Jump to comment:

• Challenges in characterising neutrophil subsets in bronchial lavage of obstructive airway disease participants using a CD16/CD62L phenotyping.
  Ravi Lokwani, Peter AB Wark, Katherine J Baines, Daniel Barker and Jodie L Simpson
  Published on: 9 April 2019
• Unravelling neutrophil heterogeneity in chronic airway disease
  Mohib Uddin, Piet L. B. Bruijnzeel and Leo Koenderman
  Published on: 1 March 2019

• Published on: 9 April 2019

  Challenges in characterising neutrophil subsets in bronchial lavage of obstructive airway disease participants using a CD16/CD62L phenotyping.

  • Ravi Lokwani, PhD student School of Medicine and Public Health, Faculty of Health and Medicine, University of Newcastle, Callaghan NSW 2308, Australia.
  • Other Contributors:
    • Peter AB Wark, Conjoint Professor
    • Katherine J Baines, Australian Lung Foundation COPD Reseach Fellow
    • Daniel Barker, Scholarly Teaching Fellow- Medical Statistics
    • Jodie L Simpson, Assistant Dean Research & Deputy Head of School of Medicine and Public Health, University of Newcastle, Australia.

  We thank Uddin and colleagues for their interest and response to our research paper on “Hypersegmented airway neutrophils and its association with reduced lung function in adults with obstructive airway disease: an exploratory study”. The neutrophil dominated inflammation in obstructive airway disease and its non-responsiveness toward classical therapies such as inhaled corticosteroids[1], validate the necessity for a more sophisticated approach[2]. The development in neutrophil-targeted therapies such as CXCR2 inhibitors have been met with safety challenges due to reductions in absolute neutrophil counts below the safety threshold [3, 4]. Besides targeting neutrophil influx, other strategies targeting neutrophil function such as the neutrophil elastase inhibitor AZ9668 were found effective in animal models of COPD but not for COPD patients[5]. Therefore, the concept of targeting disease specific or a more aggressive neutrophil subset instead of targeting the overall neutrophil population, is an alternative strategy that may more accurately target dysregulated neutrophilic inflammation in clinical practice [6].

  In this study, we excluded participants if they reported a chest infection or exacerbation of airways disease within 4 weeks of bronchoscopy, and at the time the clinician regarded them as clinically stable, excluding acute clinical infection. However, it is important to recognise that bacteria can be present in the airways in the absence of acute infection...

  Show More

  We thank Uddin and colleagues for their interest and response to our research paper on “Hypersegmented airway neutrophils and its association with reduced lung function in adults with obstructive airway disease: an exploratory study”. The neutrophil dominated inflammation in obstructive airway disease and its non-responsiveness toward classical therapies such as inhaled corticosteroids[1], validate the necessity for a more sophisticated approach[2]. The development in neutrophil-targeted therapies such as CXCR2 inhibitors have been met with safety challenges due to reductions in absolute neutrophil counts below the safety threshold [3, 4]. Besides targeting neutrophil influx, other strategies targeting neutrophil function such as the neutrophil elastase inhibitor AZ9668 were found effective in animal models of COPD but not for COPD patients[5]. Therefore, the concept of targeting disease specific or a more aggressive neutrophil subset instead of targeting the overall neutrophil population, is an alternative strategy that may more accurately target dysregulated neutrophilic inflammation in clinical practice [6].

  In this study, we excluded participants if they reported a chest infection or exacerbation of airways disease within 4 weeks of bronchoscopy, and at the time the clinician regarded them as clinically stable, excluding acute clinical infection. However, it is important to recognise that bacteria can be present in the airways in the absence of acute infection. This presence of potentially pathogenic bacteria may be implicated in neutrophil heterogeneity in the airways. Previous studies have shown associations between an increase in bacterial load of potential pathogenic bacteria and increases in neutrophils, and neutrophil chemokines such as CXCL-8 [7, 8] suggesting the presence of bacteria is linked to neutrophil activation, however further research is needed to understand the impact of bacteria on neutrophil heterogeneity.

  Immunophenotyping of our harvested bronchial lavage cells with validated makers such as CD16 and CD62L would certainly complement our existing findings. While we were not able to perform cell surface staining in this study, due to low cell numbers, we are pursuing this with blood neutrophils. While circulatory neutrophils jettison the CD62L as they move to sites of inflammation [9], airway neutrophils in COPD feature completely abrogated expression of CD62L [10, 11]. This diminished expression of CD62L on airway neutrophils poses a technical challenge in gating and characterizing dim and high CD62L population in comparison to systemic circulation model [12]. Therefore, some other markers apart from CD62L are needed to be explored for immunophenotyping of airway neutrophils.

  Uddin and colleagues also raised a very important point regarding measuring pro-neutrophilic factors present in bronchial lavage to understand the reason behind the increased presence of hypersegmented neutrophils and we concur this would be interesting to assess, though the dilutional impact of bronchial lavage has often made measuring chemokines and cytokines more variable than is seen with other body fluids such as induced sputum or blood. After migrating to the airways, neutrophils can undergo programmed cell death (apoptosis) or alternately they can undergo necrosis or NETosis, depending on the level of activation[13]. NETosis involves the formation of neutrophil extracellular traps (NET), where granular components complexed with nuclear DNA is expelled from the neutrophil to trap and kill the pathogen in its web like structure [14]. Recent studies have demonstrated an association between elevated sputum neutrophil extracellular traps (NETs) and reduced lung function in asthma and COPD [15, 16], suggesting a role for NETs in pathogenesis of chronic inflammatory airway disease. Hypersegmented neutrophils have been recently reported for their enhanced NET producing ability in head and neck squamous cell carcinoma [17]. It is interesting to speculate that the increased hypersegmented neutrophils observed in our report could also be associated with a preferential deployment of NETs in the airways. The hypersegmented neutrophils have also been reported to have a defect in intracellular killing following phagocytosis [18]. While impaired and reduced phagocytosis by neutrophils have also been reported in obstructive airway disease such as COPD [19, 20], it not clear whether this corresponds to a specific subset of neutrophils.

  In conclusion, further studies are now required not only for a better understanding of the functional behaviour of these subsets but also to understand the mechanism behind neutrophil heterogeneity in airways.

  1. Wang, M., et al., Impaired anti-inflammatory action of glucocorticoid in neutrophil from patients with steroid-resistant asthma. Respiratory research, 2016. 17(1): p. 153-153.
  2. Seys, S.F., et al., New insights in neutrophilic asthma. Curr Opin Pulm Med, 2019. 25(1): p. 113-120.
  3. Caramori, G., et al., Cytokine inhibition in the treatment of COPD. Int J Chron Obstruct Pulmon Dis, 2014. 9: p. 397-412.
  4. Rennard, S.I., et al., CXCR2 Antagonist MK-7123. A Phase 2 Proof-of-Concept Trial for Chronic Obstructive Pulmonary Disease. Am J Respir Crit Care Med, 2015. 191(9): p. 1001-11.
  5. Durham, A.L., et al., Targeted anti-inflammatory therapeutics in asthma and chronic obstructive lung disease. Translational Research, 2016. 167(1): p. 192-203.
  6. Bruijnzeel, P.L., M. Uddin, and L. Koenderman, Targeting neutrophilic inflammation in severe neutrophilic asthma: can we target the disease-relevant neutrophil phenotype? J Leukoc Biol, 2015. 98(4): p. 549-56.
  7. Wood, L.G., et al., Potentially pathogenic bacteria cultured from the sputum of stable asthmatics are associated with increased 8-isoprostane and airway neutrophilia. Free Radic Res, 2010. 44(2): p. 146-54.
  8. Kettritz, R., et al., Interleukin-8 delays spontaneous and tumor necrosis factor-alpha-mediated apoptosis of human neutrophils. Kidney Int, 1998. 53(1): p. 84-91.
  9. Mann, B.S. and K.F. Chung, Blood neutrophil activation markers in severe asthma: lack of inhibition by prednisolone therapy. Respir Res, 2006. 7: p. 59.
  10. Blidberg, K., et al., Adhesion molecules in subjects with COPD and healthy non-smokers: a cross sectional parallel group study. Respir Res, 2013. 14: p. 47.
  11. Mallia, P., et al., Neutrophil adhesion molecules in experimental rhinovirus infection in COPD. Respiratory research, 2013. 14(1): p. 72-72.
  12. Pillay, J., et al., A subset of neutrophils in human systemic inflammation inhibits T cell responses through Mac-1. J Clin Invest, 2012. 122(1): p. 327-36.
  13. Amulic, B., et al., Neutrophil function: from mechanisms to disease. Annu Rev Immunol, 2012. 30: p. 459-89.
  14. Brinkmann, V., et al., Neutrophil extracellular traps kill bacteria. Science, 2004. 303(5663): p. 1532-5.
  15. Dicker, A.J., et al., Neutrophil extracellular traps are associated with disease severity and microbiota diversity in patients with chronic obstructive pulmonary disease. J Allergy Clin Immunol, 2018. 141(1): p. 117-127.
  16. Wright, T.K., et al., Neutrophil extracellular traps are associated with inflammation in chronic airway disease. Respirology, 2016. 21(3): p. 467-75.
  17. Millrud, C.R., et al., NET-producing CD16(high) CD62L(dim) neutrophils migrate to tumor sites and predict improved survival in patients with HNSCC. Int J Cancer, 2017. 140(11): p. 2557-2567.
  18. Leliefeld, P.H.C., et al., Differential antibacterial control by neutrophil subsets. Blood Adv, 2018. 2(11): p. 1344-1355.
  19. Shanmugam, L., et al., Assessment of phagocytic activity of neutrophils in chronic obstructive pulmonary disease. Lung India, 2015. 32(5): p. 437-40.
  20. Thomas, C., P. Barnes, and L. Donnelly, Reduced phagocytosis of pathogenic bacteria by neutrophils from COPD patients. European Respiratory Journal, 2012. 40(Suppl 56): p. 387.

  Show Less

  Conflict of Interest:
  None declared.

  • Back to top
• Published on: 1 March 2019

  Unravelling neutrophil heterogeneity in chronic airway disease

  • Mohib Uddin, Translational Medical Scientist Respiratory Global Medicines Development, AstraZeneca, Gothenburg, Sweden
  • Other Contributors:
    • Piet L. B. Bruijnzeel, Professor
    • Leo Koenderman, Professor

  To the Editor:

  Human neutrophils play an immunoregulatory role in innate immunity, inflammation and resolution. Airway neutrophilia is a distinguishing feature of chronic airways disease, including COPD and severe asthma [1]. Evidence incriminating neutrophils in the immunopathogenesis of these diseases have provided a rationale for novel drugs discovery selectively targeting inflammatory pathways [1, 2]. However, the extent to which this may be linked to disease pathobiology remains unresolved.

  Advances in facets of neutrophil biology have uncovered novel observations, challenging the traditional view of the neutrophil as a short-lived, terminally differentiated homogeneous cell with a limited lifespan. Newly emerging functions of neutrophils have been reported, including neutrophil extracellular trap (NET) formation, consisting of ejected DNA microwebs that can entrap pathogens [3]. Importantly, there has been a paradigm shift in recent years with the realisation of heterogenous neutrophil subsets that may perform disparate immunomodulatory roles in homeostasis, infection and disease [4]. The group led by Koenderman and his colleagues in 2012 were the first to report the comprehensive characterisation of three distinct cellular subsets of circulating human neutrophils following endotoxin challenge in an experimental model of acute systemic inflammation in human healthy volunteers [5]. In parallel, a similar compartmentalisation has been shown in cancer immu...

  Show More

  To the Editor:

  Human neutrophils play an immunoregulatory role in innate immunity, inflammation and resolution. Airway neutrophilia is a distinguishing feature of chronic airways disease, including COPD and severe asthma [1]. Evidence incriminating neutrophils in the immunopathogenesis of these diseases have provided a rationale for novel drugs discovery selectively targeting inflammatory pathways [1, 2]. However, the extent to which this may be linked to disease pathobiology remains unresolved.

  Advances in facets of neutrophil biology have uncovered novel observations, challenging the traditional view of the neutrophil as a short-lived, terminally differentiated homogeneous cell with a limited lifespan. Newly emerging functions of neutrophils have been reported, including neutrophil extracellular trap (NET) formation, consisting of ejected DNA microwebs that can entrap pathogens [3]. Importantly, there has been a paradigm shift in recent years with the realisation of heterogenous neutrophil subsets that may perform disparate immunomodulatory roles in homeostasis, infection and disease [4]. The group led by Koenderman and his colleagues in 2012 were the first to report the comprehensive characterisation of three distinct cellular subsets of circulating human neutrophils following endotoxin challenge in an experimental model of acute systemic inflammation in human healthy volunteers [5]. In parallel, a similar compartmentalisation has been shown in cancer immunology by the discovery of myeloid-derived suppressor cells [6, 7] and the tumour-associated neutrophil subtypes, N1 and N2 [8].

  Surface marker immunophenotyping of acute inflammation-driven cellular events revealed an immunosuppressive subset characterised by a higher number of nuclear lobes (hypersegmented) [CD16high/CD62Llow] that had the capacity to suppress T-cell proliferation [5]. The other two subsets, consisted of immature neutrophils with a banded nucleus [CD16low/CD62Lhigh] and mature neutrophils with a normal segmented nucleus [CD16high/CD62Lhigh] [5]. With these novel findings in humans, this led us to first hypothesise the concept of distinct neutrophil subtypes in the airways of patients with severe neutrophilic asthma [9].

  In the recent issue of BMJ Open, Lokwani and colleagues confirm this heterogeneity theory in adult patients with obstructive airway disease (asthma, COPD and bronchiectasis) [10]. Using morphometric analysis of bronchial lavage (BL), they observed three different subsets of neutrophils which were classified by nuclear morphology, notably banded (1 lobe), normal (2-4 lobes), and hypersegmented neutrophils (>4 lobes). Elevated numbers of the hypersegmented neutrophils were observed in the BL of patients with obstructive airway disease that was associated with diminished lung function compared with healthy controls. There were a number of notable differences in the appearance of the absolute cell counts of the morphologically different subsets in the various airway diseases. As the authors report [10], increased numbers of hypersegmented neutrophils were observed in the airways of patients with COPD compared with asthma and bronchiectasis. The numbers of normal neutrophils were raised in both bronchiectasis and COPD compared to asthma. Interestingly, the highest numbers of banded neutrophils were observed in patients with bronchiectasis relative to the asthmatics. It is uncertain why the patients with COPD have an airway infiltrate dominated by hypersegmented neutrophils, while in the bronchiectasis group they have a banded-predominance of neutrophil infiltrates. These findings indicate that morphologically distinct neutrophils exist, and that these subsets are likely to confer differing immunomodulatory roles in a disease-specific context.

  Several important points should be considered regarding their observations of neutrophil heterogeneity in the airways of patients with chronic airways disease. Firstly, whether any disease-associated aberrations (infection and exacerbation) were noted before airway sampling in the susceptible patients. From much of our translational research demonstrating a preferential recruitment of hyper-segmented cells [CD16high/CD62Llow] into the circulation in response to acute inflammation [5], we would anticipate a proportional increase of these subsets in the airways following episodes of exacerbations triggered by airway infection/injury and/or inflammation.

  Secondly, what drives the spatial and temporal expansion of the hyper-segmented cells [CD16high/CD62Llow] to ingress within the airways in proportion to the other subsets is uncertain. The inflamed airways may constitute a micro-environment conducive to the preferential viability of these subsets, facilitating their unrestrained arrival and survival in these treatment-refractory diseases. These hypersegmented cells [CD16high/CD62Llow] have also been observed in blood and alveolar compartments from patients with acute respiratory distress syndrome (ARDS) exhibiting a distinct prosurvival profile [11]. It should be emphasised that the hypersegmented neutrophils [CD16high/CD62Llow] present in the circulation during acute inflammation are not an aged sub-population relative to their normal mature counterparts [CD16high/CD62Lhigh] [12] and might consist of a distinct phenotype that are generated via a separate life cycle. It is noteworthy that dysregulated human neutrophil apoptosis has also been reported in the airways of severe asthmatics [13], possibly attributed to the release of epithelial-derived pro-neutrophilic mediators, including GM-CSF [14]. Hence further characterisation in elucidating the pro-neutrophilic factors present in the serum or BL in patients with chronic airways disease that coordinate the regulation of the neutrophil subtype dominance observed (and their longevity) are warranted.

  Thirdly, appearance of the hypersegmented subsets [CD16high/CD62Llow], seem to be more prominent in patients with COPD who exhibited diminished lower lung function, in particular, worsened airflow obstruction. Moreover, they were on high-dose inhaled corticosteroids, had increased airway bacterial colonisation (40%) and had a 35.0 pack-year smoking history. A possible explanation for these observations could be confounded by indication of glucocorticoid therapy which has the capacity to delay apoptosis of neutrophils, while inducing this process in eosinophils [15]. Hence, the high daily doses of inhaled corticosteroid (ICS) in the patients with COPD is worth noting (1700 ug/day) and may have affected the enhanced proportion of the hypersegmented neutrophils observed. Recently, the production of NETs has been detected in the airways of subjects with asthma and COPD by several investigators [16]. Increased NET formation has been reported during acute exacerbations of COPD as well as in stable phases of COPD and shown to correlate significantly with the severity of airflow obstruction [17, 18] . Importantly, NET formation occurs in vivo in response to respiratory viral (e.g. Rhinovirus) and bacterial (e.g. Staphylococcus aureus) infection, as well to cigarette smoke exposure [16]. These airborne insults are common triggers for COPD exacerbations, underscoring a pathobiological link between NETs formation and COPD-related airway inflammation. The premise relating hypersegmented subset of neutrophils to the NETopathic inflammation of chronic airway disease forms an attractive clinicopathobiological proposition [16]. Could the hypersegmented subset of neutrophils observed, represent the preferential deployment of NET-forming subsets in the COPD airways and explain these observations? This relationship warrants further investigation in future studies.

  Finally, hypersegmented neutrophils are phenotypically and functionally heterogeneous to the other neutrophil subsets [5]. Recent translational findings indicate that whilst this specific subset displayed normal phagocytosing capacity, they had major defects in the intracellular killing of Staphylococcus aureus [19]. This may contribute to the bacterial colonisation of the lower airways and to the propensity for pathogen-initiated exacerbations in chronic airways disease. However, from an immunopathogenic perspective, the accumulation of augmented numbers of hypersegmented neutrophils in the inflamed airways might serve as an integrated part of the host immune response where they are deployed to combat ensuing bacterial burden.

  While the study by Lokwani and co-workers provides important evidence of morphologically different populations of neutrophils in patients with chronic airways disease, there are some limitations. For instance, immunophenotyping of the harvested BL neutrophils using validated surface markers [CD16/CD62L expression] to complement their existing observations of the hypersegmented subsets defined by morphometric analysis, would have substantiated their conclusions. Characterisation of pro-neutrophilic signals present in the inflamed airways, as already alluded to, would have potentially supported determining what drives the switch of preferential neutrophil subtype deployment into the airways in a disease-specific manner. In terms of functionality, phagocytic assays studies would have provided further information underlying the immune function and malfunction of these distinct subsets in the airways, however this may have been limited due to the harvesting of finite hypersegmented neutrophils during airway sampling. Further, the authors acknowledge, that the cross-sectional analysis in a small patient sample size is another shortcoming of their study. Going forward, real-time assessment of the cellular dynamics and transient entry of these distinct subsets neutrophils into the blood and airway compartments would provide intriguing insights regarding their potential involvement in disease activity and severity in a larger cohort of patients using the appropriate study controls (i.e. healthy smokers).
  Further translational studies addressing such issues can help us better understand the molecular mechanisms underpinning neutrophil heterogeneity and plasticity in pathophysiological settings. The growing realisation of phenotypically distinct neutrophils presents the opportunity for the development of novel therapeutics for disease-specific phenotypes in respiratory medicine.

  *Correspondence to:

  Professor Leo Koenderman,
  Department of Respiratory Medicine,
  University Medical Center Utrecht,
  Heidelberglaan Utrecht,
  The Netherlands
  L.Koenderman@umcutrecht.nl

  References:

  1. Barnes PJ. Targeting cytokines to treat asthma and chronic obstructive pulmonary disease. Nat Rev Immunol 2018;18(7):454-66. doi: 10.1038/s41577-018-0006-6
  2. Chung KF. Neutrophilic asthma: a distinct target for treatment? Lancet Respir Med 2016;4(10):765-67. doi: 10.1016/S2213-2600(16)30232-6
  3. Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular traps kill bacteria. Science 2004;303(5663):1532-5. doi: 10.1126/science.1092385
  4. Deniset JF, Kubes P. Recent advances in understanding neutrophils. F1000Research 2016;5:2912. doi: 10.12688/f1000research.9691.1
  5. Pillay J, Kamp VM, van Hoffen E, et al. A subset of neutrophils in human systemic inflammation inhibits T cell responses through Mac-1. J Clin Invest 2012;122(1):327-36. doi: 10.1172/jci57990
  6. Brandau S, Trellakis S, Bruderek K, et al. Myeloid-derived suppressor cells in the peripheral blood of cancer patients contain a subset of immature neutrophils with impaired migratory properties. J Leukoc Biol 2011;89(2):311-7. doi: 10.1189/jlb.0310162
  7. Pillay J, Tak T, Kamp VM, et al. Immune suppression by neutrophils and granulocytic myeloid-derived suppressor cells: similarities and differences. Cellular and molecular life sciences : CMLS 2013;70(20):3813-27. doi: 10.1007/s00018-013-1286-4
  8. Fridlender ZG, Sun J, Kim S, et al. Polarization of tumor-associated neutrophil phenotype by TGF-beta: "N1" versus "N2" TAN. Cancer cell 2009;16(3):183-94. doi: 10.1016/j.ccr.2009.06.017
  9. Bruijnzeel PL, Uddin M, Koenderman L. Targeting neutrophilic inflammation in severe neutrophilic asthma: can we target the disease-relevant neutrophil phenotype? J Leukoc Biol 2015;98(4):549-56. doi: 10.1189/jlb.3VMR1214-600RR
  10. Lokwani R, Wark PAB, Baines KJ, et al. Hypersegmented airway neutrophils and its association with reduced lung function in adults with obstructive airway disease: an exploratory study. BMJ open 2019;9(1):e024330. doi: 10.1136/bmjopen-2018-024330
  11. Juss JK, House D, Amour A, et al. Acute Respiratory Distress Syndrome Neutrophils Have a Distinct Phenotype and Are Resistant to Phosphoinositide 3-Kinase Inhibition. American journal of respiratory and critical care medicine 2016;194(8):961-73. doi: 10.1164/rccm.201509-1818OC
  12. Tak T, Wijten P, Heeres M, et al. Human CD62L(dim) neutrophils identified as a separate subset by proteome profiling and in vivo pulse-chase labeling. Blood 2017;129(26):3476-85. doi: 10.1182/blood-2016-07-727669
  13. Uddin M, Nong G, Ward J, et al. Prosurvival activity for airway neutrophils in severe asthma. Thorax 2010;65(8):684-9. doi: 10.1136/thx.2009.120741
  14. Uddin M, Lau LC, Seumois G, et al. EGF-induced bronchial epithelial cells drive neutrophil chemotactic and anti-apoptotic activity in asthma. PLoS One 2013;8(9):e72502. doi: 10.1371/journal.pone.0072502
  15. Hauber HP, Gotfried M, Newman K, et al. Effect of HFA-flunisolide on peripheral lung inflammation in asthma. J Allergy Clin Immunol 2003;112(1):58-63.
  16. Uddin M, Watz H, Malmgren A, et al. NETopathic inflammation in Chronic Obstructive Pulmonary Disease and Severe Asthma. Front Immunol 2019; doi:10.3389/fimmu.2019.00047
  17. Pedersen F, Marwitz S, Holz O, et al. Neutrophil extracellular trap formation and extracellular DNA in sputum of stable COPD patients. Respir Med 2015;109(10):1360-2. doi: 10.1016/j.rmed.2015.08.008
  18. Grabcanovic-Musija F, Obermayer A, Stoiber W, et al. Neutrophil extracellular trap (NET) formation characterises stable and exacerbated COPD and correlates with airflow limitation. Respir Res 2015;16:59. doi: 10.1186/s12931-015-0221-7
  19. Leliefeld PHC, Pillay J, Vrisekoop N, et al. Differential antibacterial control by neutrophil subsets. Blood Adv 2018;2(11):1344-55. doi: 10.1182/bloodadvances.2017015578

  Show Less

  Conflict of Interest:
  LK and PLBB declare no potential conflict of interest. MU is an employee of AstraZeneca and holds share in the company.

  • Back to top