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.

CSL324, a granulocyte colony-stimulating factor receptor antagonist, blocks neutrophil migration markers that are upregulated in hidradenitis suppurativa.

BACKGROUND: Neutrophils have been shown to contribute to the pathophysiology of hidradenitis suppurativa (HS), a chronic, painful and debilitating inflammatory skin disease, yet their exact role remains to be fully defined. Granulocyte colony-stimulating factor (G-CSF), a major regulator of neutrophil development and survival, can be blocked by the novel, fully human anti-G-CSF receptor (G-CSFR) monoclonal antibody CSL324. OBJECTIVES: We investigated the activation and migration of neutrophils in HS and the impact of blocking G-CSFR with CSL324. METHODS: Biopsy and peripheral blood samples were taken from participants of two studies: 2018.206, a noninterventional research study of systemic and dermal neutrophils and inflammatory markers in patients with neutrophilic skin diseases, and CSL324_1001 (ACTRN12616000846426), a single-dose ascending and repeated dose, randomized, double-blind, placebo-controlled study to assess the safety, pharmacokinetics and pharmacodynamics of CSL324 in healthy adult subjects. Ex vivo experiments were performed, including neutrophil enumeration and immunophenotyping, migration, receptor occupancy and transcriptome analysis. RESULTS: The number of cells positive for the neutrophil markers myeloperoxidase (MPO) and neutrophil elastase (NE) was significantly higher in HS lesions compared with biopsies from healthy donors (HDs) (P < 0.0001 and P = 0.0223, respectively). In peripheral blood samples, mean neutrophil counts were significantly higher in patients with HS than in HDs (2.98 vs. 1.60 × 109 L-1, respectively; P = 8.8 × 10-4). Neutrophil migration pathways in peripheral blood were increased in patients with HS and their neutrophils demonstrated an increased migration phenotype, with higher mean CXCR1 on the surface of neutrophils in patients with HS (24453.20 vs. 20798.47 for HD; P = 0.03). G-CSF was a key driver of the transcriptomic changes in the peripheral blood of patients with HS and was elevated in serum from patients with HS compared with HDs (mean 6.61 vs. 3.84 pg mL-1, respectively; P = 0.013). Administration of CSL324 inhibited G-CSF-induced transcriptional changes in HDs, similar to those observed in the HS cohort, as highlighted by expression changes in genes related to neutrophil migratory capacity. CONCLUSIONS: Data suggest that neutrophils contribute to HS pathophysiology and that neutrophils are increased in lesions due to an increase in G-CSF-driven migration. CSL324 counteracted G-CSF-induced transcriptomic changes and blocked neutrophil migration by reducing cell-surface levels of chemokine receptors.

Targeting the Human ßc Receptor Inhibits Contact Dermatitis in a Transgenic Mouse Model.

Allergic contact dermatitis (ACD) is a prevalent and poorly controlled inflammatory disease caused by skin infiltration of T cells and granulocytes. The beta common (ßc) cytokines GM-CSF, IL-3, and IL-5 are powerful regulators of granulocyte function that signal through their common receptor subunit ßc, a property that has made ßc an attractive target to simultaneously inhibit these cytokines. However, the species specificity of ßc has precluded testing of inhibitors of human ßc in mouse models. To overcome this problem, we developed a human ßc receptor transgenic mouse strain with a hematopoietic cell‒specific expression of human ßc instead of mouse ßc. Human ßc receptor transgenic cells responded to mouse GM-CSF and IL-5 but not to IL-3 in vitro and developed tissue pathology and cellular inflammation comparable with those in wild-type mice in a model of ACD. Similarly, Il3-/- mice developed ACD pathology comparable with that of wild-type mice. Importantly, the blocking anti-human ßc antibody CSL311 strongly suppressed ear pinna thickening and histopathological changes typical of ACD and reduced accumulation of neutrophils, mast cells, and eosinophils in the skin. These results show that GM-CSF and IL-5 but not IL-3 are major mediators of ACD and define the human ßc receptor transgenic mouse as a unique platform to test the inhibitors of ßc in vivo.

Cellular Microenvironment Stiffness Regulates Eicosanoid Production and Signaling Pathways.

Pathological changes in the biomechanical environment are implicated in the progression of idiopathic pulmonary fibrosis (IPF). Stiffened matrix augments fibroblast proliferation and differentiation and activates TGF-ß1 (transforming growth factor-ß1). Stiffened matrix impairs the synthesis of the antifibrogenic lipid mediator prostaglandin E2 (PGE2) and reduces the expression of the rate-limiting prostanoid biosynthetic enzyme cyclooxygenase-2 (COX-2). We now show that prostaglandin E synthase (PTGES), the final enzyme in the PGE2 biosynthetic pathway, is expressed at lower levels in the lungs of patients with IPF. We also show substantial induction of COX-2, PTGES, prostaglandin E receptor 4 (EP4), and cytosolic phospholipase A2 (cPLA2) expression in human lung fibroblasts cultured in soft collagen hydrogels or in spheroids compared with conventional culture on stiff plastic culture plates. Induction of COX-2, cPLA2, and PTGES expression in spheroid cultures was moderately inhibited by the p38 mitogen-activated protein kinase inhibitor SB203580. The induction of prostanoid biosynthetic enzyme expression was accompanied by an increase in PGE2 levels only in non-IPF-derived fibroblast spheroids. Our study reveals an extensive dysregulation of prostanoid biosynthesis and signaling pathways in IPF-derived fibroblasts, which are only partially abrogated by culture in soft microenvironments.

Blockade of the G-CSF Receptor Is Protective in a Mouse Model of Renal Ischemia-Reperfusion Injury.

Ischemia-reperfusion injury (IRI) is a complex inflammatory process that detrimentally affects the function of transplanted organs. Neutrophils are important contributors to the pathogenesis of renal IRI. Signaling by G-CSF, a regulator of neutrophil development, trafficking, and function, plays a key role in several neutrophil-associated inflammatory disease models. In this study, we investigated whether targeting neutrophils with a neutralizing mAb to G-CSFR would reduce inflammation and protect against injury in a mouse model of warm renal IRI. Mice were treated with anti-G-CSFR 24 h prior to 22-min unilateral renal ischemia. Renal function and histology, complement activation, and expression of kidney injury markers, and inflammatory mediators were assessed 24 h after reperfusion. Treatment with anti-G-CSFR protected against renal IRI in a dose-dependent manner, significantly reducing serum creatinine and urea, tubular injury, neutrophil and macrophage infiltration, and complement activation (plasma C5a) and deposition (tissue C9). Renal expression of several proinflammatory genes (CXCL1/KC, CXCL2/MIP-2, MCP-1/CCL2, CXCR2, IL-6, ICAM-1, P-selectin, and C5aR) was suppressed by anti-G-CSFR, as was the level of circulating P-selectin and ICAM-1. Neutrophils in anti-G-CSFR-treated mice displayed lower levels of the chemokine receptor CXCR2, consistent with a reduced ability to traffic to inflammatory sites. Furthermore, whole transcriptome analysis using RNA sequencing showed that gene expression changes in IRI kidneys after anti-G-CSFR treatment were indistinguishable from sham-operated kidneys without IRI. Hence, anti-G-CSFR treatment prevented the development of IRI in the kidneys. Our results suggest G-CSFR blockade as a promising therapeutic approach to attenuate renal IRI.

Easy and efficient ensemble gene set testing with EGSEA.

Gene set enrichment analysis is a popular approach for prioritising the biological processes perturbed in genomic datasets. The Bioconductor project hosts over 80 software packages capable of gene set analysis. Most of these packages search for enriched signatures amongst differentially regulated genes to reveal higher level biological themes that may be missed when focusing only on evidence from individual genes. With so many different methods on offer, choosing the best algorithm and visualization approach can be challenging. The EGSEA package solves this problem by combining results from up to 12 prominent gene set testing algorithms to obtain a consensus ranking of biologically relevant results.This workflow demonstrates how EGSEA can extend limma-based differential expression analyses for RNA-seq and microarray data using experiments that profile 3 distinct cell populations important for studying the origins of breast cancer. Following data normalization and set-up of an appropriate linear model for differential expression analysis, EGSEA builds gene signature specific indexes that link a wide range of mouse or human gene set collections obtained from MSigDB, GeneSetDB and KEGG to the gene expression data being investigated. EGSEA is then configured and the ensemble enrichment analysis run, returning an object that can be queried using several S4 methods for ranking gene sets and visualizing results via heatmaps, KEGG pathway views, GO graphs, scatter plots and bar plots. Finally, an HTML report that combines these displays can fast-track the sharing of results with collaborators, and thus expedite downstream biological validation. EGSEA is simple to use and can be easily integrated with existing gene expression analysis pipelines for both human and mouse data.

Improving the prediction of disulfide bonds in Eukaryotes with machine learning methods and protein subcellular localization.

MOTIVATION: Disulfide bonds stabilize protein structures and play relevant roles in their functions. Their formation requires an oxidizing environment and their stability is consequently depending on the redox ambient potential, which may differ according to the subcellular compartment. Several methods are available to predict cysteine-bonding state and connectivity patterns. However, none of them takes into consideration the relevance of protein subcellular localization. RESULTS: Here we develop DISLOCATE, a two-step method based on machine learning models for predicting both the bonding state and the connectivity patterns of cysteine residues in a protein chain. We find that the inclusion of protein subcellular localization improves the performance of these predictive steps by 3 and 2 percentage points, respectively. When compared with previously developed methods for predicting disulfide bonds from sequence, DISLOCATE improves the overall performance by more than 10 percentage points. AVAILABILITY: The method and the dataset are available at the Web page http://www.biocomp.unibo.it/savojard/Dislocate.html. GRHCRF code is available at http://www.biocomp.unibo.it/savojard/biocrf.html. CONTACT: piero.fariselli@unibo.it.