Microbiota e difetto immune nella patogenesi della malattia di Crohn: sviluppo di modelli per lo studio di possibili approcci correttivi non immuno-soppressivi

La malattia di Crohn(MC)\ue8 una malattia infiammatoria cronica dell\u2019intestino che pu\uf2 colpire qualsiasi tratto del tubo digerente. Nonostante siano stati effettuati numerosi studi, la patogenesi della MC non \ue8 ancora chiara e non si dispone di trattamenti in grado di guarire stabilmente la malattia. Il sistema immunitario innato sembra giocare un ruolo fondamentale nella patogenesi: \ue8 stato osservato infatti un coinvolgimento di alcuni geni dell\u2019immunit\ue0 innata ed uno squilibrato rapporto tra sistema immune mucosale e microbiota intestinale. Dati clinici e genetici portano a pensare che alla base della patogenesi stiano un difettoso funzionamento dell\u2019immunit\ue0 innata mucosale e una risposta infiammatoria adattativa compensatoria. Lo sviluppo di modelli adeguati a studiare la complessit\ue0 di questa malattia risulta difficile per la molteplicit\ue0 di fattori coinvolti, genetici e ambientali. L\u2019obiettivo di questa tesi \ue8 stato quello di sviluppare dei modelli che tenessero conto della complessit\ue0 della patologia e che permettessero di evidenziare dei profili tipici di risposta a componenti batteriche. Il primo approccio ex vivo sviluppato ha analizzato le diverse pathway di attivazione in monociti circolanti, per evidenziare l\u2019eventuale presenza di un difetto di riposta dell\u2019immunit\ue0 innata. Da questo test \ue8 emerso che i pazienti affetti da MC non sembrano presentare un difetto di risposta, bens\uec un\u2019iperattivazione delle pathways innate, testimoniata da un\u2019aumentata produzione monocitaria di TNF\u3b1. Questo stato infiammatorio, riscontrabile sia in condizione basale sia in seguito a stimolazione di alcuni recettori innati, non \ue8 riconducibile n\ue9 al genotipo di NOD2 (analizzate le principali mutazioni associate a malattia) n\ue9 all\u2019attivit\ue0 di malattia dei pazienti presi in analisi, n\ue9 alla presenza in circolo di componenti batteriche (segno di traslocazione batterica intestinale). Il secondo modello sviluppato ha analizzato direttamente la risposta immunologica mucosale nel tessuto intestinale, pi\uf9 pertinente alla patogenesi della malattia rispetto alle cellule presenti nel circolo periferico. Attraverso una cultura ex vivo di biopsie coliche in presenza o assenza di antibiotici, si \ue8 voluto valutare l\u2019influenza del microbiota sulla secrezione citochinica. Con un\u2019analisi logistica multivariata, \ue8 stato possibile dimostrare l\u2019esistenza di un profilo citochinico (G-CSF, TNF\u3b1, IL4 e IL17) in grado di discriminare la MC dalla rettocolite ulcerosa. E\u2019 stato quindi dimostrato che la MC e la rettocolite ulcerosa presentano differenti profili di risposta citochinica e che la flora batterica associata a mucosa \ue8 in grado di modulare la risposta mucosale. Con il terzo approccio \ue8 stata valutata la risposta cellulare di una linea epiteliale intestinale a diversi composti di origine microbica, utilizzando un sistema automatizzato che fornisce in tempo reale informazioni sulla crescita, adesione, morfologia e vitalit\ue0 cellulare. Da questa analisi \ue8 emerso che solo il composto purificato di derivazione micobatterica \ue8 in grado di indurre un\u2019alterazione temporanea della cinetica cellulare; questa variazione non \ue8 dovuta alla morte cellulare, ma probabilmente ad una variazione della permeabilit\ue0 del simil-epitelio ricreato in vitro. Ulteriori test sono necessari per far chiarezza sull\u2019effetto dato da questo composto sulla mucosa intestinale, visto il ruolo controverso del Mycobacterium Avium paratubercolis nella patogenesi della MC. In conclusione, in questi tre anni \ue8 stato possibile sviluppare diversi modelli per evidenziare specifici profili di risposta mucosale tipici della MC, su cui studiare l\u2019effetto di nuove strategie terapeutiche correttive, basate sulla modulazione dell\u2019interazione tra immunit\ue0 mucosale e microbiota intestinale. I risultati ottenuti sono stati oggetto di due pubblicazioni sulla rivista internazionale \u201cWorld Journal of Gastroenterology\u201d

Repositioning Of Tak-475 In Mevalonate Kinase Disease: Translating Theory Into Practice

Mevalonate Kinase Deficiency (MKD, OMIM #610377) is a rare autosomal recessive metabolic and inflammatory disease. In MKD, defective function of the enzyme mevalonate kinase (MK), due to a mutation in the MVK gene, leads to the shortage of mevalonate-derived intermediates, which results in unbalanced prenylation of proteins and altered metabolism of sterols. These defects lead to a complex multisystem inflammatory and metabolic syndrome. Although biologic therapies aimed at blocking the inflammatory cytokine interleukin-1 (IL-1) can significantly reduce inflammation, they cannot completely control the clinical symptoms that affects the nervous system. For this reason, MKD can still be considered an orphan drug disease. Cellular models for MKD can be obtained by biochemical inhibition of mevalonate-derived isoprenoids. Of note, these cells present an exaggerated response to inflammatory stimuli that can be reduced by treatment with zaragozic acid, an inhibitor of squalene synthase (SQS) able to increase the availability of isoprenoids intermediates upstream the enzymatic block. A similar action might be obtained by lapaquistat acetate (TAK-475, Takeda), a drug that underwent extensive clinical trials as a cholesterol lowering agent 10 years ago, with a good safety profile. Here we describe the preclinical evidence supporting the possible repositioning of TAK-475 from its originally intended use to the treatment of MKD and discuss its potential to modulate the mevalonate pathway in inflammatory diseases

Altered pattern of tumor necrosis factor-Alpha production in peripheral blood monocytes from Crohn's disease basic study

AIM: To evaluate the inflammatory state in Crohn's disease (CD) patients and correlate it with genetic background and microbial spreading. METHODS: By means of flow cytometry, production of tumor necrosis factor-alpha (TNF-\u3b1) was measured in peripheral blood monocytes from patients suffering from CD, ulcerative colitis (UC) and in healthy subjects after stimulation of the NOD2 and TLR pathways. CD patients were genotyped for the three most common NOD2 variants (R702W, G908R and L1007Pfs*2) and basal production of TNF-\u3b1 was correlated to NOD2 genotype. Also, production of TNF-\u3b1 was correlated to plasmatic levels of LPS Binding Protein (LBP), soluble (s) CD14 and to the activity state of the disease. RESULTS: The patients with CD were characterized by a significantly higher monocyte basal expression of TNF-\u3b1 compared with healthy subjects and UC patients, and after stimulation with Pam3CSK4 (ligand of TLR2/1) and MDP-L18 (ligand of NOD2) this difference was maintained, while other microbial stimuli (LPS, ligand of TLR4 and PolyI:C, ligand of TLR3) induced massive activation in CD monocytes as well as in UC and in healthy control cells. There was no significant difference in the production of TNF-\u3b1 between patients who carried CD-associated heterozygous or homozygous variants in NOD2 and patients with wild type NOD2 genotype. Although serum LBP levels have been shown to correlate positively with the state of activity of the disease, TNF-\u3b1 production did not show a clear correlation with either LBP or sCD14 levels in plasma. Moreover, no clear correlation was seen between TNF-\u3b1 production and activity indices in either CD or UC. CONCLUSION: Peripheral monocytes from CD express higher basal and stimulated TNF-\u3b1 than controls, regardless of NOD2 genotype and without a clear correlation with disease activity

The diagnostic challenge of very early-onset enterocolitis in an infant with XIAP deficiency

Background: Aggressive course and resistance to treatments usually characterize very early onset inflammatory bowel disease (VEO-IBD). Some VEO-IBD cases are due to monogenic immune defects and can benefit from hematopoietic stem cell transplantation (HSCT). Case presentation: We describe a Caucasian male baby who presented in the first months of life macrophage activation syndrome, followed by intractable colitis, recurrent episodes of fever and mild splenomegaly. After several immunological, genetic and clinical investigations, subsequently a therapeutic attempt with colectomy, analysis of VEO-IBD-associated genes, revealed a causative mutation in XIAP. The genetic diagnosis of a primary immune deficiency allowed curing the boy with hematopoietic stem cell transplantation. Conclusion: Our report, together with novel findings from recent literature, should contribute to increase awareness of monogenic immune defects as a cause of VEO-IBD. Comprehensive genetic analysis can allow a prompt diagnosis, resulting in the choice of effective treatments and sparing useless and damaging procedures

Neuronal dysfunction associated with cholesterol deregulation

Cholesterol metabolism is crucial for cells and, in particular, its biosynthesis in the central nervous system occurs in situ, and its deregulation involves morphological changes that cause functional variations and trigger programmed cell death. The pathogenesis of rare diseases, such as Mevalonate Kinase Deficiency or Smith–Lemli–Opitz Syndrome, arises due to enzymatic defects in the cholesterol metabolic pathways, resulting in a shortage of downstream products. The most severe clinical manifestations of these diseases appear as neurological defects. Expanding the knowledge of this biological mechanism will be useful for identifying potential targets and preventing neuronal damage. Several studies have demonstrated that deregulation of the cholesterol pathway induces mitochondrial dysfunction as the result of respiratory chain damage. We set out to determine whether mitochondrial damage may be prevented by using protective mitochondria-targeted compounds, such as MitoQ, in a neuronal cell line treated with a statin to induce a biochemical block of the cholesterol pathway. Evidence from the literature suggests that mitochondria play a crucial role in the apoptotic mechanism secondary to blocking the cholesterol pathway. Our study shows that MitoQ, administered as a preventive agent, could counteract the cell damage induced by statins in the early stages, but its protective role fades over time

Curcumin Anti-Apoptotic Action in a Model of Intestinal Epithelial Inflammatory Damage

The purpose of this study is to determine if a preventive treatment with curcumin can protect intestinal epithelial cells from inflammatory damage induced by IFNγ. To achieve this goal we have used a human intestinal epithelial cell line (HT29) treated with IFNγ to undergo apoptotic changes that can reproduce the damage of intestinal epithelia exposed to inflammatory cytokines. In this model, we measured the effect of curcumin (curcuminoid from Curcuma Longa) added as a pre-treatment at different time intervals before stimulation with IFNγ. Curcumin administration to HT29 culture before the inflammatory stimulus IFNγ reduced the cell apoptosis rate. This effect gradually declined with the reduction of the curcumin pre-incubation time. This anti-apoptotic action by curcumin pre-treatment was paralleled by a reduction of secreted IL7 in the HT29 culture media, while there was no relevant change in the other cytokine levels. Even though curcumin pre-administration did not impact the activation of the NF-κB pathway, a slight effect on the phosphorylation of proteins in this inflammatory signaling pathway was observed. In conclusion, curcumin pre-treatment can protect intestinal cells from inflammatory damage. These results can be the basis for studying the preventive role of curcumin in inflammatory bowel diseases

Curcumin anti-apoptotic action in a model of intestinal epithelial inflammatory damage

The purpose of this study is to determine if a preventive treatment with curcumin can protect intestinal epithelial cells from inflammatory damage induced by IFNγ. To achieve this goal we have used a human intestinal epithelial cell line (HT29) treated with IFNγ to undergo apoptotic changes that can reproduce the damage of intestinal epithelia exposed to inflammatory cytokines. In this model, we measured the effect of curcumin (curcuminoid from Curcuma Longa) added as a pre-treatment at different time intervals before stimulation with IFNγ. Curcumin administration to HT29 culture before the inflammatory stimulus IFNγ reduced the cell apoptosis rate. This effect gradually declined with the reduction of the curcumin pre-incubation time. This anti-apoptotic action by curcumin pre-treatment was paralleled by a reduction of secreted IL7 in the HT29 culture media, while there was no relevant change in the other cytokine levels. Even though curcumin pre-administration did not impact the activation of the NF-κB pathway, a slight effect on the phosphorylation of proteins in this inflammatory signaling pathway was observed. In conclusion, curcumin pre-treatment can protect intestinal cells from inflammatory damage. These results can be the basis for studying the preventive role of curcumin in inflammatory bowel diseases

Ex vivo response to mucosal bacteria and muramyl dipeptide in inflammatory bowel disease

To evaluate how mucosal bacteria impact on the spontaneous and muramyl dipeptide (MDP)-induced inflammation in Crohn’s disease (CD) and ulcerative colitis (UC). METHODS Colonic mucosal biopsies were collected from children with active or remissive CD, UC and controls. Twotissue samples were taken from inflamed mucosal segments (in patients with active disease) or fromnoninflamed mucosa [in patients in remission or in healthy controls (HC)]. Experiments were performed in the presence or absence of antibiotics, to assess whether the disease-associated microbiota can modulate the cytokine response ex vivo . For this purpose, each specimen was half-cut to compare spontaneous and MDP-induced inflammation in the presence of live bacteria (LB) or antibiotics. After 24 h of culture, an array of 17 cytokines was assessed in supernatants. Statistical analyses were performed to find significant differences in single cytokines or in patterns of cytokine response in the different groups. RESULTS We demonstrated that subjects with CD display aspontaneous production of inflammatory cytokines including granulocyte-colony stimulating factor (G-CSF), interleukin (IL) 6, IL8, IL10 and IL12, that was not significantly influenced by the addition of antibiotics. UC specimens also displayed a trend of increased spontaneous secretion of several cytokines, which however was not significant due to broader variability among patients. After the addition of antibiotics, spontaneous IL8 secretion was significantly higher in UC than in controls. In HC, a trend towards the weakening of spontaneous IL8 production was observed in the presence of live mucosal bacteria with respect to the presence of antibiotics. In contrast, in the presence of LB UC showed an increasing trend of spontaneous IL8 production, while MDP stimulation resulted in lower IL8 production in the presence of antibiotics. We also showed that subjects with CD seem to have a lowered production of IL8 in response to MDP in the presence of LB. Only with the addition of antibiotics, likely reducing the contribution of LB, multivariate statistical analysis could identify the combination of measures of G-CSF, tumor necrosis factor alpha, IL4 and IL17 as a good discriminator between CD and UC. CONCLUSION We showed that the presence of LB or antibiotics can significantly influence the inflammatory response ex vivo in inflammatory bowel diseases

Action of methotrexate and tofacitinib on directly stimulated and bystander-activated lymphocytes

Chronic inflammation associated with autoim- mune activation is characteristic of rheumatic diseases from childhood to adulthood. In recent decades, signi cant improvements in the treatment of these types of disease have been achieved using disease modifying anti-rheumatic drugs (DMARDs), such as methotrexate (MTX) and, more recently, using biologic inhibitors. The recent introduction of kinase inhibitors (for example, tofacitinib; Tofa) further increases the available ARDs. However, there are patients that do not respond to any treatment strategies, for whom combination therapies are proposed. The data regarding the combined action of different drugs is lacking and the knowledge of the mechanisms of ARDs and their actions upon pathogenic lymphocytes, which are hypothesized to sustain disease, is poor. An in vitro model of in ammation was developed in the current study, in which stimulated and unstimulated lymphocytes were cultured together, but tracked separately, to investigate the action of MTX and Tofa on the two populations. By analysing lymphocyte proliferation and activation, and cytokine secretion in the culture supernatants, it was estab- lished that, due to the presence of activated cells, unstimulated cells underwent a bystander activation that was modulated by the ARDs. Additionally, varying administration schedules were demonstrated to affect lymphocytes differently in vitro, either directly or via bystander activation. Furthermore, MTX and Tofa exerted different effects; while MTX showed an antiproliferative effect, Tofa marginally effected activation, although only a slight antiproliferative action, which could be potentiated by sequential treatment with MTX. Thus, it was hypothesized that these differences may be exploited in sequential therapeutic strategies, to maximize the anti-rheu- matic effect. These ndings are notable and must be accounted for, as bystander-activated cells in vivo could contribute to the spread of autoimmune activation and disease progression