PHAGOCYTIC ACTIVITY OF NEUTROPHILIC GRANULOCYTES IN CHILDREN WITH CHRONIC NONSPECIFIC LUNG DISEASES WITH POST-INFLAMMATORY PNEUMOFIBROSIS.

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Introduction. Chronic nonspecific lung diseases (CNLD) Pediatrics and pulmonology remain an urgent problem in children [4]. CNLD is characterized by recurrent infections leading to a long-lasting inflammatory process in the lungs, which can lead to an overgrowth of connective tissue in the lungs and lead to the progression of an irreversible fibrous process [7]. It is known that several cell types and signaling mechanisms are involved in the process of fibrogenesis, which, when activated, increase the secretion of inflammatory mediators [10].
One of the reasons for the development of chronic forms of lung diseases and pneumofibrosis as a pathological outcome of this inflammation may be an insufficient immune response, the key position in which belongs to neutrophil granulocytes (NG). Activation of NG is necessary to provide the first line of defense against bacterial, viral and fungal pathogens through the realization of its phagocytic function, however, in recent years, the understanding of the role of neutrophils in the realization of innate and adaptive immunity has been significantly expanded [2]. During the inflammatory process in the lungs, neutrophils are attracted to the affected area, which exhibit phagocytic activity, which is a multi-stage process that includes chemotaxis, capture of an object followed by the formation of a phagolysosome and proteolytic degradation of the absorbed object. Disorders at various stages of phagocytic reactions lead to the development of numerous pathological conditions. Therefore, the constancy of immune homeostasis depends on the adequate realization of the physiological functions of NG [14]. 
Acute neutrophilic inflammation is followed by a resolution phase important for tissue homeostasis. If these mechanisms do not work, neutrophils cause chronic inflammation characterized by the release of oxidants and proteases and leading to tissue damage, but recently they have also been considered as important participants in tissue repair [11]. Although the role of neutrophils in acute inflammation is fairly well established, their role in chronic diseases, tissue damage and repair has only just begun to be studied [13].
Neutrophils do not live long and, under normal conditions, after performing their functions in the focus of inflammation, they die. Natural death occurs due to the process of programmed cell death – apoptosis, which allows to prevent the release of cytotoxic neutrophil contents into surrounding tissues and timely eliminate dead cells by tissue macrophages [8]. Therefore, data on the apoptotic activity of neutrophils and their functional activity can be considered as one of the mechanisms that allow controlling inflammatory reactions. Violations of the mechanisms regulating apoptosis of immune cells may be a central pathogenetic factor in the initiation and/or persistence of inflammation [6].
One of the ways to disrupt the mechanisms of apoptosis of NG is a violation of changes in the activity and functions of mitochondria, the permeability of the mitochondrial membrane, which leads to mitochondrial dysfunction, therefore, it is believed that a decrease in the membrane potential of mitochondria (MPM) is one of the main indicators of the initiation of the mitochondrial pathway of apoptosis [5]. During inflammation, the lifespan of a neutrophil increases, its death is postponed to fight infection and inflammation, and these activated NG, producing reactive oxygen species, proteases exit the vascular bed into tissues, can lead to permanent tissue damage [12]. 
Given the importance and renewed interest in neutrophils as tools for immune deregulation, associated with lung disease in CNLD is a timely problem that should be addressed [15]. However, little data is known about the contribution of phagocytosis activity to the pathogenesis of inflammatory reactions in children with CNLD with post-inflammatory pneumofibrosis. In this regard, the purpose of this study was to study the phagocytic activity of blood neutrophils as an important component of innate immunity in patients with chronic heart disease.
Materials and methods. The study included observations of 70 children with CKD (52.7% of them were children with congenital lung malformations (CLM), 32.7% with chronic bronchitis (CB) and 14.5% with bronchopulmonary dysplasia (BPD)) with focal or segmental post–inflammatory pneumofibrosis (PPF), who were monitored at the Khabarovsk branch DNC FPD – OMiD Research Institute. Of these, 27 children with PPF progression (the main group) and 43 children with PPF without progression (the comparison group). The average age of the children was 6.9±0.5 years. The indicators of 23 practically healthy children, comparable in gender and age, served as a control. The research was conducted taking into account the requirements of the Helsinki Declaration "Ethical Principles of conducting medical Research with the participation of people as subjects of research", as amended in 2013 and regulatory documents "Rules of Good Clinical Practice in the Russian Federation", approved by Order No. 200 dated 04/01/2016 of the Ministry of Health of the Russian Federation. The design of the study was approved by the decision of the Ethics Committee of the Khabarovsk branch of the DNC FPD – OMiD Research Institute, the informed consent of the parents and/or legal representatives of each child to be included in the study group was obtained. In the dynamics of follow-up, patients underwent clinical and laboratory examination in accordance with the recommended standards of care for patients with bronchopulmonary pathology. Pneumofibrosis was diagnosed using multispiral computed tomography (MSCT) with a virtual bronchoscopy program and intravenous bolus contrast (according to indications) on a Toshiba Aquillion 64 tomograph with technical parameters: 100 kV and 120 Wt, with a collimation of 64* 0.5 mm, a tube rotation time of 0.35 sec., with processing at the Vitrea workstation with software for the World Bank. The criteria for adding a case to the main group were: 1. A decrease in the volume of forced lung vital capacity (FVC) according to spirometry data by 5-10% compared with the data of the previous examination, which was accompanied by an increase in clinical manifestations of the underlying disease and/or an aggravation of changes recorded on multispiral computed tomography (MSCT) of the lungs. 2. A decrease in the FVC index by less than 5% relative to the previous estimate, combined with a progressive increase in lung tissue lesions detected during MSCT and an increase in symptoms. 3. The presence of significant fibrotic changes in the structure of the pulmonary parenchyma detected by MSCT, while the FVC was less than 70% of the norm, which reflected the initially high level of lung damage [1].
The studies were conducted during clinical remission. The functional and metabolic activity of neutrophils was evaluated according to a generally accepted method, determining the phagocytic activity of neutrophils (PHAN) as the percentage of phagocytes capable of actively capturing latex particles and the phagocytic number (PH) reflecting the number of particles absorbed by one phagocyte. The metabolic potential and the ability of the cell to complete phagocytosis were evaluated in a spontaneous and stimulated HST test. The phagocytic reserve of stimulation of the NST test (NSTst-NSTsp) was calculated. The mitochondrial membrane potential was determined using a BD FCS Calibur cytometer (USA) (BD Pharmigen, USA).
Statistical processing of the material was carried out using the statistical software package "Statistica 10.0". The sample was described by calculating the median (Me) and interquartile range in the form of 1 and 3 quartiles (Q25 and Q75). The reliability of the differences between the indicators of independent samples was assessed using the Mann–Whitney criterion. The study of the relationship was carried out using the Spearman correlation coefficient. To study the relationships between the features identified in the classification of objects, a factor analysis using the principal component method was carried out. The critical value of the significance level is assumed to be 0.05. Research results and discussion of the results. The indicators of phagocytic activity and phagocytic number of neutrophils, the nitrosine tetrazolium neutrophil recovery test (NST test) in spontaneous and stimulated by latex particles variants, with the calculation of the stimulation index are presented in the table.

Table 1.

Indicators of functional activity of neutrophil granulocytes in the blood in children with chronic nonspecific lung diseases (Ме [Q 25-75])

NST stimulated test, conventional units	57 [42-71] ∆,**	36 [20, 7-59]**	22 [19-32]
Phagocytic number reduction test, units	3,3 [3, 09-3, 8] ∆,**	5,6 [3, 92-6, 4]**	9 [7, 5-14]
Phagocytic number stimulated test, units	4,7 [4, 6-5, 4] ∆,**	4,7 [4, 6-5, 4] **	13 [14, 2-16]
Phagocytic activity of neutrophils reduction. %	45 [35, 7-50]*	45,6 [16, 3-63]*	51,2 [46-67]
Phagocytic activity of neutrophils stimulated,%	47 [38-50]*	37,1 [19-50]*	53,6 [44-70]

Note:  * - p<0.05 in relation to the control group
** - p<0.001 in relation to the control group
∆ - p<0.05 in relation to the main group 
  ∆∆ - p<0.001 in relation to the main group

p<0,05  in relation to the control group
p<0,001  in relation to the control group
p<0,05  in relation to the main group
p<0,001  in relation to the main group

The phagocytic number (PH) (the average number of microbes absorbed by one blood neutrophil, normally 5-10 absorbed microbial particles) in children with CNLD with PF progression was significantly reduced both in comparison with patients of the CNLD group without PF progression (p<0.05) and with the control group (p<0.001). The phagocytic activity of neutrophils (FAN) in children with CKD in both groups, although within the normative values, was nevertheless lower than in the control group (p<0.05).
There was a significant increase in the activity of the spontaneous HST test in children of the main group, which was 1.7 times higher than in children in the comparison group (p<0.05) and 3 times higher than in children from the control group (p<0.001). The stimulated HST test was also higher in patients with CNLD, more pronounced in patients with an increase in PPF processes compared with the control (p<0.001). However, the stimulation index characterizing the multiplicity of the increase in the HST test with antigenic irritation in the main group was lower (-3 [-7 - +3] versus 8 [4, 2-9, 6], p<0.001) in the control group, indicating a decrease in the metabolic potential of phagocytes in CNLD and a mismatch in the timing of clinical and metabolic remission.
The results of factor analysis of the indicators of functional and metabolic activity of neutrophils in children with CNLD identified the main factors that serve as an internal characteristic of the processes of phagocytosis in the examined children: the stimulation index of the HST test (weight coefficient 0.85) and the level of phagocytic activity of the blood (weight coefficient 0.72).
An increase in the proportion of cells with a reduced membrane potential of mitochondria of granulocytes in children of the main group was determined compared with the control group (31.5%±6.9 and 2.2%±0.4, respectively, p<0.001), that is, during the inflammatory process in the lungs, the number of NG reflecting the initiation of the mitochondrial pathway of apoptosis significantly increases. Correlation analysis revealed a statistically significant negative relationship between the proportion of granulocytes with reduced MPM and phagocytic reserve, the stimulation index of the HST test (rs=0.31, p<0.05) and a positive one with Phagocytic number reduction test (rs= 0.35, p<0.05) and Phagocytic number stimulated test (rs= 0.34, p<0.05).
Thus, children with CNLD with the progression of fibrosis processes develop changes in the functional and metabolic activity of peripheral blood neutrophils typical for an inflammatory reaction, characterized by an increase in oxygen-dependent and a decrease in phagocytic cell activity. 
Neutrophils, as vital regulators linking the innate and adaptive immune systems, are a double-edged weapon in the immune response of the lungs, including mechanisms such as phagocytosis, degranulation, formation of extracellular traps of neutrophil cells, secretion of exosomes, release of cytokines and chemokines, and autophagy. Although neutrophils serve as strong protectors against extracellular pathogens, neutrophils and their components can trigger various cascades leading to inflammation and fibrogenesis, one such mechanism may be a violation of the oxidative metabolism of whole blood phagocytes. Earlier, on the example of children with CLM, we revealed a decrease in the functional reserve of granulocytes against the background of hyperproduction of superoxide anion and hydroxyl radicals by these cells [3]. When phagocytosis is ineffective, neutrophils release lysosomal enzymes and protease in response to stimuli. At the same time, repeated inflammation and tissue damage can contribute to the remodeling and fibrogenesis of lung tissue. 
Conclusion. Maintaining the function of neutrophils is necessary to completely eliminate the inflammatory process in the lungs. The higher level of intensity of phagocytic activity in children with CNLD is apparently explained by the fact that in this situation the immune system continues to fight pathogens, which supports the inflammatory process at some compensated level. Apparently, the appearance of tension syndrome characterizes the activation of not only the immune system, but also other body systems that determine homeostasis. The conditions in which inflammation occurs in CNLD can be accompanied by reduced microcirculation in the lungs, which leads to the development of hypoxia and the appearance of conditions of persistence and activation of anaerobic flora, a decrease in the capabilities of local immunity and reparative processes. Correction of these disorders is the main goal of measures for the prevention and treatment of children with CNLD.

About the authors

Galina Petrovna Evseeva

Khabarovsk Branch of Far Eastern Scientific Center of Physiology and Pathology of Respiration – Research Institute of Maternity and Childhood Protection, 49/1 Voronezhskaya Str., Khabarovsk, 680022, Russian Federation

Email: 1904lenok@mail.ru
SPIN-code: 8565-3889
Scopus Author ID: 119860

MD, PhD, D.Sc. (Med.), Deputy Director on Scientific Work, Main Staff Scientist of the Group of Health and Environmental Problems of Mother and Child Health

Russian Federation, 49/1 Voronezhskaya Str., Khabarovsk

Elena Vladimirovna Knizhnikova

Khabarovsk Branch of Far Eastern Scientific Center of Physiology and Pathology of Respiration – Research Institute of Maternity and Childhood Protection, 49/1 Voronezhskaya Str., Khabarovsk, 680022, Russian Federation

Email: 1904lenok@mail.ru
SPIN-code: 4640-0969
Scopus Author ID: 974052

Postgraduate student

Russian Federation, 49/1 Voronezhskaya Str., Khabarovsk

Natalia Olegovna Abdulina

Khabarovsk Branch of Far Eastern Scientific Center of Physiology and Pathology of Respiration – Research Institute of Maternity and Childhood Protection, 49/1 Voronezhskaya Str., Khabarovsk, 680022, Russian Federation

Email: 1904lenok@mail.ru
SPIN-code: 4296-9171
Scopus Author ID: 1220245

Postgraduate student

Russian Federation, 49/1 Voronezhskaya Str., Khabarovsk

Sabina Veniaminovna Pichugina

Khabarovsk Branch of Far Eastern Scientific Center of Physiology and Pathology of Respiration – Research Institute of Maternity and Childhood Protection, 49/1 Voronezhskaya Str., Khabarovsk, 680022, Russian Federation

Email: 1904lenok@mail.ru
Scopus Author ID: 409284

MD, PhD, staff scientist the group of clinical immunology and endocrinology, pulmonologist 

Russian Federation, 49/1 Voronezhskaya Str., Khabarovsk

Maria Stanislavovna Chaika

Khabarovsk Branch of Far Eastern Scientific Center of Physiology and Pathology of Respiration – Research Institute of Maternity and Childhood Protection, 49/1 Voronezhskaya Str., Khabarovsk, 680022, Russian Federation

Author for correspondence.
Email: 1904lenok@mail.ru
SPIN-code: 2403-3193
Scopus Author ID: 906633

Staff Scientist of the Group of Clinical Immunology and Endocrinology

Russian Federation, 49/1 Voronezhskaya Str., Khabarovsk

References

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