T helper subsets during the acute post-traumatic period in children

Full Text

Introduction

Severe mechanical injury is one of the leading causes of childhood disability and mortality [4]. Severe injury induces a complex host immune response to tissue damage, a parallel pro- and anti-inflammatory state associated with an increased risk of infectious complications (IC) and/or multiple organ failure (MOF) [2, 6]. A period of pronounced immunosuppression is usually observed, the pathogenesis of which is largely determined by a decrease in the level of T ymphocytes (Th) in severe trauma [8]. Also, it has been demonstrated that Treg is activated in response to massive spread causing induced injury, downregulation of Th1 responses, and T cell anergy [5]. The quantity of T helper subpopulations, such as regulatory T lymphocytes (T_reg) – CD4⁺CD127^lowCD25^high and Th17 lymphocytes (Th17), can be a significant marker in the frequency of the pathological process and predicting its outcome. Th17 and Treg cells have opposite roles in the development of autoimmune and inflammatory diseases. While Th17 cells promote autoimmunity, Treg cells serve to control it and therefore play a very important role in autoimmune pathogenesis by maintaining self-tolerance and by controlling expansion and activation of autoreactive CD4⁺T effector cells. The control of Th17/Treg balance appears also critical in the development of of many diseases, including severe trauma [9]. However, further evaluation is required to determine exact kinetic changes of CD4⁺T cells subsets count post-trauma.

The purpose of this study was to identify informative immunological criteria for the traumatic disease severity and as applicable to children. The identification relies on the assessment of Th subsets ratio – Th17/T_reg, T_127hi/T_reg, and Th17/T_127hi.

Materials and methods

The study involved 87 patients (58 boys (66.6%), 35 girls (33.4%); 331 observation sessions) with severe injury (SInj), treated at the Department of Anesthesiology and Resuscitation of the Research Institute of Emergency Pediatric Surgery and Traumatology. We used the laboratory of the National Medical Research Center for Children’s Health for laboratory studies, which were prescribed 1 to 5 times, depending on the length of stay of a given child at the ICU. The mean age of the children was 12.0 (5.75-15.0) years (Me (Q_0.25-Q_0.75)). The time options for laboratory studies were the first, third, fifth, seventh and 14th days from the day of injury. The comparison group was comprised of 34 patients (15 boys (44.1%), 19 girls (55.9%); 34 observation sessions) with mild/moderate injury (MInj) treated at the Department of Neurotrauma. The control group was comprised of 80 apparently healthy children, all of them underwent medical examination at the National Medical Research Center for Children’s Health. The children were comparable in age and sex: age – 12.41 (4.4-16.2) years (Me (Q_0.25-Q_0.75)); 47 boys (58.7%), 33 girls (41.3%).

Assessing the injury, we relied on the Injury Severity Score (ISS) and the Glasgow Coma Scale (GCS). The outcome of an SInj was assessed with the help of the Glasgow Outcome Scale (GOS) and the Severe Injury Outcomes Scale (OISS) [10]. These scales were applied to assess the condition of the patient at discharge from the hospital.

The patients in our study met the following criteria: severe injury (ISS ≥ 16), aged 1-18 years, admittance to the ICU within 72 hours. Concomitant acute inflammatory and chronic diseases were a reason for exclusion.

At the first stage, we analyzed the results from the control group, the MInj (ISS 4.0 (4.0-9.0) (Me (Q_0.25-Q_0.75)) and the SInj group (ISS 26.0 (21.0-29.0) (Me (Q_0.25-Q_0.75)). At the second stage, we analyzed the two groups from SInj formed with the help of GOS and OISS, the favorable outcome group (SInjfav, n = 47) and the unfavorable outcome group (SInjunfav, n = 40). The distribution into these groups was based on the scores: patients were allocated to the SInjfav group if they scored 4-5 points on the GOS scale and 1-2 points on the OISS scale, and to the SInjunfav group if they scored 1-3 points on the GOS scale and 3-5 points on the OISS scale.

Clinical and laboratory indicators of systemic inflammatory response syndrome and organ failure were evaluated in all patients with severe injury. Organ functioning was assessed daily after admission to the ICU using MODS (Multiple Organ Dysfunction Score) [7]. Patients with severe injury were divided into groups depending on infectious complications (IC n = 16) and the development of MODS (MODS n = 11).

We assessed the absolute cell count of T_127hi – CD4⁺CD127^higtCD25^high, Th17 – CD4⁺CD161⁺, T_reg – CD4⁺CD127^lowCD25^high and their ratio Th17/ T_reg, T_127hi/T_reg, and Th17/T_127hi in the patients. Two-platform technology enabled assessment of the quantitative indicators of the subpopulation composition of peripheral blood T lymphocytes. he absolute number of lymphocytes was calculated with the help of a Sysmex XT-2000i hematology analyzer (Sysmex Corporation; Japan). The preparation of samples for cytofluorimetric analysis included incubation of 100 µL of whole blood with 10 µL of monoclonal antibodies tagged with fluorochromes for 20 min in a dark place. The erythrocytes were lysed with BD FACS™ Lysing Solution (BD Biosciences; USA); the duration of incubation therewith in the dark at room temperature did not exceed 10-12 minutes. The resulting samples were analyzed in a Novocyte flow cytometer (ACEA Biosciences; USA). The surface markers used to determine lymphocyte subpopulations were as follows: CD45, IgG1, IgG2a, CD3, CD4, CD25, CD127, CD161 (Beckman Coulter, USA; BD Biosciences, USA; SONY corp., Japan).

We used MS Excel 2016 (Microsoft corp.; USA), Statistica 10 (StatSoft, Inc.; USA) to process the data obtained. The results are presented as a median (Me) and quartiles (Q_0.25-Q_0.75). Mann–Whitney U test and Kruskal–Wallis test enabled comparison of differences in the attributes. The conclusions were considered significant at p < 0.05 (*).

Results and discussion

Using the nonparametric Kruskal–Wallis test, we compared the differences in Th17/T_reg, T_127hi/ T_reg, and Th17/T_127hi ratio in children with injury over time in different groups: Control group, Minj and Sinj groups (Table 1, Figure 1). For Sinj group, a pronounced increase of Th17/T_reg ratio in the acute post-traumatic period with an decrease to 14 days was revealed groups (Figure 1A). The values of T_127hi/ T_reg and Th17/ T_127hi in the first day for indicators of patients with MInj correspond to the values of control group and significantly differ from patients with SInj in the 3-5^th day and in the 5-14^th day after the injury respectively (Figure 1B, C).

 

Figure 1. Dynamics of Th17/T_reg (A), T_127hi/T_reg (B), and Th17/T_127hi (C) ratio in the critical period of injury in children

Note. Me (Q_0.25-Q_0.75) Min-Max; the significance is represented by letters accoding to pairwise comparation through the Kruskal–Wallis test; comparison groups: Cntr – control group; ISS < 16 – MInj group; ISS > = 16 – SInj group by day after severe injury.

 

Using the nonparametric Mann–Whitney test, we compared the differences in Th subsets ratio in children with severe injury over time in different groups: with and without MOF, with and without IC and outcome groups (Table 1).

 

TABLE 1. Dynamics of Th17/T_reg, T_127hi/T_reg, AND Th17/T_127hi ratio in the critical period of severe injury in children (SInj Group) in comparation with control group and MInj Group and in SInj group depending of IC, mof development and outcome prediction of oiss, Me (Q_0.25-Q_0.75)

Factor		Control	MInj	SInj, day after injury
				1	3	5	7	14
n		80	34	68	87	35	74	67
Th17/Treg	ratio c. u.	1.78 (1.32-2.31)	1.78 (1.11-2.48)	2.58 (1.83-3.36)	2.45 (1.82-3.43)	2.47 (1.50-3.43)	1.88 (1.34-2.84)	1.95 (1.49-2.63)
MOF Th17/Treg	N			2.59 (1.61-3.34)	2.52 (1.80-3.61)	2.50 (1.67-3.86)	2.04 (1.36-2.92)	2.07 (1.54-2.72)
	Y			2.45 (2.21-3.57)	2.28 (2.04-2.97)	2.33 (1.13-2.59)	1.45 (1.18-2.08)	1.49 (1.34-1.99)
IC Th17/Treg	N			2.77 (2.09-3.42)	2.57 (1.94-3.75)	2.47 (1.41-3.20)	2.13 (1.46-2.98)*	2.02 (1.53-2.63)
	Y			2.03 (1.65-2.48)	2.24 (1.77-2.65)	3.21 (2.18-4.56)	1.28 (1.00-1.82)*	1.52 (1.17-2.38)
OISS Th17/Treg	F			2.61 (1.60-3.28)	2.27 (1.77-3.44)	2.38 (1.90-3.85)	2.22 (1.27-3.05)	2.20 (1.77-2.92)*
	UF			2.50 (1.90-4.51)	2.65 (1.99-3.29)	2.54 (1.13-2.88)	1.81 (1.39-2.15)	1.58 (1.32-2.46)*
T127hi/Treg	ratio c. u.	1.20 (1.03-1.84)	1.11 (1.01-1.73)	1.55 (1.08-2.46)	1.87 (1.09-2.73)	1.89 (1.23-2.92)	1.55 (0.96-2.39)	1.62 (1.08-2.30)
MOF T127hi/Treg	N			1.53 (1.04-2.23)	1.89 (1.13-2.77)	1.90 (1.40-2.95)	1.63 (1.14-2.40)	1.73 (1.15-2.47)
	Y			1.87 (1.23-3.01)	1.20 (0.82-2.14)	1.22 (0.98-1.89)	0.94 (0.83-2.11)	1.27 (0.99-1.71)
IC T127hi/Treg	N			1.70 (1.16-2.69)*	2.11 (1.13-2.87)**	1.89 (1.25-2.89)	1.70 (1.11-2.44)*	1.78 (1.15-2.47)*
	Y			1.00 (0.82-1.38)*	1.20 (0.83-1.68)**	2.10 (1.11-3.73)	1.09 (0.77-1.46)*	1.26 (0.78-1.40)*
OISS T127hi/Treg	F			1.70 (1.10-2.34)	1.69 (1.02-2.51)	1.88 (1.44-2.91)	1.67 (1.01-2.38)	2.01 (1.16-2.82)
	UF			1.41 (1.04-2.74)	2.05 (1.18-2.82)	1.89 (1.06-2.89)	1.45 (0.98-2.31)	1.44 (1.05-1.95)
Th17/T127hi	ratio c. u.	1.49 (1.03-1.85)	1.52 (1.12-1.79)	1.47 (1.21-2.10)	1.40 (1.09-1.99)	1.18 (0.94-1.40)	1.25 (0.96-1.64)	1.24 (0.96-1.50)
MOF Th17/T127hi	N			1.48 (1.21-2.10)	1.38 (1.09-1.96)	1.16 (0.91-1.36)	1.25 (0.94-1.58)	1.20 (0.96-1.56)
	Y			1.36 (1.24-1.61)	1.44 (0.95-2.52)	1.27 (1.26-1.45)	1.46 (1.08-1.82)	1.32 (0.99-1.44)
IC Th17/T127hi	N			1.42 (1.14-2.09)	1.37 (1.07-1.91)	1.18 (0.89-1.33)	1.25 (0.96-1.63)	1.19 (0.91-1.50)
	Y			1.62 (1.44-2.20)	2.03 (1.26-2.56)	1.22 (1.00-1.66)	1.27 (0.97-1.86)	1.32 (1.12-1.53)
OISS Th17/T127hi	F			1.43 (1.20-1.80)	1.43 (1.10-2.03)	1.27 (0.90-1.52)	1.33 (0.96-1.62)	1.24 (0.96-1.47)
	UF			1.60 (1.26-2.24)	1.29 (1.09-1.86)	1.08 (1.00-1.27)	1.20 (0.96-1.69)	1.24 (0.97-1.51)

Note. Mann–Whitney U test; *, p <0,05; **, p <0,01; comparison groups: MOF group (N, no; Y, yes); Infectious complication (N, no; Y, yes); Outcome (F, favorable outcome; UF, unfavorable outcome); c. u., conventional units.

 

Significant differences in Th17/T_reg ratio level were found in IC groups – on 7^th day, in OISS groups on 14^th day from, and no reliable differences in groups with and without MOF throughout the observation period. Significant differences in T_127hi/T_reg ratio level were found in IC groups from 1^st to 3^rd day and from 7^th to 14^th day, in IC and OISS – no reliable differences. Also, there were no significant differences in the level of Th17/T_127hi for all comparison groups in children with severe trauma (Table 1, Figure 2C-F, see 3^rd page of cover). Even though the levels of Th subsets ratio in the groups were determined in a wide range of values, patients with MOF had lower median concentrations within from 7^th to 14^th day for Th17/T_reg and from 3^rd to 14^th day – for T_127hi/T_reg than patients without MOF (Table 1, Figure 2A, B, see 3^rd page of cover). This is due to differences in the kinetics of the levels of Th subsets in the acute post-traumatic period in children. The level of T_127hi lymphocytes reach normal values by 5^th day, unlike Th17 and T_reg, the level of which remains reduced for up to 7-14 days. The patients with MOF and/or with IC had significantly lower median concentrations of Th17 and T_reg within 1-7 days after admission to the ICU than patients without MOF/IC (Figure 2, see 3^rd page of cover). Zhang et al. demonstrated that the level of Th17 showed increased initially and then decreased in patients with thoracic trauma. The frequency of Th17 was significantly increased in traumatic patients compared to healthy controls on the day after admission [11]. Another studies have shown that on trauma patients found elevated Th17/CD4⁺Treg ratios in trauma patients who developed sepsis, furthermore the ratio of Th17 cells to CD4⁺Tregs was skewed in favor of Th17 cells in non-surviving patients [1, 3].

 

Figure 2. Heat maps show the kinetics of changes in perepheral blood T127hi, Treg, Th17 absolut cell count levels and Th17/Treg, T127hi/T_reg, and Th17/T_127hi ratio levels in the critical period of severe injury in children depending of MOF (A, B), infectious complication (C, D) development and outcome prediction of OISS (E, F)

 

Conclusions

Therefore, we have characterized and analyzed the dynamics of Th subsets ratio – Th17/T_reg, T_127hi/T_reg, and Th17/T_127hi in the critical period of severe injury in children, which may play an important role in the post-traumatic immunosuppression, and thereby the recovery from trauma. The lower-level T_127hi/T_reg ratio in trauma patients admitted to the ICU is significantly associated with develop the infectious complications and MOF. The lower-level Th17/T_reg is significantly associated with develop the infectious complications and outcome of the traumatic disease.

Compliance with ethical standards

The study was conducted in accordance with the Declaration of Helsinki and approved by the Committee on Biomedical Ethics of Institute of Urgent Children Surgery and Traumatology (Protocol No. 2 of 26.05.2020). All study participants signed an informed consent.

Acknowledgments

The authors express their gratitude to all patients who participated in the study, as well as colleagues from the Department of Combined Trauma, Anesthesiology and Intensive Care Unit of Institute of Urgent Children Surgery and Traumatology.

About the authors

R. Sh. Zakirov

National Medical Research Center for Children's Health; Institute of Urgent Children Surgery and Traumatology

Author for correspondence.
Email: biochemik@bk.ru
ORCID iD: 0000-0002-3101-0207

Clinical Laboratory Physician, Clinical Diagnostic Laboratory; Researcher, Laboratory of Immunology, Cytochemistry and Biochemistry

Russian Federation, 2/1, Lomonosovsky prospect., Moscow, 119296; 22 Bolshaya Polyanka st., Moscow, 119180

S. V. Petrichuk

National Medical Research Center for Children’s Health

Email: cito@list.ru
ORCID iD: 0000-0003-0896-6996

PhD, MD (Biology), Professor, Chief Research Associate, Laboratory of Experimental Immunology and Virology

Russian Federation, 2/1, Lomonosovsky prospect., Moscow, 119296

Ekaterina V. Freidlyn

National Medical Research Center for Children’s Health

Email: frdl-cito@mail.ru
ORCID iD: 0000-0002-5645-5181

Research Laboratory Assistant, Laboratory of Experimental Immunology and Virology

Russian Federation, 2/1, Lomonosovsky prospect., Moscow, 119296

Daria G. Kuptsova

National Medical Research Center for Children’s Health

Email: dg.kuptsova@gmail.com
ORCID iD: 0000-0001-7771-3314

Junior Researcher, Clinical Laboratory Physician, Laboratory of Experimental Immunology and Virology

Russian Federation, 2/1, Lomonosovsky prospect., Moscow, 119296

Olga G. Yanyushkina

Institute of Urgent Children Surgery and Traumatology

Email: spartak-lfc@mail.ru
ORCID iD: 0000-0002-6227-466X

Researcher, Department of Multiple Trauma and Intensive Care Unit

22 Bolshaya Polyanka st., Moscow, 119180

Olga V. Karaseva

National Medical Research Center for Children’s Health; Institute of Urgent Children Surgery and Traumatology

Email: karaseva.o@list.ru
ORCID iD: 0000-0001-9418-4418

PhD, DM, Professor, Deputy Director for Research, Chief of Department of Multiple Trauma and Intensive Care Unit, Head of the Department of Emergency Surgery and Trauma in Children

2/1, Lomonosovsky prospect., Moscow, 119296; 22 Bolshaya Polyanka st., Moscow, 119180

References

Supplementary files