effect of this compound was most likely the targeting of TBK-1-mediated mechanisms in the GC reaction. This approach may have therapeutic potential in RA and in other GC-associated autoantibody-driven inflammatory diseases.

GM-CSF primes cardiac inflammation in a mouse model of Kawasaki disease.

Kawasaki disease (KD) is the leading cause of pediatric heart disease in developed countries. KD patients develop cardiac inflammation, characterized by an early infiltrate of neutrophils and monocytes that precipitates coronary arteritis. Although the early inflammatory processes are linked to cardiac pathology, the factors that regulate cardiac inflammation and immune cell recruitment to the heart remain obscure. In this study, using a mouse model of KD (induced by a cell wall Candida albicans water-soluble fraction [CAWS]), we identify an essential role for granulocyte/macrophage colony-stimulating factor (GM-CSF) in orchestrating these events. GM-CSF is rapidly produced by cardiac fibroblasts after CAWS challenge, precipitating cardiac inflammation. Mechanistically, GM-CSF acts upon the local macrophage compartment, driving the expression of inflammatory cytokines and chemokines, whereas therapeutically, GM-CSF blockade markedly reduces cardiac disease. Our findings describe a novel role for GM-CSF as an essential initiating cytokine in cardiac inflammation and implicate GM-CSF as a potential target for therapeutic intervention in KD.

Maintaining dendritic cell viability in culture.

When mouse dendritic cells (DCs) are isolated from tissues, purified and placed in a nutritive culture they die more rapidly than would be expected from their normal turnover in vivo. This can distort culture assays of DC function. We therefore tested several approaches to prolonging DC survival in culture. Of several cytokines tested granulocyte-macrophage colony stimulating factor was most effective at preserving the viability of conventional DCs (cDCs) but was ineffective for plasmacytoid DCs (pDCs). Surprisingly, Fms-like tyrosine kinase 3 ligand, crucial for DC development, produced only a marginal improvement in DC survival in culture, and interleukin-3, reported to prevent apoptosis of human pDCs, produced only a minor improvement in survival of mouse DCs. Genetic manipulation of cell death pathways was also tested, to avoid activation effects exerted by cytokine signalling. The isolation of DCs from mice overexpressing Bcl-2 was especially effective in maintaining pDC viability but gave a lesser improvement in cDC viability. DCs isolated from Bim(-/-)Noxa(-/-) mice also showed improved culture survival, but in this case with pDCs showing the least improvement.

Factors influencing lid pressure on the cornea.

PURPOSE: To investigate the effects of eyelid pressure on corneal topography in different angles of gaze and under different eye movement conditions. METHODS: Ten young subjects with healthy eyes were recruited for the study. Corneal topography of the right eye was measured with a videokeratoscope before and after four 15-minute visual tasks (downward gaze of 25 degrees or 45 degrees , both with no eye movements or 1 Hz eye movements). The four tasks were conducted on four separate mornings. RESULTS.: All subjects showed significant regions of topographic change after each of the four tasks. In general, eye movements were found to cause greater topographic changes than tasks without eye movements, and the larger angle of downward gaze led to greater changes in topography. The topographic changes were located close to the lid margin position during each task. CONCLUSIONS: The pressure exerted by the eyelid is capable of altering corneal topography. These changes are enhanced when the eye looks downward and when the eye moves laterally.