NEUTROPHIL EXTRACELLULAR TRAPS AS MARKERS OF INFECTIOUS AND ASEPTIC INFLAMMATION

Alexander N. Kazimirskii; Казимирский Александр Николаевич; Jean M. Salmasi; Салмаси Жеан Мустафаевич; Gennady V. Poryadin; Порядин Геннадий Васильевич; Marina I. Panina; Панина  Марина Ивановна; Anna E. Kim; Ким Анна Эрнестовна; Lyudmila S. Rogozhina; Рогожина Людмила Сергеевна

doi:10.46235/1028-7221-16602-NET

NEUTROPHIL EXTRACELLULAR TRAPS AS MARKERS OF INFECTIOUS AND ASEPTIC INFLAMMATION

Authors: Kazimirskii A.N.¹, Salmasi J.M.¹, Poryadin G.V.¹, Panina M.I.¹, Kim A.E.¹, Rogozhina L.S.¹
Affiliations:
1. Pirogov Russian National Research Medical University, Ostrovityanova St.1, Moscow 117997, Russian Federation
Section: Joint Immunology Forum 2024
URL: https://rusimmun.ru/jour/article/view/16602
DOI: https://doi.org/10.46235/1028-7221-16602-NET
ID: 16602

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Abstract

One of the important problems of medicine is the search for new reliable markers of inflammation that would allow us to determine the type of inflammatory process. The aim of the study was to identify specific markers of infectious and aseptic inflammation based on the analysis of morphological variants of neutrophil extracellular traps (NETs). The study included 26 patients with various nosological forms of inflammation in the abdominal cavity (12 with acute appendicitis, 8 with acute cholecystitis, 6 with a diagnosis of pancreatitis/pancreonecrosis) after surgery. The study also included 20 patients with postcovid and a group of 10 volunteers no clinical manifestations of the disease, but at the same time had contacts with flu patients. Neutrophils were isolated using gradient centrifugation. Fluorescence microscopy with the dye SYBR Green (Evrogen, Russia). NETs in the morphological form of neutrophils web structure was found in the blood of surgical patients with acute appendicitis who had a favorable course of the postoperative period. In a group of volunteers who were exposed to influenza but did not have symptoms of an infectious disease, we also found neutrophils web structure. In all patients with abdominal inflammation complicated by abdominal abscess, in addition to neutrophil web structure, abnormal forms of NETs were recorded, in particular, NETs in the form of single filaments. In the postcovid patients an absolute absence of neutrophilis web structure was revealed. In all postcovid patients, NETs are found only in the form of single filaments. The results of subsequent experiments showed that collagen peptides are inducers of the formation of NETs in the form of single filaments. Conclusion. The detection of neutrophils web structure is a sign of an infectious inflammatory process. NETs in the morphological form of single filaments are markers of aseptic inflammation, as well as a sign of endothelial damage.

Keywords

markers of inflammation, infectious inflammation, aseptic inflammation, neutrophil extracellular traps, NETs, neutrophil web-like structures, single filaments.

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NEUTROPHIL EXTRACELLULAR TRAPS AS MARKERS OF INFECTIOUS AND ASEPTIC INFLAMMATION INTRODUCTION

At present, one of the important problems of medicine is the search for new objective and reliable, but at the same time easily detectable, markers of inflammation that would make it possible to determine the nature of the phlogogenic factor and the type of inflammatory process in order to choose the right treatment tactics, assess the effectiveness of anti-inflammatory therapy, justify the need for surgical intervention, and determine the prognosis of the disease.

The reliability of detecting a marker of inflammation may be determined by its involvement in key processes that trigger the protective responses of innate immunity. This earliest and most fundamental process is the response of neutrophils to incoming pathogens or to damage to their own tissues.

Neutrophils form neutrophil extracellular traps (NETs) when pathogens of various nature enter the body, primarily infectious ones (bacteria, viruses), as well as in response to damaged structures of their own body. Despite a large number of studies on NETs, morphological variants of these extracellular neutrophil structures have not yet been characterized. Our studies describe several variants of NETs that may be related to the type of inflammatory process.

Aim: Determination of specific markers of infectious and aseptic inflammation based on the analysis of morphological variants of neutrophil extracellular traps.

PATIENTS & METHODS

Patients. The study included 26 patients treated in 51 Moscow City Clinical Hospitals with various nosological forms of the inflammatory process in the abdominal cavity (12 patients with acute appendicitis, 8 patients with acute cholecystitis, 6 patients diagnosed with pancreatitis/pancreatic necrosis) after surgery. The study also included 20 patients with post-COVID and a group of 10 volunteers who considered themselves healthy, but at the same time had contact with influenza patients.

Methods. A vacutainer with EDTA was used to collect blood samples to prevent clotting. Isolation of neutrophils from venous blood treated with EDTA was carried out by the traditional method using gradient centrifugation. To do this, the blood was diluted 4 times with a sodium-phosphate buffer solution of 50 mM, pH 7.4, and layered on a double density gradient of ficolla-verographin. The density of the upper gradient layer is 1.077 g/cm3 and the lower one is 1.190 g/cm3. After centrifugation (1600 rpm, 30 min), a 98-100% purity of neutrophils accumulates at the gradient boundary. Neutrophils were twice washed of ficoll impurities with sodium-phosphate buffer solution (50 mM, pH 7.4). Blood cell deposition was carried out by centrifugation (1200 rpm, 15 min). Isolated neutrophils in RPMI-1640 medium were used in short-term culture experiments. The viability of the isolated neutrophils was at least 95% (test with 0.1% trypan blue solution).

Immunofluorescence detection of neutrophil extracellular traps

Fluorescence microscopy was used to detect and count neutrophil extracell traps [2]. Detection of neutrophil extracellular traps was carried out using the fluorescent dye SYBR Green (Evrogen, Russia), which specifically interacts with double-stranded DNA. Microscopy, counting and photoregistration of cells and extracellular structures were carried out at a magnification of ×1000. The results were expressed as a percentage, as the ratio of the number of extracellular traps to the total number of cells in the field of view.

Statistical processing The obtained results were processed in Statistica 12.0 (StatSoft, Inc.). The data is presented as an average (M)±standard error of the mean (m). Quantitative traits were compared using the Mann-Whitney rank U test and Kruskal-Wallis analysis of variance. The differences were considered statistically significant at p<0.05 values.

Results

Markers of infectious inflammation

Qualitative analysis of NETs in patients with inflammatory diseases makes it possible to identify characteristic morphological signs of NETs in inflammation of infectious origin. At present, the morphological forms of neutrophil extracellular traps and their functional activity in inflammatory diseases have not been practically studied. In our research, we have identified 4 main morphological forms of neutrophil traps - neutrophil networks, single filaments, fibers and veils, of which only one extracellular structure, the net-like structure, is functionally active, that is, capable of capturing and binding pathogens.

Neutrophil extracellular traps in the morphological form of neutrophil networks were found in the blood of surgical patients diagnosed with acute appendicitis, who had a favorable course of the disease and the postoperative period. Moreover, the examined patients did not have any other abnormal forms (threads, fibers, and veils) of neutrophil extracellular traps. In a group of volunteers who did not have any clinical manifestations of influenza disease, but were in constant contact with patients, we also found neutrophil networks in the complete absence of abnormal forms of NETs. The morphological forms of NETs found in patients from the Department of Abdominal Surgery and in volunteers who did not get sick with influenza are summarized in Table 1.

Table 1

Morphological forms of neutrophil extracellular traps (NETs) in patients with acute appendicitis with a favorable course and infected with influenza virus volunteers, but without obvious symptoms

Groups of patients	The number of NETs, %	Morphological forms of NETs
Groups of patients	The number of NETs, %	Web-like structures	Single filaments	Fibers	Clouds
Acute appendicitis, uncomplicated (n=6)	10,19±0,73	++++	-	-	-
Infected with the flu virus, but not sick volunteers (n=10)	8,58±0,51	++++	-	-	-

Note: ++++ - a significant number of neutrophil extracellular structures; + - minimum (less than 10% of the total) neutrophil extracellular structures.

It should be noted that neutrophil extracellular traps in the morphological form of neutrophil networks found in these two groups of examined surgical patients and infected volunteers had extremely high functional activity of neutrophil networks. Comparison of these results suggests that neutrophil extracellular traps in the morphological form of neutrophil networks may be markers of infectious inflammation. The data of other researchers confirm our observations that neutrophil networks are characteristic of the acute infectious process in the body [6, 7].

Complicated forms of abdominal inflammation - a variety of morphological types of NETs.

The study of neutrophil extracellular traps in patients with various nosological forms of the inflammatory process shows that in addition to neutrophil networks, some surgical patients have NETs in the morphological form of single threads. Thus, in all patients with appendicitis complicated by an abscess of the abdominal cavity, in addition to neutrophil networks, neutrophil extracellular structures in the form of single filaments were found. Such neutrophilic extracellular structures in the form of single filaments were also found in some patients with cholecystitis and pancreatitis (Table 2).

Table 2

Morphological forms of NETs in patients with various nosological forms of the inflammatory process in the abdominal cavity and in patients with postcovid syndrome

Groups of patients	The number of NETs, %	Morphological forms of NETs
Groups of patients	The number of NETs, %	Web-like structures	Single filaments	Fibers	Clouds
Acute appendicitis complicated by abdominal abscess (n=6)	13,26±0,95	+++	+	+	+
Acute cholecystitis (n=8)	15,66±2,81	++	+	+	-
Acute pancreatitis/pancreonecrosis (n=6)	12,64±2,73	+	+	+	+
Post covid (n=20)	6,55±0,94	-	+	-	-

Note: ++++ - a significant number of neutrophil extracellular structures; + - minimum (less than 10% of the total) neutrophil extracellular structures.

It should be noted that in all patients with abdominal inflammation complicated by abdominal abscess, in addition to neutrophil networks, abnormal forms of NEL were also recorded, in particular, NEL in the morphological form of single threads. The group of patients with post- covid syndrome, selected as a comparison group within the framework of this study, demonstrates the absolute absence of neutrophil networks. In all studied patients with post- covid, NETs is found only in the form of single filaments [3]. The pathogenesis of post- covid is associated with virus-induced damage to the vascular endothelium, leading to the development of an inflammatory process [3, 5, 8]. Contact interactions of neutrophils with fibroblasts can support the activation of neutrophil leukocytes and induce the NETs formation in the form of single filaments.

Markers of aseptic inflammation (Filamentous neutrophil extracellular traps - markers of aseptic inflammation)

In experiments on cell culture of neutrophils of healthy donors with fibroblasts, we found the formation of NETs in the form of single filaments coming out of the nucleus of neutrophils. The results of subsequent experiments showed that collagen peptides are inducers of NETs formation in the morphological form of single strands (Fig. 1).

Fig.1 Neutrophil extracellular traps formation in filamentous form by neutrophils of healthy donors under the influence of collagen peptides.

On the vertical axis - neutrophil extracellular traps (in %)

On the horizontal axis - the concentration of collagen peptides (mcg/ml).

Thus, our assumption that collagen localized on the surface of fibroblasts can induce the NETs formation in the form of single filaments has found its experimental confirmation. The physiological significance of this effect still needs to be clearly interpreted, but the prospects for its practical application are already quite clear. Literature data show that NETs may be involved not only in the initial stages of inflammation, but also in subsequent fibrosis [9]. In patients with tumor diseases, collagen has been shown to induce the formation of neutrophil extracellular traps [10] and promote the infiltration of activated lymphocytes into tumor tissue, but the morphological forms of NETs have not been determined.

Conclusion

Determining the cause and type of inflammation is important in medicine, as this principle of classification is necessary for making a diagnosis and determining treatment tactics. A system of classification of types of inflammation based on the determination of the parameters of adaptive immunity is proposed. This classification uses combinations of different cytokines [11]. The results of our study demonstrate the effectiveness of the chosen approach to the search for specific markers of inflammation. The detection of neutrophil networks in the blood of patients is a sign of an infectious inflammatory process. This conclusion is to some extent confirmed by the data of other studies that have found similar web-like structures [4].

At the same time, neutrophil networks are also formed under the influence of apoptosing cells infected with viruses or intracellular microorganisms [1]. Neutrophil extracellular traps in the morphological form of single filaments, according to the results of our studies, are markers of aseptic inflammation, as well as a sign of endothelial damage.