Intestinal myofibroblast-specific Tpl2-Cox-2-PGE2 pathway links innate sensing to epithelial homeostasis

Tumor progression locus-2 (Tpl2) kinase is a major inflammatory mediator in immune cell types recently found to be genetically associated with inflammatory bowel diseases (IBDs). Here we show that Tpl2 may exert a dominant homeostatic rather than inflammatory function in the intestine mediated specifically by subepithelial intestinal myofibroblasts (IMFs). Mice with complete or IMF-specific Tpl2 ablation are highly susceptible to epithelial injury-induced colitis showing impaired compensatory proliferation in crypts and extensive ulcerations without significant changes in inflammatory responses. Following epithelial injury, IMFs sense innate or inflammatory signals and activate, via Tpl2, the cyclooxygenase-2 (Cox-2)- prostaglandin E2 (PGE2) pathway, which we show here to be essential for the epithelial homeostatic response. Exogenous PGE2 administration rescues mice with complete or IMF-specific Tpl2 ablation from defects in crypt function and susceptibility to colitis. We also show that Tpl2 expression is decreased in IMFs isolated from the inflamed ileum of IBD patients indicating that Tpl2 function in IMFs may be highly relevant to human disease. The IMF-mediated mechanism we propose also involves the IBD-associated genes IL1R1, MAPK1, and the PGE2 receptor-encoding PTGER4. Our results establish a previously unidentified myofibroblast-specific innate pathway that regulates intestinal homeostasis and may underlie IBD susceptibility in humans

A Novel Immunological Assay for Hepcidin Quantification in Human Serum

Contains fulltext : 81054.pdf (publisher's version ) (Open Access)BACKGROUND: Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes a specific and non-operator demanding immunoassay for hepcidin quantification in human sera. METHODS AND FINDINGS: An ELISA assay was developed for measuring hepcidin serum concentration using a recombinant hepcidin25-His peptide and a polyclonal antibody against this peptide, which was able to identify native hepcidin. The ELISA assay had a detection range of 10-1500 microg/L and a detection limit of 5.4 microg/L. The intra- and interassay coefficients of variance ranged from 8-15% and 5-16%, respectively. Mean linearity and recovery were 101% and 107%, respectively. Mean hepcidin levels were significantly lower in 7 patients with juvenile hemochromatosis (12.8 microg/L) and 10 patients with iron deficiency anemia (15.7 microg/L) and higher in 7 patients with Hodgkin lymphoma (116.7 microg/L) compared to 32 age-matched healthy controls (42.7 microg/L). CONCLUSIONS: We describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility

Study of hepcidin

Iron is an essential element for the survival of all living organisms due to its involvement in basic biological procedures. However, free iron in excess is also highly toxic for the cell and therefore for the whole organism. Furthermore, mammals do not possess a special excretion pathway for its removal from the body. Therefore, the strict regulation of iron metabolism is necessary in order to avoid both its deficiency and overload. This means that cell types that are involved in iron absorption (enterocytes), utilization (erythrocytes), recycling (macrophages) and storage (hepatocytes) must work together so as to maintain its homeostasis. Recently it was found that this is achieved through the presence of a common hormonal regulator, hepcidin. Hepcidin is a small cationic peptide that was first discovered due to its antimicrobial properties. It is produced mainly by the liver in the form of an 84-aa propeptide (prohepcidin), which is enzymatically cleaved resulting to the active 25-aa form. Moreover, two more hepcidin isoforms of 20 and 22 amino acid residues have also been described. Τhe 20 and 25-aa peptides have been found both in serum and urine, while the 22 form was found only in urine. Hepcidin has a characteristic loop structure that is stabilized through the presence of 4 disulfide bonds between the molecule’s eight cysteine residues. Of highly importance is the fact that the last 5 amino acids at the N-terminal of hepcidin25 seem to be responsible for its iron-regulating activity. Hepcidin’s role in the human body is the inhibition of iron absorption from the intestine and iron recycling from macrophages. This function is achieved through its binding to its receptor, ferroportin (FPN1), promoting thus its internalisation and degradation in lysosomes. Ferroportin is expressed in enterocytes, macrophages and hepatocytes, where it is the cell’s sole iron exporter. Therefore, high levels of hepcidin result in the increase of ferroportin degradation and the sequestration of iron inside the cell. In contrast, low hepcidin levels result in the increased presence of ferroportin molecules at the cell surface and, therefore, in the induction of iron absorption and recycling. The regulation of hepcidin synthesis is controlled by many factors. Induction of hepcidin expression has been observed in inflammation, hypoxia and increased iron stores, while its inhibition has been reported in situation where iron stores are deplete and erythropoiesis is increased. The previous data suggest that hepcidin is the final common mediator in the regulation of iron homeostasis in response to both iron stores and erythropoiesis. Furthermore, studies have highlightened hepcidin’s crucial role in the pathogenesis of serious disorders of iron homeostasis in humans, like anaemia of chronic disease and hemochromatosis. Its quantification in biological fluids, as serum and urine, could provide further insight in its contribution to these diseases. During the past few years several such methods have been published. Most of them are based on the use of mass spectrometry (SELDITOF-MS ή LCMS/MS), but although results are hopeful, these techniques are characterised so far as semi-quantitative and demand the utilization of specialized equipment. There are two methods so far that are antibody-based· one is considered semi-quantitative and the other one detects prohepcidin, rendering its use controversial. The development of a facile ELISA assay would prove to be a potentially valuable tool for both research and diagnostic approaches. The first aim of this study was the production of human recombinant hepcidin isoforms of both 20- and 25-aa, with the use of a eukaryotic expression system, and the further evaluation of their activity...Ο σίδηρος αποτελεί απαραίτητο στοιχείο για την επιβίωση όλων των ζωντανών οργανισμών λόγω της συμμετοχής του σε βασικές βιολογικές διεργασίες. Όμως, όταν βρίσκεται σε περίσσεια είναι δυνατόν να αποδειχτεί εξαιρετικά τοξικός για το κύτταρο και κατ’ επέκταση για όλο τον οργανισμό. Επιπλέον, δεν υπάρχει κάποιος ειδικός απεκριττικός μηχανισμός για την απομάκρυνση του από το σώμα. Επομένως, για να αποφευχθεί τόσο η έλλειψη όσο και η συσσώρευση του είναι απαραίτητη η αυστηρή ρύθμιση του μεταβολισμού του. Αυτό σημαίνει ότι οι διάφοροι κυτταρικοί τύποι που συμμετέχουν στην απορρόφηση (εντεροκύτταρα), στη χρησιμοποίηση (ερυθροκύτταρα), στην ανακύκλωση (μακροφάγα) και στην αποθήκευση (ηπατοκύτταρα) του σιδήρου θα πρέπει να συνεργάζονται μεταξύ τους για τη διατήρηση της ομοιόστασης του. Σχετικά πρόσφατα βρέθηκε ότι αυτό επιτυγχάνεται μέσω ενός κοινού ορμονικού ρυθμιστή, της ηπατιδίνης. Η ηπατιδίνη είναι ένα μικρό κατιονικό πεπτίδιο, το οποίο αρχικά μελετήθηκε για τις αντιμικροβιακές του ιδιότητες. Παράγεται κατά κύριο λόγο στο ήπαρ με τη μορφή ενός προπεπτιδίου μεγέθους 84 αμινοξέων (προ-ηπατιδίνη), από το οποίο μετά από ενζυματική διάσπαση προκύπτει το ενεργό πεπτίδιο των 25 αμινοξέων. Επιπλέον, έχουν περιγραφεί και άλλες δύο μορφές της ηπατιδίνης μεγέθους 20 και 22 αμινοξέων. Από αυτές, τα πεπτίδια των 20 και 25 αμινοξέων ανιχνεύονται τόσο στον ορό όσο και στα ούρα, ενώ αυτό των 22 μόνο στα ούρα. Χαρακτηριστική είναι η δομή της ηπατιδίνης, που σχηματίζει φουρκέτα, η οποία σταθεροποιείται μέσω 4 δισουλφιδικών δεσμών μεταξύ των οκτώ κυστεϊνών του μορίου. Ιδιαίτερα σημαντικό είναι, επίσης, το γεγονός ότι τα 5 αμινοξέα του αμινοτελικού άκρου της ηπατιδίνης25 φαίνεται να είναι υπεύθυνα για τον σιδηρορυθμιστικό της ρόλο. Ο λειτουργικός ρόλος της ηπατιδίνης στον ανθρώπινο οργανισμό είναι η αναστολή της απορρόφησης του σιδήρου από το έντερο και της ανακύκλωσης του από τα μακροφάγα. Η λειτουργία αυτή επιτυγχάνεται μέσω της πρόσδεσης της στον υποδοχέα της, την φερροπορτίνη (FPN1), επάγοντας με αυτόν τον τρόπο την ενδοκυττάρωση και την αποικοδόμηση του στα λυσοσώματα. Η φερροπορτίνη είναι ο μοναδικός εξαγωγέας σιδήρου από το κύτταρο και εκφράζεται σε εντεροκύτταρα, μακροφάγα και εντεροκύτταρα. Επομένως, αύξηση της ηπατιδίνης έχει σαν αποτέλεσμα την αύξηση της αποικοδόμησης της φερροπορτίνης και τον εγκλωβισμό του σιδήρου στο εσωτερικό του κυττάρου. Αντίθετα, μείωση της ηπατιδίνης οδηγεί σε αυξημένο αριθμό μορίων φερροπορτίνης στην κυτταρική επιφάνεια και άρα επαγωγή τόσο της πρόσληψης όσο και της ανακύκλωσης του στον οργανισμό. Η ρύθμιση της σύνθεσης της ηπατιδίνης εξαρτάται από μια πληθώρα παραγόντων. Πιο συγκεκριμένα έχει παρατηρηθεί επαγωγή της σε καταστάσεις φλεγμονής, υποξίας και αυξημένων αποθηκών σιδήρου, ενώ αντίθετα καταστολή της σε καταστάσεις μειωμένων αποθηκών σιδήρου και αυξημένων αναγκών ερυθροποίησης. Όλα τα παραπάνω δεδομένα υποδεικνύουν την ηπατιδίνη σαν τον τελικό κοινό διαμεσολαβητή στη ρύθμιση της ομοιόστασης του σιδήρου, τόσο με βάση τις αποθήκες σιδήρου, όσο και με βάση τις ανάγκες ερυθροποίησης. Επιπλέον, μελέτες έχουν καταδείξει τον καίριο ρόλο της ηπατιδίνης στην παθογένεση σοβαρών διαταραχών της ομοιόστασης του σιδήρου στον άνθρωπο, όπως είναι η αναιμία της χρονίας νόσου και η αιμοχρωμάτωση. Η ποσοτικοποίηση της σε βιολογικά υγρά, όπως ο ορός και τα ούρα, θα μπορούσε να προσφέρει σημαντικές πληροφορίες όσον αφορά στην συμβολή της σε αυτές τις παθήσεις..

Innate Sensing through Mesenchymal TLR4/MyD88 Signals Promotes Spontaneous Intestinal Tumorigenesis

MyD88, an adaptor molecule downstream of innate pathways, plays a significant tumor-promoting role in sporadic intestinal carcinogenesis of the Apc(min/+) model, which carries a mutation in the Apc gene. Here, we show that deletion of MyD88 in intestinal mesenchymal cells (IMCs) significantly reduces tumorigenesis in this model. This phenotype is associated with decreased epithelial cell proliferation, altered inflammatory and tumorigenic immune cell infiltration, and modified gene expression similar to complete MyD88 knockout mice. Genetic deletion of TLR4, but not interleukin-1 receptor (IL-1R), in IMCs led to altered molecular profiles and reduction of intestinal tumors similar to the MyD88 deficiency. Ex vivo analysis in IMCs indicated that these effects could be mediated through downstream signals involving growth factors and inflammatory and extracellular matrix (ECM)-regulating genes, also found in human cancer-associated fibroblasts (CAFs). Our results provide direct evidence that during tumorigenesis, IMCs and CAFs are activated by innate TLR4/MyD88-mediated signals and promote carcinogenesis in the intestine

A Novel Immunological Assay for Hepcidin Quantification in Human Serum

Background: Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes a specific and non-operator demanding immunoassay for hepcidin quantification in human sera. Methods and Findings: An ELISA assay was developed for measuring hepcidin serum concentration using a recombinant hepcidin25-His peptide and a polyclonal antibody against this peptide, which was able to identify native hepcidin. The ELISA assay had a detection range of 10-1500 mu g/L and a detection limit of 5.4 mu g/L. The intra-and interassay coefficients of variance ranged from 8-15% and 5-16%, respectively. Mean linearity and recovery were 101% and 107%, respectively. Mean hepcidin levels were significantly lower in 7 patients with juvenile hemochromatosis (12.8 mu g/L) and 10 patients with iron deficiency anemia (15.7 mu g/L) and higher in 7 patients with Hodgkin lymphoma (116.7 mu g/L) compared to 32 age-matched healthy controls (42.7 mu g/L). Conclusions: We describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility

IKK beta in intestinal mesenchymal cells promotes initiation of colitis-associated cancer

The importance of mesenchymal cells in inflammation and/or neoplastic transformation is well recognized, but their role in the initiation of these processes, particularly in the intestine, remains elusive. Using mouse models of colorectal cancer, we show that IKK beta in intestinal mesenchymal cells (IMCs) is critically involved in colitis-associated, but not spontaneous tumorigenesis. We further demonstrate that IMC-specific IKK beta is involved in the initiation of colitis-associated cancer (CAC), as in its absence mice develop reduced immune cell infiltration, epithelial cell proliferation, and dysplasia at the early stages of the disease. At the molecular level, these effects are associated with decreased early production of proinflammatory and protumorigenic mediators, including IL-6, and reduced STAT3 activation. Ex vivo IKK beta-deficient IMCs show defective responses to innate immune stimuli such as LPS, as shown by decreased NF-kappa B signaling and reduced expression of important NF-kappa B target genes. Collectively, our results reveal a hitherto unknown role of mesenchymal IKK beta in driving inflammation and enabling carcinogenesis in the intestine

Intestinal myofibroblast-specific Tpl2-Cox-2-PGE2 pathway links innate sensing to epithelial homeostasis

Tumor progression locus-2 (Tpl2) kinase is a major inflammatory mediator in immune cell types recently found to be genetically associated with inflammatory bowel diseases (IBDs). Here we show that Tpl2 may exert a dominant homeostatic rather than inflammatory function in the intestine mediated specifically by subepithelial intestinal myofibroblasts (IMFs). Mice with complete or IMF-specific Tpl2 ablation are highly susceptible to epithelial injury-induced colitis showing impaired compensatory proliferation in crypts and extensive ulcerations without significant changes in inflammatory responses. Following epithelial injury, IMFs sense innate or inflammatory signals and activate, via Tpl2, the cyclooxygenase-2 (Cox-2)-prostaglandin E2 (PGE2) pathway, which we show here to be essential for the epithelial homeostatic response. Exogenous PGE2 administration rescues mice with complete or IMF-specific Tpl2 ablation from defects in crypt function and susceptibility to colitis. We also show that Tpl2 expression is decreased in IMFs isolated from the inflamed ileum of IBD patients indicating that Tpl2 function in IMFs may be highly relevant to human disease. The IMF-mediated mechanism we propose also involves the IBD-associated genes IL1R1, MAPK1, and the PGE2 receptor-encoding PTGER4. Our results establish a previously unidentified myofibroblast-specific innate pathway that regulates intestinal homeostasis and may underlie IBD susceptibility in humans

Inter-assay variation.

<p>Inter-assay variation.</p