Multi-dimensional flow cytometry analysis reveals increasing changes in the systemic neutrophil compartment during seven consecutive days of endurance exercise

PLoS One. 2018 Oct 30;13(10):e0206175. doi: 10.1371/journal.pone.0206175. eCollection 2018.

Abstract

Endurance exercise is associated with a transient increase in neutrophil counts in the peripheral blood. Here we investigate the impact of intensified endurance exercise on the neutrophil compartment. We hypothesized that intensified endurance exercise leads to mobilization of neutrophil subsets, which are normally absent in the blood. Furthermore, we followed the potential build-up of neutrophil activation and the impact on overnight recovery of the neutrophil compartment during a seven-day cycling tour. The neutrophil compartment was studied in 28 healthy amateur cyclists participating in an eight-day strenuous cycling tour. Blood samples were taken at baseline, after 4 days and after 7 days of cycling. The neutrophil compartment was analyzed in terms of numbers and its phenotype by deep phenotyping of flow cytometry data with the multi-dimensional analysis method FLOOD. Repeated endurance exercise led to a gradual increase in total neutrophil counts over the days leading to a 1.26 fold-increase (95%CI 1.01-1.51 p = 0.0431) in the morning of day 8. Flow cytometric measurements revealed the appearance of 2 additional neutrophil subsets: CD16brightCD62Ldim and CD16dimCD62Lbright. A complex change in neutrophil phenotypes was present characterized by decreased expression of both CD11b and CD62L and marked increased expression of LAIR-1, VLA-4 and CBRM1/5. The changes in expression were found on all neutrophils present in the blood. Strikingly, in strong contrast to our findings during acute inflammation evoked by LPS challenge, these neutrophils did not upregulate classical degranulation markers. In fact, our FLOOD analysis revealed that the exercise induced neutrophil phenotype did not overlap with the neutrophil subsets arising upon acute inflammation. In conclusion, during multiple days of endurance exercise the neutrophil compartment does not regain homeostasis overnight. Thereby our study supports the concept of a build-up of inflammatory cues during repeated endurance exercise training, causing a prolonged change of the systemic neutrophil compartment.

MeSH terms

  • Adult
  • Bicycling
  • Blood Cell Count
  • CD11b Antigen / metabolism
  • Female
  • Flow Cytometry / methods*
  • GPI-Linked Proteins / metabolism
  • Gene Expression Regulation
  • Healthy Volunteers
  • Humans
  • Integrin alpha4beta1 / metabolism
  • L-Selectin / metabolism
  • Male
  • Middle Aged
  • Neutrophils / cytology*
  • Neutrophils / immunology*
  • Phenotype
  • Physical Endurance / physiology*
  • Receptors, IgG / metabolism
  • Receptors, Immunologic / metabolism

Substances

  • CD11b Antigen
  • FCGR3B protein, human
  • GPI-Linked Proteins
  • ITGAM protein, human
  • Integrin alpha4beta1
  • Receptors, IgG
  • Receptors, Immunologic
  • leukocyte-associated immunoglobulin-like receptor 1
  • L-Selectin

Grants and funding

This research received funding from the Netherlands Organisation for Scientific Research (NWO) in the context of a private public partnership Technology Area COAST. Website: www.ti-coast.com; grant number 053.21.112; grant recipient: Leo Koenderman. The commercial partners had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The partnership provided support in the form of salaries for authors SvS and GHT, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the "author contributions" section for authors SvS and GHT, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.