A growing number of studies and research projects on the gut-brain axis have been initiated in past years. Several studies have shown that the presence of dysbiosis or pathologies in the gastrointestinal tract, like Crohn's disease and ulcerative colitis, can contribute to the development of neurodegenerative disorders. The gut microbiome consists of a large population of microorganisms where bacteria represent the majority with over 500 distinct species. Almost all bacteria have a similar external structure, where the main constituent is peptidoglycan (PGN). Bacteria and host can produce enzymes that degrade the bacterial cell wall, liberating PGN and other fragments, such as muramyl dipeptide (MDP), in the intestinal lumen and in blood circulation. Peripheral blood is destined to pass through the liver, which filters it, extracting nutrients, metabolizing drugs, and detoxifying exogenous molecules. PGN and MDP are examples of conserved Pathogen-Associated Molecular Patterns (PAMP), small molecules conserved in different microorganisms, that allow the innate immune system to recognize pathogens and protect the host from infection, through the action of Pattern Recognition Receptors (PRR). Membrane PRRs are widely represented by toll-like receptors (TLR), while the intracellular receptor by nucleotide oligomerization domain (NOD)-like receptors (NLR) that have been extensively studied for their ability to produce inflammatory cytokines when they bind PGN and MDP. This study investigates how the intestinal immune system reacts to bacterial fragments, demonstrating the presence and location of PGN and cells involved in the degradation of it, in the intestinal tissue of swine and humans. Initially, a HE staining was performed to deepen the morphology of several human and porcine intestinal tissue samples. With the help of immunohistochemistry, the detection of PGN and MDP proved the presence of bacterial fragments in the liver, used as a tissue-positive control. Liver contains bacterial fragments inside the hepatocytes, with a major concentration near the central vein of the hepatic lobule and the portal tract. In the intestinal tissue, bacterial fragments are located in the lamina propria, in the germinal center and in the apical side of the Lieberkühn crypts. Another purpose of this study is to localize intestinal immune cells and PGLYRP2. Several immunohistochemical staining’s proved that PGLYRP2 is present in and between the crypts. Plasma cells and macrophages are instead placed in lamina propria, in the basal part of the crypts and in the germinal center. It was also evidenced that IgA and IgG are widely present in the lamina propria, between the crypts, as well as being present in fewer numbers in the secondary lymphatic organs. Ultimately, use of MDP conjugates in immunofluorescence revealed the presence of specific plasma cells that produce antibody against MDP in lamina propria, inside the cytoplasm of plasma cells and in the basal part of the Lieberkühn crypts.
Negli ultimi anni sono stati avviati un numero crescente di studi e progetti di ricerca sull'asse intestino- cervello. Diverse evidenze scientifiche hanno dimostrato che la presenza di disbiosi o patologie nel tratto gastrointestinale, come il morbo di Crohn e la colite ulcerosa, può contribuire allo sviluppo di disturbi neurodegenerativi. Il microbioma intestinale è costituito da una vasta popolazione di microrganismi dove i batteri rappresentano la maggioranza con oltre 500 specie distinte. Quasi tutti i batteri hanno una struttura esterna simile, dove il costituente principale è il peptidoglicano (PGN). Batteri e ospite possono produrre enzimi che degradano la parete cellulare batterica, liberando PGN e altri frammenti, come il muramil dipeptide (MDP), nel lume intestinale e nella circolazione sanguigna. Il sangue periferico è destinato a passare attraverso il fegato, che lo filtra, estraendo nutrienti, metabolizzando farmaci e detossificando molecole esogene. PGN e MDP sono esempi di Pathogen-Associated Molecular Patterns (PAMP) conservati, piccole molecole conservate in diversi microrganismi, che consentono al sistema immunitario innato di riconoscere i patogeni e proteggere l'ospite dalle infezioni, attraverso l'azione dei Pattern Recognition Receptors (PRR). I PRR di membrana sono ampiamente rappresentati dai recettori toll-like (TLR), mentre il recettore intracellulare dai recettori nucleotidici oligomerizzati (NOD)-like (NLR) che sono stati ampiamente studiati per la loro capacità di produrre citochine infiammatorie quando legano PGN e MDP. Questo studio indaga come il sistema immunitario intestinale reagisce ai frammenti batterici, dimostrando la presenza e la posizione di PGN e delle cellule coinvolte nella sua degradazione, nel tessuto intestinale di suini e umani. Inizialmente, una colorazione HE è stata eseguita per approfondire la morfologia di diversi campioni di tessuto intestinale umano e suino. Con l'aiuto dell'immunoistochimica, la rilevazione di PGN e MDP ha dimostrato la presenza di frammenti batterici nel fegato, utilizzato come tessuto di controllo positivo. Il fegato contiene frammenti batterici all'interno degli epatociti, con una concentrazione maggiore vicino alla vena centrale del lobulo epatico e al tratto portale. Nel tessuto intestinale, i frammenti batterici si trovano nella lamina propria, nel centro germinativo e nel lato apicale delle cripte di Lieberkühn. Un altro scopo di questo studio è localizzare le cellule immunitarie intestinali e PGLYRP2. Diverse colorazioni immunoistochimiche hanno dimostrato che PGLYRP2 è presente nelle e tra le cripte. Le plasmacellule e i macrofagi sono invece posizionati nella lamina propria, nella parte basale delle cripte e nel centro germinativo. È stato inoltre dimostrato che IgA e IgG sono ampiamente presenti nella lamina propria, tra le cripte, oltre a essere presenti in numero inferiore negli organi linfatici secondari. Infine, l'uso di coniugati MDP nell'immunofluorescenza ha rivelato la presenza di specifiche plasmacellule che producono anticorpi contro MDP nella lamina propria, all'interno del citoplasma delle plasmacellule e nella parte basale delle cripte di Lieberkühn.
Analisi qualitativa nella colorazione istologica e nella fluorescenza per la rilevazione di peptidoglicano e dipeptide muramico: uno studio immunologico per localizzare frammenti batterici e risposta dell’ospite nell’intestino.
LORENZI, SARA
2023/2024
Abstract
A growing number of studies and research projects on the gut-brain axis have been initiated in past years. Several studies have shown that the presence of dysbiosis or pathologies in the gastrointestinal tract, like Crohn's disease and ulcerative colitis, can contribute to the development of neurodegenerative disorders. The gut microbiome consists of a large population of microorganisms where bacteria represent the majority with over 500 distinct species. Almost all bacteria have a similar external structure, where the main constituent is peptidoglycan (PGN). Bacteria and host can produce enzymes that degrade the bacterial cell wall, liberating PGN and other fragments, such as muramyl dipeptide (MDP), in the intestinal lumen and in blood circulation. Peripheral blood is destined to pass through the liver, which filters it, extracting nutrients, metabolizing drugs, and detoxifying exogenous molecules. PGN and MDP are examples of conserved Pathogen-Associated Molecular Patterns (PAMP), small molecules conserved in different microorganisms, that allow the innate immune system to recognize pathogens and protect the host from infection, through the action of Pattern Recognition Receptors (PRR). Membrane PRRs are widely represented by toll-like receptors (TLR), while the intracellular receptor by nucleotide oligomerization domain (NOD)-like receptors (NLR) that have been extensively studied for their ability to produce inflammatory cytokines when they bind PGN and MDP. This study investigates how the intestinal immune system reacts to bacterial fragments, demonstrating the presence and location of PGN and cells involved in the degradation of it, in the intestinal tissue of swine and humans. Initially, a HE staining was performed to deepen the morphology of several human and porcine intestinal tissue samples. With the help of immunohistochemistry, the detection of PGN and MDP proved the presence of bacterial fragments in the liver, used as a tissue-positive control. Liver contains bacterial fragments inside the hepatocytes, with a major concentration near the central vein of the hepatic lobule and the portal tract. In the intestinal tissue, bacterial fragments are located in the lamina propria, in the germinal center and in the apical side of the Lieberkühn crypts. Another purpose of this study is to localize intestinal immune cells and PGLYRP2. Several immunohistochemical staining’s proved that PGLYRP2 is present in and between the crypts. Plasma cells and macrophages are instead placed in lamina propria, in the basal part of the crypts and in the germinal center. It was also evidenced that IgA and IgG are widely present in the lamina propria, between the crypts, as well as being present in fewer numbers in the secondary lymphatic organs. Ultimately, use of MDP conjugates in immunofluorescence revealed the presence of specific plasma cells that produce antibody against MDP in lamina propria, inside the cytoplasm of plasma cells and in the basal part of the Lieberkühn crypts.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14239/28515