The clinical significance of the MIF homolog d-dopachrome tautomerase (MIF-2) and its circulating receptor (sCD74) in burn

Abstract

Background: We reported earlier that the cytokine macrophage migration inhibitory factor (MIF) is a potential biomarker in burn injury. In the present study, we investigated the clinical significance of the newly discovered MIF family member d-dopachrome tautomerase (DDT or MIF-2) and their common soluble receptor CD74 (sCD74) in severely burned patients.

Methods: DDT and sCD74 serum levels were measured 20 severely burned patients and 20 controls. Serum levels were correlated to the abbreviated burn severity index (ABSI) and total body surface area (TBSA) followed by receiver operating characteristic (ROC) analysis. Data were supported by gene expression dataset analysis of 31 burn patients and 28 healthy controls.

Results: CD74 and DDT were increased in burn patients. Furthermore, CD74 and DDT also were elevated in septic non-survivors when compared to survivors. Serum levels of DDT showed a positive correlation with the ABSI and TBSA in the early stage after burn, and the predictive character of DDT was strongest at 24h. Serum levels of CD74 only correlated with the ABSI 5 days after injury.

Conclusions: DDT may assist in the monitoring of clinical outcome and prediction of sepsis during the early post-burn period. Soluble CD74 and MIF, by contrast, have limited value as an early predictor of death due to their delayed response to burn.

Keywords: Biomarker; Burn; CD74; DDT; MIF; Sepsis.

Fig. 1. Flowchart

Flowchart according to the Strobe recommendations

Fig. 2. Serum levels of DDT and sCD74 in burn patients

Serum levels of DDT and sCD74 were measured in burn patients by ELISA over a time period of 120 hours. Healthy patients without burn injury served as controls. A Serum levels of DDT are highest at the time of admission and decrease within the first 12 hours. B Serum levels of sCD74 increase over time. * p <0.05, ** p <0.01, and *** p <0.001. Data represent mean ± SEM

Fig. 3. Serum levels of DDT and sCD74 of survivors and non-survivors

DDT and sCD74 serum levels were compared between surviving burn patients, non-surviving burn patients, and healthy controls. We further compared the levels between patients who died before 120 hours (patient 6, 16, 19) and patients who died between 120 hours and 10 days (“non-survivors (>5 days)”). A On admission, serum levels of DDT are significantly increased in non-survivors when compared to survivors and healthy controls. The DDT serum levels of survivors also are significantly higher when compared to controls. After a temporary decrease, DDT levels again increase in non-survivors. In survivors, DDT serum levels remain low with no statistical difference to healthy controls. B Patients 6, 16, and 19 who died within 5 days after admission show a rapid increase pre mortem when compared to patients who died after 5 days (“non-survivors (>5 days)”). C Serum levels of sCD74 are comparable in the early stage after burn injury but increase in non-survivors after 24 hours. Serum CD74 levels of survivors are slightly higher than healthy controls (statistically not significant). D Except for patient 6, patients who died within 5 days after admission (patients 16, 19) show higher levels of sCD74 when compared to patients who died after 5 days (“non-survivors (>5 days)”). Significant difference between non-survivors and survivors: ** p <0.01, *** p <0.001. Significant difference between non-survivors and healthy control: ## p <0.01, ### p <0.001. Significant difference between non-survivors and healthy control/survivor and healthy control: §§§ p <0.001. Data represent mean ± SEM

Fig. 4. Correlation between DDT/sCD74 serum levels and ABSI/TBSA

DDT and sCD74 serum levels were correlated with ABSI and TBSA at each time point by Spearman’s rank correlation. While CD74 showed only one significant correlation, DDT was positively associated with ABSI and TBSA at 0h and 24h. Only significant correlations are shown with their respective p-value and Spearman r value. The complete correlation data is found in Supplementary Figure 1–4. A DDT and ABSI at 0h. B DDT and TBSA at 0h. C DDT and ABSI at 24h. D DDT and TBSA at 24h. E sCD74 and ABSI at 120h. ABSI – abbreviated burn severity index; TBSA – total burn surface area

Fig. 5. Impact of TBSA on DDT and sCD74 serum levels

To evaluate the impact of burn size on DDT and sCD74 serum levels, we divided burn patients into three groups: TBSA <20%, TBSA 20–40%, and TBSA >40%. We compared the serum levels between three groups A DDT serum levels increased with burn size. Significant differences are already seen at 0h. In the group of TBSA <20%, DDT levels normalized after five days. B Serum CD74 serum levels also increased with the TBSA. The differences, however, became more evident in the later stages. * p <0.05, ** p <0.01, *** p <0.001. Significant difference between healthy and TBSA >40%/TBSA 20–40%: # p <0.05. Significant difference between TBSA >40% and TBSA <20%/healthy control: § p <0.05. Data represent mean ± SEM. TBSA – total burn surface area

Fig. 6. Predictive value of DDT and sCD74

The predictive character of DDT and sCD74 serum levels was calculated by ROC curve analysis at the five different time points. For each curve, AUC and p-values are illustrated. Significant AUC curves for DDT are found at 24h, 48h, and 120h, whereas the only significant AUC curve for sCD74 was observed at 120h. A AUC curve at 0h. B AUC curve at 12h. C AUC curve at 24h. D AUC curve at 48h. E AUC curve at 120h. ROC – receiver operating characteristics; AUC – area under the curve

Fig. 7. Gene array data of DDT and CD74 expression in burn patients

Our ELISA data were supported by analysis of a gene array dataset. Gene expression of burn samples was measured at an early (49.93 ± 9.26h) and late time point (430.5 ± 23.62h). A DDT gene expression in burn patients compared to healthy controls. B CD74 gene expression in burn patients compared to healthy controls. C DDT gene expression in survivors and non-survivors at the early time point. D CD74 gene expression in survivors and non-survivors at the early time point. E DDT gene expression in survivors and non-survivors at the late time point. F CD74 gene expression in survivors and non-survivors at the late time point. G ROC curve analysis for CD74 and DDT expression at the early time point with their respective AUC and p-values. H ROC curve analysis for CD74 and DDT expression at the late time point with their respective AUC and p-values. ROC – receiver operating characteristics; AUC – area under the curve