Neutrophil subsets in airway inflammation and hyperreactivity

Neutrophils are part of the first lines of defence against invading microbes. They play an essential role in antimicrobial host defence by recognizing microorganisms through the various receptors that can be expressed on their surfaces. The known function of neutrophils in the airway inflammatory process has changed over time, from being an expendable cell limited to phagocytosis, releasing enzymes and other cytotoxic agents, to a dedicated cell that can release mediators with a more precise role in the defence and disease development. Identifying pathways that are active during inflammation and its resolution is central for understanding the mechanisms behind disease development and conjure a foundation for the development of new therapeutic strategies. Novel subsets with different functions of already classified cells are continuously discovered. In line with this, four different neutrophil subsets have been identified based on their expression of CD 16 and CD62L. These subsets reflect different stages of cell maturity and activity. The dim/high neutrophils are considered imma­ture as they are recently derived from the bone marrow. The high/high neutrophils are considered mature and the high/dim neutrophils are activated. The dim/dim neutrophils are believed to be active in a stage just before apoptosis.

In paper I, we studied the distribution of neutrophil subsets in blood, nasal biopsies and nasal lavage in healthy controls and allergic patients. We saw that healthy subjects and allergic patients harboured high/high neutrophils in blood and nasal biopsies. High/dim neutrophils were found in the nasal biopsies and to some extent in the nasal lavage, whereas the dim/dim neutrophils were only found in the nasal lavage. However, among the allergic patients the distribution in nasal biopsies was skewed towards high/dim neutrophils. The high/high and high/dim neutrophils were used in functional assays to study their effects on T-cells and eosinophils. The high/dim neutrophils had the ability to prime the CD4+ T-cells and function as a chemotactic factor for the eosinophils. This shows that the high/ dim neutrophil might have a role in the development of allergy.

In paper II, neutrophils from healthy subjects and allergic asthma patients were obtained and stimulated in order to study markers crucial for their clearance. Stimulated neutrophils from patients with asthma had much higher expression of "don't eat me" markers than neutrophils from healthy subjects. The former neu­trophils also had reduced production ofCCL3, CCL4 and CLL20, which affected the migration rate of monocytes. The findings may explain the prolonged duration of an infection that allergic asthmatics often experience.

Paper III-IV characterised neutrophil subsets in blood before and after an inhaled allergen provocation. The fraction high/high neutrophils decreased and the high/ dim neutrophils increased as a result of the challenge. To evaluate the effects of high/high and high/dim neutrophils on airways; human bronchi or mice trachea were co-cultured with the different subsets. The functional changes caused by the co-cultures were then evaluated in a myograph. The high/dim neutrophils increased the response towards bradykinin. They also enhanced contraction induced by nerve-mediated stimulation. The increase in the bradykinin response was related to a release of TNFɑ that subsequently upregulated the bradykinin receptor 2. The nerve-mediated airway hyper-responsiveness in conjunction with high/dim neutrophils was due to production of IL-1β that caused an increase of substance Pin the nerves via COX-2. These new findings may lead to a better understand­ing of the role of neutrophils in severe asthma, and potentially to new treatments.

In summary, the thesis demonstrates that the distribution of the neutrophil subsets differs between healthy subjects and allergic individuals and that the different subsets might have diverse effects on the airways.

List of scientific papers

I. Are bro J, Ekstedt S, Hjalmarsson E, Winqvist 0, Kumlien Georen S, Cardell LO. A possible role for neutrophils in allergic rhinitis revealed after cellular sub­classification. Sci Rep. 2017;7:43568.
https://doi.org/10.1038/srep43568

II. Sandra Ekstedt, Ellen Tufvesson, Bjermer L, Susanna Kumlien Georen and Lars Olaf Cardell. "Don't eat me" markers indicate that neutrophils from asthmatic patients resist phagoptosis, explaining the occurrence of prevailing airway infections. [Manuscript]

III. Ekstedt S, Stenberg H, Tufvesson E, Diamant Z, Kumlien Georen S, et al. The potential role of CD 16highCD62Ldim neutrophils in the allergic asthma. Allergy. 2019 Nov;74(11):2265-2268.
https://doi.org/10.1111/all.13861

IV. Ekstedt S, Safholm J, Kumlien Georen S, Cardell LO. Dividing neutrophils in subsets, reveals a significant role for activated neutrophils in the develop­ment of airway hyperreactivity. Clin Exp Allergy. 2019 Mar;49(3):285-291.
https://doi.org/10.1111/cea.13311

V. Sandra Ekstedt, Olivia Larsson, Susanna Kumlien Georen and Lars Olaf Cardell. CD 16highCD62Ldim neutrophils induce nerve mediated airway hyper­reactivity. [Manuscript]