Neutrophils as critical orchestrators of chronic inflammation

Abstract

Neutrophils are the first key effector innate immune cells recruited toward inflammatory sites. Through the release of neutrophilic extracellular traps (NETs), the production of reactive oxygen species (ROS), degranulation and phagocytosis, neutrophils play a central role in the rapid elimination of invading pathogens. Recently, increasing attention has been given to the role of neutrophils in chronic inflammation, challenging the dichotomy between innate and adaptive immune responses. In chronic inflammatory conditions, neutrophils generally display a hyperinflammatory phenotype via dysregulated pathogen defense mechanisms. Excessive neutrophil activation may result in aberrant cell death, uncontrolled oxidative burst or NET formation and sustained release of inflammatory mediators such as proteases and inflammatory cytokines. Therefore, neutrophils contribute to the development of a sustained inflammatory environment and cause collateral tissue damage. In addition to their direct inflammatory effects, neutrophils further orchestrate inflammation and tissue remodeling by actively engaging in crosstalk with other cells within the immune microenvironment, such as endothelial cells, monocytes, platelets, and T and B cells. This review summarizes the current knowledge of the emerging role of neutrophils in the context of chronic inflammation. The key characteristics of neutrophils and their interactions with distinct cell types are discussed within the initial part of the review, whereas the second part focuses on their contributions to the pathophysiology of immune-driven diseases, including rheumatoid arthritis, atherosclerosis, inflammatory bowel disease, systemic lupus erythematosus, chronic obstructive pulmonary disease, and fibrotic disorders. Increasing knowledge on neutrophil behavior in the context of chronic inflammation may offer novel insights into disease pathology and, potentially, the identification of novel therapeutic targets.

Keywords: Chronic inflammation; Innate immune response; Neutrophil.

Fig. 1

Neutrophil effector functions in inflammation. Panel 1 Neutrophils produce reactive oxygen species intracellularly, aiding in pathogen killing, and extracellularly, causing damage to the surrounding tissue. Crucial for ROS production is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX 2), which comprises 6 distinct subunits located at the cytosolic side of the plasma membrane. The membrane subunit flavocytochrome b558 contains p22^phox and the catalytic core gp91^phox. Upon stimulation of neutrophils, the cytosolic subunits p47^phox (NCF1), p67^phox (NCF2), and p40^phox (NCF4) and the small GTPases Rac1 and Rac2 are recruited toward flavocytochrome b558 to form a functional electron transfer system. Panel 2 Neutrophils are able to expel web-like chromatin structures called neutrophil extracellular traps (NETs). The release of NETs (or NETosis) can occur in a NOX2-dependent or NOX2-independent manner, referred to as suicidal or vital NETosis, respectively. Suicidal, lytic or NOX2-dependent NET release can be initiated by various stimuli, such as pathogens, phorbol 12-myristate 13-acetate (PMA), cholesterol, antibodies and cytokines. These stimuli result in remodeling of chromatin and histone modification mediated by enzymes, including myeloperoxidase (MPO), neutrophil elastase (NE), and peptidylarginine deiminase (PAD4). In addition, among cytoplasmic enzymes, NE, cathepsin G (CG), and azurocidin (AZU) also cause remodeling of F-actin, which contributes to NET release. As a consequence, NETosis coincides with the expulsion of many proteins, including MPO, CG, NE, matrix metalloproteinases (MMPs), proteinase 3 (PR3) and citrullinated histones. Panel 3 Vital NETosis occurs independently of NOX2 through vesicular trafficking and is traditionally triggered by pathogens, lipopolysaccharide (LPS), or activated platelets. NETs containing mitochondrial DNA can be expelled in a nonlytic manner; however, contrary to the previous mechanisms, this process is dependent on NOX activity. Panel 4 Apoptosis is a tightly regulated form of programmed cell death and is dysregulated in many chronic inflammatory diseases. The intrinsic apoptotic pathway is induced by intracellular stress signals that activate B-cell lymphoma 2 (BCL-2) homology domain 3 (BH3)-only proteins. These proteins subsequently activate the BCL-2-associated X protein (BAX)/BCL-2 antagonist/killer (BAK) complex, causing mitochondrial membrane permeabilization and the release of cytochrome C within the cytoplasm. Cytochrome C, together with apoptotic protease-activating factor 1 (APAF1), cleaves procaspase-9, which subsequently results in the activation of procaspase-3 and procaspase-7 (i.e., effector caspases) and associated apoptotic cell death. The extrinsic apoptotic pathway is initiated by the binding of external factors, such as tumor necrosis factor (TNF), Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL). This pathway is mediated by the activation of caspases 8 and 10, which are in turn able to convert procaspase-3 and procaspase-7

Fig. 2

Neutrophils at the intersection of immunity. Neutrophils contribute to chronic inflammation through their ability to interact with various cell types. Panel 1 Neutrophil extravasation (called “diapedesis”) is mediated by multiple ligand‒receptor interactions with neutrophils and endothelial cells. Crucial mediators within this process include integrins on neutrophils [e.g., lymphocyte function associated antigen-1 (LFA-1 or CD11a/CD18 or αLβ2), macrophage-1 antigen (Mac-1 or CD11b/CD18 or αMβ2) and very late antigen-4 (VLA-4 or α4β1)] and adhesion molecules, containing immunoglobulin domains, on endothelial cells [e.g., intercellular adhesion molecule 1/2 (ICAM-1/-2) and vascular cell adhesion molecule 1 (VCAM-1)]. Panel 2 Neutrophils influence macrophage polarization toward either a pro- or anti-inflammatory phenotype (M1 or M2, respectively). Polarization toward the “M1 phenotype” is mediated by the release of neutrophil-derived peptides such as heparin-binding protein (HBP), human neutrophil peptides 1–3 (HNP1–3) and S100A8/A9. In contrast, anti-inflammatory reprogramming is mediated by the release of microvesicles by neutrophils and is characterized by the suppression of nuclear factor kappa light chain enhancer of activated B cells (NF-kB) signaling. As a consequence, the expression of proinflammatory cytokines is downregulated, while the expression of anti-inflammatory cytokines such as interleukin-10 (IL-10), IL-1Ra and transforming growth factor-β (TGF-β) is upregulated. Panel 3 Chronic inflammation results in a prothrombotic state called “immunothrombosis”, wherein mutual activation of thrombocytes, leukocytes and coagulation factors plays a central role. Crucial for this interaction is the binding of P-selectin glycoprotein ligand 1 (PSGL-1) on neutrophils with P-selectin on thrombocytes, which results in the release of chemokines [e.g., CXCL7] and danger-associated molecular patterns (DAMPs) [e.g., high mobility group box 1 (HMGB1)]. Panel 4 In addition to their role in initial immune responses, neutrophils influence the activity of adaptive immunity by interacting with B and T cells. B-cell-stimulating mediators produced by neutrophils include B lymphocyte stimulator (Blys), also known as BAFF, and “a proliferation-inducing ligand” (APRIL), which binds with B-cell maturation antigen (BCMA). In addition, neutrophils appear to possess antigen-presenting capacities [e.g., expression of major histocompatibility complex II (MHCII)] in specific circumstances and may thus contribute to the activation of the T-cell receptor (TCR) and steer adaptive immunity

Fig. 3

Neutrophils in rheumatoid arthritis (RA). Neutrophils in the synovial fluid of patients with RA display a hyperinflammatory phenotype characterized by the upregulation of CD66b, CD11b, CD15 and major histocompatibility complex class II (MHCII) and the downregulation of CXC chemokine receptor 1/2 (CXCR1/2). They show increased protein expression of tumor necrosis factor (TNF), interleukin-6 (IL-6), IL-1β, CXC chemokine ligand 8 (CXCL8), and granulocyte macrophage-colony stimulating factor (GM-CSF). Neutrophils produce high levels of neutrophil extracellular traps (NETs), which are mediated by the activity of myeloperoxidase (MPO), neutrophil elastase (NE) and peptidylarginine deiminase 4 (PAD4), causing the release of citrullinated peptides, such as citrullinated vimentin (CitVIM) and citrullinated histones 3 and 4 (CitH3/4), into the extracellular environment. These citrullinated peptides are taken up by fibroblast-like synoviocytes (FLSs) through the receptor for advanced glycation end products (RAGE) and toll-like receptor 9 (TLR9) and are presented to T cells through MHCII. Subsequently, activated T helper 17 (Th17) and Th21 cells, which produce IL-17 and IL-21, respectively, stimulate the transformation of B cells into autoantibody-producing plasma cells. The key autoantibodies found in RA include rheumatoid factor (RF) and anti-citrullinated protein autoantibodies (ACPAs). ROS produced by neutrophils activate synovial macrophages (through TLR4) and FLSs, which in turn stimulate inflammation by producing high levels of TNF, IL-6, IL-1β and CXCL8. In addition to ROS and NETs, neutrophils within the synovial fluid of patients with RA release receptor activator of nuclear factor κB ligand (RANKL) and matrix metalloproteinases 8 and 9 (MMP8/9), resulting in osteoclast activation and cartilage degradation, respectively