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Медицинская иммунология Medical Immunology (Russia)/
2021, Т. 23, № 4, Краткие сообщения Meditsinskaya Immunologiya
стр. 629-634 . ' . 2021, Vol. 23, No 4, pp. 629-634
© 2021, СПбРО РААКИ SnOVt COmmUniCtttlOnS © 2021, SPb RAACI
РЕГУЛЯТОРНЫЕ Т-КЛЕТКИ ЛИМФАТИЧЕСКИХ УЗЛОВ У Muc2-" МЫШЕЙ С HELICOBACTER SPP.
Ачасова К.М., Гвоздева О.В., Кожевникова Е.Н., Литвинова Е.А.
ФГБНУ «Научно-исследовательский институт нейронаук и медицины», г. Новосибирск, Россия
ФГБУН«Сибирский федеральный научный центр агробиотехнологий РАН», р. п. Краснообск, Новосибирская
обл., Россия
Резюме. Иммунные процессы, связанные с формированием устойчивости к патогенам в кишечнике, зависят от микробиома. Поддержание гомеостаза в кишечнике обеспечивают регуляторные Т-клетки. При воспалительных заболеваниях кишечника (ВЗК) наблюдают как нарушение функции Т-регуляторных клеток, так и изменения микрофлоры. Развитие осложнений при этих заболеваниях сопровождается различными инфекциями. Однако некоторые представители патобионтов, например Helicobacter spp., могут влиять на Т-регуляторные клетки. Одной из генетических моделей для изучения ВЗК являются мыши с нокаутом гена Muc2. У таких мышей, как и у людей с ВЗК, эпителиальные и иммунные клетки кишечника находятся в тесном взаимодействии с микрофлорой. Полагают, что иммунные клетки лимфатических узлов Muc2-/- мышей чувствительны к изменению сформировавшейся у них микрофлоры, даже если в ее состав входят патобионты. В данном исследовании было показано влияние присутствия Helicobacter spp. на количество и процент различных типов лейкоцитов и Т-регуляторных клеток в мезентериальных лимфатических узлах Muc2-/- мышей. Количество CD45+CD19+, CD45+CD3+, CD45+CD3+CD4+, CD45+CD3+CD8+-клеток в мезентериальных лимфатических узлах мышей Muc2-/- был достоверно выше, чем у мышей дикого типа Muc2+/+. Однако наличие инфекции у Muc2-/- мышей отменяло увеличение количества CD45+CD19+, CD45+CD3+, CD45+CD3+CD4+, CD45+CD3+CD8+-клеток. У мышей дикого типа Muc2+/+ инфекция не оказывала достоверного эффекта на клетки в мезентериальных лимфатических узлах. Такое изменение в снижении иммунных клеток в мезентериальных лимфатических узлах под действием Helicobacter spp. может быть связано с активацией регуляторных Т-клеток. Действительно, было показано, что наличие врожденной инфекции Helicobacter spp. вызывало увеличение количества регуляторных Т-клеток (CD45+CD4+CD25+FoxP3+) в мезентериальных лимфатических узлах. Известно, что регуляторные Т-клетки обеспечивают противовоспалительные реакции в кишечнике. Таким образом, увеличение регуляторных Т-клеток способствует снижению всех типов иммунных клеток в мезентериальных лимфатических узлах мышей Muc2-/- с инфекцией Helicobacter spp., что способствует улучшению жизнедеятельности этих мышей и, возможно, уменьшает воспалительные реакции в кишечнике. Это может быть свидетельством того, что некоторые патобионты, приобретенные с рождения, могут быть активаторами регуляторных механизмов иммунитета и, тем самым, оказывать благоприятное воздействие на хозяина.
Ключевые слова: Т-регуляторные лимфоциты, муцин 2, микрофлора, Helicobacter, мыши
Адрес для переписки:
Литвинова Екатерина Анатольевна
ФГБУН «Сибирский федеральный научный центр
агробиотехнологий РАН»
630501, Россия, Новосибирская область,
Новосибирский район, р. п. Краснообск.
Тел.: 8 (383) 335-98-55.
Факс: 8 (383) 335-97-54.
E-mail: litvinovaea@physiol.ru
Образец цитирования:
К.М. Ачасова, О.В. Гвоздева, Е.Н. Кожевникова, Е.А. Литвинова «Регуляторные Т-клетки лимфатических узлов у Muc2-/ мышей с Helicobacter spp.» //Медицинская иммунология, 2021. Т. 23, № 4. С. 629-634. doi: 10.15789/1563-0625-LNR-2268 © Ачасова К.М. и соавт., 2021
Address for correspondence:
Litvinova Ekaterina A.
Siberian Federal Scientific Centre of Agrobiotechnology,
Russian Academy of Sciences
630501, Russian Federation, Novosibirsk region,
Krasnoobsk settlement.
Phone: 7 (383) 335-98-55.
Fax: 7 (383) 335-97-54.
E-mail: litvinovaea@physiol.ru
For citation:
K.M. Achasova, O.V. Gvozdeva, E.N. Kozhevnikova, E.A. Litvinova "Lymph node regulatory T-cell in Muc2-/ mice with Helicobacter spp.", Medical Immunology (Russia)/Meditsinskaya Immunologiya, 2021, Vol. 23, no. 4, pp. 629-634. doi: 10.15789/1563-0625-LNR-2268 DOI: 10.15789/1563-0625-LNR-2268
LYMPH NODE REGULATORY T-CELL IN Muc2-- MICE WITH HELICOBACTER SPP.
Achasova K.M., Gvozdeva O.V., Kozhevnikova E.N., Litvinova E.A.
Research Institute of Neurosciences and Medicine, Novosibirsk, Russian Federation
Siberian Federal Scientific Centre ofAgrobiotechnology, Russian Academy of Sciences, Krasnoobsk settlement, Novosibirsk region, Russian Federation
Abstract. The immune processes associated with the formation of resistance to pathogens in the intestine depend on the microbiome. The maintenance of homeostasis in the intestine is provided by regulatory T-cells. In inflammatory bowel disease (IBD), both a disturbance of the T-regulatory function and changes in microflora are observed. Aggravation of the disease is accompanied by various infections. However, pathobionts such as Helicobacter spp., can affect regulatory T-cells. One of the genetic models for studying IBD is Muc2 knockout mice. In these mice, as in humans with IBD, intestinal epithelial and immune cells closely interact with the microflora. It is believed that the immune cells of the lymph nodes Muc2-/- mice are sensitive to changes in the microflora formed in them. In this study, the effect of Helicobacter spp. on the number and percentage of different types of leukocytes and T regulatory cells in the mesenteric lymph nodes of Muc2-/- mice was studied. The number of CD45+CD19+, CD45+CD3+, CD45+CD3+CD4+, CD45+CD3+CD8+-cells in the mesenteric lymph nodes of Muc2-/- mice was significantly higher to compare with wild-type Muc2+/+ mice. However, the presence of infection in Muc2-/- mice canceled the increase in the number of CD45+CD19+, CD45+CD3+, CD45+CD3+CD4+, CD45+CD3+CD8+-cells. In wild-type Muc2+/+ mice, infection had no significant effect on cells in mesenteric lymph nodes. This change in the decrease in immune cells in the mesenteric lymph nodes under the Helicobacter spp. may be associated with the activation of regulatory T-cells. Indeed, it has been shown that the presence of a congenital Helicobacter spp. infection increased of the number of regulatory T-cells (CD45+CD4+CD25+FoxP3+) in the mesenteric lymph nodes. Well known that regulatory T-cells mediate anti-inflammatory responses in the gut. Thus, an increase in regulatory T-cells promotes a decrease in all types of immune cells in the mesenteric lymph nodes of Muc2-/- mice infected with Helicobacter spp. It could provide an improvement in the vital functions of these mice and possibly reduces inflammatory responses in the intestine. This may indicate that some congenital pathobionts activate of the regulatory mechanisms of immunity and, thereby, have a beneficial effect on the host.
Keywords: regulatory T-cells, mucine 2, microflora, Helicobacter, mice
Laboratory animals were obtained with financial support of the Budgetary Funding for Basic Scientific Research (Grant #0538-2019-0005). FC analysis was supported by the Russian Science Foundation (RSF) (Grant #20-64-47020), State Assignment (No. 05332019-0003).
Introduction
Microflora plays an important role in the formation of intestinal lymphoid tissue, to increase resistance to pathogens [8]. Regulatory T-cells controlled by symbiotic microflora are involved in intestinal homeostasis [4, 9]. It is known that in germfree vs. conventional mice (GF) regulatory T-cells have less suppressive activity [13]. Certain bacterial species can influence on the function of regulatory T-cells. Bacteroides fragilis stimulates the production of anti-inflammatory cytokine IL-10 by CD4+-cells, preventing development of inflammation in mice [14]. The intestinal colonization of such bacteria in GF mice stimulates the differentiation of IL-10-producing
CD4+FoxP3+T-cells [14]. Clostridium spp. leads to increased number of regulatory T-cells in the gut lamina propria and stimulates IL-10 production [3]. The differentiation of regulatory T-cells can also be influenced by bacterial metabolites. Butyrate has an effect on the expression of the FoxP3 transcription factor gene by CD4+T-cells in mouse colon as well as in vitro. At the same time, dietary butyrate helps to ameliorates mouse induced colitis [7].
Muc2 knockout mice (Muc2-/-) are one of the genetic models for studying intestinal inflammation [5]. At the same time, colitis in Muc2-/-mice has similar features to ulcerative colitis in humans [15]. Muc2-/- mice are more susceptible to various pathogens, due to impaired production of mucin2. In the presence of pathogens, these mice exhibit acute intestinal inflammation [5]. Thus, Muc2-/- mice as IBD model can be used to study a relationship between changes in intestinal microflora and intestinal inflammation.
Previously in our laboratory have been shown that mice with impaired gut barrier function and mutation in the gene Muc2 (Muc2-/- Kaiso-/-), are more sensitive to treatment with antibiotics, and the elimination of Helicobacter spp, compared to mice with normal gut barrier function (C57BL/6). Helicobacter spp. are considered pathobionts, i.e. show their pathogenic properties only in the presence of defects in immune function, or under special environmental conditions. Also Helicobacter spp. can induce regulatory T-cells [10]. Member of the genus Helicobacter (H. hepaticus) enable mechanisms that maintain a non-pathogenic, symbiotic relationship with the host organism [6]. Thus, the bacteria Helicobacter spp. found in host from birth, can act as symbionts, influencing the formation of the immune system.
Due to the lacked mucin2, the intestinal epithelial and immune cells are in closer contact with the microflora. It can be assumed that the immune cells in regional lymph nodes of Muc2-/- mice are sensitive to changes in microbiota composition. In this study we examined an effect of inoculated Helicobacter spp. on number and percentage of different types of leukocytes and T-regulatory cells in the mesenteric lymph nodes (LN) of Muc2-/- mice.
Materials and methods
The study was carried out on sexually mature female knockout Muc2 gene knockout mice (Muc2-/-) and their littermates with normal gene function (Muc2+/+). Mice were free of species-specific pathogens (SPF) recommended by the Federation of European Laboratory Animal Science Associations
(FELASA), except for Helicobacter spp. Three to six females were housed in individually ventilated cages (Optimice, USA) under an artificial light regimen (14L: 10D), at a temperature of 20-22 °C. Animals received chop diet (Ssniff, Germany) and sterile drinking water ad libitum.
The study was carried out on four groups of mice (two genotypes with and without Helicobacter spp., n = 7 per each group). Mesenteric lymph node cells were isolated and stained with PE-anti-CD3s, FITC-anti-CD4, PE / Cy7-anti-CD8a and PE-anti-CD3s, FITC-anti-CD19, PE / Cy7-anti-CD4 and APC-anti-CD25, AlexaFluor488-anti-FoxP3 and True-NuclearTM Fix (BioLegend, USA) and analyzed on a Guava easyCyte 8HT Flow Cytometer (Merk, Germany).
Data statistical distribution was not normal; the analysis of the data was performed by using nonparametric criteria.
Results and discussion
The effect of the groups on the cell number in mesenteric LNs (mLNs) was found (Kruskal—Wallis test H(3,25) = 16.08, p < 0.01). The cell number in mLN of mutant mice born without infection was higher than in Muc2-/- mice with infection, as well as in Muc2+/+ mice of the corresponding infection status (Mann-Whitney U-test Z = 3.03 and Z = 2.85, p < 0.01; Figure 1A). A similar effect was observed for the number of lymphocyte subsets such as CD19+, CD3+, CD3+CD4+, CD3+CD8+ (Figure 1A).
The study of regulatory T-cell subpopulations revealed an effect of the group on the percentage of CD25+FoxP3+-cells among CD4+-cells (Kruskal-
А
40
Lymphocytes CD19+
CD3* CD3+CD4+ СйЗ*С08*
Muc2"'" □ Muc2"'" with infection
CD25+Foxp3+ gated on CD4+
CD4+CD25+
CD4*Foxp3+
gated on lymphocytes
Muc2+'+
Muc2+'+ with infection
Figure 1. Lymphocytes in mLNs of Muc2"'" and Muc2+,+ mice
Note. A, cell number of lymphocytes and different lymphocyte subsets in the mLNs. B, percentage of regulatory T-cell subpopulations. *, p < 0.05; **, p < 0.01; Mann-Whitney U-test.
B
Wallis test H(3,25) = 9.15, p < 0.05). The percentage of CD25+FoxP3+-cells among CD4+-cells in Muc2-/-mice with vs. without infection was higher (MannWhitney U-test Z = 2.78, p < 0.01; Figure 1B). The increased number of such cells was probably due to upregulated expression of the FoxP3 protein in CD4+-cells (effect of the Kruskal-Wallis test H(3.25) = 9.47, p < 0.05). The percentage of CD4+FoxP3+-cells among lymphocytes was higher in Muc2-/- mice with infection compared to mice without infection (Mann-Whitney U-test Z = 2.92, p < 0.01; Figure 1B). At the same time, no such effect was found for the percentage of CD4+CD25+-cells.
Thus, the presence of Helicobacter spp. in Muc2-/-mice was associated with increased mLN percentage of regulatory T-cells ensuring anti-inflammatory responses in the intestine.
A study of lymphocytes in mLNs showed that Muc2-/- mice bearing Helicobacter spp. had increased percentage of CD25+FoxP3+ regulatory T-cells compared to mice without infections. These differences are likely due to increased expression of the
FoxP3 protein in CD4+-cells. Possibly, Helicobacter spp. activates the regulatory function of T-cells in mice, which can prevent the development of extremely strong immune responses to host microflora. It was shown that tolerogenic properties of Helicobacter spp. particularly were due to its potential to stimulate the production of the anti-inflammatory cytokine IL-10 [2, 11] and activate regulatory T-cells [1, 10, 12]. It is possible that the pathobiont Helicobacter spp. can favorably influence the state of Muc2-/- mice by stimulating the regulatory function. In our study, we found that Helicobacter spp.-free Muc2-/-mice had prolapse at an earlier age (2-3-months old) and significantly more frequently than in mice with infection. It was also noted that the fertility of Muc2-/- mice without Helicobacter spp. is lower than in mice with infection (unpublished data).
Thus, the presence of Helicobacter spp. in Muc2-/-mice, it was associated with an increase in regulatory T-cells, which provide anti-inflammatory responses in the intestine and improve the vital functions of mice with a pathology of thinned mucus in the intestine.
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Авторы:
Ачасова К.М. — младший научный сотрудник ФГБНУ «Научно-исследовательский институт нейронаук и медицины», г. Новосибирск; младший научный сотрудник ФГБУН«Сибирский федеральный научный центр агробиотехнологий РАН», р. п. Краснообск, Новосибирская обл., Россия
Гвоздева О.В. — к.б.н., научный сотрудник ФГБНУ «Научно-исследовательский институт нейронаук и медицины», г. Новосибирск; научный сотрудник ФГБУН «Сибирский федеральный научный центр агробиотехнологий РАН», р. п. Краснообск, Новосибирская обл., Россия
Authors:
Achasova K.M., Junior Research Associate, Research Institute of Neurosciences and Medicine, Novosibirsk; Junior Research Associate Siberian Federal Scientific Centre of Agrobiotechnology, Russian Academy of Sciences, Krasnoobsk settlement, Novosibirsk region, Russian Federation
Gvozdeva O.V., PhD (Biology), Research Associate, Research Institute of Neurosciences and Medicine, Novosibirsk; Research Associate Siberian Federal Scientific Centre of Agrobiotechnology, Russian Academy of Sciences, Krasnoobsk settlement, Novosibirsk region, Russian Federation
Кожевникова Е.Н. — к.б.н., заведующая лабораторией ФГБНУ«Научно-исследовательский институт нейронаук и медициныi»; ведущий научный сотрудник ФГБУН «Сибирский федеральный научный центр агробиотехнологий РАН», р. п. Краснообск, Новосибирская обл., Россия
Литвинова Е.А. — к.б.н., заведующая лабораторией ФГБУН «Сибирский федеральный научный центр агробиотехнологий РАН», р. п. Краснообск, Новосибирская обл.; ведущий научный сотрудник ФГБНУ «Научно-исследовательский институт нейронаук и медицины», г. Новосибирск, Россия
Поступила 15.03.2021 Отправлена на доработку 31.05.2021 Принята к печати 03.06.2021
Kozhevnikova E.N., PhD (Biology), Head of Laboratory, Research Institute of Neurosciences and Medicine, Novosibirsk; Leading Researcher Siberian Federal Scientific Centre of Agrobiotechnology, Russian Academy of Sciences, Krasnoobsk settlement, Novosibirsk region, Russian Federation
Litvinova E.A., PhD (Biology), Head of Laboratory, Siberian Federal Scientific Centre of Agrobiotechnology, Russian Academy of Sciences, Krasnoobsk settlement, Novosibirsk region; Leading Researcher Research Institute of Neurosciences and Medicine, Novosibirsk, Russian Federation
Received 15.03.2021 Revision received 31.05.2021 Accepted 03.06.2021
