MiR-155 modulates the inflammatory phenotype of intestinal myofibroblasts by targeting SOCS1 in ulcerative colitis

Abnormal levels of microRNA (miR)-155, which regulate inflammation and immune responses, have been demonstrated in the colonic mucosa of patients with inflammatory bowel diseases (IBD), although its role in disease pathophysiology is unknown. We investigated the role of miR-155 in the acquisition and maintenance of an activated phenotype by intestinal myofibroblasts (IMF), a key cell population contributing to mucosal damage in IBD. IMF were isolated from colonic biopsies of healthy controls, ulcerative colitis (UC) and Crohn's disease (CD) patients. MiR-155 in IMF was quantified by quantitative reverse transcription-PCR in basal condition and following exposure to TNF-alpha, interleukin (IL)-1 beta, lipopolysaccharide (LPS) or TGF-beta 1. The effects of miR-155 mimic or inhibitor transfection on cytokine release and suppressor of cytokine signaling 1 (SOCS1) expression were assessed by enzyme-linked immunosorbent assay and western blot, respectively. Regulation of the target gene SOCS1 expression by miR-155 was assessed using luciferase reporter construct. We found that miR-155 was significantly upregulated in UC as compared with control-and CD-derived IMF. Moreover, TNF-alpha and LPS, but not TGF-beta 1 and IL-1 beta, significantly increased miR-155 expression in IMF. Ectopic expression of miR-155 in control IMF augmented cytokines release, whereas it downregulated SOCS1 expression. MiR-155 knockdown in UC-IMF reduced cytokine production and enhanced SOCS1 expression. Luciferase reporter assay demonstrated that miR-155 directly targets SOCS1. Moreover, silencing of SOCS1 in control IMF significantly increased IL-6 and IL-8 release. In all, our data suggest that inflammatory mediators induce miR-155 expression in IMF of patients with UC. By downregulating the expression of SOCS1, miR-155 wires IMF inflammatory phenotype

Ruolo di TAK1 (TGF-beta activated kinase 1) nel processo di fibrogenesi nel corso della malattia di Crohn

Crohn's disease is a chronic inflammatory disease that can affect any segment of the intestinal tract, with a peak of incidence between 25 and 35 years of age affecting 2-7/100000 people per year. More than 33% of patients suffering from Crohn's disease develop intestinal stenosis, preferentially in the small intestine, within 10 years of diagnosis and the only treatment available is surgery. Recurrences occur in 75% of cases. The repairing process of an organ after a chronic damage, like Crohn's disease, is a delicate balance between the production of new extracellular matrix and destruction of the excess. When this balance is lost, the physiological process of repair leads to fibrosis characterized by overgrowth and scarring of tissue with the loss of normal functions. Sub-epithelial myofibroblasts are the main cells involved in deposition of extracellular matrix in the bowel and their role is now under investigation by many groups looking at several pathogenetic processes such as inflammation and cancer. TAK1 is a MAP3K activated by mitogen and inflammatory stimuli and involved in various intracellular signalling cascades. Among several cytokines able to activate TAK1 there is also TGF-?, a known profibrogenic cytokine, that shows high expression in the tissue affected by excessive fibrosis in Crohn's disease. Recently, the role of TAK1 has been associated with cardiac hypertrophy and renal fibrosis, this prompted us to investigate its role in the fibrogenic process in Crohn's disease. To study the involvement of TAK1 in fibrogenetic Crohn's disease we initially determined the expression levels and phosphorylation status of the protein in the ileal mucosa obtained from patients undergoing surgical resection for stenosis and in normal ileum specimens obtained from patients undergoing colectomy for cancer. The data obtained through western blotting analysis showed an increase in TAK1 expression in fibrotic tissue associated to an increased phosphorylation state, which characterizes its activation. Immunohistochemical studies, through co-localization with ?SMA, demonstrated a massive presence of myofibroblasts in the mucosa of patients with Crohn's disease, that expressed TAK1 and its phosphorylated form, mainly in the periglandular region. The functional characterization of TAK1 was therefore performed on primary ISEMFs extracted from regions of Crohn’s associated fibrosis and normal ileum. Cells derived from fibrotic tissues maintained the disease phenotype in vitro and showed higher levels of TAK1 expression and greater activated status compared to control cells. By immunocytochemical and western blotting studies we observed that TGF-? induced TAK1 phosphorylation in intestinal myofibroblasts. For this reason we started functional studies of gene silencing by RNA interference using an adenoviral vector. Collagen 1? mRNA was analyzed by Real Time PCR in ISEMF after TGF-? stimulus or silencing with the viral vector. The expression of collagen was higher in cells derived from Crohn’s disease specimens than in controls; however TGF-? induced more collagen expression in control cells. Tak1 silenced cells showed significantly reduced basal collagen expression and prevented TGF-?-induced collagen up-regulation. Using a TAK1 inhibitor we confirmed the data obtained with siRNA at both the RNA level and the protein level. In addition, myofibroblasts treated with TAK1 inhibitor or siRNA showed a reduced migratory capacity in a wound healing assay. To assess whether TAK1 can be used as an early marker of fibrogenetic process in Crohn's disease, we correlated its expression in a population of patients with Crohn's disease in remission and without clinical signs of stenosis, with markers of fibrosis. Preliminary data showed that the increased expression of Tak1 in Crohn’s mucosa correlated with the expression of two important fibrotic markers, collagen 1? and ?SMA. In conclusion, the results of this study support the role of TAK1 in intestinal myofibroblasts during the TGF-? -induced fibrogenesis process in Crohn’s disease and further studies might indicate whether it is a suitable therapeutic target as well as a prognostic tool.Il morbo di Crohn è una malattia infiammatoria cronica che può colpire qualsiasi tratto dell’apparato digerente, con un picco di incidenza di 2-7/100000 abitanti per anno, nella fascia di età compresa tra i 25 ed i 35 anni. Più di un terzo dei pazienti colpiti da malattia di Crohn sviluppa stenosi intestinale, preferenzialmente al piccolo intestino, entro 10 anni dalla diagnosi e l’unico trattamento ad oggi efficace è l’asportazione chirurgica del tratto coinvolto con una frequenza di recidiva del 75%. La riparazione di un organo che subisce un danno cronico, come nel caso della malattia di Crohn, dipende dal delicato equilibrio fra la produzione di nuova matrice extracellulare e la distruzione di quella prodotta in eccesso. Quando questo equilibrio si perde la fisiologica riparazione sfocia nel processo fibrogenico caratterizzato dall’ispessimento ed indurimento di un tessuto, accompagnato dalla perdita delle sue normali funzioni. Le cellule che partecipano principalmente alla deposizione di matrice extracellulare nell’intestino sono i miofibroblasti subepiteliali (ISEMFs). TAK1 è una MAP3K attivata da mitogeni e stimoli infiammatori coinvolta in svariate cascate di segnale intracellulari. Tra le varie citochine in grado di attivarla vi è il TGF?, nota citochina profibrogenetica, i cui livelli di espressione sono aumentati nei segmenti intestinali sede di fibrosi del morbo di Crohn. Recentemente il ruolo di TAK1 è stato associato allo sviluppo dell’ipertrofia cardiaca e della fibrosi renale. Per tali ragioni abbiamo voluto investigare il suo ruolo nella fibrosi del morbo di Crohn. Per studiare il coinvolgimento di TAK1 nel processo fibrogenetico della malattia di Crohn ne è stata paragonata l’espressione e lo stato di fosforilazione nella mucosa ileale ottenuta da pazienti con malattia di Crohn, sottoposti a resezione chirurgica per stenosi, rispetto a segmenti di ileo sano ricavato da pazienti sottoposti a colectomia per cancro. I dati ottenuti mediante analisi di western blotting hanno mostrato un aumento dell’espressione di TAK1 in corso di fibrosi accompagnata dalla maggiore fosforilazione della chinasi che ne dimostra l’attivazione. Studi di immunoistochimica hanno permesso, mediante co-localizzazione con ?SMA, di osservare la distribuzione di TAK1 nel tessuto e la sua preponderante espressione in miofibroblasti subepiteliali. Difatti, come atteso, la mucosa derivata dai pazienti con malattia di Crohn ha mostrato una abbondante presenza di miofibroblasti. L’abbondanza dell’espressione dell’?SMA nella mucosa di Crohn è stata inoltre osservata mediante analisi di western blottin (figura 5.1C). L’espressione di TAK1 e della sua forma fosforilata è stata osservata soprattutto nella regione perighiandolare, in corrispondenza dei miofibroblasti, e aumentata nei segmenti fibrotici. La caratterizzazione di TAK1 è stata dunque effettuata su miofibroblasti primari estratti da mucosa fibrotica di Crohn e tessuto controllo. Cellule derivate da tessuto fibroso hanno mostrato maggiori livelli di espressione proteica di TAK1 ed un maggiore stato di attivazione rispetto a cellule controllo. Studi di immunoistochimica e western blotting hanno permesso di osservare che il TGF? in grado di attivare TAK1 nei miofibroblasti intestinali. Per questo motivo abbiamo intrapreso degli studi funzionali di silenziamento genico mediante RNA interference utilizzando un vettore adenovirale per trasdurre miofibroblasti intestinali. Abbiamo analizzato il trascritto del collagene 1?, come marcatore della produzione di matrice extracellulare, mediante Real Time PCR in cellule isolate da pazienti con morbo di Crohn e controllo, stimolate con TGF?. L’espressione del collagene è risultata aumentata in cellule di Crohn rispetto ai controlli, e lo stimolo con il TGF? ne ha stimolato in maniera significativa l’espressione solo in cellule controllo. Il silenziamento di Tak1 ha inibito l’incremento del collagene 1? indotto dal TGF? ed ha ridotto l’espressione in cellule di Crohn a valori paragonabili ai controlli. L’uso di un inibitore di TAK1 ha confermato ed esteso, grazie al saggio dell’incorporazione di 3H prolina, i dati ottenuti nella valutazione dell’espressione del collagene 1? a livello genico anche a livello di proteine. Inoltre. In saggi di wound healing i miofibroblasti trattati con l’inibitore di TAK1 hanno mostrato una ridotta mobilità. Infine, per valutare se TAK1 possa essere considerato un marker precoce del processo fibrogenetico nella malattia di Crohn, abbiamo paragonato la sua espressione, correlandola a marcatori della fibrosi, in una popolazione di pazienti con morbo di Crohn, in fase di remissione e senza manifestazioni cliniche di stenosi, con soggetti sani e con colite ulcerosa. I dati preliminari da noi ottenuti hanno mostrato un aumento dell’espressione di Tak1 nella mucosa di Crohn rispetto ai controlli e tale espressione correlava con l’espressione del collagene 1? e di ?SMA. In conclusione i risultati che abbiamo ottenuto supportano un ruolo centrale di TAK1 nella attivazione dei miofibroblasti intestinali indotta dal TGF? in corso di malattia di Crohn e pertanto questa chinasi potrebbe rappresentare un marcatore precoce della evoluzione in senso fibrotico della malattia che un potenziale target terapeutico

Essential oil of Lindera neesiana fruit: Chemical analysis and its potential use in topical applications

The composition of the essential oil of Lindera neesiana Kurz fruit was examined by GC-MS, (1)H, (13)C and bidimensional NMR techniques (HMQC, HMBC, COSY, TOCSY). Forty compounds were identified, representing approximately 86% of the oil: Z-citral (15.08%), E-citral (11.89%), eucalyptol (8.75%), citronellal (6.72%), alpha-pinene (6.63%) and beta-pinene (5.61%) were the major components. The essential oil of L. neesiana fruit showed significant antimicrobial activity against Staphylococcus aureus and Candida albicans at non-cytotoxic doses in human keratinocytes, suggesting possible topical applications. GRAPHICAL ABSTRACT: The essential oil of Lindera neesiana was investigated by GC-MS and NMR techniques. Its biological activities suggest possible topical applications

Rectal Administration of Lactobacillus casei DG Modifies Flora Composition and Toll-Like Receptor Expression in Colonic Mucosa of Patients with Mild Ulcerative Colitis.

BACKGROUND: An imbalance in gut microbiota seems to contribute to the development of chronic inflammatory disorders of the gastrointestinal tract, such as ulcerative colitis (UC). Although it has been suggested that probiotic supplementation is an effective approach to colitis, its effects on intestinal flora and on mucosal cytokine balance have never been explored. AIM: To evaluate the effect of Lactobacillus casei (L. casei) DG, a probiotic strain, on colonic-associated microbiota, mucosal cytokine balance, and toll-like receptor (TLR) expression. METHODS: Twenty-six patients with mild left-sided UC were randomly allocated to one of three groups for an 8-week treatment period: the first group of 7 patients received oral 5-aminosalicylic acid (5-ASA) alone, the second group of 8 patients received oral 5-ASA plus oral L. casei DG, and the third group of 11 patients received oral 5-ASA and rectal L. casei DG. Biopsies were collected from the sigmoid region to culture mucosal-associated microbes and to assess cytokine and TLR messenger RNA (mRNA) levels by quantitative real-time polymerase chain reaction (RT-PCR). RESULTS: 5-ASA alone or together with oral L. casei DG failed to affect colonic flora and TLR expression in a significant manner, but when coupled with rectally administered L. casei DG, it modified colonic microbiota by increasing Lactobacillus spp. and reducing Enterobacteriaceae. It also significantly reduced TLR-4 and interleukin (IL)-1\u3b2 mRNA levels and significantly increased mucosal IL-10. CONCLUSIONS: Manipulation of mucosal microbiota by L. casei DG and its effects on the mucosal immune system seem to be required to mediate the beneficial activities of probiotics in UC patients

Relationship between virulence factor genes in bovine Staphylococcus aureus subclinical mastitis isolates and binding to anti-adhesin antibodies

Staphylococcus aureus is the most common aetiologic agent of contagious bovine mastitis. It is characterized by a wide array of virulence factors. The differences among strains jeopardize the development of effective vaccines against Staph. aureus mastitis. We tested the immunogenicity of a peptide subunit vaccine coding for three different adhesion factors, fibrinogen-binding protein (Efb), fibronectin-binding protein A (FnbpA) and clumping factor A (ClfA). Then we evaluated the influence of some virulence factors on the ability of specific anti-adhesin antibodies to react with sixteen Staph. aureus strains isolated from bovine subclinical mastitis. Immunization with the recombinant adhesins stimulated a strong humoural (IgG and IgA) and mucosal IgA immune response in all animals tested. Hyperimmune serum recognized with diverse efficiency the sixteen Staph. aureus strains and this circumstance correlated well with the level of expression of adhesins. Among the different virulence factors considered to classify strains, spa gene polymorphisms showed the strongest influence on isolate reactions to hyperimmune serum. Our results indicate the importance of a disease- and environment-specific analysis of isolates. Thus, as opposed to other pathogens to obtain an effective vaccine we should characterize multiple strains and identify the prevalent virulence factors expressed

Clostridium difficile TxA(C314) and SLP-36kDa enhance the immune response toward a co-administered antigen

This study evaluated the in vivo adjuvant activity of two peptides derived from Clostridium difficile: a fragment of the receptor-binding domain of toxin A (TxA(C314)) and a fragment of the 36 kDa surface-layer protein (SLP-36kDa) from strain C253. Their ability to affect the magnitude, distribution and polarization of the immune response against fibronectin-binding protein A (FnbpA), a protective vaccine antigen against Staphylococcus aureus, was evaluated using two different routes of immunization: intranasal and subcutaneous. It was shown that (i) the route of immunization affected the magnitude of the immune response; (ii) both peptides enhanced the production of circulating anti-FnbpA IgG and IgA; (iii) following mucosal immunization TxA(C314) was more effective than SLP-36kDa at inducing antibody in the gastrointestinal tract; (iv) the adjuvant influenced the Th1/Th2 balance; and (v) TxA(C314) was more effective than SLP-36kDa in inducing a cell-mediated response. These studies provide insight into the ability of different C. difficile-derived peptides to differentially affect and polarize the activity of the immune system and on their potential use as adjuvants in newly developed vaccines

Mucosa-Associated Microbiota and Risk of Chronic and/or Relapsing Pouchitis After Restorative Proctocolectomy for Ulcerative Colitis

Background Pouchitis can be classified as occasional or relapsing and about 5% of patients develops a chronic inflammatory condition. Chronic pouchitis can lead to the pouch failure. Aims of our study were to identify possible relationship between chronic/relapsing pouchitis and mucosa-associated microbiota, systemic and local inflammation, local cytokines network and toll like receptors expression. Patients and methods In this prospective study 32 consecut- ive patients who underwent restorative proctocolectomy, coming for follow up endoscopy in our out-patient department were recruited in 2007. Clinical disease activity was classified using Pouchitis Disease Activity Index. During pouch endoscopy biopsies from the ileal pouch were obtained to culture bacteria adherent to the mucosa and perform routine histology. Systemic inflammatory status was graded according to blood cell count, ESR, CRP, and albuminemia. Local inflammatory status was assessed with faecal lactoferrin levels analyzed by quantitative ELISA. Serum and mucosal levels of IL-1\u3b2, IL-6 and TNF-\u3b1 were measured with immunometric assays. The mucosal expression of toll-like receptors for bacterial lipopolysaccharide (TLR4) and peptidoglycan (TLR2) were measured by quantitative Real Time RT-PCR. After a median follow up of 23 (IQR 20-24) months the patients were contacted for a reassessment of current and past disease activity. Univariate logistic regression, non parametric statistics and survival analysis were performed. Results Clinical diagnosis of pouchitis (PDAI>7) had been made in 10 patients in 2007. During the follow up 3 of these patients failed to recover and thus had chronic pouchitis while other 3 of them had relapsing episodes of pouchitis. In patients with chronic/relapsing pouchitis mucosal TLR2 and TLR4 were more expressed than in those with no or only one episode of pouchitis (p=0.036 and p=0.016, respectively). The number of CFU of mucosa-associated Clostridiaceae spp was higher in patients with chronic/relapsing pouchitis than in those with no or only one episode of pouchitis (p=0.031). The presence of Clostridiaceae spp adherent to the ileal mucosa was associated to a significant risk of chronic/relapsing pouchitis [OR: 14 (95% CI 0.887- 224.021), p=0.045]. Survival analysis showed that the presence of Clostridiaceae spp wasassociated to earlier pouchitis relapse (p=0.019). Conclusion Chronic/relapsing pouchitis is associated to increased expression of mucosal TLR2 and TLR4. The populations of mucosa- associated Clostridiaceae spp seemed to play a role in the pathogenesis of chronic/relaps- ing pouchitis