Longitudinal characterization of olfactomedin-4 expressing neutrophils in pediatric patients undergoing bone marrow transplantation

Abstract

Sepsis is an important cause of morbidity and mortality in pediatric patients. Increased expression of olfactomedin-4 (OLFM4), a glycoprotein contained within a subpopulation of neutrophils, has been associated with complicated course in sepsis. The factors that regulate OLFM4 expression are unknown. Here, we followed children undergoing bone marrow transplantation (BMT) to document the percentage of neutrophils that express OLFM4 over time. This population was selected because of the ability to observe nascent neutrophils following engraftment, perform frequent blood sampling, and the children are at high risk for clinical complications that may associate with changes in percentage of OLFM4+ neutrophils. We found a surprising degree of variability of OLFM4 expression between patients. In the weeks following initial neutrophil recovery we also saw great variability in OLFM4 expression within individual patients, indicating that multiple external factors may modify OLFM4 expression. We identified decreased expression of CD64 (a marker associated with response to infection), in OLFM4+ neutrophils. This is the first study to demonstrate fluctuation in OLFM4 expression within patients and provides insight into possible mechanisms for OLFM4 regulation in nascent neutrophils.

Fig 1. Percentage of OLFM4+ neutrophils in patients undergoing bone marrow transplantation.

A. Box and whisker plots showing median, 25^th to 75^th percentile for each sample from healthy controls and from patients undergoing BMT. B. Last measured percentage of OLFM4+ neutrophils before myeloablative regiment and first measurement following engraftment. C. Box and whisker plots for percentage of OLFM4+ neutrophils each week following engraftment. D. Box and whisker plot showing average OLFM4 expression for all time points following engraftment for each patient grouped by race. One way analysis of variance of the groups showed statistically significant differences (denoted by *) between Arabic and Caucasian (p = 0.002) and African American and Caucasian (p = 0.01).

Fig 2. Variation in OLFM4 expression in patients following bone marrow transplantation.

Four different patients were selected to show variation in percentage of OLFM4+ neutrophils. Arrows demark time points discussed further in the text. ANC = absolute neutrophils count (black), AONC = absolute OLFM4 neutrophil count (green) and %OLFM4+ = is the percentage of neutrophils that express OLFM4 (red). As demonstrated here, some patients had a stable ANC and AONC throughout their course (A). Others, showed an increase in only OLFM4+ neutrophils (B) or only OLFM4- neutrophils (C). Others, had a simultaneous increase in both (D).

Fig 3. Neutrophil surface markers comparing OLFM4+ and OLFM4- neutrophils.

Box and whisker plots showing median fluorescence intensity as well as 25^th and 75^th percental for each group of cells. *p<0.001.

Fig 4. Plasma OLFM4 concentration relative to absolute number of OLFM4+ neutrophils.

Three patients comparing number of OLFM+ neutrophils and plasma OLFM4 concentration.

Fig 5. Three comparisons for change in OLFM4+ neutrophils relative to clinical change.

Example of a patient who had a clinical change on week 6 post BMT. Arrows depict the three comparisons we made, 1-week prior to clinical change to week of clinical change. 2-Week of clinical change to week after clinical change. 3-Baseline level to week of clinical change.