Plasma fractalkine is a sustained marker of disease severity and outcome in sepsis patients

Patient characteristics

Characteristics of all sepsis patients are shown in Table 1. From a total of 1,103 patients, 786 (71.3 %) survived until day 30 after ICU admission,
whereas 305 (27.7 %) patients did not; 12 patients were lost to follow up. The lung
was the most common source of infection, followed by abdominal and urinary tract infections;
survivors and non-survivors had similar sites of infection. As expected, non-survivors
presented with more severe disease, as reflected by higher APACHE IV and SOFA scores,
more organs failing and more shock. Thirty-day mortality occurred in 70.2 % of patients
in the ICU.

Table 1. Clinical characteristics and outcome of sepsis patients stratified according to survival
status 30 days after admission

Fractalkine levels are elevated in sepsis patients during the first 4 days after ICU
admission and are associated with severity of disease

Fractalkine levels, measured in plasma obtained within 24 hours after admission (day
0) and at days 2 and 4, were consistently elevated in patients with sepsis relative
to healthy controls (Fig. 1a, P 0.0001 for all time points). On longitudinal analysis there was no effect of time
on plasma fractalkine levels (P?=?0.43). In contrast, in sepsis patients during the first 4 days of the ICU stay
there was a time-dependent decline in plasma IL-6, IL-8 and IL-10 (Additional file
1: Figure S1, P 0.0001 for all analytes), which are well-known cytokine markers in sepsis 29]. Sustained elevated plasma fractalkine levels after admission for sepsis were confirmed
upon separate analysis of the two contributing ICUs (data not shown). Fractalkine
concentrations did not differ between sepsis patients with different sources of infection
(Fig. 1b).

Fig. 1. Fractalkine levels are elevated in sepsis and correlate with organ failure. Blood
was drawn from patients within 24 hours of admission to the ICU (day 0) and on days
2 and 4. a Fractalkine levels were elevated in patients compared to healthy volunteers (HV) at all time points, and did not differ between days 0 and 4. b Fractalkine levels were not influenced by the primary source of infection. c Admission fractalkine levels in patients with increasing numbers of failing organs
at admission. Fractalkine levels were higher in patients with shock on admission (d) and in patients who developed shock 24 hours after admission (e). Box and whisker diagrams depict the median and lower quartile, upper quartile, and their respective
1.5 IQR as whiskers (as specified by Tukey). Gray boxes represent sepsis; open box or dotted lines represent healthy volunteers. ***P 0.001, *P 0.05

Admission fractalkine levels increased with increasing number of organs failing on
admission (Fig. 1c, r?=?0.42, P 0.001). Patients presenting with shock had significantly higher fractalkine levels
than those without shock (Fig. 1d). Of the 730 patients not presenting with shock on admission, 90 (12.3 %) developed
shock later during their stay on the ICU; admission fractalkine levels were higher
in those septic patients who developed shock while on the ICU when compared with patients
who did not (Fig. 1e).

Fractalkine levels are higher in non-survivors

To assess the relationship between fractalkine levels and sepsis mortality, measurements
were partitioned in survivors and non-survivors at day 30 after ICU admission. Fractalkine
levels on admission and 2 and 4 days thereafter were significantly higher in non-survivors
compared to survivors (Fig. 2a, P 0.001 for all time points). In a sub-analysis restricted to patients presenting
with shock, fractalkine remained significantly associated with mortality (Additional
file 2: Figure S2). To further investigate the association between fractalkine and 30-day
mortality, admission fractalkine levels were partitioned into quartiles for which
log-rank tests were performed. Quartile 3 (50–75 %, 25.5–58.7 pg/ml) and quartile
4 (75–100 %, 58.7–6329.0 pg/ml) of fractalkine levels had statistically significantly
reduced survival (P 0.01 and P 0.001, respectively), compared to quartile 1 (Fig. 2b). Admission fractalkine levels remained significantly associated with 30-day mortality
after adjustment for age, Charlson comorbidity index, body mass index (BMI), admission
type and APACHE IV scores in a logistic regression model (odds ratio (OR), 1.37 (95 %
CI, 1.19–1.58) for each log increase in fractalkine, P 0.0001). Similarly, when fractalkine levels were included in a logistic regression
analysis together with SOFA scores, the association with 30-day mortality persisted
(OR, 1.25, 95 % CI, 1.09–1.44, for each log increase in fractalkine, P?=?0.001). Moreover, plasma levels on days 2 and 4 remained independently associated
with 30-day mortality after adjustment for SOFA scores for those particular days (OR
1.40, 95 % CI 1.20–1.63, P?=?0.001 and OR 1.31, 95 % CI 1.08–1.59, P?=?0.006, respectively). The ROC AUC of fractalkine was 0.65 (95 % CI 0.61–0.69).
The Youden index determined a fractalkine level of 38.2 pg/ml to be the optimal cutoff.
At this cutoff, fractalkine had 54 % sensitivity and 71 % specificity for predicting
mortality at 30 days after ICU admission. Fractalkine in addition to the APACHE IV
score, improved classification of patients who were deceased at 30 days after admission
and those who were not; the NRI was 0.27 (95 % CI 0.12–0.41, P?=?0.0003) and the IDI was 0.02 (95 % CI 0.006–0.02, P?=?0.002).

Fig. 2. Fractalkine plasma levels are associated with 30-day mortality. Blood was drawn within
24 hours of admission to the ICU (day 0) and days 2 and 4. a Fractalkine levels of survivors and non-survivors at day 30, presented as box and whiskers as specified by Tukey. b Kaplan–Meier plots of survival time up to 30 days after ICU admission for quartiles
(Q1 4.0–13.4 pg/ml, Q2 13.4–25.5 pg/ml, Q3 25.5–58.7 pg/ml, Q4 58.7–6329 pg/ml) of
admission fractalkine levels. Dotted lines in box and whisker plots represent the median level in healthy volunteers (HV). ***P 0.001, **P 0.01

The association between elevated fractalkine concentrations and mortality persisted
when outcomes were 7-day, 90-day or 1-year mortality (Additional file 3: Figure S3). Together these data suggest that higher fractalkine levels are associated
with short-term mortality, and that this association drives differences in long-term
mortality.

Comparison of fractalkine release with that of soluble E-selectin

Several studies have identified endothelial cells as a major source of fractalkine
4], 30], 31]. Considering that it is difficult to assess whether the endothelium produces fractalkine
during human sepsis in vivo, we compared the kinetics of fractalkine release into
the circulation relative to that of the established specific endothelial cell activation
marker E-selectin 32], 33] in a controlled human setting of systemic inflammation induced by bolus intravenous
injection of LPS. Intravenous LPS induced a rise in fractalkine levels with similar
kinetics to the increase in soluble E-selectin (Fig. 3a, P 0.0001). The initial increase in soluble E-selectin coincided with the rise in fractalkine
levels, and fractalkine and soluble E-selectin levels measured during the first 5 hours
after LPS injection had strong positive correlation (Fig. 3a; r?=?0.79, P 0.0001). The release of IL-6 occurred faster and was more transient, and IL-6 levels
did not correlate with fractalkine levels (Additional file 4: Figure S4). We also measured soluble E-selectin levels in the patient samples used
in Figs. 1 and 2. While soluble E-selectin levels were clearly elevated in patients with sepsis when
compared with healthy controls (Fig. 3b, P 0.001), confirming previous reports 34], 35], soluble E-selectin was not associated with increased mortality at day 30 (Fig. 3c); admission soluble E-selectin was, in contrast with fractalkine, lower in non-survivors
(P?=?0.02). The ROC AUC of soluble E-selectin for 30-day mortality was 0.55 (95 % CI
0.51–0.59). Fractalkine outperformed soluble E-selectin in predicting 30-day mortality
(P?=?0.001).

Fig. 3. Soluble E-selectin correlates with fractalkine release after intravenous injection
of endotoxin in healthy humans in vivo but is not associated with increased mortality
in sepsis patients. a Fractalkine release after intravenous injection of endotoxin (lipopolysaccharide,
4 ng/kg body weight) into five healthy subjects compared with E-selectin release.
Right panel shows correlation between fractalkine levels and E-selectin measured during the first
5 hours after endotoxin administration (shaded gray). Data are expressed as mean?±?standard
error of the mean. b Soluble E-selectin levels were elevated in patients compared to healthy volunteers
(HV) at all time points. c Soluble E-selectin levels of survivors and non-survivors at day 30, presented as
box and whiskers as specified by Tukey. ***P 0.001, **P 0.01, *P 0.05

Elevated plasma fractalkine levels are not specific for infection

Considerable research has focused on discriminating infectious from non-infectious
sources of critical illness. To determine whether fractalkine levels can provide diagnostic
value we analyzed fractalkine levels in 344 patients consecutively admitted to the
ICU with suspected CAP. Of these, 271 patients were classified as having CAP (these
were also part of the sepsis cohort described above), whereas in 73 patients the CAP
diagnosis was retrospectively refuted (no-CAP controls) 17]. Clinical characteristics of patients with and without CAP are depicted in Table 2. these patients were largely similar in demographics, comorbidities, severity of
disease and outcome. Patients with CAP more often had shock and had longer lengths
of stay in the ICU. The ICU mortality and 30-day mortality did not differ between
groups. Fractalkine levels were similar in patients with and without CAP on admission
and at 2 and 4 days thereafter (Fig. 4a). Fractalkine levels were higher in patients with CAP and without CAP who presented
with shock than in those who did not (Fig. 4b); similarly, fractalkine levels were higher in patients with and without CAP who
did not survive until day 30, when compared with those who did (Fig. 4c).

Table 2. Clinical characteristics and outcome of patients with community-acquired pneumonia
(CAP) and no-CAP controls

Fig. 4. Fractalkine levels in patients with community-acquired pneumonia (CAP) and critically ill patients with no CAP. Blood was drawn from patients within 24 hours
of admission to the ICU (day 0) and at days 2 and 4. a Fractalkine plasma levels in patients with suspected CAP upon ICU admission and classified
in retrospect as having CAP or no CAP. Differences between groups were not significant.
b Fractalkine levels were higher in patients with shock on admission (b) and in non-survivors (at day 30) (c) in both CAP and no-CAP patients. Box and whisker diagrams depict the median and lower quartile, upper quartile, and their respective
1.5 IQR as whiskers (as specified by Tukey). Dotted lines represent the median level in healthy volunteers (HV). ***P 0.001, *P 0.05