Study of immunomodulatory properties of helicobacter pylori in an experimental model of multiple sclerosis

Multiple sclerosis (MS) consists an autoimmune T-cell mediated disease of the central nervous system (CNS). A combination of genetic, as well as environmental factors, is regarded to trigger the onset of the disease. Although various infectious agents have been linked with increased frequency of MS, on the basis of epidemiological observations, a specific causative agent that triggers CNS autoimmunity, is still to be identified. Recently, Helicobacter pylori (H.pylori, Hp), a common pathogen that colonizes the gastric mucosa of approximately 50% of the population in the developed countries, was epidemiologically linked with various extra-gastric diseases, also in the CNS, such as primary open-angle glaucoma, Alzheimer’s disesase, Guillain-Barrè syndrome, mild cognitive impairment and MS. However, experimental evidence on the field is lacking. The aim of the present thesis was to investigate possible immunomodulatory effects of Hp antigen administration, in the course of experimental autoimmune encephalomyelitis (EAE), a widely used mouse model for MS. The present thesis provided evidence that intraperitoneal administration of Hp antigen ameliorates the severity of actively-induced EAE. The protective effect seemed to be mediated by marked down-regulation of MHC-II molecules on various subsets of antigen-presenting cells (APCs), without affecting T-effector phenotype and cytokine production from T-cells. Furthermore, in the setting of the present thesis, Hp-antigen mediated protection against EAE was not attributed to induction of T-regulatory phenotype. In the setting of a different immunization approach, consisting of whole inactivated Hp administered simultaneously with adjuvant known to prime antigen-specific T-cell population, Hp did not prove to be encephalitogenic. In the context of the present thesis, differencial intestinal microbiota profile was compared between a resistant and a susceptible TCRMOG transgenic mouse strain for spontaneous EAE. Mice resistant to spontaneous EAE exhibited reduced intrinsic capacity to elicit Th17 immune responses, as well as lower antigen-presenting capacity. Interestingly, mice resistant to spontaneous EAE exhibited reduced presence of intestinal Lactobacilli species, compared to mice susceptible to spontaneous EAE, following molecular analysis with a panel of intestinal commensal microbiota. The present thesis provided for the first time evidence of immunomodulatory effect of Hp antigen administration in EAE, thereby providing implications regarding the role of this common human pathogen in MS.Η Πολλαπλή Σκλήρυνση (ΠΣ) αποτελεί χρόνια φλεγμονώδη νόσο Τα-λεμφοκυτταρικής (TL) αρχής, χαρακτηριζόμενη, στην πλειονότητα των περιπτώσεων, από κλινικά επεισόδια εξάρσεων και υφέσεων. Παθογενετικά για τη νόσο θεωρείται ότι ευθύνεται ένας συνδυασμός γενετικών και περιβαλλοντικών παραγόντων. Παρά τη συσχέτιση της νόσου με έναν αριθμό λοιμωδών παραγόντων στη βάση επιδημιολογικών παρατηρήσεων, αποκλειστικός αιτιολογικός παράγοντας που να πυροδοτεί αυτοανοσία στο ΚΝΣ, δεν έχει αναγνωριστεί. Πρόσφατα, το Ελικοβακτηρίδιο του Πυλωρού (ΕΠ), ένα ευρύτατα διαδεδομένο παθογόνο που αποικίζει το γαστρικό βλεννογόνο περίπου 50% του πληθυσμού στον ανεπτυγμένο κόσμο, συνδέθηκε επιδημιολογικά με έναν αριθμό εξω-γαστρικών εκδηλώσεων, συμπεριλαμβανομένων νοσημάτων του ΚΝΣ, όπως γλαύκωμα, νόσος Alzheimer’s, σύνδρομο Guillain-Barrè, ήπια γνωστική διαταραχή και ΠΣ. Εντούτοις, πειραματικές ενδείξεις αναφεορικά με το πεδίο αυτό δεν υφίστανται στη βιβλιογραφία. Σκοπός της παρούσης διδακτορικής διατριβής ήταν να μελετηθούν οι ανοσοτροποποιητικές ιδιότητες της χορήγησης αντιγόνου ΕΠ σε μοντέλο πειραματικής αυτοάνοσης εγκεφαλομυελίτιδας (ΠΑΕ), το ευρύτερα χρησιμοποιούμενο πειραματικό μοντέλο ΠΣ. Η μελέτη παρέχει ενδείξεις ότι η χορήγηση αντιγόνου ΕΠ ενδοπεριτοναϊκά δρα προστατευτικά έναντι της ΠΑΕ σε πειραματόζωα. Η προστατευτική δράση φαίνεται να ασκείται μέσω σημαντικής μείωσης στην έκφραση αντιγονοπαρουσιαστικών μορίων MHC-II στην επφάνεια διαφόρων τύπων αντιγονοπαρουσιαστικών κυττάρων, δίχως να επηρεάζεται το προφίλ των παραγόμενων κυτταροκινών και ο Τα-ρυθμιστικός (Treg) φαινότυπος. Με βάση διαφορετική προσέγγιση ανοσοποίησης, δια της υποδόριας χορήγησης ολικού αδρανοποιημένου ΕΠ, φάνηκε ότι η χορήγηση ΕΠ υπό τις αναφερόμενες συνθήκες ανοσοποίησης δεν είναι εγκεφαλιτιδόνος. Στα πλαίσια της παρούσας διδακτορικής διατριβής μελετήθηκε συγκριτικά το προφίλ μικροβιακής χλωρίδας του εντέρου μεταξύ δυο φυλών διαγονιδιακών πειραματοζώων, εκ των οποίων η μια παρουσιάζει προδιάθεση στην εμφάνιση αυτόματης ΠΑΕ, ενώ η άλλη εμφανίζει αντίσταση. Πειραματόζωα με αντίσταση έναντι της αυτόματης ΠΑΕ παρουσίασαν μειωμένη ικανότητα επαγωγής Th17 ανοσιακού φαινότυπου, συγκριτικά με πειραματόζωα με προδιάθεση έναντι αυτόματης ΠΑΕ και μειωμένη ανιγονοπαορουσιασική ικανότητα. Πειραματόζωα με αντίσταση έναντι της αυτόματης ΠΑΕ παρουσίασαν επίσης ελαττωμένη παρουσία στελεχών Lactobacilli στο έντερο, επί συγκριτικής μοριακής ανάλυσης μικροβιακής χλωρίδας του εντέρου. Η παρούσα διδακτορική διατριβή παρέχειχε για πρώτη φορά ενδείξεις ανοσοστροποποιητικής δράσης χορήγησης αντιγόνου ΕΠ στην ΠΑΕ, υποδεικνύοντας ενδεχόμενο ρόλο του κοινού αυτού παθογόνου στην ΠΣ

Prevention, Intervention and Care of Neurodegenerative Diseases

Chronic neurodegenerative diseases encompass a wide spectrum of disorders and affect millions of people worldwide [...

Selective Accumulation of Pro-Inflammatory T Cells in the Intestine Contributes to the Resistance to Autoimmune Demyelinating Disease

<div><p>Myelin-specific, pro-inflammatory T_H17 cells are widely regarded as the drivers of experimental autoimmune encephalomyelitis (EAE), an animal model for Multiple sclerosis (MS). The factors, responsible for the generation and maintenance of T_H17 cells as well as their participation in the pathogenic cascade leading to the demyelinating disease, have been studied extensively. However, how these harmful autoreactive cells are controlled <i>in vivo</i> remains unclear. By comparing TCR transgenic mice on a disease susceptible and a disease resistant genetic background, we show here that pathogenic T_H17 cells are sequestered within the intestine of spontaneous EAE resistant B10.S mice. Disease resistant B10.S mice harbored higher frequencies of T_H17 cells in the intestine compared to EAE susceptible SJL/J mice. Moreover, transferred T_H17 cells selectively migrated to intestinal lymphoid organs of B10.S mice. The sequestration of T_H17 cells in the gut was partially dependent on the gut homing receptor α4β7-mediated adhesion to the intestine. Administration of α4β7 blocking-antibodies increased the peripheral availability of T_H17 cells, resulting in increased EAE severity after immunization in B10.S mice. Together, these results support the concept that the intestine is a check-point for controlling pathogenic, organ-specific T cells.</p></div

Absence of spontaneous EAE in RR B10.S mice.

<p>A. Incidence of spontaneous EAE in a cohort of RR SJL/J (n = 14) or RR B10.S mice (n = 17). B. Frequencies of CD3⁺ CD4⁺ T cells in spleen, pooled axillary and inguinal lymph nodes (pLN), mesenteric lymph nodes (mLN), Peyer's patches (PP) and small intestinal lamina propria (siLP) were measured by flow cytometry. C. Frequencies of Foxp3⁺ CD3⁺ CD4⁺ regulatory T cells in the indicated organs were measured by flow cytomtery. Results are from n  = 4–6 mice per group. Data were pooled from 2–3 different experiments (B–C). Error bars indicate SEM (B–C).</p

Integrin α4β7 mediated accumulation of T_H17 cells in the intestine of RR B10.S mice.

<p>A. Enhanced migration of T_H17 cells to the intestine. Flow cytometric analysis of transferred CFSE-labeled T_H17 cells in various lymphoid organs. Data represent the percentage of IL-17⁺ T cells in the gated CFSE⁺ CD4⁺ population. **, p<0.01 (Mann-Whitney U test). Results are from n = 5–6 mice per group. Data were pooled from 2–3 different experiments. B. Blockade of α4β7 increases peripheral availability of IL-17-producing T cells. RR B10.S mice were treated weekly twice with anti-α4β7 or isotype control antibodies for two consecutive weeks. Splenocytes were stimulated <i>in vitro</i> with anti-CD3/anti-CD28 antibodies. After a 72 hours culture period, production of IL-17 was measured by ELISA. Bars show mean + SEM. **, p<0.01 (Mann-Whitney U test). n = 11–12 mice per group. Data were from 3 independent experiments. C. Effect of blockade of α4β7 on EAE pathogenesis. RR B10.S mice were immunized with rMOG in CFA and treated weekly twice with anti-α4β7 or isotype control antibodies. Mean clinical scores are depicted. *, p<0.05. n = 9–11 mice per group. Data were from 3 independent experiments.</p

Activation of T cells is not affected by lower MHC class II expression on APCs of B10.S mice.

<p>A. Expression of the MHC class II molecule I-A^s on B cells was determined by flow cytometry in various lymphoid organs of RR SJL/J and B10.S mice. Bar graphs show the mean fluorescent intensity (MFI) + SEM of I-A^s on the gated B220⁺ population. *, p<0.05; **, p<0.01 (Mann-Whitney U test). Results are from n = 5–7 mice per group from 3 different experiments. B. Splenocytes from RR SJL/J or RR B10.S mice were isolated and their proliferative response to the indicated concentrations of rMOG or anti-CD3 tested <i>in vitro</i>. Data are shown as mean counts per minute (cpm) with error bars indicating SEM. Data were pooled from 3 different experiments.</p

Optical Coherence Tomography and Optical Coherence Tomography with Angiography in Multiple Sclerosis

Multiple sclerosis (MS) is an inflammatory and neurodegenerative, potentially disabling disease of the central nervous system. OCT (Optical Coherence Tomography) and OCT-A (Optical Coherence Tomography with Angiography) are imaging techniques for the retina and choroid that are used in the diagnosis and monitoring of ophthalmological conditions. Their use has recently expanded the study of several autoimmune disorders, including MS. Although their application in MS remains unclear, the results seem promising. This review aimed to provide insight into the most recent OCT and OCT-A findings in MS and may function as a reference point for future research. According to the current literature, the retinal nerve fibre layer (RNFL) and ganglion cell-inner plexiform complex (GC-IPL) are significantly reduced in people with MS and are inversely correlated with disease duration. The use of OCT might help distinguish between MS and neuromyelitis optica spectrum disorders (NMOSD), as the latter presents with more pronounced thinning in both the RNFL and GC-IPL. The OCT-A findings in MS include reduced vessel density in the macula, peripapillary area, or both, and the enlargement of the foveal avascular zone (FAZ) in the setting of optic neuritis. Additionally, OCT-A might be able to detect damage in the very early stages of the disease as well as disease progression in severe cases

B10.S T_H17 cells fail to accumulate in peripheral lymphoid organs.

<p>A. Flow cytometric analysis of IL-17 expression by CD4⁺ T cells in the gut associated lymphoid tissues. Data represent the percentage of cytokine producing cells in the gated CD4⁺ population. *, p<0.05; (Mann-Whitney U test). B. Flow cytometric analysis of IL-17 expression by T cells in the peripheral lymphoid organs. Data represent the percentage of cytokine producing cells in the gated CD4⁺ population. **, p<0.01 (Mann-Whitney U test). Results are from n = 5–6 mice per group. Data were pooled from 2–3 different experiments (A–B). Error bars indicate SEM (A–B). C. Cytokine production by splenocytes from RR SJL/J or B10.S mice. After a 72 hours culture period, production of IL-17 by RR SJL/J or B10.S spleen cells in response to medium alone or anti-CD3/anti-CD28 stimulation were measured by ELISA. Bars depict mean + SEM. **, p<0.01 (Mann-Whitney U test).</p