133 Systemic Immune Response in Burn Patients of study participation, patient discharge and death. In addition, we acknowledge that the difference in age and gender between burn patients and healthy controls represents a limitation of this work, as aging and gender can affect the immune response [70,71]. Unfortunately, the group size was insufficient to analyze these differences in more detail. Supervised gating of flow cytometry data can be challenging due to biological variation and the fact that manual gating relies on the researcher’s prior knowledge causing bias in the analysis [72]. To overcome this, we took a combined approach of both supervised and unsupervised analysis of our data, and showed that they largely reached the same outcome. To better understand the behavior of these immune cells, it would be interesting to study functionality of these cells after burn injury. Furthermore, we are curious to see if the observed systemic immune response is reflected by the local immune response to burn injury and will pursue this aim in the near future. Information on the immunological mechanisms driving burn-induced inflammation and pathophysiology is very limited. Because of the excessive and persistent inflammation, it could be beneficial for burn wound patients to use anti-inflammatory drugs [73]. Directed therapy that either decreases the influx of neutrophils or supports the suppressive arm of the immune system, might lower the risk of complications caused by the systemic inflammation, which in turn should improve wound healing. Because of variation between burn wounds, patients and differences in the intensity of the burn-induced immune response, treatment should be empirical and personalized to improve the outcome. Taken together, we showed that the burn-induced leukocytosis is mainly due to an increase of neutrophils and monocytes and that burn injury caused a long-lasting influx of immature neutrophils. The persistent elevated levels of pro-inflammatory cytokines and the shifts in neutrophil and lymphocyte composition suggest that the immune system remains in a long-term pro-inflammatory state rather than switching to a resolving state. Because these immune reactions are likely to strengthen one another and keep the inflammation going, we need to search for ways to resolve inflammation in an early stage in order to improve burn treatment, prevent secondary complications, and reduce length of hospital stay. MATERIALS AND METHODS Subject Recruitment and Sample Collection Twenty burn wound patients admitted to the intensive care unit (ICU) of the Burn Center of the Red Cross Hospital in Beverwijk, the Netherlands were included in this study after written consent was obtained from the patient or a legal representative 4
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