Background
Chronic obstructive pulmonary disease (COPD) is characterised by excessive airway inflammation in response to the inhalation of noxious particles [
1]. Some COPD patients suffer with chronic bacterial colonisation of the airways [
2], while acute respiratory tract infections caused by new bacterial species also occur [
3]. Bacterial presence increases the levels of inflammation in the airways of COPD patients [
4]. Non-typeable
Haemophilus influenza (NTHi) is a common pathogen found in the lungs of COPD patients [
5].
Inhaled corticosteroids (ICS) are the mainstay of anti-inflammatory treatment for COPD [
1]. ICS combined with long acting beta agonists (LABA) reduce exacerbation rates, improve lung function and increase quality of life compared to LABA alone [
6,
7]. However, the effects of ICS vary between individuals, and there is growing evidence of greater clinical benefit in COPD patients with higher blood eosinophil counts [
6,
7]. The molecular mechanisms for the differential response between individuals remain unclear.
Corticosteroids bind to the cytoplasmic glucocorticoid receptor (GR); this complex translocates into the nucleus where it suppresses pro-inflammatory gene transcription (transrepression) or activates anti-inflammatory gene expression (transactivation) [
8]. Phosphorylation within the GR N-terminus occurs during GR activation, with serine (S) 211 and 226 phosphorylation associated with GR-ligand nuclear translocation and nuclear export respectively [
9]. Mitogen activated protein kinases (MAPKs), including p38 MAPK, can modulate GR phosphorylation, although this effect varies between cell types [
10‐
12].
Studies using the bacterial endotoxin lipopolysaccharide (LPS) to stimulate COPD alveolar macrophages have shown that the effects of corticosteroids vary between cytokines, with CXCL8 in particular being less responsive to corticosteroid suppression [
13‐
15]. LPS activates Toll like receptor 4 (TLR4) signaling, while bacteria such as NTHi cause more complex inflammatory responses. It has been reported that a subset of NTHi stimulated cytokines from COPD alveolar macrophages, including CXCL8, showed no suppression with corticosteroids [
16]. These results implicate NTHi as a cause of corticosteroid insensitive inflammatory responses in COPD, thus contributing to the between individual clinical variation in response to ICS treatment.
Macrophages display plasticity, changing their characteristics in response to extracellular stimuli. The traditional model of M1/M2 macrophage polarisation states that M1 macrophages are pro-inflammatory, while M2 macrophages have anti-inflammatory and tissue repair functions. This is an oversimplification as further subtypes of macrophages have been identified [
17]. Nevertheless, it has been reported that COPD lung macrophages have unique characteristics, with increased M2 and reduced M1 gene expression levels compared to controls [
18]. Oxidative stress, inflammation and bacterial exposure may all influence macrophage polarisation [
19].
We report an in depth characterisation of the effects of NTHi exposure on COPD macrophages. Firstly, we studied the effects of corticosteroids on macrophage cytokine production caused by NTHi exposure, in order to confirm the corticosteroid insensitivity previously reported [
16]. We then investigated mechanisms by which NTHi can influence GR function, focusing on GR phosphorylation and the modulation of GR function by NTHi induced p38 MAPK activation. We also investigated the effects of NTHi exposure on COPD macrophage polarisation.
Discussion
NTHi caused increased cytokine secretion by COPD alveolar macrophages that was poorly responsive to corticosteroids, with CXCL8 being completely corticosteroid resistant. This corticosteroid insensitivity was present in both COPD and control macrophages. NTHi caused GR S226 phosphorylation, which is known to be associated with enhanced nuclear export [
22]. GR S226 phosphorylation after NTHi exposure was regulated by p38 MAPK activation. p38 MAPK inhibition enhanced GR nuclear localisation, suggesting a synergistic interaction between p38 MAPK inhibitors and corticosteroids. However, combination treatment with a p38 MAPK inhibitor and corticosteroid caused increased anti-inflammatory effects compared to either drug alone in an additive rather than synergistic manner.
Acute and chronic infection with NTHi is a common feature in COPD patients [
5]. Using an in vitro macrophage model, our results suggest that corticosteroids do not sufficiently address the innate immune response caused by NTHi in COPD patients. In contrast, there is emerging evidence that ICS effects are greater in COPD patients with higher levels of blood eosinophils [
23,
24]. Overall, one can propose that ICS more effectively target eosinophilic and/or T2 inflammation in COPD [
6,
7], rather than inflammation caused by bacterial infection.
LPS from
E.coli has frequently been used to study corticosteroid effects on COPD macrophages [
13,
15,
25]. However,
E.coli is not a clinically relevant respiratory pathogen in COPD. Live NTHi stimulates TLR4 and TLR2 through the ligands LOS and outer membrane protein P6 respectively [
26,
27], and also causes inflammatory responses through activation of other cellular signalling mechanisms [
28]. Corticosteroid inhibition varies between cytokines secreted by LPS stimulated COPD macrophages, with a reduced effect on CXCL8 [
13‐
15]. We observed no significant inhibition of CXCL8 secretion after NTHi exposure, in keeping with Cosio et al., who reported no inhibition of CXCL8 as well as IL-1β and IL-6 [
16]. We observed approximately 40% inhibition of IL-6; these numerical differences between the studies may be due to methodological differences. Nevertheless, both studies report either a partial or no effect of corticosteroids on a subset of cytokines including CXCL8 and IL-6. CXCL8 is a neutrophil chemoattractant, with increased levels in COPD compared to control lungs [
29]. The poor suppression of NTHi stimulated CXCL8 secretion by corticosteroids is likely to be a clinically relevant observation, highlighting an inflammatory pathway in COPD patients with airway bacterial colonisation that can promote neutrophilic inflammation despite corticosteroid treatment.
The effects of corticosteroids were similar in COPD patients compared to smokers, with any differences being small in magnitude and inconsistent. These findings are similar to previous observations using LPS stimulated macrophages [
13‐
15].
The effects of corticosteroids vary between LPS stimulated genes in mouse macrophages [
30], due to variation in GR-ligand complex activity at different pro-inflammatory gene promotor regions according to the transcription factors involved. For CXCL8, a high degree of NF-κB activation decreases corticosteroid effects [
31]; this might be important in our study, as we demonstrated NF-κB activation after NTHi exposure.
NHTi exposure caused GR S226 phosphorylation. There are numerous phosphorylation sites within the GR N-terminus with serine (S) S211 and S226 having functional importance and roles in subcellular localisation [
9,
32]; GR-ligand nuclear translocation is associated with phosphorylation of S211, and S226 phosphorylation known to cause nuclear GR export [
22]. We observed that GR S226 phosphorylation was associated with decreased nuclear GR expression when cells were exposed to NTHi plus corticosteroid compared to corticosteroid alone. p38 MAPK can phosphorylate GR serine residues [
10,
11]. This effect is cell type dependent [
12]. We observed that p38 MAPK inhibition reduced the effect of NTHi on GR S226 phosphorylation, and thereby opposed the effect of NTHi on GR nuclear localisation.
In PBMCs from COPD patients, p38 MAPK inhibition reduced S211 phosphorylation in the presence of corticosteroid [
12]. Our different results highlight potential differences that can occur due to cell type and stimulus and it would be interesting to replicate these finding using alveolar macrophages obtained from bronchoalveolar lavage. PBMCs from severe asthma patients showed a reduction of GR nuclear localisation that was associated with increased GR S226 phosphorylation [
33], supporting the association of S226 phosphorylation and reduced corticosteroid effects.
The enhancement of GR nuclear localisation by BIRB-796 after macrophage exposure to both corticosteroid and NTHi is potentially a synergistic interaction between drug classes that may increase the anti-inflammatory effects of corticosteroids. We observed that BIRB-796 combined with dexamethasone caused significantly greater inhibition of all cytokines compared to either drug used alone. IR analysis showed this effect to be additive rather than synergistic. However, neither drug alone had a statistically significant effect on CXCL8, while the combination achieved significant inhibition (48.4%), suggestive of more than an additive effect although the IR failed to meet the criteria for synergy. The failure to demonstrate synergy on cytokine production may be due to experimental design, as combining full dose response curves is the optimum methodology [
34,
35]. Nevertheless, these findings suggest that combining these drug classes can increase anti-inflammatory effects during bacterial infection.
Acute polarisation to a pro-inflammatory macrophage phenotype occurs in response to diverse bacteria [
36]. NTHi caused an upregulation of TNF-α and CXCL8 transcription, with CXCL8 transcription being persistently increased at 24 h. This prolonged CXCL8 production is likely to contribute to persistent neutrophilic airway inflammation in COPD patients colonised with NTHi. CD38 is involved in intracellular calcium regulation, macrophage phagocytosis and cytokine release [
37]. NTHi upregulated CD38 gene expression levels in COPD alveolar macrophages, which may also enhance pro-inflammatory responses. In contrast, CD14 mediates the inflammatory response to bacterial LPS, and NTHi downregulation of CD14 agrees with previous findings in COPD monocytes [
38]. This is a mechanism that may limit the pro-inflammatory response.
Bacteria can regulate macrophage programming to give an immunoregulatory phenotype aiding survival within the host [
36]. NTHi decreased HLA-DR gene expression levels, which could favor immune tolerance and NTHi persistence. Pons et al. reported reduced HLA-DR expression in COPD compared to control alveolar macrophages, although bacterial airway colonization was not investigated [
39]. COPD macrophages display defective phagocytosis of pathogens and efferocytosis of apoptotic cells [
40,
41]. CD36 is involved in efferocytosis [
42], while mannose receptor (CD206) is involved in both phagocytosis and efferocytosis [
41]. CD163 is an innate immune sensor of bacteria, and higher CD163 expression has been reported in COPD macrophages [
43]. The downregulation of CD36, CD206 and CD163 by NTHi reported here may contribute to bacterial persistence in the airways and facilitate defective macrophage function in COPD. The increased IL-10 production caused by NTHi may also promote in bacterial persistence, as this immunoregulatory cytokine can facilitate chronic infections such as mycobacteria [
44].
Overall, these gene expression experiments indicate that NTHi causes phenotypic changes in COPD alveolar macrophages that promote prolonged neutrophilic inflammation through CXCL8 production, and may also favour bacterial persistence, and reduced phagocytosis and efferocytosis. These hypothesis generated by gene expression experiments remain to be confirmed in functional experiments.
Acknowledgements
This report is independent research supported by National Institute for Health Research South Manchester Respiratory and Allergy Clinical Research Facility at University Hospital of South Manchester NHS Foundation Trust. The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health.
We would also like to acknowledge Dr. Nicola High (University of Manchester, Oxford Road, Manchester) for providing the NTHi strain R2846.