The definition of neutrophil extracellular traps and the concentration of short-chain fatty acids in salmonella-induced inflammation of the intestine against the background of vancomycin and Bacteroides fragilis

Results. The introduction of vancomycin contributed to an increase in the number of Sytox+-cells in scrapings of the intestinal mucosa and in the blood by 55 % and 2.5 times (group II). With the combined administration of vancomycin and S. enteritidis (III group), S. typhimurium (IV group), the mean Sytox+-cell counts in mucosal scrapings increased by 30 % and 2.4 times, and in the blood by 30 % (group IV ), there was also a decrease in the number of NETs by 40 % (group IV). The introduction of B. fragilis against the ґbackground of pretreatment with vancomycin and infection with salmonella showed a decrease of Sytox+-cells in the scrapings of the intestinal mucosa by 43 % and 53 %, and in the blood by 46 % and 58 % (V and VI groups), and the number of NETs in scrapings from the intestinal mucosa and in the blood significantly increased by 43 % and 40 % (V group), and by 2.3 and 2.0 times (group VI). When infecting the rats with S. typhimurium against the background of pretreatment with vancomycin and the introduction of B. fragilis, the concentration of acetate in the samples increased by 2 times; propionate – 6 times and butyrate – 3 times.

Intestinal microbiome is a unique system responsible for maintaining the body's homeostasis.This constancy is ensured by the interaction of representatives of microflora with gut-associated lymphoid tissue (GALT) through a certain group of molecules -short-chain fatty acids (SCFA) [1].These acids are metabolic products of anaerobic bacteria that are capable of influencing chemotaxis, apoptosis, T-cell differentiation, the transcriptional activity of the FFAR2, Foxp3 and RORγt genes and activation of the receptors for acetate, propionate and butyrate (GPR43, GPR41 and GPR109a (HCA2)).One of the main producers of SCFA in GALT are bacteroides [2][3][4].We showed previously that the administration of B.fragilis against the background of vancomycin pretreatment and salmonella infection increases the mRNA level of the FFAR2 gene, and also increases the transcriptional activity of the Foxp3 gene directing T-cell differentiation toward Treg, which contributed to a decrease in inflammatory reactions in the intestine and recovery of the microbiome development of experimental gastroenteritis [5].
Meanwhile, the effect of changes in the level of SCFA on the functional activity of some of the main cells of innate immunity -neutrophils, practically nothing is known.A recent discovery related to the ability of neutrophils to distinguish lipopolysaccharides from different bacterial agents and selectively release extracellular traps (NETs) is of great interest in studying the mechanisms of interaction of pathogenic microorganisms with immune system cells and their ability to influence the formation of NETs the concentration of SCFA, as a factor affecting the inhibition of inflammatory reactions in the intestine [6,7].
Salmonella are one of the inducers of the formation of NETs.Interestingly, there is the fact that taking antibiotics can also stimulate the formation of NETs, which leads to inhibition of the growth of pathogenic microorganisms [16].Therefore, the study of the nature of generation of NETs with the administration of S. typhimurium, S. enteritidis, vancomycin and Bacteroides fragilis is of particular interest.

The aim
To study the features of the formation of NETs in the blood and GALT with salmonella-induced inflammation against the background of the administration of vancomycin and B. fragilis, and to determine the concentration of SCFA in the luminal microflora of rats by means of chromatography-mass spectrometry.

Materials and methods of research
The experiments were carried out on 60 male Wistar rats of 3 months age who were divided into 6 groups (10 each): I - Control (intact rodents) II -Vancomycin (animals that received oral administration of vancomycin at a dose of 50 mg/kg); III -Vancomycin + S. enteritidis (animals that received vancomycin at oral dose of 50 mg/kg and received bacterial load of S. enteritidis in an amount of 3×10 8 CFU/ml every other day); IV -Vancomycin + S. typhimurium (animals that received vancomycin at the dose of 50 mg/kg per day and received a bacterial load of S. typhimurium in the amount of 3×10 8 CFU/ml every other day); V -Vancomycin + S. enteritidis + B. fragilis (animals received by oral administration of vancomycin at a dose of 50 mg/kg, a day later -a bacterial load of S. еnteritidis in the amount of 3×10 8 CFU/ml and the next day of B. fragilis 3×10 8 CFU/ml); VI -Vancomycin + S. typhimurium + B. fragilis (animals that received vancomycin at oral dose 50 mg/kg, a day later -a bacterial load of S. typhimurium in the amount of 3×10 8 CFU/ml and the next day -B.fragilis in 3×10 8 CFU/ml).On the fifth day the rats were removed from the experiment in accordance with the principles of euthanasia.Infection of animals was carried out by daily cultures of salmonella grown on 1.5 % of MPA, obtained from the Museum of strains of microorganisms of the Ukrainian Center for Disease Control and Monitoring of the Ministry of Health of Ukraine.Bacteroid cultures were grown on nutrient media prepared according to methodological recommendations "Laboratory diagnostics of purulent inflammatory diseases caused by asporogenous anaerobic microorganisms" (Kharkiv, 2000).Bacterial suspensions were standardized using a DEN-1B (Biosan) densitometer by McFarland (McF).To introduce the S. enteritidis, S. typhimurium and B. fragilis to rats, we prepared suspensions in the concentration of 1.0 McF standards, which corresponds to the concentration of 3×10 8 CFU/ml.
The evaluation of macroscopic manifestations of ileum deficiency was performed on a scale from 0 to 8 in accordance with the criteria of Morris et al. in the modification of Jagtap [17]: (0) no visible changes, (1) there are areas of hyperemia, (2) there are lesions having a diameter of 1 mm or less, (3) lesions having a diameter of 2 mm or less (number <5), (4) there is a defect having a diameter of 2 mm or less (number 5-10), (5) a defect having a diameter of 2 mm or less (>10), ( 6) there is a lesion, having a diameter of more than 2 mm (number <5), (7) there are lesions having a diameter greater than 2 mm (number 5-10); (8) lesions having a diameter greater than 2 mm (>10).
As a material for conducting the chromatography-mass-spectrometric study to determine the concentration of SCFA (acetate, propionate and butyrate), a luminal microflora of rats was used and only in comparison groups IV and VI.
Sample preparation process: fecal samples (2 g) were taken into sterile tubes, 1ml of bidistilled water was added to each by the glass pipette, it was mixed well.Then 1 ml of 0.1 N hydrochloric acid and 2 ml of diethyl ether were added.The stirring was continued for 25 minutes and it was left for 10 minutes for complete phase separation.Using medical sterile syringes for 1000 μl, the upper phase was taken, transferred to numbered centrifuge microtubes [18].To determine the concentration of SCFA, we developed a new methodology for performing of chromatography-mass spectrometric analysis (CMSA).The analysis was carried out using liquid chromatography-mass spectrometеr (LC MS): Agilent 1260 Infinity HPLC System (USA) (degasser, binary pump, autosampler, column thermostat); one-wave Agilent 6120 mass spectrometer with electrospray ionization (ESI); OpenLAB CDS Software.In the CMSА study, the following tests were used: 1) Binary gradient-A: Determination of the average number of Sytox + and NETs cells in scrapes taken from the mucous membrane of the ileum of the intestine and in the blood (groups I-VI) was carried out with the immunofluorescence microscope PrimoStar (ZEISS, Germany) in the ultraviolet excitation spectrum of 390 nm (FITC).Digital image analysis was performed using the AxioVision 4.7.2 system (Imaging Associates Ltd, Germany).The material was taken with a sterile plastic curette from the mucous membrane of the ileum of the intestine and transferred to a sterile slide glass.The drug was fixed with 95 % ethanol for 5 minutes.The excess of ethanol after fixation was removed with the help of filter paper, not allowing the preparation to dry in air.To identify the NETs, the fluorescent dye SYTOX TM Green nucleic acid stain (Invitrogen, USA) was used, according to the instructions attached to the kit.The drug was additionally stained for 30 seconds by Romanovsky-Giemsa and light and fluorescent microscopy was performed on a ×10 lens in the field of view.In the automatic mode, areas with statistically significant fluorescence characteristic of these cells were determined.Immune-positive cells were studied using the computer program ImageJ (NIH, USA).The morphometric and densitometric characteristics of immune-positive cells were counted.Cells with a circularity ≤0.5 (4π×[Area]/[Perimeter] 2 ) were referred to neutrophils forming NETs.
The statistical analysis of the results was carried out using Microsoft Excel 2016 and StatSoft Statistica v12 (NAXXR712D833214FAN5).To calculate the reliability of the differences between the mean values of the concentration of short chain fatty acids the non-parametric Mann-Whitney criterion (U-test) and the Student's t-criterion for calculating the average number of Sytox + and NETs cells were used.Statistically significant differences were considered at a value of P < 0.05.

Results and its discussion
The results obtained in the course of the studies on the quantitative determination of Sytox + -cells and NETs in scrapings from the mucous membrane of the ileum of the intestine and in the blood showed that when vancomycin (group II) was administered to experimental animals, an increase in the number of Sytox + cells by 55 % and by 2.5 times (P < 0.05) compared with the control group.When analyzing the data of groups III and IV, which underwent pretreatment with vancomycin, and after infection with S.enteritidis and S. typhimurium, the average value of Sytox + -cells in scrapings from the ileal mucosa increased significantly by 30% and by 2.4 times, and in the blood by 30% and only in the IV group (P < 0.05), than with the single administration of vancomycin (II group).Also, in scrapings from the ileal mucosa, there was a decrease in the number of NETs by 40 % (group IV) (P < 0.05) (Table 1).Calculation of the average number of Sytox + -cells in scrapings from the intestinal mucosa in the V and VI groups, where the animals received bacterial strain of B. fragilis against the background of vancomycin pretreatment and the introduction of S. enteritidis, S. typhimurium, A -the content of acetate in animals in the group S. typhimurium + Vancomycin.A1 -the content of acetate in animals in the group S. typhimurium + Vancomycin + B. fragilis.

B B1
Original research decreased by 43 % and 53 %, and in the blood -by 46 % and 58 % (P < 0.05) in relation to the results obtained in the analysis of groups III and IV.However, the number of NETs in scrapings from the ileal mucosa and in the blood increased significantly in the V group by 43 % and 40 %, and in the VI group by 2.3 and 2.0 times, respectively (P < 0.05) than in III and IV groups (Table 2).
In the course of the studies, we determined the concentrations of SCFA (acetyl, propionate and butyrate) in the luminal microflora of rats when they were infected with S. typhimurium against the background of the preliminary administration of vancomycin, as well as in combination with B. fragilis.Thus, in the infection of rats S. typhimurium against the background of pretreatment with vancomycin and in the combined administration of B. fragilis, the average concentration of acetate in the samples increased significantly -2-fold (Fig. 1); propionate -6 times (Fig. 2) and butyrate -3 times, respectively (P < 0.05) (Fig. 3), rather than infecting S.typhimurium animals against the background of the preliminary administration of vancomycin (Table 3).
The formation of NETs (netosis) can take place in three versions: a) cell lysis-suicidal netosis; b) vesicular secretion of nuclear DNA without destruction of MTC neutrophils -vital netosis; c) formation of NETs from mitochondrial DNA [19,20].At present, the most studied model is suicidal netosis, which lasts for 2-4 hours.Its onset is characterized by neutrophil activation, through the recognition of pathogens, which leads to the induction of the NADPH oxidase (NOX) complex, through protein kinase C, and to an increase in cytosolic Ca + [21][22][23].These cations serve as a co-factor for peptidyl-arginase deaminase 4 (PAD4) and a nuclear enzyme that deaminates histones, modifies amino acids, which leads to decondensation of chromatin and loss of positive charges necessary for the interaction of histones with DNA [24,25].Active forms of oxygen contribute to the destruction of the nuclear membrane into small granules and to the mixing of chromatin with cytoplasmic proteins and granular toxins.The formation of NETs depends on the transport of elastase and myeloperoxidase from the granules to the nucleus.This results in the release of chromatin and lysis of the cell with the release of DNA as extracellular traps (NETs) [26,27].The second type of ECT generation (5-60 min), proceeding without loss of the nuclear or plasma membrane and independent of ROS, is vital netosis.This process is carried out due to three morphological modifications: the growth of the nuclear envelope and the release of vesicles; decondensation of the nucleus; destruction of the nuclear membrane and release of DNA [28][29][30].This variant of NETosis is mediated by the effect on Toll-like receptors (TLRs) and complement receptors of the C3 protein [31][32][33].In addition, the formation of NETs by neutrophils can be facilitated by the interaction of the glycoprotein Ib in platelets with β2-integrin (CD18), by activating ERK, PI3K and src-kinases [34].Thus, non-nuclear neutrophils retain the ability to phagocytosis pathogenic microorganisms, regardless of the loss of DNA.The third, ROS-dependent mechanism of NETosis, is the release of mitochondrial DNA, instead of nuclear, by recognizing the lipopolysaccharide (LPS) of bacteria or C5a, resulting in the formation of NETs for 15 min [35][36][37][38].
Our results showed that the introduction of bacteroides changes significantly the concentration of SCFA in the intestine and this is accompanied by an increase in the formation of NETs.This is confirmed by other studies on the effect of SCFA on effector functions of neutrophils [39].SCFA, when interacting with neutrophils, can affect the production of pro-inflammatory cytokines (TNF-α and IL-17) and neutrophil chemoattractants (CXCL1 and   CXCL8) by initiating the FFAR2 receptor, which results in the induction of neutrophil chemotaxis [40][41][42].The inhibitory effect of neutrophils on the production of TNF-α has been described by Tedelind (2007) who showed that this process was mediated by the Toll-like receptor (TLR) agonists and SCFA.When human neutrophils were incubated with TLR2 agonists and SCFA, the production of CXCL8 was noted, which proves the direct effect of free fatty acids on neutrophil activity [43].The experiments carried out to study the neutrophil functions have shown that under the influence of SCFA, they are capable of releasing reactive oxygen species and nitrogen oxide, which can affect their life cycle.In his works Sina (2009) demonstrated changes in migration of mouse neutrophils under the influence of propionate [44].At the same time, Le Poul (2003) suggests that intensive neutrophil migration may be due to the presence of formyl-methionyl-leucyl-phenylalanine (fMLP) [45].In the studies on different experimental models (dextran sulfate-sodium colitis [46], T-cell-dependent colitis [47,48], thioglycollate-induced peritonitis [49], acute renal failure [50]), the researchers determined that activation of SCFA reduces the restructuring of the neutrophil cytoskeleton and the intensity of inflammatory responses.This fact is confirmed by the investigation of Mishiro (2013) that was conducted on mouse models with dextran sulfate-sodium colitis and rectal administration of butyrate in which the weakening of the rearrangement of the cytoskeleton of neutrophils and reduction in inflammation were also observed [51].The results obtained by Kim (2013), likewise show a reduction in inflammatory responses due to the interaction of neutrophils with SCFA and epithelial cells of the intestine (GPR41), when C. rodentium mice are infected with the subsequent introduction of acetate.However, the results obtained by other scientists showed that SCFAs not only did not influence but sometimes even inhibited phagocytosis and the formation of reactive oxygen species by neutrophils [52,53].Also, another group of scientists found that with the activation of caspase, propionate and butyrate are capable to launch a neutrophil apoptosis, which is independent of protein kinases and activation of SCFA receptors [54].Similar studies on SCFA, conducted by Maslovsky (2009), demonstrated the effect of acetate on the induction of neutrophil apoptosis, but, unlike the Aoyama (2010) studies, they indicated the direct involvement of the FFAR2 receptor in this process [55,56].Some of the findings indicate the involvement of SCFA in the infectious process.Thus, scientists, who used a subcutaneous model of opportunistic infection, caused by pathogenic microorganisms A. actinomycetemcomitans, demonstrated that the presence of SCFA did not affect the number of leukocytes, but they reduced significantly the production of cytokines, phagocytic activity and caused the death of bacteria, but did not lead to a change in effector mechanisms of neutrophils.A similar effect that was not associated with the activation of FFAR2 and the main SCFA receptor expressed on the surface of neutrophils was also observed in the stimulation of neutrophils by bacteria, which may have been due to the inhibition of histonedeacetylases [57][58][59].Of particular interest are the results of studies obtained by Brinkmann (2004), who showed that the infection of S. typhimurium and S.enteritidis, the main agents of gastroenteritis development, against the background of the dysbiotic changes in the intestinal microflora, can enhance the formation of NETs due to the action of superoxidedismutase [60][61][62].

Conclusions
1.The administration of vancomycin caused an increase in the number of Sytox + cells in scrapings of the ileal mucosa and in the blood of rats.In pre-treatment of animals with vancomycin and infection of S.enteritidis and S.typhimurium, there was also an increase in the content of Sytox + cells in scrapings from the ileal mucosa and in the blood, and the number of NETs decreased.The administration of B.fragilis to experimental animals receiving S. enteritidis and S. typhimurium against the background of vancomycin pretreatment leads to a decrease in the number of Sytox + cells in scrapings from the ileal mucosa and in the blood, but induces the generation of NETs, which increases significantly the effectiveness of phagocytosis in the development of gastroenteritis salmonella etiology.
2. The conducted chromatography-mass spectrometric analysis showed that the administration of B. fragilis to animals pretreated with vancomycin followed by infection with S. typhimurium causes an increase in the level of SCFA concentration, which contributes to a decrease
Prospects for further research are the study of the transcriptome of the immune response genes in the intestinal lymphoid tissue of rats with salmonella-induced inflammation.

Table 1 .
Chromatograms of butanoic acid in the fecal samples.Average number of Sytox+ -neutrophiles and NETs in scrapings from the intestinal mucosa and in the rat blood (Total Density (mean) + S.E.M.) in the field of view C -the content of butyrate in animals in the group S. typhimurium + Vancomycin.C1 -the content of butyrate in animals in the group S. typhimuriu m + Vancomycin + B. fragilis.C C1 Оригінальні дослідження Pathologia.Volume 15.No. 1, January -April 2018 ISSN 2306-8027 http://pat.zsmu.edu.ua

Table 2 .
Determination of the average number of Sytox+ neutrophiles and NETs cells in rats upon administration of S. enteritidis, S. typhimurium and B. fragilis against the background of vancomycin pretreatment (Total Density (mean) + S.E.M.) in the field of view : the reliability of differences in the parameters of p < 0.05 in relation to the groups S. enteritidis + Vancomycin and S. typhimurium + Vancomycin. *

Table 3 .
Determination of the average content of SCFA in the luminal microflora of rats, g/ml