Abstract
Bronchiectasis is a heterogenous disease with multiple underlying causes. The pathophysiology is poorly understood but neutrophilic inflammation and dysfunctional killing of pathogens is believed to be key. There are, however, no licensed therapies for bronchiectasis that directly target neutrophilic inflammation. In this review, we discuss our current understanding of neutrophil dysfunction and therapeutic targeting in bronchiectasis. Immunometabolic reprogramming, a process through which inflammation changes inflammatory cell behaviour by altering intracellular metabolic pathways, is increasingly recognised across multiple inflammatory and autoimmune diseases. Here, we show evidence that much of the neutrophil dysfunction observed in bronchiectasis is consistent with immunometabolic reprogramming. Previous attempts at developing therapies targeting neutrophils have focused on reducing neutrophil numbers, resulting in increased frequency of infections. New approaches are needed and we propose that targeting metabolism could theoretically reverse neutrophil dysfunction and dysregulated inflammation. As an exemplar, 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK) activation has already been shown to reverse phagocytic dysfunction and neutrophil extracellular trap (NET) formation in models of pulmonary disease. AMPK modulates multiple metabolic pathways, including glycolysis which is critical for energy generation in neutrophils. AMPK activators can reverse metabolic reprogramming and are already in clinical use and/or development. We propose the need for a new immunomodulatory approach, rather than an anti-inflammatory approach, to enhance bacterial clearance and reduce bronchiectasis disease severity.
Abstract
Immunometabolic alternation in neutrophils may contribute to excess inflammation in bronchiectasis. Targeting AMPK can modulate immunometabolism and improve neutrophil function, and is a potential therapeutic approach for bronchiectasis. https://bit.ly/3iB7Deb
Introduction
Bronchiectasis is the final outcome of a range of infectious, inflammatory, autoimmune, allergic and genetic conditions [1, 2]. It is a heterogeneous disease and there is no single established pathophysiology underlying its onset [3–5].
In 1986, Cole et al. [6] proposed the “vicious cycle” model, shown in figure 1a, to describe the pathophysiology of bronchiectasis. Their model describes multiple cycles of airway tissue destruction that impairs mucociliary clearance leading to opportunistic bacterial colonisation. Chronic infection results in persistent neutrophilic airway inflammation and damages neighbouring airway tissues, thus repeating the vicious cycle. It has been long believed that breaking the cycle would be the right approach to therapy, but this has met with limited success [7–9]. Flume et al. [10] further adapted this model in 2018, creating the “vicious vortex” model. The vicious vortex (figure 1b) is a modified concept, suggesting that progress can be initiated at any step and does not follow a sequence like the vicious cycle, with interaction between all components of the vortex. This would explain why a single intervention such as antibiotic treatment does not modify the course of the disease and why, since the cycle is never “broken”, that bacterial loads return to baseline as soon as antibiotics are stopped [11]. Implicit in the vortex concept is the idea that we need to find interventions that target multiple components of pathophysiology [10].
Antibiotic treatment is the mainstay of therapy for bronchiectasis but there are limited therapies that target host inflammatory and epithelial cells (which constitute the majority of the vicious cycle/vicious vortex components). Identifying therapies that can modulate inflammation and mucociliary dysfunction is a major objective of drug development in bronchiectasis. In this review, we look at known inflammatory pathways in bronchiectasis and review the limited success of past approaches to anti-inflammatory treatment. We discuss the emerging role of immunometabolism in chronic inflammation and the potential of targeting immunometabolism in bronchiectasis.
Neutrophil dysfunction during inflammation in pulmonary diseases
Neutrophil dysfunction is a common feature of several airway diseases including chronic obstructive pulmonary disease (COPD), bronchiectasis and cystic fibrosis (CF). Impaired neutrophil chemotaxis has been demonstrated in COPD. Sapey et al. [12] showed increased movement speed in neutrophils during chemotaxis, but decreased accuracy in movement towards the target. Normally, neutrophils move towards a target by sensing chemo-attractants, with receptors such as C-X-C chemokine receptor type 1 (CXCR1) or type 2 (CXCR2) and formyl peptide receptor 1 (FPR1) expressed on the neutrophil surface. For further understanding, refer to the review by Rosales [13] and work by Xie et al. [14] on the heterogeneity of neutrophils and how this influences neutrophil function and development. Comparisons between COPD and control groups show no difference in these receptors, but phosphoinositide 3-kinase (PI3K) inhibition in COPD neutrophils rescues the effect by decreasing neutrophil speed and increasing accuracy. It has been suggested that phosphatidylinositol-3,4,5-triphosphate production by PI3K polarises neutrophils and leads to pseudopod formation, allowing neutrophils to move towards chemo-attractants. In view of the overlap between COPD and bronchiectasis, whether there is defective chemotactic targeting of neutrophils in bronchiectasis merits further study [15].
Upon arrival in the bronchiectatic airway, neutrophils encounter a pro-inflammatory environment with high concentrations of mediators such as C-X-C chemokine ligand 8 (CXCL8), interleukin (IL)-1β, tumour necrosis factor-α (TNF-α) and, in particular, neutrophil serine proteases such as neutrophil elastase (NE) [16, 17].
Whether neutrophil dysfunction in bronchiectasis is caused by this inflammatory airway milieu, or whether bronchiectasis patients have intrinsic neutrophil defects, is not known; however, this has been investigated (with inconsistent results) by comparing neutrophil function in peripheral blood between bronchiectasis patients and controls. King et al. [18] have shown no significant difference in phagocytosis and oxidative burst between patients and controls, while Pasteur et al. [19] and Ruchaud-Sparagano et al. [20] reported similar findings, with no evidence of reduced phagocytosis in peripheral neutrophils of bronchiectasis patients. The Pasteur et al. [19] study showed normal CD11b expression in bronchiectasis patients. These findings were later contradicted by Bedi et al. [21], who found that blood neutrophils of bronchiectasis patients had impaired phagocytosis, deficient killing of Pseudomonas aeruginosa, delayed apoptosis and increased CD11b expression compared to controls, leading them to describe blood neutrophils of bronchiectasis patients as “reprogrammed”. The term reprogramming is typically defined by changes in metabolic pathways, although the mechanisms of this reprogramming were not investigated in the Bedi study.
As bronchiectasis is a heterogeneous disease, inconsistent results across studies are not unusual and we speculate that neutrophil dysfunction may be limited to subsets of patients. Indeed, different metabolic states influencing the functional capabilities of neutrophils could be the reason for the disparity in these findings. Furthermore, the ex-vivo conditions in which functional studies are performed are artificial and may not replicate conditions in the airway. Borregaard et al. [22] have shown that neutrophils with different glucose availability have altered lactate production rates and biochemical pathways producing adenosine triphosphate (ATP). The limitation to these studies was that the phagocytic abilities of neutrophils in different nutrient states was not analysed.
Watt et al. [23] found fewer apoptotic airway neutrophils in bronchiectasis patient sputum, but higher numbers of necrotic neutrophils as compared to induced sputum from healthy controls. Increased necrosis suggests disrupted resolution of inflammation. Potential mechanisms for a reduction in apoptotic airway neutrophils and an increase in necrotic cells include secondary necrosis, pyroptosis or necroptosis, from a failure of efferocytosis, primary necrosis induced by, for example, bacterial toxins, or indeed artifacts of the preparation of neutrophils from inflamed airways. Bedi et al. [21] have shown reduced spontaneous neutrophil apoptosis in bronchiectasis neutrophils, but the mechanisms are unknown. Data from CF suggests delayed apoptosis has multiple mechanisms, including CF transmembrane conductance regulator (CFTR) dependent and independent pathways [24–26]. Multiple inflammatory signals upregulate survival signals within neutrophils. An example in CF is proliferating cell nuclear antigen (PCNA), a cyclin-dependent kinase inhibitor which prevents neutrophil apoptosis and is upregulated by lipopolysaccharide (LPS), TNF-α and other pro-inflammatory cytokines which are known to be elevated in bronchiectasis [27, 28]. Neutrophil death can also be delayed in bacterial infection [29].
Much of our knowledge of the pathophysiology of bronchiectasis comes from CF and the extensive knowledge of neutrophil function and pathology in CF has been reviewed by Cantin et al. [30]. In a recent study, circulating neutrophils in CF were found to be metabolically reprogrammed, which induced the neutrophils to be more pro-inflammatory upon entering the lungs. This was demonstrated by the role of low levels of LPS exposure in increasing markers of Warburg metabolism. Increased LPS, presumably due to Gram-negative infection in the CF lung, also activates the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome (which activates caspase-1) and converts pro-IL-1β to IL-1β, which is pro-inflammatory. IL-1β associates with severe disease outcomes for CF, but also with non-CF bronchiectasis. CF murine models have shown that NLRP3 inhibition rescues this effect by enhancing P. aeruginosa clearance, as well as decreasing lung inflammation [31]. In health, neutrophils usually counter bacterial infections via phagocytosis [32–34]; however, they are also capable of releasing neutrophil extracellular traps (NETs), consisting of chromatin with neutrophil granular proteins and platelets, to trap and immobilise bacteria. The process of NET formation and release is known as “NETosis” [12, 35–44]. Although the literature describing links between NETosis and human disease is growing, there remains controversy over the extent to which in vitro observations occur in vivo and, in particular, over the role of NETs in bacterial killing and control in vivo, which remain highly disputed [45]. NETosis has been reported to associate with respiratory diseases. In CF, P. aeruginosa exposure causes macrophage migration-inhibitory factor (MIF) to induce NETosis via mitogen-activated protein kinase (MAPK) and increased MIF levels measured in CF patients associate with decreased lung function [46]. A CFTR-dependent reduction in neutrophil apoptosis increases neutrophil survival and this increases NET production in CF, which becomes pro-inflammatory to macrophages (a defect that could be rescued by CFTR modulators) [47].
These observations are highly relevant to bronchiectasis because elevated IL-1β in association with bacterial infection is a feature of bronchiectasis [48] and, as in CF, NET formation has recently been shown to be associated with clinical outcomes in this disease. Our group recently described pregnancy zone protein (PZP) as a novel NET-associated protein [49] and NET-related proteins as driving differences between the proteomics profiles of sputum from mild and severe cases [50]. The presence of PZP and other markers of NETs was associated with disease severity such as higher bronchiectasis severity index score, frequent exacerbations, chronic lung infection, decreased quality of life (QoL) and increased mortality. The levels of NETs in sputum also increased in samples with increased neutrophil-related markers such as IL-10, CXCL8 and IL-1β. PZP is also anti-inflammatory and, altogether, this suggests the presence of multiple immune-modulating factors in the bronchiectatic airways which can influence disease outcomes [49, 50].
Current therapies
Unfortunately, there are no licenced drugs for bronchiectasis. Antibiotics are the mainstay of therapy; however, antibiotic resistance is a global healthcare crisis and therefore nonantibiotic therapies are urgently needed.
The recent WILLOW study (www.clinicaltrials.gov; identifier NCT03218917) has given a strong indication of the importance of neutrophil serine proteases in bronchiectasis [51]. WILLOW is a Phase 2 clinical study for the drug brensocatib (INS1007), a dipeptidyl peptidase-1 (DPP1) inhibitor (figure 2, drug 1) which prevents activation of NE, proteinase 3 and cathepsin G in neutrophil granules during maturation in the bone marrow. By doing so, it greatly inhibits neutrophil serine protease activity in sputum in bronchiectasis patients and is found to prolong the time to next exacerbation at two doses compared with a placebo. Larger studies are planned.
Mechanisms of current anti-inflammatory therapies in bronchiectasis in trials. NE: neutrophil elastase; ESL-1: E-selectin ligand-1; ICAM-1: intercellular adhesion molecule-1; CXCR1/2: C-X-C chemokine receptor type 1/2; CXCL8: C-X-C chemokine ligand 8.
Before WILLOW, there were multiple unsuccessful approaches to developing anti-inflammatory therapies in bronchiectasis. A clinical trial [52] tested the anti-inflammatory drug AZD5069 (a CXCR2 antagonist) in bronchiectasis patients (figure 2, drug 2). Since CXCR2 is the receptor for CXCL8 during neutrophil recruitment, antagonising CXCR2 should reduce airway neutrophil recruitment and decrease neutrophilic inflammation. This was demonstrated successfully with a significant decrease in neutrophilic inflammation, but pneumonia increased in just 28 days with no evident clinical benefits and, consequently, the trial programme was abandoned [52]. Similarly, a clinical trial in CF using a leukotriene B4 (LTB4) receptor antagonist demonstrated an increase in exacerbation and a worsening of forced expiratory volume in 1 s (FEV1). A follow-up study using murine models demonstrated increased P. aeruginosa infection in the lungs following BIIL 284 anti-inflammatory treatment, due to reduced neutrophilic inflammation (figure 2, drug 3) [53]. These two anti-inflammatory approaches target neutrophil migration and chemotaxis, which contribute to the amounts of neutrophils at a site, but do not influence the bactericidal functions of those neutrophils. This suggests that simply removing neutrophilic inflammation might not be the best solution and that there should be modulation of neutrophil function. DPP1 inhibition is not expected to affect neutrophil recruitment but modulates inflammation and NETosis.
The results with DPP1 inhibition contrast with those of prior direct inhibitors of NE. [54, 55]. AZD9668 was tested in 22 bronchiectasis patients and compared to 16 patients receiving placebo. The study found an increase in FEV1 of 100 mL with active treatment versus placebo and some inflammatory cytokines were reduced; however, patients' QoL did not improve significantly [56]. A further study used a synthetic NE inhibitor (BAY 85-8501) [57] in 94 patients (figure 2, drug 4). Patients in the treatment group showed significant reductions in neutrophilic inflammation after treatment, with decreased CXCL8 and reduced NE activity in blood after zymosan challenge. However, patients' FEV1 and QoL were not significantly improved. Perhaps direct inhibition of NE alone is not enough or possibly these studies were too small and short in duration to demonstrate an effect.
New potential approaches to bronchiectasis treatment
So far, we have reviewed different neutrophil dysfunctions found across respiratory diseases and discussed therapeutic approaches to target them. However, there are still potential approaches that remain unexplored. Neutrophil reprogramming refers to the switch in metabolic pathways, due to modified reaction rates of biochemical pathways, leading to extensive changes in cellular behaviour. For example, the study by McElvaney et al. [31] discussed above describes elevated Warburg metabolism following LPS exposure, leading to pro-inflammatory markers being released by neutrophils. Reprogramming remains a controversial term with regard to neutrophils, as it implies a pre-set “programme” in neutrophil behaviour that is rewired during inflammatory response. The extent to which neutrophils can be “deprogrammed” is not clear. Whatever terminology is used, it is clear that metabolic changes in neutrophils impact their function and therefore therapeutic approaches for preventing or reversing adverse metabolic changes within neutrophils represent a potential therapeutic avenue.
In cellular energetics, glucose uptake into cells enters glycolysis to produce pyruvate for the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) for ATP production. However, during metabolic reprogramming pyruvate enters anaerobic glycolysis to produce lactate and glycolysis also interfaces with the pentose phosphate pathway (PPP), producing reduced nicotinamide adenine dinucleotide phosphate (NADPH) (figure 3) and contributing to reactive oxygen species (ROS) production [58, 59]. This pathway switching leads to a pro-inflammatory phenotype of neutrophils and this will be further elaborated upon later.
Summary of the main biochemical pathways involved in neutrophil metabolism and cell signalling, as well as the role of 5' AMP-activated protein kinase (AMPK) in various pathways. Glycolysis is drawn in blue, the pentose phosphate pathway in green and the tricarboxylic acid (TCA) cycle in brown. Fatty acid synthesis and oxidation are shown in yellow and glycogenesis in orange. AMPK involvement is highlighted using various symbols. ACC: acetyl-CoA carboxylase; ADP: adenosine diphosphate; AMP: adenosine monophosphate; ATP: adenosine triphosphate; 1,3-BPG: 1,3-biphosphoglycerate; cAMP: cyclic AMP; CAP1: adenylyl cyclase-associated protein 1; CAT: carnitine acyltransferase; CPT: carnitine palmitoyltransferase; DHAP: dihydroxyacetone phosphate; ETC: electron transport chain; FAS: fatty acid synthesis; F1,6BP: fructose 1,6-biphosphate; F2,6BP: fructose 2,6-biphosphate; F6P: fructose 6-phosphate; FAD: oxidised flavin adenine dinucleotide; FADH2: reduced flavin adenine dinucleotide; G3P: glyceraldehyde 3-phosphate; G1P: glucose 1-phosphate; G6P: glucose 6-phosphate; GDP: guanosine diphosphate; GTP; guanosine triphosphate; GSK3β: glycogen synthase kinase 3β; HIF-1α: hypoxia-inducible factor 1α; IKK: IκB kinase; IRAK: interleukin-1 receptor-associated kinase; LPS: lipopolysaccharide; NAD+: oxidised nicotinamide adenine dinucleotide; NADH: reduced nicotinamide adenine dinucleotide; NADP+: oxidised nicotinamide adenine dinucleotide phosphate; NADPH: reduced nicotinamide adenine dinucleotide phosphate; PFK1/2: phosphofructokinase 1/2; PGK: phosphoglycerate kinase; PHD2: prolyl hydroxylase domain protein 2; PKA: protein kinase A; PKM: pyruvate kinase; PPP: pentose phosphate pathway; SDH: succinate dehydrogenase; TCA: tricarboxylic acid; UDP: uridine diphosphate.
Rates of metabolic pathways are regulated by multiple enzymes, but 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK) is considered the “master regulator” of multiple metabolic pathways. It functions as an energy sensor but is also linked to multiple inflammatory pathways. In acute respiratory distress syndrome (ARDS) [60] and acute lung injury (ALI) [61–63], multiple studies have demonstrated increased NET formation that contributes to severe disease, in a similar manner to bronchiectasis. These results also showed inflammatory factors such as high-mobility group box 1 (HMGB1) [60] and resistin [61] causing AMPK inhibition, which was associated with increased disease severity. AMPK inhibition was also shown to cause severe disease in emphysema [64, 65] and murine models for idiopathic pulmonary fibrosis (IPF) showed reduced AMPK activity in IPF regions [66]. Although not yet studied directly in bronchiectasis, this data is highly relevant as our recent proteomics study also showed resistin and HMGB1 as among the most upregulated proteins in severe bronchiectasis [50].
Altogether, these studies suggest the potential for a novel therapy. AMPK activation has been shown to improve phagocytosis in macrophages and neutrophils and has a positive impact on disease outcome [60, 63, 65, 67–70]. These studies have also provided evidence, across various lung pathologies, for “immunometabolic reprogramming” and suggest the importance of adequate AMPK activity for function and combating diseases. As a starting point, it would thus be crucial to look for inhibited AMPK activity in bronchiectasis.
5' AMP-activated protein kinase
As noted above, AMPK is an energy sensor kinase that regulates the cellular AMP:ATP ratio, by activating during periods of high AMP/ATP ratio in order to replenish cellular ATP levels [71]. Mammalian AMPK is comprised of three subunits (α, β and γ). The α-subunit contains its main phosphorylation site (T172), which is phosphorylated during activation by upstream kinases (e.g. liver kinase B1 (LKB1) and calcium/calmodulin dependent protein kinase kinase II (CAMKK2)) [72].
AMPK binds to free AMP and ATP to modulate cellular energy. In low energy states, increased intracellular AMP increases binding to AMPK. This changes the AMPK allosteric conformation for T172–AMPK phosphorylation by upstream kinases. As it has multiple targets, AMPK is involved in many pathways to increase cellular ATP for cell functioning. In periods of high ATP:AMP ratio, less AMP binds to AMPK and lower AMPK activation leads to decreased stimulation and slower rates in catabolic pathways [72–75].
AMPK also has other serine and tyrosine residues as phosphorylation sites for other kinases, which also modulate AMPK activity. For example, glycogen synthase kinase 3β (GSK3β) inhibits AMPK activity by phosphorylating T479 and causing T172 dephosphorylation [62, 76].
AMPK in metabolic pathways
Glucose generates ATP via glycolysis, yielding pyruvate at the end [77]. Usually, pyruvate enters mitochondria and is converted into Acetyl-CoA before entering the TCA cycle to produce reduced nicotinamide adenine dinucleotide (NADH) and reduced flavin adenine dinucleotide (FADH2). NADH and FADH2 enter ETC to produce ATP during oxidative phosphorylation [3].
Meanwhile, anaerobic respiration takes place in the cytoplasm by converting pyruvate to lactate, which recycles NADH by converting glyceraldehyde 3-phosphate (G3P) into 1,3-biphosphoglycerate (1,3-BPG) [3]. Oxidative phosphorylation is widely believed to be more efficient in producing ATP, as one cycle of oxidative phosphorylation has a net production of 36 ATP units, while one cycle of anaerobic respiration has a net production of two ATP units [77].
In the post-prandial state, increased insulin secretion targets insulin receptors on cells and induces GLUT4 translocation to the cell surface [78]. However, AMPK activation can also induce GLUT4 translocation to the cell surface independent of insulin. AMPK also facilitates glycolysis by activating phosphofructokinase (PFK) 2, thus producing fructose 2,6-biphosphate (F2,6BP) and activating PFK 1 (figure 3) [68, 71, 73, 74, 77, 79, 80].
Glycolysis also interfaces with multiple pathways. During increased glucose uptake, glucose 6-phosphate (G6P) enters glycogenesis to synthesise glycogen for energy storage [81]. AMPK has a carbohydrate-binding module on its β-subunit which seems to be involved in sensing glycogen stores in the cell. However, its exact function in neutrophils has not been fully elucidated [73–75]. G6P can also enter the PPP, which produces different substrates for other cellular processes, including G3P and fructose 6-phosphate (F6P), which re-enters glycolysis (figure 3) [82].
Metabolic pathways in neutrophils
Neutrophils are mainly glycolytic as they have a small number of mitochondria [83, 84] that are not involved in ATP production and have low enzymatic activity. Neutrophil mitochondria function is required for apoptosis and chemotaxis of neutrophils but not bacterial killing [83]. GLUT1, GLUT3 and GLUT4 isoforms are expressed on the neutrophil cell surface, and GLUT1 and GLUT3 expression is upregulated during cell activation, presumably to meet increased glucose requirements [85].
Metabolic reprogramming has been demonstrated in neutrophilic inflammation. Azevedo et al. [86] have shown a metabolic switch in neutrophils to the PPP during phorbol 12-myristate 13-acetate (PMA)-induced NETosis. This is due to increased NADPH production activating NADPH oxidase (Nox) to generate ROS for NETosis. NADPH also interfaces with glutathione disulfide to produce glutathione for hydrogen peroxide detoxification (figure 3) [82, 87, 88]. However, their experiments did not demonstrate how the metabolic switch would occur physiologically [86].
Another study further examined the metabolic requirements of neutrophils during NETosis [89] and showed two metabolic phases. Phase 1 of NETosis is independent of glucose uptake and requires about 3 h to form NETs. However, Phase 2 only takes 15 min and requires glucose uptake and glycolysis to release NETs extracellularly. Combining both studies, neutrophils probably switch to the PPP intracellularly for ROS production and NET formation, but switch back to glycolysis for NET extrusion.
Although neutrophil mitochondria are not actively involved in oxidative phosphorylation [84], the TCA cycle still occurs in order to produce metabolic substrates for other functions, such as autophagy [90, 91]. Excess citrate produced from the TCA cycle can be transported out of the mitochondria via the citrate shuttle. Cytoplasmic citrate is broken down into acetyl-CoA and oxaloacetate. Acetyl-CoA carboxylase (ACC), which can be inhibited by AMPK via phosphorylation [92, 93], then converts acetyl-CoA into malonyl-CoA during fatty acid synthesis (FAS), producing triglycerides and palmitate (figure 3) [77].
It should be noted, however, that a preliminary study from 1976 [94] found that neutrophils incorporated more palmitate and glucose into their triglycerides than lymphocytes. Triglycerides are a source of fatty acids for phosphatidylcholine synthesis during phagocytosis, which provides phospholipids for cell membrane maintenance [95]. This difference in lipid metabolism was speculated to better meet neutrophil requirements during phagocytosis [94]. Additionally, TNF-α can also influence arachidonic acid metabolism in neutrophils via phospholipase A2, which induces the turnover of various phospholipids [96, 97]. This further suggests the importance of adequate lipid synthesis in neutrophils during inflammation, in order to keep up with its bactericidal needs. All these studies give an insight into how immunometabolism links to antimicrobial function.
AMPK in neutrophils during inflammation
AMPK inhibits GSK3β via Akt phosphorylating at S9-GSK3β [98, 99]. However, GSK3β can also inhibit AMPK via TLR4–IκB kinase (IKK)β stimulation. IKKβ phosphorylates S485-AMPK leading to GSK3β phosphorylation of T479-AMPK, dephosphorylating T172-AMPK and thereby inhibiting AMPK activity (figure 3, pathway A) [62].
The pro-inflammatory protein resistin is associated with increased severity in ARDS [61], CF [100] and bronchiectasis [50], and was originally found to be produced by adipocytes, which caused insulin resistance in murine models [101, 102]. However, human resistin is mainly released by neutrophils [103] and macrophages [104] during inflammation and can inhibit AMPK. A similar effect has been seen with LPS and, since LPS and resistin both target TLR4, the TLR4–IKKβ–GSK3β pathway is currently the proposed pathway for AMPK inhibition (figure 3, pathway A) [62, 76].
Neutrophils stimulated with LPS and resistin also release increased concentrations of the factor HMGB1, a danger-associated molecular pattern (DAMP) that also targets TLR4 on neutrophils. [61, 62]. HMGB1-TLR4 stimulates ROS and pro-inflammatory cytokine production. Elevated levels of resistin, LPS and HMGB1 during inflammation all stimulate the TLR4 pathway to suppress AMPK activity. As AMPK enhances glycolysis via PFK 2, TLR4 suppression is likely to delay glycolysis and promote the PPP, as well as increase ROS production to promote NET formation [61].
Resistin can also target adenylyl cyclase-associated protein 1 (CAP1) in monocytes and has been shown binding to CAP1 to increases intracellular cyclic AMP (cAMP), activating protein kinase A (PKA) and NF-κB. NF-κB translocates to the nucleus, inducing inflammatory cytokine transcription [105]. Interestingly, PKA also phosphorylates S485-AMPK [106] and S173-AMPK [107], thereby dephosphorylating T172-AMPK for inhibition (figure 3, pathway A). High resistin levels have been detected in CF [100] and in bronchiectasis sputum, where it was one of the most abundant and differentially expressed proteins between those bronchiectasis patients chronically infected with P. aeruginosa and those who were not [49].
Even though hypoxia is known to promote AMPK activity via hypoxia-inducible factor 1α (HIF-1α), it might be insufficient to overcome the effects of resistin/HMGB1 and hence hypoxic areas in the lung or hypoxia are still present in bronchiectasis and ARDS patients. Altogether, these results suggest a strong AMPK-inhibitory environment during inflammation in bronchiectasis, which could be worsened during prolonged lung inflammation. While there are licensed AMPK-activating drugs, they have not been trialled for use in bronchiectasis, although their potential will be discussed in the following sections.
AMPK and inflammatory dysfunction in bronchiectasis
NETosis appears to be a key feature of bronchiectasis pathophysiology. This poses a problem because certain species, such as P. aeruginosa, Haemophilus influenzae and Staphylococcus aureus, are NET-resistant but susceptible to phagocytosis [5, 108–113]. The exact mechanism causing such a switch from phagocytosis to pro-inflammatory NETosis is still unknown. However, there is a strong mechanistic basis to believe metabolic reprogramming may underlie this switch in neutrophil behaviour. HMGB1 might inhibit AMPK, block phagocytosis in macrophages and promote NETosis [36]. Whether HMGB1 also blocks neutrophil phagocytosis is not known and the roles of HMGB1 in bronchiectasis have also not been studied.
Furthermore, studies have demonstrated improved phagocytosis in neutrophils when treated with the AMPK activators metformin [69] and 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) [70]. Experience in other diseases and recent data on the inflammatory airway environment in bronchiectasis supports the concept that the bronchiectasis airway is an AMPK inhibitory environment, which encourages metabolic reprogramming leading to increased anaerobic glycolysis and the PPP. This increases NADPH production and generates ROS, encouraging NETosis. We propose that increasing AMPK activity in neutrophils would decrease NETosis and encourage phagocytosis instead, a less pro-inflammatory mechanism of bacterial control which is reported to be impaired in bronchiectasis [21]. This supports the potential of AMPK activators as novel therapies for bronchiectasis. However, before using them as experimental drugs for bronchiectasis, future experiments should consider incorporating AMPK activators to confirm their potential in reversing neutrophil dysfunction for bronchiectasis.
Potential therapies/targetable pathways
There are additional therapeutic targets in metabolic pathways which could theoretically reverse bronchiectasis-associated inflammatory changes, many of which also interact with AMPK. HIF-1α is targetable since there is a switch to the pro-inflammatory succinate HIF-1α pathway and active HIF-1α activates lactate dehydrogenase (LDH), further encouraging anaerobic glycolysis [59, 114–116]. AMPK and HIF-1α also activate each other [117–119] and, therefore, therapies targeting the HIF-1α pathway have similar benefits to those for AMPK activators. As mentioned above, inhibition of neutrophil serine proteases is being evaluated in bronchiectasis and can be achieved through DPP1 inhibition, direct competitive inhibition or by supplementing endogenous proteinase inhibitors such as α1-antitrypsin. While these therapies aim to restore the proteinase:anti-proteinase balance, they also have important potential effects on immunometabolic pathways. NE knockout mice have shown increased AMPK activity, thereby decreasing ACC 2 activity. This decreases the production of malonyl-CoA and reduces inhibition of carnitine palmitoyltransferase 1 (CPT1), the enzyme involved in fatty acid oxidation (FAO), thus increasing the rate of FAO (figure 3) and potentially providing adequate lipids for membrane maintenance and phagocytosis. Furthermore, without α1-antitrypsin countering NE activity, AMPK activity decreases and inflammation concurrently increases [120]. These findings suggest NE in the bronchiectatic lung might also be AMPK inhibitory, potentially contributing to depressed neutrophil phagocytosis in the lungs of severe bronchiectasis patients. As such, directly countering NE activity with α1-antitrypsin, or reducing NE activity through DPP1 inhibition, may neutralise NE-mediated immune dysfunction and restore AMPK activity.
Impaired mucociliary clearance is also one of the features of bronchiectasis pathophysiology but remains untargeted as an avenue for treatment. Studies summarised by Burgel and Nadel [121] discuss the involvement of epidermal growth factor receptor (EGFR) signalling in contributing to mucin production. Pathogens from the environment invading epithelial cells with Toll-like receptors (TLRs) leads to ligand binding on the EGFR for cell signalling, to produce immune responses such as CXCL8 for neutrophil recruitment, mucin production for mucociliary clearance and ROS production. As such, the EGFR pathway should also be studied in the context of bronchiectasis to understand if exacerbations were also contributed by EGFR. More importantly, AMPK can also inhibit EGFR signalling [122–124], providing another link between immunometabolism and key target pathways.
Conclusions
Despite recent breakthroughs in bronchiectasis, there is still a pressing need to look for new treatments. In this review, we have shown how factors such as bacterial infection, resistin, pro-inflammatory cytokines and HMGB1 might induce immunometabolic reprogramming in neutrophils and recapitulate many of the features of bronchiectasis (including decreased chemotaxis, phagocytosis and bacterial killing by neutrophils), while increasing NETs and cytokine release. Incomplete inflammatory resolution further increases leukocyte recruitment and accumulation without efficient bacterial killing, leading to the well-characterised vicious cycle. A graphical summary of bronchiectasis pathophysiology with pro-inflammatory cytokines causing neutrophil reprogramming is given in figure 4.
Summary review of bronchiectasis pathophysiology with pro-inflammatory cytokines causing neutrophil reprogramming and how treatment could target dysfunctional neutrophils by restoring 5' AMP-activated protein kinase (AMPK) activity. AMP: adenosine monophosphate; LPS: lipopolysaccharide; NET: neutrophil extracellular trap; NE: neutrophil elastase.
AMPK activation, DPP1 inhibition and additional approaches may reverse metabolic reprogramming, enhancing neutrophil function while reducing inflammatory-mediator release. Current and future studies will address whether this represents an effective approach to restoring neutrophil function and reducing inflammation in bronchiectasis.
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Footnotes
Conflict of interest: Y.H. Giam has nothing to disclose.
Conflict of interest: A. Shoemark has nothing to disclose.
Conflict of interest: J.D. Chalmers reports grants and personal fees from AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline and Insmed, personal fees from Chiesi, Novartis and Zambon, and grants from Gilead Sciences, outside of the submitted work.
Support statement: This work was supported by the Chief Scientist Office of the Scottish Government (Senior Clinical Fellowship to J.D. Chalmers). Funding information for this article has been deposited with the Crossref Funder Registry.
- Received August 15, 2020.
- Accepted January 12, 2021.
- Copyright ©The authors 2021. For reproduction rights and permissions contact permissions{at}ersnet.org
References
