Increased serum levels of high mobility group protein B1 and calprotectin in pre‐eclampsia

To determine whether women with pre‐eclampsia have serum levels of biomarkers indicative of an elevated systemic inflammatory response.

immunologic response and promote persistent inflammatory conditions by activating pattern-recognition receptors. 6 DAMPs are self-molecules primarily released as a result of non-programmed cell death in the first few hours of an injury. They mediate innate immune responses by recruiting and activating immune cells. They are also referred to as alarmins. 7 DAMPs have been suggested to be crucial mediators in pre-eclampsia. 2 High mobility group protein B1 (HMG-1) and S100 proteins are DAMPs that are hypothesized to be involved in the pathophysiology of pre-eclampsia. 2 As a DNA-binding protein, HMG-1 has been detected in a variety of eukaryotic cell nuclei. 8 This potential immunomodulatory factor can induce cells to produce a variety of cytokinesincluding IL-6 and IL-8-and acts as a critical extracellular mediator in inflammatory processes 9 Calprotectin belongs to the S100 calgranulin subfamily and is a 24-kDa heterodimer composed of protein S100-A9 and protein S100-A8. 10 This protein is secreted mainly from neutrophils, has the ability to bind calcium and zinc ions, and represents a biomarker of active inflammation. Calprotectin and HMG-1 are regarded as late inflammatory mediators in many acute and chronic inflammatory responses. These two molecules are reported to be involved in promoting the inflammatory response in acute or chronic infection, autoimmunity, and cancer development. [11][12][13] The toll-like receptors are transmembrane proteins that enable extracellular and endosomal recognition of microbes or other infectious components.
TLRs exhibit the most diverse repertoire of DAMP ligands amongst the innate immune receptors. Toll-like receptor 4 (TLR4) is involved in the intracellular signaling pathway that leads to activation of the innate immune system. 9 As an association has been reported between pre-eclampsia and systemic inflammation 4,14 ; the aim of the present study was to evaluate calprotectin, HMG-1, and TLR4 among women with pre-eclampsia and determine whether the serum concentrations of these biomarkers were associated with clinical characteristics. The pre-eclampsia group included women who met the American College of Obstetricians and Gynecologists definition of this condition. 15 Pre-eclampsia was characterized as elevated blood pressure (≥140/90 mm Hg) on two occasions, measured at least 4 hours apart, after 20 weeks of pregnancy in a woman with previously normal blood pressure, plus the presence of proteinuria (≥300 mg per urine collection or a dipstick reading of 1 + ). 15 In the absence of proteinuria, preeclampsia was defined as new-onset hypertension plus the onset of any of the following factors during pregnancy: platelet count less than 100 000/mL, elevated serum concentrations of creatinine and liver transaminases, pulmonary edema, or cerebral or visual symptoms. 15 The duration of hypertension was calculated from the participants' prenatal care records.

| MATERIALS AND METHODS
The control group comprised pregnant women without a diagnosis of pre-eclampsia. Women with full-term healthy pregnancies were recruited from among hospitalized individuals, who were ready to delivery, whereas women with premature healthy pregnancies were recruited when attending outpatient services.
Exclusion criteria for all participants were a medical history of chronic hypertension, renal disease, pre-existing diabetes mellitus, hemostatic disease, and acute or chronic infection. Patients who were in active labor or experienced premature rupture of membranes were also excluded from the present study.
All participants were induced to deliver either vaginally or via cesarean delivery according to their symptoms. Blood sampling was checked up to 48 hours before delivery. Premature healthy pregnancies in the control group were assessed via blood sampling at a matched number of pregnancy weeks before delivery. Clotted blood samples were centrifuged at 800 g for 5 minutes. The serum was collected and stored at −80°C until enzyme-linked immunosorbent assays were performed. Serum concentrations of the three biomarkers were measured using commercial kits according to the manufacturer's instructions. Calprotectin was evaluated using a Human S100A8/S100A9 Heterodimer Immunoassay kit (R&D Systems, Minneapolis, MN, USA), whereas HMG-1 and TLR4 were assessed using a Human ELISA Kit for HMG-1 and TLR4, respectively (Cloud-Clone, Wuhan, China).
The data were analyzed using SPSS version 17.0 (SPSS, Chicago, IL, USA). Non-parametric statistical analysis was used to verify normal distribution of the data. Continuous variables with normal distribution were presented as mean ± SD; these factors were compared using the Student t test. Variables with non-normal distribution were presented as the median (interquartile range); differences between such variables were calculated using the non-parametric Mann-Whitney U test. Spearman correlation coefficients were also calculated. Statistical significance was defined as a two-sided P<0.05.

| RESULTS
The present study included 25 women in the pre-eclampsia group and 30 women in the control group. The characteristics of the participants are outlined in Table 1 The serum levels of TLR4 were measured to investigate if this signaling pathway was potentially related to the increased levels of inflammatory mediators in the pre-eclampsia group (Fig. 1). The concentration of TLR4 did not differ significantly between the groups (P=0.057); however, TLR4 concentrations tended to be higher in the pre-eclampsia group than in the control group (22.83 ± 8.46 μg/L vs 18.83 ± 6.79 μg/L). Unlike calprotectin, HMG-1 and TLR4 levels were not associated with either the duration of pregnancy hypertension or pregnancy duration at delivery.

| DISCUSSION
The findings of the present study were three-fold. First, serum concentrations of calprotectin and HMG-1 were elevated among women with pre-eclampsia. Second, high calprotectin levels were positively associated with the duration of hypertension in pregnancy but negatively associated with pregnancy duration at delivery in this group.
Third, a nonsignificant trend was observed for raised serum TLR4 concentrations among women with pre-eclampsia.
The current finding of elevated serum concentrations of calprotectin in the pre-eclampsia group was consistent with previous research. Braekke et al. 16 discovered substantially elevated plasma calprotectin levels among pre-eclamptic women versus normotensive pregnant women; however, no statistically significant between-group differences were found for the levels of calprotectin in amniotic fluid or umbilical venous plasma. In the present study, calprotectin levels increased as the duration of pregnancy hypertension lengthened.
To the best of our knowledge, the current analysis was the first to report an association between calprotectin levels and the duration of pregnancy hypertension or pregnancy duration at delivery. One previous study 17 found that calprotectin concentration was higher among patients with severe pre-eclampsia than among those with mild preeclampsia. Further clarification of the association between calprotectin elevation and the clinical presence of pre-eclampsia has not yet been reported.
As a proinflammatory factor, calprotectin has the ability to trigger an inflammatory reaction. 11 Elevated circulating levels of calprotectin are indicative of leukocyte activation and an excessive systemic inflammatory response in pre-eclampsia. 18 Sustained inflammation could lead to vascular endothelial injury, immune dysfunction, and worsening inflammatory response. Furthermore, calcium-binding and zinc-binding proteins have previously been shown to inhibit the expression of matrix metalloproteinases, which play an important role in the infiltration of the placental villi and uterine spiral artery remodeling in the early stages of pregnancy. 19   Thus, it has been speculated 19,20 that this molecule is involved in the onset of hypertension during pregnancy. In the present study, the relationship between serum calprotectin levels and pregnancy duration among women with pre-eclampsia could not be clarified. Preeclampsia has been shown to be associated with persistent hypoxia in the placenta and shallow placental implantation. 2 with control individuals. 28 In the present study, although the difference between the two groups was not statistically significant, there was a tendency toward increased serum TLR4 levels among women with pre-eclampsia.
Previously, 29 HMG-1 has been shown to modulate the function of regulatory T cells through the TLR4 pathway and to exacerbate the inflammatory response and organ damage in experimental models of inflammation. Pre-eclampsia is associated with an excessive inflammatory response and immune activation. Thus, the TLR4 pathway is likely to play a role in the pathogenesis of this pregnancy-related condition.
Currently, it is unknown whether calprotectin affects the inflammatory and immune responses by activating TLR4. Nonetheless, it would be of considerable interest to explore whether TLR4 might constitute a novel target for pre-eclampsia therapeutic strategies.
A limitation of the present study was the cross-sectional design, which prevented analysis of HMG-1, calprotectin, and TLR4 concentrations for the duration of pregnancy. Future studies should therefore ascertain the expression of DAMPs throughout pregnancy in a large population and determine their roles in pre-eclampsia.
In conclusion, the findings of the present study suggested that DAMPs could play important roles in the pathogenesis of pre-eclampsia.
Calprotectin could be a potential biomarker to monitor pre-eclampsia, especially when it develops at an early stage of pregnancy; however, this hypothesis must be confirmed in future studies. Additional research will be necessary to explore the roles of HMG-1, calprotectin, and TLRs in insufficient placenta implantation or in provoking an abnormal immune response. The findings of such studies would aid evaluation of new targets for the diagnosis and treatment of pre-eclampsia.

AUTHOR CONTRIBUTIONS
JL contributed to the design of the study, data collection, data analysis, and writing the manuscript. LH contributed to obtaining institutional review board approval, data collection, and revising the manuscript.
SW contributed to data collection and revising the manuscript. ZZ contributed to the design of the study, data interpretation, and revising the manuscript.

ACKNOWLEDGMENTS
The present study was supported by the National Natural Science

CONFLICTS OF INTEREST
The authors have no conflicts of interest.