Early Mixed Inammatory Response in Children with Sepsis

Objective: The systemic inammatory and immune response directly inuence the prognosis of patient with sepsis. By detecting the pro-/anti-inammatory and immune biomarkers, we aim to understand the early systemic inammatory response and immune status, and explore the potential biomarkers for diagnosis and treatment for pediatric patients with sepsis. Methods: 62 pediatric patients with sepsis and 48 pediatric patients without sepsis, between 9 months and 13 years old were included. The HLA-DR expression on CD14 + monocytes, the proportion of CD4 + CD25 + Foxp3 + Treg cells and IL-27 + CD4 + cells were analyzed by ow cytometry. The Foxp3, CTLA-4, GITR, IL-10, L-17A, IL-17F and IL-27 mRNA levels in CD4 + cells were evaluated by Real-time PCR. And Cytokines IL-4, IFN-γ and TGF-β were measured by enzyme-linked immunosorbent assay. Results: Serum IFN-γ (cid:0) transcription levels of IL-17A and IL-17F were higher than that in patients without sepsis (p<0.01), while TGF-β was lower (p<0.05). There was no signicant difference in IL-4 level between pediatric patients with and without sepsis. The HLA-DR expression was signicantly lower than that in pediatric patients without sepsis (p<0.01), but the expression rate of HLA-DR in two groups are greater than 30%. Compared to pediatric patients without sepsis, there was a lower proportion of CD4 + CD25 + Treg cells in pediatric patients with sepsis (p<0.01), but with a higher of Foxp3, CTLA-4 and IL-10 mRNA (p<0.01). The IL-27 mRNA and the proportion of CD4 + IL-27 + cells were signicantly raised in pediatric patients with sepsis than that in pediatric patients without sepsis (p<0.01). Conclusion: response dominates. The IL-27 may exert bi-directional regulation in the mixed immune responses, and can be detected as a potential biomarker for determination of immune status for pediatric patients with sepsis. Also, although the immune function inuenced at early stage in pediatric patients with sepsis, the expression rate of HLA-DR was greater than 30%, so treatments of immune regulation could not be necessary at early stage for pediatric patients with sepsis. inammation and immune status of patient with sepsis determine the progression of sepsis. In the current study, both pro-inammation and anti-inammation factors in the the status of CD4 + CD25 + Treg cells were analyzed in pediatric patients with and without sepsis. Results indicate the mixed immune response presented at early stage in pediatric sepsis patients, IL-27 may exert bi-directional regulation in early immune responses, an elaborate regulation of immune status regarding on IL-27 expression can be used as an effective therapeutic strategy in of pediatric sepsis patients. the signicantly while the anti-inammatory TGF-β signicant decreased. These results indicate that both the pro- and anti-inammatory responses presented at early stage in pediatric sepsis patients, the pro-inammatory response dominants. In this study, we also investigated the CD4 + CD25 + Treg ratio and the expression of specic genes Foxp3, CTLA-4, GITR and IL10 in CD4 + T cells. Although no signicant difference shown in GITR, while the transcription levels in CD4 + T cells of Foxp3, CTLA-4, and IL-10 are signicant higher in sepsis patients than those in controls, combination with the results of HLA-DR expression on CD14 monocytes, our studies suggest the compensatory anti-inammatory responses presented in children with sepsis at early stage. The immune disorder in sepsis is caused by a dynamic alteration between systemic inammatory response syndrome (SIRS) and Compensatory anti-inammatory response syndrome (CARS), and the anti-inammatory factors released by CARS are protective reaction to SIRS, which led to complicated immune process and imbalanced immune response Systematic surveillance and evaluation of the immune status of patients are vital to early sepsis patients. Combination with above the results of HLA-DR expression, this study implies that the therapeutic strategies at early stage in pediatric patient with sepsis


Introduction
Sepsis is a leading cause of death in children with serious diseases [1], with its high morbidity and mortality in pediatric patients, sepsis aroused the worldwide consideration in modern critical care medicine [2].
Sepsis occurs as a generalized in ammatory cascade including severe sepsis, septic shock, and multiorgan failure [3], The overwhelming or persistent release of pro-in ammatory mediators from systemic in ammatory response syndrome (SIRS) and anti-in ammatory mediators from compensatory antiin ammatory response syndrome (CARS) result in in ammatory imbalance and immunologic dissonance [4]. Researches indicate patient's prognosis related to the in ammatory response and immune status in early stage, so early detection and treatment of sepsis is vital to prevent widespread tissue injury, morbidity and death [5].
The dissonant immune response in sepsis involves abnormal CD4 and CD8 T cell response [6]. CD4 T cell plays important role in primary CD8 T cell response, formation of functional CD8 T cell memory and e cient isotype switching of B cells [7]. By producing cytokines, activating and altering the migration of other immune cells, CD4 T cell can effectively respond to infection [7]. The activation of Th1 CD4 cells results in the production of cytokines, such as IL-2 and IFN-γ, IL-10, which in turn activates macrophages.
While the activated Th2 CD4 cells results in various different immune responses by producing cytokines such as IL-4, IL-5, and IL-13 [7,8]. Therefore, the observation of T cell immunity at early stage conduces to understand the regulation of the immune status and identify novel strategies in pediatric sepsis patient.
As a member of IL-12 family of cytokines, IL-27 signals through a heterodimeric receptor complex consisting of the common IL-6 receptor chain gp130 and a unique IL-27 receptor α chain (IL-27R) WSX-1 [9]. By acting on various immune cells including T cells, B cells, macrophages and dendritic cells, IL-27 presents as pleiotropic cytokine in both pro-in ammatory and anti-in ammatory [10,11]. Studies has suggested that IL-27 could be a diagnostic biomarker for predicting bacterial infection in sepsis patients [12], and the blocking of IL-27 function could be used as a therapeutic strategy of sepsis [13,14].
HLA-DR, a glycosylated cell surface transmembrane protein expressed on antigen-presenting cell (monocytes, mHLA-DR), is essential for activation of CD4-or CD8 T cells to initiate the speci c immune response [15,16]. mHLA-DR is considered as a reliable biomarker in critical infection patients, a lower HLA-DR than 30% means that patient's immune function is suppressed [17][18][19]. Evidences indicate the mortality increased 30-fold when mHLA-DR expression reduced by 30% [20,21].
Currently, few of biomarkers had been evaluated for sepsis to different potential sources of infections [22,23]. In this study, we are aiming to observe the early immune status in pediatric patients with sepsis and the performance of serval biomarkers, by which providing the reference to the diagnosis and treatment to pediatric patients with sepsis.

Material And Methods
Patients 62 pediatric patients with sepsis in the pediatric intensive care unit (PICU) of Guizhou Provincial People's Hospital between January 2015 and May 2017 were studied as sepsis group, including 42 boys and 20 girls aged from 1 to 13 years (mean 6.27 years). The diagnosis of sepsis was conducted according to the recommended criteria for pediatric sepsis [24]. Another 48 pediatric patients consisted of 36 boys and 12 girls aged from 9 months to 14 years (mean 5.75 years) without sepsis, who underwent surgery (foreskin circumcision: 32; non-incarcerated hernia: 16) in the pediatric surgery department, were studied as control group. Pediatric patients with autoimmune diseases, immune de ciency, genetic metabolic disorders and tumor were excluded from this study. Pediatric patients who accepted the treatments with drug or blood products that in uence the immune system, or died within 24 hours after admission were excluded as well.

The Diagnostic Criteria Of Sepsis
Sepsis was de ned as the life-threatening organ dysfunction caused by a dysregulated host response to infection according to the Sepsis-3 criteria [24]: increase in Sequential Organ Failure Assessment score by ≥ 2 at day 1 and suspicion of infection.

Blood Sample Collection
Venous blood was immediately obtained after admission in hospital or diagnosis of sepsis. 1 ml of blood was used for the detection of various in ammatory or anti-in ammatory factors in serum, and 8 ml was used for detection of cell sorting, analysis and gene expression.

The Detection Of Pro/anti-in ammatory Cytokines And Hladr
The HLA-DR expression on CD14 + monocytes, the proportion of CD4 + CD25 + Foxp3 + regulatory T cells

Statistical analysis
Data was presented as mean ± standard deviation (SD). Software SPSS 17.0 (SPSS Inc., USA) was utilized in the current study. Differences between groups were analyzed by Two-tailed t test or Mann-Whitney test. p ≤ 0.05 was considered as statistically signi cant, and p < 0.01 was considered as remarkably statistically signi cant.

Results
Pro-in ammatory responses at an early stage of sepsis The differential expression of selected pro-in ammatory factors was detected: the serum level of IFN-γ (99.29 ± 16.01) was signi cantly higher in patients with sepsis than that in control group (55.39 ± 8.35) (p < 0.01). And the transcription levels of IL-17A and IL-17F in CD4 + T cells of patients with sepsis were signi cantly higher than that in control group (p < 0.01) ( Table 1).
The expression of HLA-DR on the surfaces of CD14 + monocytes was also detected. It was 75.36 ± 6.77 in patients with sepsis, signi cantly lower than that of control (95.13 ± 2.69) (p < 0.01). But both were higher than 30% (Table 1 and Fig. 1).
Anti-in ammatory responses at early stage of sepsis The differential expression of selected markers of the anti-in ammatory responses were detected. The serum level of TGF-β (1139.45 ± 161.82) was signi cantly lower in patients with sepsis than that in control (1772.76 ± 397.79) (p < 0.05). However, there was no signi cant difference between sepsis patients and control regarding to IL-4 serum level ( Table 1). The peripheral CD4 + CD25 + Foxp3 + Treg ratio in patients with sepsis was signi cantly lower than that in controls (p < 0.01) (Fig. 2 and Table 1). However, compared to patients without sepsis, the expression of Treg speci c genes in CD4 + T cells, Foxp3, CTLA-4 and IL-10 were signi cantly higher in patients with sepsis (p < 0.01). There was no signi cantly difference of mRNA level of GITR in CD4 + T between in sepsis group and control group (Table 1).

IL-27 expression at early stage of sepsis IL-27 gene expression in CD4 + T cells was higher in patients of sepsis comparing to that in control group
(p < 0.01). Meanwhile, the ratio of CD4 + IL-27 + cells was signi cantly higher in patients with sepsis comparing to that in control group (p < 0.01) as well ( Fig. 3 and Table 1).

Discussion
The pathogenic mechanism of sepsis involves the systemic in ammatory network, gene polymorphisms, and immune dysfunction [25], systemic in ammation and immune status of patient with sepsis determine the progression of sepsis. In the current study, both pro-in ammation and anti-in ammation factors in the serum, the status of CD4 + CD25 + Treg cells were analyzed in pediatric patients with and without sepsis. Results indicate the mixed immune response presented at early stage in pediatric sepsis patients, IL-27 may exert bi-directional regulation in early immune responses, an elaborate regulation of immune status regarding on IL-27 expression can be used as an effective therapeutic strategy in of pediatric sepsis patients.
HLA-DR molecules are central to the speci c immune response, the decreasing of HLA-DR on monocytes connects with complications of infections and progression of sepsis [26]. And reduction by 30% of HLA-DR expression can be considered as immune suppression [27]. In this study, the expression of HLA-DR on CD14 monocytes in sepsis patients is lower than that in control group, but both are higher than 30% (shown in table 1). Which suggests that although the early immune function is stimulated by the in ammatory response, it is not suppressed.
Comparing to patients without sepsis, the pro-in ammatory factor IFN-γ in serum, IL17A / IL-17F secreted by Th17 cells, the ratio IFN-γ/ IL-4 in pediatric sepsis patients signi cantly increased, while the antiin ammatory factor TGF-β signi cant decreased. These results indicate that both the pro-and antiin ammatory responses presented at early stage in pediatric sepsis patients, the pro-in ammatory response dominants. In this study, we also investigated the CD4 + CD25 + Treg ratio and the expression of speci c genes Foxp3, CTLA-4, GITR and IL10 in CD4 + T cells. Although no signi cant difference shown in GITR, while the transcription levels in CD4 + T cells of Foxp3, CTLA-4, and IL-10 are signi cant higher in sepsis patients than those in controls, combination with the results of HLA-DR expression on CD14 monocytes, our studies suggest the compensatory anti-in ammatory responses presented in children with sepsis at early stage. The immune disorder in sepsis is caused by a dynamic alteration between systemic in ammatory response syndrome (SIRS) and Compensatory anti-in ammatory response syndrome (CARS), and the anti-in ammatory factors released by CARS are protective reaction to SIRS, which led to complicated immune process and imbalanced immune response [28]. Systematic surveillance and evaluation of the immune status of patients are vital to early sepsis patients.
Combination with above the results of HLA-DR expression, this study implies that the therapeutic strategies at early stage in pediatric patient with sepsis should base on the effective treatment to proin ammatory reaction and suppressing the damage resulting from the immune response to infection. IL-27, as a new member of IL-12 family, plays duel-functions in the immune and in ammation reactions [10,29]. IL-27 exerts duel-regulation effects on Th1 cells [30]. Though IL-27 cannot induce the production of Foxp3 + Treg, it can promote Tr1 cell development and differentiation through IL-10 [31,32]. While IL-10 participates in IL-17 suppression by IL-27 and prevents Foxp3 + Treg cells differentiating from CD4 + T through down-regulating IL-6 and TGF-β expression. Therefore, IL-27 plays a critical role in balancing the immune response. In addition, IL-27 can also induce major histocompatibility complex (MHC) molecule expression in non-immune cells to endow them with the ability to perform antigen presentation. In the current study, the increased expression of IL-27, IFN-γ, IL-17A, IL-17F and IL-10 in CD4 + T cells were observed in the pediatric sepsis at early stage. The serum level of TGF-β and ratio of CD4 + CD25 + Treg cells in patients were lower in sepsis patients than that in controls. These ndings imply that IL-27 might promote the release of IFN-γ and IL-17, repress the TGF-β expression and differentiation of Treg cells.
Meanwhile, IL-27 might increase the expression of IL-10, Foxp3 + and CTLA-4, by which could enhance the immune suppression effect of Treg, and balance immune response. Therefore, by the duel effects on the immune system, IL-27 could be used as a novel target for the treatment of pediatric sepsis, but how to utilize its advantages remains to be further studied.
In sum, our results collectively show that both pro-in ammation and anti-in ammation response presented at early stage in pediatric sepsis patients, while pro-in ammation response predominate in this stage. Although the immune status of pediatric patients with sepsis declined in early stage of sepsis, it was not immunosuppressed, therefore, we should focus on the treatment of anti-in ammation rather than the enhancement of immune at early stage. As a duel-regulation cytokine, IL-27 balances the duel immune response by regulating the differentiation of immune cells and secretion of other cytokines in early stage of pediatric sepsis patients, and can be used as a potential therapeutic strategy for pediatric sepsis patients. However, in consideration of limitations of this study, several aspects should be further discussed and explored in future. Firstly, the immuno-logical system is in uenced by age-related factors, immune status in individual pediatric patients should be further individually analyzed. Secondly, our conclusions about in ammatory response based on the cytokines we examined in this study, more classic in ammatory cytokines should be detected to con rm the in ammatory response status in early stage of pediatric sepsis patients.

Declarations
Ethic approval and Informed consent Ethical approval for this study was obtained from the Human Ethics Committee of Guizhou Provincial People's Hospital ([2015]014). The Written informed consent was signed by the guardian of each patient before study.

Statements
We con rm that the manuscript has been read and approved by all named authors, and the order of authors listed in the manuscript has been approved by all of us. All authors declare that they have no con ict of interests.

Funding
This study was supported by Science and Technology Fund of Guizhou Provincial Health Commission (gzwjkj2019-1-067). Figure 3 Fluorescence-activated cell sorting (FACS) analysis of CD4+ IL-27+cells. Percentages of CD4+ CD27+ cells in blood samples from sepsis patients (patients) or patients without sepsis (controls) were determined respectively. Representative histograms of IL-27+ population("M2") within CD4+ cells are shown in gure.