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Медицинская иммунология Medical Immunology (Russia)/
2020 Т. 22, № ^ Оригинальные статьи Meditsinskaya Immunologiya
стр. 335-346 ^ , . . . . 2020, Vol. 22, M 2, pp. 335-346
© 2020, спб ро рааки Original articles © 2020, spb raaci
ОСОБЕННОСТИ СУБПОПУЛЯЦИОННОГО СОСТАВА РЕГУЛЯТОРНЫХ Т-ЛИМФОЦИТОВ И МИКРОБИОТЫ КИШЕЧНИКА ПРИ СИНДРОМЕ РАЗДРАЖЕННОГО КИШЕЧНИКА
Ермоленко Е.И.1, 2, Кудрявцев И.В.1, 4, Соловьева О.И.3, Карасева А.Б.1, Цапиева А.Н.1, Кондратенко Ю.Д.2, Котылева М.П.1, Серебрякова М.К.1, Шумихина И.А.3, Суворов А.Н.1, 2
1ФГБНУ «Институт экспериментальной медицины», Санкт-Петербург, Россия
2 ФГБОУВО «Санкт-Петербургский государственный университет», Санкт-Петербург, Россия
3 ФГБОУ ВО «Северо-Западный государственный медицинский университет имени И.И. Мечникова» Министерства здравоохранения РФ, Санкт-Петербург, Россия
4 ФГБОУ ВО «Первый Санкт-Петербургский государственный медицинский университет имени академика И.П. Павлова» Министерства здравоохранения РФ, Санкт-Петербург, Россия
Резюме. При помощи метагеномного анализа (16 S rRNA) выявлены особенности состава кишечного микробиоценоза у больных синдромом раздраженного кишечника (СРК): 1) увеличение представительства Actinobacteria, в том числе Bifidobacterium spp., Firmicutes, в том числе принадлежащих к семействам Streptococcaceae (Streptococcus), Lachnosperaceae(Dorea), Veillonellaceae (Dialister), Proteobacteria (семейства Enterobacteriaceae и Desulfovibrionaceae); 2) уменьшение популяции Bacteroidetes, в том числе представителей семейств Prevotellacea (Prevotella spp.), Bacteroidaceae (Bacteroides spp.), фирмикутных бактерий, относящихся к семействам Clostridiaceae и Ruminococcaceae (Fecalibacterium spp.). Проточная цитометрия при исследовании субпопуляционного состава T-регуляторных ^reg) лимфоцитов выявила у больных СРК увеличение количества CD45R0+CD62L+ клеток центральной памяти (СМ), способных регулировать процессы созревания и дифференцировки лимфоцитов в лимфоидной ткани. Обнаружено снижение экспрессии экзонуклеаз CD39 и CD73, что может оказывать существенное влияние на их активность. Отмечено снижение эффекторных клеток памяти (ЕМ) ^eg.
Изменения в уровне экспрессии экзонуклеаз CD39 и CD73 находились в обратной корреляционной связи с содержанием протеобактерий и представительством родов Bifidobactrium и Faecalibacterium. Количественное содержание СМ Treg находилось в прямой корреляционной связи с содержанием Dorea spp.
Полученные результаты могут указывать на нарушения в процессах дифференцировки ^eg, которые тесно связаны с изменениями ключевых компонентов кишечного микробиоценоза при СРК.
Ключевые слова: кишечный микробиоценоз, метагеномный анализ, проточная цитометрия субпопуляции Т-лимфоцитов
Адрес для переписки:
Ермоленко Елена Игоревна
ФГБНУ «Институт экспериментальной медициныI»
197376, Россия, Санкт-Петербург,
ул. Акад. Павлова, 12.
Тел.: 8 (812) 234-68-68.
Факс: 8 (812) 234-94-89.
E-mail: Lermolenko1@yandex.ru
Образец цитирования:
Е.И. Ермоленко, И.В. Кудрявцев, О.И. Соловьева, А.Б. Карасева, А.Н. Цапиева, Ю.Д. Кондратенко, М.П. Котылева, М.К. Серебрякова, И.А. Шумихина, А.Н. Суворов «Особенности субпопуляционного состава регуляторных Т-лимфоцитов и микробиоты кишечника при синдроме раздраженного кишечника» //Медицинская иммунология, 2020. Т. 22, № 2. С. 335-346. doi: 10.15789/1563-0625-POS-1905 © Ермоленко Е.И. и соавт., 2020
Address for correspondence:
Ermolenko Elena I.
Institute of Experimental Medicine
197376, Russian Federation, St. Petersburg,
Acad. Pavlov str., 12.
Phone: 7(812) 234-68-68.
Fax: 7 (812) 234-94-89.
E-mail: Lermolenko1@yandex.ru
For citation:
E.I. Ermolenko, I.V. Kudryavtsev, O.I. Solovyova, A.B. Karaseva, A.N. Tsapieva, Yu.D. Kondratenko, M.P. Kotyleva, M.K. Serebryakova, I.A. Shumikhina, A.N. Suvorov "Profile of subpopulation composition of regulatory T lymphocytes and intestinal microbiota in patients with irritable bowel syndrome", Medical Immunology (Russia)/ Meditsinskaya Immunologiya, 2020, Vol. 22, no. 2, pp. 335-346. doi: 10.15789/1563-0625-POS-1905 DOI: 10.15789/1563-0625-POS-1905
PROFILE OF SUBPOPULATION COMPOSITION OF REGULATORY T LYMPHOCYTES AND INTESTINAL MICROBIOTA IN PATIENTS WITH IRRITABLE BOWEL SYNDROME
Ermolenko E.I.a' b, Kudryavtsev I.V.a' d, Solovyova O.I.c,
Karaseva A.B.a, Tsapieva A.N.a, Kondratenko Yu.D.b, Kotyleva M.P.a,
Serebryakova M.K.a, Shumikhina I.A.c, Suvorov A.N.a' b
a Institute of Experimental Medicine, St. Petersburg, Russian Federation b St. Petersburg State University, St. Petersburg, Russian Federation c North-Western State I. Mechnikov Medical University, St. Petersburg, Russian Federation d First St. Petersburg State I. Pavlov Medical University, St. Petersburg, Russian Federation
Abstract. The following specificcharacteristics of the composition of intestinal microbiota in patients with irritable bowel syndrome (IBS) were identified using a metagenomic analysis (16 S rRNA): 1) an increase in the representation of Actinobacteria, including Bifidobacterium spp., Firmicutes, including representatives of Streptococcaceae (Streptococcus), Lachnosperaceae (Dorea), Veillonellaceae (Dialister), Proteobacteria (Enterobacteriaceae and Desulfovibrionaceae families); 2) a decrease in the population of Bacteroidetes, including representatives of the families Prevotellacea (Prevotella spp.), Bacteroidaceae (Bacteroides spp.). Firmicutes belonging to the families Clostridiaceae and Ruminococcaceae (Fecalibacterium spp.).
Flow cytometry in the study of the subpopulation composition of T regulatory (Treg) lymphocytes in patients with IBS revealed an increase in the number of CD45R0+CD62L+ central memory cells (CM), which can regulate the processes of maturation and differentiation of lymphocytes in lymphoid tissue. A decrease in the expression of exonucleases CD39 and CD73 was detected, which can have a significant effect on their activity. A reduction in effector memory cells (EM) Treg was observed.
Changes in the expression level of exonucleases CD39 and CD73 were inversely correlated with the content of Proteobacteria and the representation of the genera Bifidobacterium spp. and Faecalibacterium spp. The content of CM Treg was directly correlated with the content of Dorea spp.
The results may be indicative of impairment in the processes of Treg differentiation, which are closely related to changes in key components of intestinal microbiocenosis in IBS.
Keywords: intestinal microbiocenosis, metagenomic analysis, flow cytometry of a subpopulation of T lymphocytes
The work is supported by the grant of RPF 16-1510085.
Introduction
Irritable bowel syndrome (IBS) is one of the most common functional diseases of the gastrointestinal tract, which affects more than 40% of gastroenterology patients. Significant heterogeneity of the disease is a predictor of the low effectiveness of treatments prescribed to these patients, which in turn leads to the progression of symptoms, a decrease in quality of life, and to an increase in time on sick leave [1, 5, 13, 19, 34].
Studies in recent years have revealed the multifactor nature ofthe pathogenesislBS, the presence ofdisorders of neurohumoral mechanisms of the gastrointestinal tract and the whole body. The cellular composition and morphofunctional characteristics of the intestinal mucosa in IBS have been thoroughly studied. This
disease, a condition in the gastrointestinal tract, is more often interpreted not as dysbiosis, but as "low-grade inflammation" [4, 37]. The microbiota of IBS patients demonstrated an increase in the representation of phylum Proteobacteria, changes in the ratio of individual representatives of phylum Bacteroidetes and Firmicutes, a decrease in biodiversity [25, 26, 36]. Most often, IBS increased the population of enterobacteria [36], lipopolysaccharides (LPS) of the cell wall, which can have a direct effect on immunocompetent cells, interacting with TLR4 receptors on their surface [20, 29].
Also, unsaturated fatty acids (butyric, propionic and acetic), which are produced by other representatives ofthe intestinal microbiocenosis, including those ofthe Bacteroidetes and Firmicutes phyla, play an important role in regulating the immune response in patients with IBS [14, 21, 32, 33]. All of the above factors affect the polarization of the immune response.
In IBS, significant changes in the composition of immunocompetent cells at the local and system levels have been identified [3, 7, 17]. The content of mast cells, intraepithelial lymphocytes and enterochromaffin cells (endocrine cells producing 80% of all serotonin synthesized in the human body) is elevated in the cellular infilatrate in the intestine of IBS patients. Locally and in the peripheral blood of patients, there has been an increase in the number of inflammatory markers: C-reactive protein [11], proinflammatory cytokines — IL-1, IL-6 and TNFa, while the amount of regulatory and anti-inflammatory cytokine IL-10 is generally reduced [3]. These changes in the state of the mucosa, the composition of the microbiota, cytokine status in IBS are accompanied by activation of T lymphocytes [17] circulating in the blood of patients.
Previously, we have shown [18], that the relative content of follicular T helpers (Tfh) with the phenotypes CXCR3-CCR6-CCR4 decreased in IBS patients, while the level of Tfh populations with the properties of Th17 (DN Tfh17 and Tfh17.1) demonstrated a statistically significant increase. These changes in Tfh composition suggest there are changes in how the specific humoral immune response in IBS patients functions, which was supported by reports of an increase in the population of B-lymphocytes previously detected in IBS by other authors [7].
Despite the fact that the exact causes of development and pathogenesis of IBS remain unknown, the involvement of Treg in this pathological process is of interest, as it is the pool of Treg cells that is able to act as regulators of inflammation and as suppressors in the development of allergic and autoimmune reactions of the body.
The main objective of this study was to analyze the subpopulation composition of Treg lymphocytes in peripheral blood in patients with IBS, taking into account the analysis of the gut microbiome.
Materials and methods
Patient characteristics
Twenty-two patients fulfilling the Rome Criteria V for irritable bowel syndrome with diarrhea (IBS-D were selected for this clinical trial. The subjects included 8 men (32%) and 17 women (68%). The mean age in the group was 37.0±8.05 (24-52) years. Patients were monitored in the North-Western State I. Mechnikov
MedicalUniversity.Allpatientshave signedaninformed consent.
The control group was made up of 30 healthy volunteers of similar age and gender, and same proportion of male/ female subjects as in the group of subjects with IBS.
The object of the study was blood samples obtained by the peripheral vein puncture and collected in vacuum tubes with the addition of K3EDTA. All studies were performed with the informed consent of the subjects and in accordance with the Helsinki Declaration ofthe World Medical Association "Ethical Principles for Medical Research Involving Human Subjects" with amendments from 2000 and "Rules of Clinical Practice in the Russian Federation" approved by the Order of the Ministry of Health of the Russian Federation 19.06.2003 No. 266. All laboratory tests were carried out on the day of the blood collection.
Study of the composition of the gut microbiota
The composition of intestinal lumen microbiotawas identified in the samples of faeces frozen on the day of the material's collection. Metagenomic analysis was used for the analysis. Libraries of hypervariable regions V3 and V4 of the 16S RNA gene were analyzed on MiSeq (Illumina, USA). DNA were isolated from faeces using the kit DNK-EKSPRESS by Litekh (Moscow, Russia). The standard method recommended by Illumina based employing two rounds of PCR was used to prepare the libraries. The first round was the amplification of a 16S RNA gene fragment, adding to the target fragment of the adapter nucleotide sequences included in the PCR primers (Table 1).
The second round of PCR, sample preparation, sequencing and bioinformatic processing was performed at the Resource Center "Biobank" of the St. Petersburg State University. The quality of raw DNA readings was assessed by Fastqc (http://www. bioinformatics.babraham.ac.uk/projects/fastqc). The selection of operatiionsl taxonomic units (OTEs) based on similar sequences of 16S rRNA was carried out by the CD-HIT-OTU-Miseq software. CD-HIT-OTU-Miseq allows to select OTE by the sequence of terminal regions without the need to merge paired sequences. Clustering was carried out by comparing the DNA regions R1 and R2 with good reading quality of 200 and 180 nucleotide pairs, respectively, 97% of the homology of the selected sites and cut-
TABLE 1. SEQUENCE OF PRIMERS FOR ANALYSIS OF THE SEQUENCES OF 16S rRNA GENES
Sequence of the primers of V3 and V4 regions of 16S rRNA used Amplicon size
Forward (341) tcgtcggcagcgtcagatgtgtataagagacagcctacgggnggcwgcag 464
Reverse (785) gtctcgtgggctcggagatgtgtataagagacaggactachvgggtatctaatcc
offs 0.00001 by quantity. The OTE annotation was implemented using Greengenes database version 13.5. OTE presented in less than 5% of samples were dismissed as noise.
Study of the phenotypes of immune cells
Phenotypes of immune cells were determined by flow cytofluorimetry in venous blood obtained by puncture of a peripheral vein and collected in vacuum tubes with the addition ofK3EDTA. Blood samples were taken on the same day as the feces. The preparation of peripheral blood samples and the adjustment of the flow cytofluorimeter was carried out in accordance with the recommendations set forth by S.V Khaidukov et al. [15]. The following panel of monoclonal antibodies conjugated with various fluorochromes was used to identify the main populations of regulatory T lymphocytes and assess their expression level of CD39 and CD73: CD39-FITC (clone A1, cat. 328206, BioLegend, Inc., USA), CD25-PE (clone B1.49.9, cat. A07774, Beckman Coulter), USACD62L-ECD (clone DREG56, cat. IM2713U, Beckman Coulter), USACD45R0-PC5.5 (clone UCHL1, cat. IM2712U, BeckmanCoulter), USACD4-PC7 (cloneSFCI12T4D11 (T4), cat 737660, Beckman Coulter), USACD8-APC (clone B9.11, cat. IM2469, Beckman Coulter), USACD3-APC-Alexa Fluor 750 (clone UCHT1, cat. A94680), CD73-Pacific Blue (clone AD2, cat. 344012, BioLegend,Inc.)and CD45-KromeOrange (clone J33, cat. ABeckman Coulter, USAUSA96416, Beckman Coulter, USA). This set of monoclonal antibodies was used to stain 100 |l of peripheral blood in accordance with the manufacturer's recommendations. To identify the main Treg populations, as well as to analyze their expression of CD39 and CD73, an algorithm (gating strategy) was used, which was described in detail earlier [24].
Removal of red blood cells from samples was carried out following a no-wash procedure using a lysing solution Versa Lyse (cat. No. No A09777), to 975 ml of which 25 ml of IOTest 3 Fixative Solution was added ex tempera (Cat. No A07800). After the destruction of red blood cells, the samples were once washed off with an excess of saline solution at 330g for 7 minutes, after which the supernatant was removed, and the cell sediment was resuspended in a saline solution with a pH of7.2-7.4containing 2 % paraformaldehyde (Sigma-Aldrich, USA). The samples were analyzed on the Navios™ cytofluorimeter (Beckman Coulter, USA), equipped with three 405, 488 and 638 nm diode lasers.
Statistical analysis
Comparative analysis was conducted using ANOVA with post hoc HSD test for unequal sample sizes in Program Statistica 8. Proportions were compared using the Yates correction. Spearman's correlation was used to define statistical relationships between the test paramaters. Differences of p < 0.05 were considered significantreliable in all statistical analyses.
Results
Study of the features of the composition of the intestinal microbiocenosis
The analysis of the composition of the gastrointestinal microbiome in patients with IBS was carried out by comparing it with the microbiome of healthy volunteers with similar gender and age characteristics.
Metagenomic analysis showed that at the filum level there is an increase in the representation of Actinobacteria, Firmacutes and Proteobacteria. At the same time, a decrease in the Bacteroidetes population was noted (Figure 1).
IBS patients
Healthy volunteers
% 0.00
I Actinobacteria ¡ Bacteroídets
20.00 40.00 60.00 80.00 100.00
S F¡rm¡cutes □ Proteobacteria
□ Unknown И Verrucoiracroba
IBS patients |
* \ \\\ \
Healthy volunteers
% 0.00 20.00 40.00 60.00 80.00 100.00
^ Bacteroidaceae S3 Ruminococcaceae ■ Lachnospiraceae
□ Prevotellaceae ■ Enterobacteriaceae □ Veillonellaceae
□ Clostridiaceae H Unknown
Figure 1. Features of the composition of the gut microbiota in IBS patiens at the phylum level
Note. The arrows indicate statistically significant differences in the representation of individual phyla . i p, decrease; t, an increase compared to the group of healthy volunteers. The picture does not show values less than 1%.
Figure 2. Features of the composition of the intestinal microbiota in IBS at the family level
Note. The arrows indicate statistically significant differences in the representation of individual families. i, decrease; t, increase in comparison with the healthy volunteers group. The figure does not show values less than 1%.
An analysis of the representation of bacterial families present in the gut mircobiota of healthy volunteers and IBS patients allowed to clarify the taxonomic features of the composition of the population. Thus, the phylum Bateroidetes in healthy people was distinguished by a high content of Bacteroides and Proteobacteria. The representation of the families Enterobacteriaceae, Streptococcaceae and Veilonellaceae in the intestine of patients with IBS was increased, together with a reduction in Clostridiaceae, Prevotellaceae, Ruminococcaceae and Bacteroidaceae (Figure 2, Table 2).
Analysis of the intestinal microbiome at the level of individual genera of bacteria revealed: a decrease in the representation of Bacteroidetes and Faecalibacterium spp. against the background of an increase in representatives of the genera Dorea, Bifidobacterium, Desulfovibrio, Streptococcus and Dialister (Table 2).
Analysis of subpopulation composition of peripheral blood T lymphocytes
Content of major populations of T lymphocytes of peripheral blood of IBS patients
The study of cluster differentiation of the main populations of T lymphocytes showed that peripheral blood samples obtained from IBS patients and healthy volunteers are not statistically different in terms of the absolute content of CD3+, CD3+CD4+ and CD3+CD8+ cells (data not shown). The relative
content of regulatory T cells with the phenotype CD4+CD25bright also did notstatistically differ between the groups (63 cells/ |l (42-84) in patients with IBS and 57 cells/|l (44-73) in healthy volunteers (p = 0.658). The relative cell content of this population did not change within the general population of peripheral blood lymphocytes (3.09% (2.62-3.74) vs 3.38% (2.71-3.88) at the p = 0.213), and when analyzing the relative content of Tregs among CD3+CD4+ T helpers 6.48% (5.86-7.80) vs 6.78% (6.05-8,34) at p = 0.335.
Analysis of subpopulation composition of regulatory T lymphocytes of peripheral blood of IBS patients by level of expression of CD45R0 and CD62L
In order to detect individual peripheral blood Treg subpopulations, the level of expression on the surface of cells of two antigens was evaluated: CD45R0 (short form of antigen CD45, indicating differentiation of T lymphocytes into central or effector memory cells) and CD62L (adhesion molecule allowing T lymphocytes to enter peripheral lymphoid organs through high endothelial venules) [24]. Based on this analysis three main populations of Treg were identified: naive or thymic cells with CD45R0-CD62L+ phenotype and two populations of peripheral Treg — central and effector memory cells with CD45R0+CD62L+ and CD45R0+CD62L- phenotypes, respectively. The analysis of the relative and absolute content of these cell populations is shown in Figure 3. Thus, there was
TABLE 2. REPRESENTATION OF STATISTICALLY SIGNIFICANT TAXA IN THE FAECES OF IBS PATIENTS AND HEALTHY VOLUNTEERS IN %
Taxons Name of the taxons Healthy volunteers Patients with IBS Changes as compared to the volunteers
Actinobacteria 0.13 1.24 t
Phylum Firmacutes 31.71 49.69 t
Proteobacteria 1.68 4.02 t
Bacteroidetes 41.09 26.92 i
Enterobacteriaceae 0.43 2.84 t
Veilonellaceae 3.74 20.0 t
Clostridiaœae 0.67 1.72 i
Family Prevotellaceae 6.19 2.71 i
Ruminococcaceae 13.52 13.16 i
Bacteroidaceae 27.44 20.24 i
Streptococcaceae 0.15 0.64 t
Fecalibacterium 2.5 0.8 i
Bacteroides 39.1 21.2 i
Dorea 0.00085 0.00211 t
Genus Bifidobacterium 0.00086 0.01475 t
Desulfovibrio 0.000291 0.002832 t
Streptococcus 0.00291 0.017398 t
Dialister 0.079716 0.11864 t
a significant increase (p = 0.026) in the proportion of Treg with phenotype CD45R0+CD62L+ in IBS patients from 57.45% (49.57-60.15) to 63.24% (55.96-66.76), which was accompanied by a decrease in the level of CD45R0+CD62L"Treg in circulation (p < 0.001), from 11.34% (9.15-14.77) to 6.88% (4.24-8.44).
Analysis of the expression of effector molecules CD39 and CD73 by regulatory T lymphocytes of peripheral blood of IBS patients
As a part of further studies of Treg phenotype in IBS patients the expression of two membrane-associated enzymes was analyzed: CD39 (or E-NTP Dase1, Ectonucleoside triphosphate diphosphohydrolase-1) and CD73 (or Ecto5'NTase, ecto-5'-nucleotidase) with nucleotidase activity. It has been shown that in patients with IBS and in healthy volunteers there is not statistically significant difference in relative content of CD39 Treg (30.21% (8.49-47.02) and 40.10% (30.4150.00), respectively, at p = 0.228), respectively, and of CD73 Treg (4.46% (2.66-5.47) and 4.81% (3.536.01), respectively, at p = 0.220).
However, in analyzing the expression of these surface molecules on various subpopulations of Treg of identified on the basis of CD45R0 and CD62L coexpression, significant differences were found between the groups compared, which concerned naive or thymic CD45R0"CD62L+Treg (Figure 4). Thus, an almost twofold reduction in the content of CD39+ naive Treg and CD73-positive cells of this population in patients with IBS has been demonstrated: from 14.36% (6.34-18.78) to 6.25% (1.68-9.28, p < 0,001) and from 5.44% (3.93-7.18) to 2.87% (0.88-3.98, p < 0,001), respectively.
The summarized results obtained in the study of cluster differentiation of Treg lymphocytes are presented in the Table 3.
Correlation between the indicators of cluster differentiation of lymphocytes and the representation of individual taxons of bacteria in IBS patients
Spearman's correlation test used to analyze data for the the group of patients with IBS revealed a direct correlation (r = 0.483744, p < 0.05) between representatives of the Dorea spp. and CD45R0+CD62L+Treglymphocytesofcentralmemory (Figure 5).
A negative correlation was established between the representation of phylum Proteobacteria, genera Faecalibacterium and Bifidobacterium, and Treg lymphocytes of central memory, on which two or one of the following enzymes was expressed: diphosphohydrolase and nucleotidase (CD73+ and/or CD39+ molecules) (Figures 6-8).
Discussion
IBS is one of the most common human gastrointestinal diseases, at the same time it is difficult to diagnose since the intestinal pathology is closely associated with psycho-somatic disorders. For that reason it is extremely important to discover characteristic pathogenesis mechanisms and specific properties of innate immune cell differentiation. This work was used to trace the correlation between the expression of marker molecules on the surface of immunocompetent cells and changes in the composition of microbiocenosis. It is no secret that the microbiota of patients with IBS is subject to significant disbiotic changes, however, the available in the literature data on the structure of microbiota in patients with IBS are quite contradictory.
The increase of the Proteobacteria quantity, including the Enterobacteriaceae family, has previously been noted by many authors, [25, 26, 27, 28, 36]. This study showed that the growth of enterobacteriae population is accompanied by an increase in the content of Deltaproteobacteria, — Desulfovibrio, capable of partially neutralizing toxic and radioactive compounds as well as of causing superficial destruction of other bacteria [16, 35], as well as cause superficial destruction of other bacteria. In addition to the usual controlled shifts, an increase of bacteria belonging to the genera Weilonella, Lahnospiria, Streptococcus, Bifidobacteria and decrease of Bacteroides spp is present. Numerous studies of gut microbiota in IBS have noted an increase in some types of microorganisms with "pro-inflammatory" activity [24, 26], including representatives of the Dorea, Ruminococcus and Clostridium genera. It should be noted, that the presence of Dorea (a member of the Lachnospiraceae family) was closely associated with an increase in pro-inflammatory cytokine production.
TABLE 3. REPRESENTATION OF STATISTICALLY SIGNIFICANT INDICATORS IN THE EVALUATION OF Treg SUBPOPULATIONS IN THE PERIPHERAL BLOOD OF PATIENTS WITH IBS AND HEALTHY VOLUNTEERS
Indicator markers Healthy volunteers Patients with IBS Change compared to healthy volunteers
CD45R0+CD62L+, % 57.45* 63.24 t
CD45R0+CD62L-, % 11.34 6.88 i
CD45R0-CD62L+CD39+, % 14.36 6.25 i
CD45R0-CD62L+CD73+, % 5.44 2.78 i
Note. *, % from total amount of Treg.
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p = 0.236
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uu°l
00
- Q )P
O LO
Q O
p = 0.376
0
15
15
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10
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Figure 4. Expression of CD39 and CD73 by various subpopulations of regulatory peripheral blood T cells in IBS patients
Note. The upper row (from left to right) is the content of CD39+ cells relative to CD45R0-CD62L+, CD45R0+CD62L+ and CD45R0+CD62L-Treg, respectively. The bottom row of histograms (from left to right) is the content of CD73+ cells relative to CD45R0-CD62L+, CD45R0+CD62L+ and CD45R0+CD62L-Treg, respectively. Black, patients with IBS (n = 25); white, healthy volunteers (n = 30). The results are presented as median and interquantile range (Me (Q0 25-Q0 75). The nonparametric Mann-Whitney test was used to compare the samples.
The ability of these bacteria to metabolize sialic acids could be accompanied by increase of permeability and further development of inflammatory reactions in the intestine [31]. The ability of this kind of bacteria to form a significant compared to other representatives of the gut microbiota quantity of hydrogen and carbon
dioxide largely explains the symptoms of bloating and pain in the intestines characteristic of IBS [38].
It has been shown that the increase in streptococcus content led to stimulation of the production of proinflammatory cytokine IL-6 and the degradation of mucin [26], so the increase in the representation of
Scattering diagram for Dorea u naive Tregs fromt reg 134v*22c
„„,, _Dorea = -0,0021+0,0001*x
0,014 ■■■ **[* ***1 ■■■■ f *f**
0,012-----
0,010-----
0,008 -----
520,006 -----
I naive Tregs: Dorea: r = 0,4108; p = 0,0806 | naive Tregs
Figure 5. Direct correlation (r = 0.483744, p < 0.05) between the content of naive Tregs (% of Treg) and the representation of Dorea spp. (%)
Note. Naive or thymic cells (CD45RA+CD62L+% relative to the total number of naive T helpers with phenotype CD45RA+CD62L+.
these bacteria in IBS patients, identified in this work, was predictable.
As in other reports on the composition of intestinal microbiota of patients with IBS a decrease in beneficial resident F. prausnitzii bacteria producing butyrate has been found in this work. Many authors associate the increase in these bacteria with a favorable prognosis for the progression and course of non-specific ulcerative colitis and Crohn's disease [2]. There is a correlation between the presence of F. prausnitzii (together with Butyricococcus pullicaecorum and other butyrate-producing bacteria) in the mammalian gastrointestinal tract and a decrease in TNFa and IL-8 levels, as well as an increase in production of IL-10 [6, 9, 22].
The excessive increase in bifidobacteria, as well as the increase in the representation of Actinobacteria was unexpected. However, these changes can be associated with the launch of a compensatory reaction of the organism to the formation of a low-grade inflammation site in the intestine of patients with IBS. Further studies may involve an analysis of species and strains of Bifidobacterium spp.
Treg play a leading role in regulating the functional activity of immune system cells in both peripheral lymphoid and in inflamed tissues [10]. The analysis of the relative and absolute content of Treg in the peripheral blood of IBS patients did not demonstrate any differences in these indicators when compared with the control group, which is fully consistent with the results given in the literary sources [12].
The analysis of our results, based on the study of the subpopulation composition of Treg, has identified a statistically significant increase in the population of CD45R0+CD62L+CM cells, which can regulate the maturation and differentiation of lymphocytes in
0,22 0,20 0,18 0,16 „ 0,14 £ 0,12 I 0,10 g 0,08 CL 0,06
-0,02-0,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 CM Tregs 73*39*
Figure 6. Correlation (r = -0.459851, p < 0.05) between the cluster differentiation indices of CD45RA-62L+CD39+CD73+ lymphocytes (% relative to the total number of T helper cells) and Proteobacteria representation (%) in patients with IBS
lymphoid tissue, including lymph nodes and Peyer's patches.
At the same time, we have noted a significant decrease in both the relative and absolute content of EM Tregs, the main function of which is believed to be regulating immune reactions occurring in peripheral tissues, including connective tissue ofthe intrstibal wall. A drop in the circulation of these cells may be related both to an increase of their migration to inflamed tissues and to abnormalities in the mechanisms of their differentiation in lymphoid organs related to a decrease in mature and Tregs capable of systematic migration to the periphery.
Moreover, sources indicate that the expression of homing molecules on CD4+ lymphocytes in IBS increases, which are associated with an increase in CD levels of 62L and z4-7-integrin [23]. However, the changes described by the authors also seem to apply to Treg, as we have shown in the course of the study.
One of the most important mechanisms of suppression of inflammation, which is realized by Treg both in lymphoid tissue and in the site of inflammation, is the degradation of pro-inflammatory extracellular ATP to adenosine, which has a wide spectrum of anti-inflammatory action and acts on the effector cells of innate and acquired immunity [8]. When comparing CD39+ and CD39-Treg in vitro, it was shown that the presence of CD39 is closely related to the high ability of these cells to show their suppressor properties due to the efficient expression of the transcription factor FoxP3, as well as the regulatory molecules CTLA4, GARP, and LAP [28].
In the course of our research we found a decrease in the density of CD39 and CD73 expression on the surface of "naive" Treg, which can significantly affect the functions performed by these cells. It
0,009
Scattering diagram for Faecalibacterium и 73*39* T cells fromt reg 134v*22c Faecalibacterium = 0,0033-0,0009*x
0,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 73*39* T cells
0,14 0,12 0,10 I 0,08
js
J 0,06 s
oB 0,04 0,02 0,00 -0,02
0
Scattering diagram for Bifidobacterium и 73-39* T cells fromt reg 134v*22c Faecalibacterium = 0,0217-0,0005*x
10
20
30
40
50
I 73-39* T cells: Bifidobacterium: r = -0.3339; p = 0.1624 19+ T cells
60
Figure 7. Correlation (r = -0.460728, p < 0.05) between the indicators of cluster differentiation of lymphocytes CD39+CD73+ of T cells and representation of the genus Faecalibacterium in patients with IBS
should be especially noted that CD45R0-Treg, which have passed only the stage of antigen-independent differentiation in the thymus, is capable of recognizing only the body's own antigens and not the antigens of exogenous origin [30]. Apparently, this very population of cells plays a leading role in maintaining tolerance to autoantigens and prevents the development of autoimmune processes in the body. The results obtained may indicate abnormalities in the processes of Treg differentiation, which may lead to the disruption of peripheral tolerance in IBS.
The composition of the intestinal microbiota and the characteristics of cluster differentiation of subpopulations of T lymphocytes were evaluated in patients of IBS simultaneously. Therefore, the negative correlations between the decrease in CM lymphocytes functioning as suppressors of the inflammatory response and the increase in proteobacteria and the maingas producers of the genus Dorea spp. seems very significant and important.
On the other hand, the identified negative correlations between regulatory cell populations and bifidobacteria and fecal bacteria, which are usually correlated with positive changes in the state of intestinal microbiota are difficult to explain. It is possible that in the latter case there is a compensatory
Figure 8. Correlation (r = -0.456460, p < 0.05) between the indicators of cluster differentiation of lymphocytes CD39+CD73+ of T cells and the representation of Bifidobacterium (%) in patients with IBS
reaction of the intestinal microbiota to the chronic inflammation developing in the gut.
The discovered tendency to trigger inflammatory reactions, noted earlier [18], and the discovered abnormalities in systematic and local regulation of immunological processes associated with intestinal dysbiosis create the preconditions for the possible development of an autoimmune pathology associated with an increase in the number of naive thymic lymphocytes and possible deficiencies of immunological tolerance to their own antigens and antigens to representatives of resident microbiota.
The results of the study suggest that changes in the level of individual microorganisms in the microbiota and the subpopulation composition of Treg cells are related, and the correction ofthe abnormal composition of microbiota of the intestine may represent a new strategy to change the immune status of patients with IBS. In addition, the identified patterns open up new diagnostic possibilities to determine the degree of progression of dysbiosis in IBS using flow cytometry. On the other hand, the identified new features of the microbiota of patients with IBS can also contribute to a more accurate diagnosis of the disease, opening up additional prospects for controlling its course.
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Авторы:
Ермоленко Е.И. — д.м.н., доцент, заведующая лабораторией биомедицинской микроэкологии отдела молекулярной микробиологии ФГБНУ «Институт экспериментальной медицины»; профессор кафедры физиологии ФГБОУ ВО «Санкт-Петербургский государственный университет», Санкт-Петербург, Россия
Кудрявцев И.В. — к.б.н., старший научный сотрудник отдела иммунологии ФГБНУ «Институт экспериментальной медицины»; доцент кафедры иммунологии ФГБОУ ВО «Первый Санкт-Петербургский государственный медицинский университет имени академика И.П. Павлова» Министерства здравоохранения РФ, Санкт-Петербург, Россия
Соловьева О.И. — к.м.н., доцент кафедры внутренних болезней, клинической фармакологии и нефрологии ФГБОУ ВО «Северо-Западный государственный медицинский университет имени И.И. Мечникова» Министерства здравоохранения РФ, Санкт-Петербург, Россия
Authors:
Ermolenko E.I., PhD, MD (Medicine), Associate Professor, Head, Laboratory of Biomedical Microecology, Department of Molecular Microbiology, Institute of Experimental Medicine; Professor, Department of Physiology, St. Petersburg State University, St. Petersburg, Russian Federation
Kudryavtsev I.V., PhD (Biology), Senior Research Associate, Department of Immunology, Institute of Experimental Medicine; Associate Professor, Department of Immunology, First St. Petersburg State I. Pavlov Medical University, St. Petersburg, Russian Federation
Solovyova O.I., PhD (Medicine), Associate Professor, Department of Internal Diseases, Clinical Pharmacology and Nephrology, North-Western State I. Mechnikov Medical University, St. Petersburg, Russian Federation
Карасева А.Б. — научный сотрудник отдела молекулярной микробиологии ФГБНУ «Институт экспериментальной медицины», Санкт-Петербург, Россия
Цапиева А.Н. — научный сотрудник отдела молекулярной микробиологии ФГБНУ «Институт экспериментальной медицины», Санкт-Петербург, Россия
Кондратенко Ю.Д. — программист лаборатории «Центр алгоритмической биотехнологии», Институт трансляционной биомедицины ФГБОУ ВО «Санкт-Петербургский государственный университет», Санкт-Петербург, Россия
Котылева М.П. — научный сотрудник отдела молекулярной микробиологии ФГБНУ «Институт экспериментальной медицины», Санкт-Петербург, Россия
Серебрякова М.К. — научный сотрудник отдела иммунологии ФГБНУ «Институт экспериментальной медицины», Санкт-Петербург, Россия Шумилина И.А. — старший лаборант кафедры внутренних болезней, клинической фармакологии и нефрологии ФГБОУ ВО «Северо-Западный государственный медицинский университет имени И.И. Мечникова» Министерства здравоохранения РФ, Санкт-Петербург, Россия
Суворов А.Н. — д.м.н., член-корр. РАН, заведующий отделом молекулярной микробиологии ФГБНУ «Институт экспериментальной медицины»; заведующий кафедрой фундаментальных проблем медицины и медицинской технологии ФГБОУ ВО «Санкт-Петербургский государственный университет», Санкт-Петербург, Россия
Поступила 20.11.2019 Принята к печати 11.12.2019
Karaseva A.B., Research Associate, Department of Molecular Microbiology, Institute of Experimental Medicine, St. Petersburg, Russian Federation
Tsapieva A.N., Research Associate, Department of Molecular Microbiology, Institute of Experimental Medicine, St. Petersburg, Russian Federation
Kondratenko Yu.D., Software Engineer, Center for Algorithmic Biotechnology, Institute of Translational Biomedicine, St. Petersburg State University, St. Petersburg, Russian Federation
Kotyleva M.P., Research Associate, Department of Molecular Microbiology, Institute of Experimental Medicine, St. Petersburg, Russian Federation
Serebryakova M.K., Research Associate, Department of Immunology, Institute of Experimental Medicine, St. Petersburg, Russian Federation
Shumikhina I.A., Senior Laboratory Assistant, Department of Internal Diseases, Clinical Pharmacology and Nephrology, North-Western State I. Mechnikov Medical University, St. Petersburg, Russian Federation
Suvorov A.N., PhD, MD (Medicine), Corresponding Member, Russian Academy of Sciences, Head, Department of Molecular Microbiology, Institute of Experimental Medicine; Head, Department of Fundamental Medicine and Medical Technologies, St. Petersburg State University, St. Petersburg, Russian Federation
Received 20.11.2019 Accepted 11.12.2019
