Evaluation of the Relationships between Intestinal Regional Lymph Nodes and Immune Responses in Viral Infections in Children

Viral infections increase the risk of developing allergies in childhood, and disruption of mucosal homeostasis is presumed to be involved. However, no study has reported a role for viral infections in such disruption. In this study, we clarified the mechanism of immunoglobulin A (IgA) overproduction in viral infections. Autopsies were performed on 33 pediatric cases, IgA and interferon (IFN)β levels were measured, and histopathological and immunohistochemical examinations were conducted. Furthermore, we cultured human cells and measured IFNβ and IgA levels to examine the effect of viral infections on IgA production. Blood IgA levels in viral infections were higher than in bacterial infections. Moreover, IFNβ levels in most viral cases were below the detection limit. Cell culture revealed increased IgA in gastrointestinal lymph nodes, especially in Peyer’s patches, due to enhanced IFNβ after viral stimulation. Conversely, respiratory regional lymph nodes showed enhanced IgA with no marked change in IFNβ. Overproduction of IgA, identified as an aberration of the immune system and resulting from excessive viral infection-induced IFNβ was observed in the intestinal regional lymph nodes, particularly in Peyer’s patches. Further, increased IgA without elevated IFNβ in the respiratory system suggested the possibility of a different mechanism from the gastrointestinal system.

Primary Site of Coxsackievirus B Replication in the Small Intestines: No Proof of Peyer’s Patches Involvement

Background: Enterovirus (EV) infections are associated with a broad range of diseases. Since the first experimental infection of primates with poliovirus (PV), tonsils and the Peyer’s patches (PPs) have been believed to be the primary replication sites of EVs. Our aim was to localize different viral markers in the small intestines (SI) of coxsackievirus B (CVB) orally and intraperitoneally (i.p.) infected mice. Methods: Transverse sections of SIs of both infected and control male outbred mice were collected at different intervals post-infection (p.i) and analyzed for presence of interferon-alpha (IFN-α) and viral protein VP1 by immunohistochemistry and in situ hybridization (ISH). Fluorescent marker, eGFP, was identified in cryosections of mice infected with eGFP-CVB3. Results: In the infected SIs, we observed enlarged germinating centers (GCs) in the PPs; IFN-α was detected in the PPs and mucosal layer of the SIs. However, VP1, viral RNA and the eGFP were absent in the GCs of PPs at all stages of infection irrespective of the virus strains used. Conclusions: Virus was present in the epithelial cells but not in GCs of the PPs of the murine SIs. Our results do not support the hypothesis of EV replication in the PP especially in the GCs.

Aging-Related Impairments to M Cells in Peyer’s Patches Coincide With Disturbances to Paneth Cells

The decline in mucosal immunity during aging increases susceptibility, morbidity and mortality to infections acquired via the gastrointestinal and respiratory tracts in the elderly. We previously showed that this immunosenescence includes a reduction in the functional maturation of M cells in the follicle-associated epithelia (FAE) covering the Peyer’s patches, diminishing the ability to sample of antigens and pathogens from the gut lumen. Here, co-expression analysis of mRNA-seq data sets revealed a general down-regulation of most FAE- and M cell-related genes in Peyer’s patches from aged mice, including key transcription factors known to be essential for M cell differentiation. Conversely, expression of ACE2, the cellular receptor for SARS-Cov-2 virus, was increased in the aged FAE. This raises the possibility that the susceptibility of aged Peyer’s patches to infection with the SARS-Cov-2 virus is increased. Expression of key Paneth cell-related genes was also reduced in the ileum of aged mice, consistent with the adverse effects of aging on their function. However, the increased expression of these genes in the villous epithelium of aged mice suggested a disturbed distribution of Paneth cells in the aged intestine. Aging effects on Paneth cells negatively impact on the regenerative ability of the gut epithelium and could indirectly impede M cell differentiation. Thus, restoring Paneth cell function may represent a novel means to improve M cell differentiation in the aging intestine and increase mucosal vaccination efficacy in the elderly.

IL-4-Producing Vγ1+/Vδ6+ γδ T Cells Sustain Germinal Center Reactions in Peyer’s Patches of Mice

The mucosal immune system is the first line of defense against pathogens. Germinal centers (GCs) in the Peyer’s patches (PPs) of the small intestine are constantly generated through stimulation of the microbiota. In this study, we investigated the role of γδ T cells in the GC reactions in PPs. Most γδ T cells in PPs localized in the GCs and expressed a TCR composed of Vγ1 and Vδ6 chains. By using mice with partial and total γδ T cell deficiencies, we found that Vγ1+/Vδ6+ T cells can produce high amounts of IL-4, which drives the proliferation of GC B cells as well as the switch of GC B cells towards IgA. Therefore, we conclude that γδ T cells play a role in sustaining gut homeostasis and symbiosis via supporting the GC reactions in PPs.

EFFECT OF IMUNOPHAN ON MORPHOMETRIC PARAMETERS AND HISTOLOGICAL STRUCTURE OF PEYER'S PATCHES IN SMALL INTESTINE OF RATS UNDER IMMUNOSUPPRESSION INDUCED BY CYCLOPHOSPHAN

The aim of the study is to analyze morphometric parameters and histological structure of Peyer's patches in the small intestine of rats after imunofan administration under immunosuppression caused by cyclophosphan. Materials and Methods. The study was carried out on 36 white male rats (210–250 gramms). The animals were divided into two groups. Rats of Group 1 were injected with cyclophosphamide on the 1st day of the experiment (200 mg/kg). On the 2nd, 4th, 6th, 8th and 10th days they received imunofan (0.7 μg/kg). Group 2 consisted of intact rats. The animals were withdrawn from the experiment on the 7th, 30th, and 90th days after imunofan administration. The authors measured the length and width of Peyer's patches, and the distance from the ileocecal angle to the first patch. We also examined the height and width of lymph nodes and internodal zones on histological sections. Results. On the 7th and 30th days of our study the length and width of Peyer's patches increased by 18.71 %/16.94 % and 3.56 %/24.22 % respectively in mature animals, compared with the intact rats. At the same time, the distance from the ileocecal angle to the 1st Peyer’s patch decreased by 6.15 % and 19.80 %. The micrography showed that the height and width of the lymph nodes of Peyer's patches in experimental animals increased by 29.79 %/12.73 % (Day 7) and 10.49 %/12.46 % (Day 30) in comparison with the intact rats. The linear sizes of internodal zones increased by 39.35 %/10.33 % (Day 7) and 4.77 %/4.25 % (Day 30). By the 90th day, the macroscopic parameters of Peyer's patches, the height and width of their lymph nodules and internodal zones approxomated those of intact animals. Conclusion. When using imunofan under experimental immunosuppression, macro- and micromorphometric parameters of Peyer's patches of the small intestine of mature rats gradually approximated those in intact rats, which indicated the leveling of cyclophosphan immunosuppressive effect. Key words: rats, small intestine, Peyer's patches, imunofan, cyclophosphan. Цель исследования – изучение морфометрических параметров и гистологического строения пейеровых бляшек тонкой кишки крыс после введения имунофана на фоне иммуносупрессии, вызванной циклофосфаном. Материалы и методы. Исследование проводилось на 36 белых крысах-самцах массой 210–250 г. Животные были разделены на две группы. В I группе крысам в 1-е сут эксперимента вводился циклофосфан в дозе 200 мг/кг, на 2, 4, 6, 8, 10-е сут – имунофан в дозе 0,7 мкг/кг массы тела; II группу составили интактные крысы. Животные выводились из эксперимента на 7, 30, 90-е сут после завершения введения имунофана. Измерялись длина и ширина пейеровых бляшек, а также расстояние от илеоцекального угла до первой из них, а на гистологических срезах – высота и ширина их лимфатических узелков и межузелковых зон. Результаты. У половозрелых животных на 7-е и 30-е сут наблюдения длина и ширина пейеровых бляшек увеличивалась на 18,71 и 16,94 % и 3,56 и 24,22 % соответственно по сравнению с данными интактных крыс, а расстояние от илеоцекального угла до первой из них уменьшалось на 6,15 и 19,80 %. При микроскопическом исследовании высота и ширина лимфатических узелков пейеровых бляшек были больше по сравнению с данными интактных крыс на 29,79 и 12,73 % (7-е сут), 10,49 и 12,46 % (30-е сут), а линейные размеры межузелковых зон – на 39,35 и 10,33 % (7-е сут), 4,77 и 4,25 % (30-е сут). К 90-м сут макроскопические параметры пейеровых бляшек, а также высота и ширина их лимфатических узелков и межузелковых зон приближались к данным интактных животных. Выводы. При использовании имунофана на фоне экспериментальной иммуносупрессии значения исследуемых макро- и микроморфометрических параметров пейеровых бляшек тонкой кишки половозрелых крыс в ходе эксперимента постепенно приближались к аналогичным показателям интактных крыс, что свидетельствует о нивелировании иммуносупрессивного эффекта циклофосфана. Ключевые слова: крысы, тонкая кишка, пейеровы бляшки, имунофан, циклофосфан.

Maf is a regulator of differentiation for gut immune epithelial cell Microfold cell (M cell)

Microfold cells (M cells) are a specialized subset of epithelial intestinal cells responsible for immunosurveillance of the gastrointestinal tract. M cells are located in the Peyer's patches and are crucial for monitoring and the transcytosis of antigens, microorganisms, and pathogens via their mature receptor GP2. A mature M cell with Gp2 receptor aids in the uptake of antigens, which are passed through the single layer of epithelium and presented to underlying antigen-presenting cells and processed further down-stream with B cells, T cells, and dendritic cells. Recent studies revealed several transcription factors and ligands responsible for the development and differentiation of mature M cells however, an exhaustive list of factors remains to be elucidated. Our recent work on the epigenetic regulation of M cell development found 12 critical transcription factors that were controlled by the polycomb recessive complex 2. Musculoaponeurotic fibrosarcoma transcription factor (Maf) was identified as a gene regulated by the polycomb repressive complex (PRC2) during the development of M cells. In this paper, we explore Maf's critical role in M cell differentiation and maturation. Maf falls under the purview of RANKL signaling, is localized in the Peyer's patches of the intestine, and is expressed by M cells. Given that, complete knockout of the Maf gene leads to a lethal phenotype, organoids isolated from Maf knockout mice and treated with RANKL exhibited impaired M cell development and a significant decrease in Gp2 expression. These findings reveal that Maf is an important regulator for M cell development and differentiation.

Histochemical Studies on Post Natal Development of Gut Associated Lymphoid Tissue (GALT) of Intestine of Pati Duck (Anas platyrhynchos domesticus) of Assam

Background: The Pati duck constitutes a major indigenous duck variety in the state of Assam. The study of the gut associated lymphoid tissue (GALT) of Pati duck of Assam is of great value in regard to normal academic and bio-medical research aspects. It is also pre-requisite for correct diagnosis and evaluating the treatment of certain diseases like duck virus enteritis, duck cholera, aflatoxicosis, botulism etc, caused by different types of pathogens, food poisoning and food allergy. Methods: The study was conducted on Pati duck of Assam in where 45 numbers of ducks were selected by irrespective of sex at different stages of development. The birds were divided into five groups depending on its age viz., 1st week, 4th week, 16th week, 24th week and 42nd weeks old. The pieces of gut having lymphoid tissue or Peyer’s patches were collected immediately after slaughter. Samples were made cryosections (-20oC) at 10µm in thickness and were temporally stored at (-22oC). The histochemical staining was done after that. Result: The acid phosphatase activity was weak in the lymphoid follicles of 1st and 4th week of age of Pati duck, while its activity was moderate in 16th, 24th and 42th week of age.The alkaline phosphatase activity was moderate reaction in the lymphoid follicles of 1st and 4th week of age of Pati duck, while its activity was intense in 16th, 24th and 42th week of age of duck. The lymphoid follicles of both Peyer’s patches and solitary lymphoid nodules were showed moderate activity for adenosine tri-phosphatase activity in 1st and 4th week old Pati duck and strong activity in 16th, 24th and 42th week of age of Pati duck, respectively. The lymphoid nodules of intestine showed strong reaction for non-specific esterase activity in all the age groups of Pati duck.

Distinct B cell subsets in Peyer’s patches convey probiotic effects by Limosilactobacillus reuteri

Background Intestinal Peyer’s patches (PPs) form unique niches for bacteria-immune cell interactions that direct host immunity and shape the microbiome. Here we investigate how peroral administration of probiotic bacterium Limosilactobacillus reuteri R2LC affects B lymphocytes and IgA induction in the PPs, as well as the downstream consequences on intestinal microbiota and susceptibility to inflammation. Results The B cells of PPs were separated by size to circumvent activation-dependent cell identification biases due to dynamic expression of markers, which resulted in two phenotypically, transcriptionally, and spatially distinct subsets: small IgD+/GL7−/S1PR1+/Bcl6, CCR6-expressing pre-germinal center (GC)-like B cells with innate-like functions located subepithelially, and large GL7+/S1PR1−/Ki67+/Bcl6, CD69-expressing B cells with strong metabolic activity found in the GC. Peroral L. reuteri administration expanded both B cell subsets and enhanced the innate-like properties of pre-GC-like B cells while retaining them in the sub-epithelial compartment by increased sphingosine-1-phosphate/S1PR1 signaling. Furthermore, L. reuteri promoted GC-like B cell differentiation, which involved expansion of the GC area and autocrine TGFβ-1 activation. Consequently, PD-1-T follicular helper cell-dependent IgA induction and production was increased by L. reuteri, which shifted the intestinal microbiome and protected against dextran-sulfate-sodium induced colitis and dysbiosis. Conclusions The Peyer’s patches sense, enhance and transmit probiotic signals by increasing the numbers and effector functions of distinct B cell subsets, resulting in increased IgA production, altered intestinal microbiota, and protection against inflammation.