Critical COPD respiratory illness is linked to increased transcriptomic activity of neutrophil proteases genes

Neutrophil related genes

ELANE (Elastase, neutrophil expressed), CTSG (cathepsin G), and PRTN3 (proteinase 3) are neutrophil serine proteases (NSPs) produced during neutrophil development in bone marrow and stored in the azurophilic granules of mature neutrophils. NSPs contribute to the neutrophil oxygen-independent system-mediated protection of the host against invading pathogens [7]. On the other hand, NSPs play a critical role in neutrophil-associated lung inflammatory and tissue-destructive diseases, including COPD. NSPs have a broad substrate specificity and degrade a variety of extra-cellular matrix proteins including elastin, collagen (type I–IV), fibronectin, laminin and proteoglycans [8]. High concentrations of NSPs are found in purulent secretions of COPD patients[9]. Intra-tracheal instillation of mice with human neutrophil elastase or proteinase 3 leads to tissue destruction and airspace enlargement [10]. Neutrophil elastase may also impair host defence interfering with muco-ciliary clearance of bacteria and phagocytosis of pathogens. In turn, both elastase and cathepsin G impair T-cell function through cleavage of CD2, CD4 and CD8 on the surface of T-cells [8]. Patients lacking α1-Pi, the main physiological inhibitor of neutrophil elastase, are at greater risk of developing emphysema [11]. CTSD (Cathepsin D) is also present in azurophil granules. By using a mice model, Bracke et al.. have recently demonstrated that cigarette smoke induces the expression of CTSD in pulmonary macrophages [12], supporting the role of cathepsins in the respiratory compromise associated to COPD . MPO (Myeloperoxidase) constitutes the major component of neutrophil azurophilic granules. MPO is important in bacterial killing, but also drives inflammatory reactions and tissue oxidation. By using a mice model of influenza infection, Sugamata R et al.. found that the absence of MPO reduced inflammatory damage with suppression of leakage of total proteins in bronchoalveolar lavage fluid associated with alteration of claudins in the lung [13]. Activated neutrophils (MPO + cells) are found in severe COPD [14]. In this sense, inhibition of MPO may be a novel and useful therapeutic treatment for COPD [15]. In turn, AZU1 (azurocidin 1), BPI (bactericidal/permeability-increasing protein) and alpha-defensins (such as DEFA3, defensin, alpha 3, neutrophil-specific), are as NSPs, major constituents of neutrophil azurophilic granules. The protein encoded by AZU1 is an antibiotic protein, with monocyte chemotactic and antibacterial activity. It is also an important multifunctional inflammatory mediator. Bactericidal/permeability-increasing protein (BPI) encodes a lipopolysaccharide binding protein with bactericidal activity on gram-negative organisms. Defensins show activities against Gram-positive and Gram-negative bacteria, fungi, yeast, and enveloped viruses, but, as NSPs, also play important roles in promoting inflammation in the lungs, potentially contributing to lung injury [16] . Along with NSPs, defensins may also be released upon neutrophil stimulation. Both have been described to affect the integrity of the epithelial layer, decrease the frequency of ciliary beat, increase the secretion of mucus, and induce the synthesis of epithelium-derived mediators that may influence the amplification and resolution of neutrophil-dominated inflammation [8].

A number of other gene transcripts related to neutrophils were identified in our analysis as more represented in the ICU group: LTF (lactotransferrin), TCN1 (transcobalamin I (vitamin B12 binding protein, R binder family)) as proteins which form part of secondary granules in neutrophils. S100A12 (S100 calcium binding protein A12) which codifies a protein proposed to be involved in specific calcium-dependent signal transduction pathways and which regulatory effect on cytoskeletal components may modulate various neutrophil activities. STXBP2 (Syntaxin binding protein 2) is involved in neutrophil degranulation. MPP1 (Membrane protein, palmitoylated 1, 55 kDa), which participates in the regulation of neutrophil chemotaxis. FCAR (Fc fragment of IgA, receptor for): this protein interacts with IgA-opsonized targets and triggers several immunologic defense processes, including phagocytosis. ITGAX (Homo sapiens cDNA, FLJ99683) which mediates adherence of neutrophils and monocytes to stimulated endothelium cells, and in the phagocytosis of complement coated particles. FCAR: this gene encodes a receptor for the Fc region of IgA, present on the surface of myeloid lineage cells such as neutrophils, monocytes, macrophages, and eosinophils, and triggers several immunologic defence processes, including phagocytosis, antibody-dependent cell-mediated cytotoxicity, and stimulation of the release of inflammatory mediators.

This “neutrophil signature” supports the notion of the existence at the systemic level of neutrophils ready to fight against infection but also ready to produce mediators which are able to induce tissue injury. Participation of these molecules in COPD pathogenesis had already been described at the respiratory level, but this work is the first suggesting an important activity of these enzymes at the systemic level. Migration of these cells from the blood vessels could certainly help to clear the causative microbe, but also to damage the pulmonary parenchyma, contributing to the explanation of the severity of respiratory conditions of these patients. Finding this “neutrophil signature” was possible since we used whole blood instead of PBMCs for the transcriptomic study [4]. This signature is probably related to the fact that the vast majority of patients in the ICU group showed bacterial infection, and neutrophils are major actors in antibacterial defence. In fact, critically ill patients in our cohort showed significantly higher total counts of neutrophils in blood than non-critically ill ones. On the other hand, a limitation of our work is that we did not isolated neutrophils for the gene expression profiling assays. Further works targeting specific cell types would contribute to the definition of the exact contribution of neutrophils and other leukocytes to the gene expression signatures linked to severe respiratory illness in patients with COPD.

Other genes showing increased expression in the ICU group

C8G (Complement component 8, gamma polypeptide) forms part of C8, protein which plays a central role in assembly of the "membrane attack complex" (MAC) of complement. The MAC is a macromolecular pore that targets and lyses pathogens that challenge the host. Deposition of these pores on human cells contributes to tissue damage. Interestingly, MPO products seem to be able to activate complement [17], indicating a potential link between MPO over-expression and induction of MAC formation. CD24: This gene encodes a sialoglycoprotein that is expressed on mature granulocytes and in many B cells. Other interesting gene relatively up-regulated in critical patients was VEGFA (Vascular endothelial growth factor A). This protein is a glycosylated mitogen that specifically acts on endothelial cells and has various effects, including mediating increased vascular permeability, inducing angiogenesis, vasculogenesis and endothelial cell growth, promoting cell migration, and inhibiting apoptosis. Circulating levels of VEGF are up-regulated in patients with acutely exacerbated COPD and decrease after recovery from exacerbation [18]. Respiratory levels of VEGF have been shown to negatively correlate with pulmonary function in stable COPD, which suggests its important role in COPD airway remodelling [19]. ELN (Elastin): This gene encodes a protein that is one of the two components of elastic fibbers, and participates in extracellular matrix organization, such as those taking place after severe tissue injury. MUC1 (Mucin 1, cell surface associated) play an essential role in forming protective mucous barriers on epithelial surfaces and in the response to hypoxia. Finally, we identified increased transcript levels in the ICU group of a group of genes involved in antibacterial defence: PGLYRP1 (peptidoglycan recognition protein 1), CEBPE (CCAAT/enhancer binding protein (C/EBP), epsilon), CHIT1 (chitinase 1 (chitotriosidase)) and CYBA (cytochrome b-245, alpha polypeptide).