Helen L. Wright

Neutrophil function in inflammation and inflammatory diseases

In inflammatory conditions such as RA, the neutrophil has tended to be dismissed as a short-lived, terminally differentiated, irrelevant bystander cell. However, this is clearly not the case. A better understanding of the complex heterogeneous pathways and processes that constitute RA, in parallel with a more sophisticated knowledge of neutrophil biology has identified many potential roles for these cells in the persistence of inflammation and progression of joint damage, which should not be underestimated. Not only are neutrophils found in high numbers within the rheumatoid joint, both in synovial tissue and in joint fluid, they have a huge potential to directly inflict damage to tissue, bone and cartilage via the secretion of proteases and toxic oxygen metabolites, as well as driving inflammation through antigen presentation and secretion of cytokines, chemokines, prostaglandins and leucotrienes. Drugs already used to treat RA down-regulate many neutrophil functions, including migration to the joint, degranulation and production of inflammatory mediators, and these cells should be considered as important targets for the development of new therapies in the future.

The multifactorial role of neutrophils in rheumatoid arthritis.

Of all cells implicated in the pathology of rheumatoid arthritis (RA), neutrophils possess the greatest cytotoxic potential, owing to their ability to release degradative enzymes and reactive oxygen species. Neutrophils also contribute to the cytokine and chemokine cascades that accompany inflammation, and regulate immune responses via cell–cell interactions. Emerging evidence suggests that neutrophils also have a previously unrecognised role in autoimmune diseases: neutrophils can release neutrophil extracellular traps (NETs) containing chromatin associated with granule enzymes, which not only kill extracellular microorganisms but also provide a source of autoantigens. For example, citrullinated proteins that can act as neoepitopes in loss of immune tolerance are generated by peptidylarginine deiminases, which replace arginine with citrulline residues, within neutrophils. Indeed, antibodies to citrullinated proteins can be detected before the onset of symptoms in patients with RA, and are predictive of erosive disease. Neutrophils from patients with RA have an increased tendency to form NETs containing citrullinated proteins, and sera from such patients contain autoantibodies that recognize these proteins. Thus, in addition to their cytotoxic and immunoregulatory role in RA, neutrophils may be a source of the autoantigens that drive the autoimmune processes underlying this disease.

Analysis of SF and plasma cytokines provides insights into the mechanisms of inflammatory arthritis and may predict response to therapy

OBJECTIVES Biologic drugs have revolutionized the care of RA, but are expensive and not universally effective. To further understand the inflammatory mechanisms underlying RA and identify potential biomarkers predicting response to therapy, we measured multiple cytokine concentrations in SF of patients with inflammatory arthritides (IAs) and, in a subset of patients with RA, correlated this with response to TNF-α inhibition. METHODS SF from 42 RA patients and 19 non-RA IA patients were analysed for 12 cytokines using a multiplex cytokine assay. Cytokines were also measured in the plasma of 16 RA patients before and following treatment with anti-TNF-α. Data were analysed using Mann-Whitney U-test, Spearmans rank correlation and cluster analysis with the Kruskal-Wallis test with Dunns post-test analysis. RESULTS RA SF contained significantly elevated levels of IL-1β, IL-1ra, IL-2, IL-4, IL-8, IL-10, IL-17, IFN-γ, G-CSF, GM-CSF and TNF-α compared with other IA SF. RA patients who did not respond to anti-TNF therapy had elevated IL-6 in their SF pre-therapy (P < 0.05), whereas responders had elevated IL-2 and G-CSF (P < 0.05). Plasma cytokine concentrations were not significantly modulated by TNF inhibitors, with the exception of IL-6, which decreased after 12 weeks (P < 0.05). CONCLUSIONS Cytokine profiles in RA SF vary with treatment and response to therapy. Cytokine concentrations are significantly lower in plasma than in SF and relatively unchanged by TNF inhibitor therapy. Concentrations of IL-6, IL-2 and G-CSF in SF may predict response to TNF inhibitors.

RNA-Seq Reveals Activation of Both Common and Cytokine-Specific Pathways following Neutrophil Priming

Neutrophils are central to the pathology of inflammatory diseases, where they can damage host tissue through release of reactive oxygen metabolites and proteases, and drive inflammation via secretion of cytokines and chemokines. Many cytokines, such as those generated during inflammation, can induce a similar “primed” phenotype in neutrophils, but it is unknown if different cytokines utilise common or cytokine-specific pathways to induce these functional changes. Here, we describe the transcriptomic changes induced in control human neutrophils during priming in vitro with pro-inflammatory cytokines (TNF-α and GM-CSF) using RNA-seq. Priming led to the rapid expression of a common set of transcripts for cytokines, chemokines and cell surface receptors (CXCL1, CXCL2, IL1A, IL1B, IL1RA, ICAM1). However, 580 genes were differentially regulated by TNF-α and GM-CSF treatment, and of these 58 were directly implicated in the control of apoptosis. While these two cytokines both delayed apoptosis, they induced changes in expression of different pro- and anti-apoptotic genes. Bioinformatics analysis predicted that these genes were regulated via differential activation of transcription factors by TNF-α and GM-CSF and these predictions were confirmed using functional assays: inhibition of NF-κB signalling abrogated the protective effect of TNF-α (but not that of GM-CSF) on neutrophil apoptosis, whereas inhibition of JAK/STAT signalling abrogated the anti-apoptotic effect of GM-CSF, but not that of TNF-α (p<0.05). These data provide the first characterisation of the human neutrophil transcriptome following GM-CSF and TNF-α priming, and demonstrate the utility of this approach to define functional changes in neutrophils following cytokine exposure. This may provide an important, new approach to define the molecular properties of neutrophils after in vivo activation during inflammation.

Human neutrophils in auto-immunity

Human neutrophils have great capacity to cause tissue damage in inflammatory diseases via their inappropriate activation to release reactive oxygen species (ROS), proteases and other tissue-damaging molecules. Furthermore, activated neutrophils can release a wide variety of cytokines and chemokines that can regulate almost every element of the immune system. In addition to these important immuno-regulatory processes, activated neutrophils can also release, expose or generate neoepitopes that have the potential to break immune tolerance and result in the generation of autoantibodies, that characterise a number of human auto-immune diseases. For example, in vasculitis, anti-neutrophil cytoplasmic antibodies (ANCA) that are directed against proteinase 3 or myeloperoxidase are neutrophil-derived autoantigens and activated neutrophils are the main effector cells of vascular damage. In other auto-immune diseases, these neutrophil-derived neoepitopes may arise from a number of processes that include release of granule enzymes and ROS, changes in the properties of components of their plasma membrane as a result of activation or apoptosis, and via the release of Neutrophil Extracellular Traps (NETs). NETs are extracellular structures that contain chromatin that is decorated with granule enzymes (including citrullinated proteins) that can act as neo-epitopes to generate auto-immunity. This review therefore describes the processes that can result in neutrophil-mediated auto-immunity, and the role of neutrophils in the molecular pathologies of auto-immune diseases such as vasculitis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We discuss the potential role of NETs in these processes and some of the debate in the literature regarding the role of this phenomenon in microbial killing, cell death and auto-immunity.

Interferon gene expression signature in rheumatoid arthritis neutrophils correlates with a good response to TNFi therapy

OBJECTIVE The aim of this study was to use whole transcriptome sequencing (RNA-Seq) of RA neutrophils to identify pre-therapy gene expression signatures that correlate with disease activity or response to TNF inhibitor (TNFi) therapy. METHODS Neutrophils were isolated from the venous blood of RA patients (n = 20) pre-TNFi therapy and from healthy controls (n = 6). RNA was poly(A) selected and sequenced on the Illumina HiSeq 2000 platform. Reads were mapped to the human genome (hg19) using TopHat and differential expression analysis was carried out using edgeR (5% false discovery rate). Signalling pathway analysis was carried out using Ingenuity Pathway Analysis (IPA) software. IFN signalling was confirmed by western blotting for phosphorylated signal transducer and activator of transcription (STAT) proteins. Response to TNFi was measured at 12 weeks using change in the 28-item DAS (DAS28). RESULTS Pathway analysis with IPA predicted activation of IFN signalling in RA neutrophils, identifying 178 IFN-response genes regulated by IFN-α, IFN-β or IFN-γ (P < 0.01). IPA also predicted activation of STAT1, STAT2 and STAT3 transcription factors in RA neutrophils (P < 0.01), which was confirmed by western blotting. Expression of IFN-response genes was heterogeneous and patients could be categorized as IFN-high or IFN-low. Patients in the IFN-high group achieved a better response to TNFi therapy [ΔDAS28, P = 0.05, odds ratio (OR) 1.4 (95% CI 1.005, 1.950)] than patients in the IFN-low group. The level of expression of IFN-response genes (IFN score) predicted a good response [European League Against Rheumatism (EULAR) criteria] to TNFi using receiver operating characteristic curve analysis (area under the curve 0.76). CONCLUSION IFN-response genes are significantly up-regulated in RA neutrophils compared with healthy controls. Higher IFN-response gene expression in RA neutrophils correlates with a good response to TNFi therapy.

Effects of IL-6 and IL-6 blockade on neutrophil function in vitro and in vivo

OBJECTIVES Reports on the regulation of neutrophil function by IL-6 are often conflicting. Therapeutic inhibition of IL-6 in RA is associated with occasional neutropenia, but the mechanisms underlying this observation are poorly understood. This study investigated interactions between IL-6, the anti-IL-6 receptor agent tocilizumab (TCZ) and neutrophils in vitro and in vivo. METHODS Neutrophils were isolated from healthy controls and incubated in vitro with pharmacologically relevant concentrations of IL-6 or TCZ. Neutrophils were also isolated from RA patients, including a cohort following TCZ therapy. Apoptosis was measured by annexin V/propidium iodide (PI) flow cytometry; phagocytosis was measured by incubating apoptotic neutrophils with THP-1-derived macrophages; chemotaxis was measured using cell migration through hanging-cell inserts towards IL-8 and cell surface proteins, including adhesion molecules CD11b (αMβ2 integrin) and CD62L (L-selectin) were measured by flow cytometry. RESULTS IL-6 (10-100 ng/ml) did not affect the rate of neutrophil apoptosis, priming of the respiratory burst or adhesion molecule expression nor act as a neutrophil chemoattractant. However, IL-6 enhanced signal transducer and activator of transcription 3 (STAT3) activation and neutrophil migration towards IL-8. TCZ in vitro did not induce apoptosis or phagocytosis of neutrophils, nor did it have a significant effect upon apoptosis or cell surface molecule expression. Neutrophil functions in ex vivo neutrophils from RA patients receiving TCZ treatment were unaffected. CONCLUSION Therapeutic blockade of IL-6, while inducing a transient neutropenia, does not directly affect neutrophil functions associated with host defence. TCZ-associated neutropenia cannot be explained by direct induction of apoptosis by TCZ, induction of apoptosis following depletion of IL-6, nor increased phagocytosis of neutrophils.

Changes in expression of membrane TNF, NF-κB activation and neutrophil apoptosis during active and resolved inflammation

Background Tumour necrosis factor (TNF) is central to the pathophysiological process of rheumatoid arthritis (RA), whether as soluble cytokine or membrane-expressed pro-TNF (mTNF). Objectives To determine whether neutrophils, which can express TNF, are activated in the blood of patients with RA compared with healthy controls. To investigate, by focusing on mTNF expression, if the functions of RA neutrophils change in response to therapeutic TNF inhibition. Methods TNF was measured by flow cytometry and qPCR in neutrophils from 20 patients with RA before and after the start of TNF inhibitor therapy. Apoptosis was measured by morphology, and western blotting of pro- and antiapoptotic proteins in cell lysates. Nuclear factor κB (NF-κB) activation was determined by western blotting of phosphorylated NF-κB (p65). Results Before treatment RA neutrophils exhibited increased TNF mRNA expression, elevated mTNF levels and NF-κB activity compared with controls. They also underwent delayed apoptosis as shown by altered expression of anti- and proapoptotic proteins, such as Mcl-1 and caspases. Neutrophil TNF expression returned to baseline levels during successful treatment with anti-TNF biological agents, and there was a close correlation between clinical disease improvement and changes in neutrophil function. Conclusions Neutrophils express elevated levels of TNF in RA and the transcription factor, NF-κB, a target of TNF, is activated. This mechanism could lead to a self-sustained inflammatory process. These data point to an important role of neutrophils in the abnormal TNF signalling pathways activated in RA and provide new evidence that neutrophils actively contribute to altered cytokine signalling in inflammatory diseases.

Low-density granulocytes: functionally distinct, immature neutrophils in rheumatoid arthritis with altered properties and defective TNF signalling

Our aim was to determine whether rheumatoid arthritis (RA) low‐density granulocytes (LDGs) are functionally different from RA neutrophils. LDGs from 32 RA patients were characterized using flow cytometry and quantitative PCR (qPCR). RNA sequencing (RNA‐Seq) was carried out on paired RA LDGs and neutrophils (n = 4) and validated using qPCR. Functional assays included chemotaxis, phagocytosis, reactive oxygen species (ROS) production, cell‐cycle analysis, apoptosis, neutrophil extracellular trap (NET)osis, and measurement of cytokine production (n ≥ 5 paired RA LDGs/neutrophils). RA LDGs had a substantially altered transcriptome, expressing >5000 genes at significantly different levels compared with RA neutrophils, including elevated levels of transcripts for granule proteins [including elastase and myeloperoxidase (MPO)] and cell‐cycle genes [including cyclin‐dependent kinase (CDK)2, CDK4, and CDK6]. Approximately 1% of RA LDGs stained positive for the G2/S phase of the cell cycle. RA LDGs had a significantly lower constitutive rate of apoptosis compared with RA neutrophils and did not respond to TNF‐α in culture. Expression of transcripts for cytokines and cytokine receptors was lower in RA LDGs. NET formation was lower in LDGs in response to PMA compared with RA neutrophils. Chemotaxis and phagocytosis was lower in RA LDGs compared with neutrophils. RA LDGs produced significantly lower amounts of ROS in response to fMLP following priming with TNF‐α. Expression of TNFR1 and ‐2 mRNA and protein was significantly lower in LDGs. We conclude that RA LDGS are functionally different from RA neutrophils, representing an immature neutrophil population within peripheral blood. Their enhanced survival properties and decreased TNF signaling are likely to have important consequences for disease pathology and response to therapy.

A lack of confirmation with alternative assays questions the validity of IL-17A expression in human neutrophils using immunohistochemistry.

We identified IL-17A-positive neutrophils in Wolbachia-positive Onchocerca volvulus nodules using an antibody that has previously reported IL-17A-positive neutrophils in several inflammatory conditions. However, we could not detect IL-17A using a range of alternative assays. Our data question the IL-17A antibody specificity and the ability of human neutrophils to express IL-17A.