Listeria monocytogenes Infection in Macrophages Induces Vacuolar-Dependent Host miRNA Response

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen, causing serious illness in immunocompromised individuals and pregnant women. Upon detection by macrophages, which are key players of the innate immune response against infection, L. monocytogenes induces specific host cell responses which need to be tightly controlled at transcriptional and post-transcriptional levels. Here, we ask whether and how host miRNAs, which represent an important mechanism of post-transcriptional regulation in a wide array of biological processes, are altered by a model pathogen upon live infection of murine bone marrow derived macrophages. We first report that L. monocytogenes subverts the host genome-wide miRNA profile of macrophages in vitro. Specifically, we show that miR-155, miR-146a, miR-125a-3p/5p and miR-149 were amongst the most significantly regulated miRNAs in infected macrophages. Strikingly, these miRNAs were highly upregulated upon infection with the Listeriolysin-deficient L. monocytogenes mutant Δhly, that cannot escape from the phagosome thus representing a vacuolar-contained infection. The vacuolar miRNA response was significantly reduced in macrophages deficient for MyD88. In addition, miR-146a and miR-125a-3p/5p were regulated at transcriptional levels upon infection, and miR-125a-3p/5p were found to be TLR2 responsive. Furthermore, miR-155 transactivation in infection was regulated by NF-κB p65, while miR-146a and miR-125a-3p/5p expression was unaffected in p65-deficient primary macrophages upon L. monocytogenes infection. Our results demonstrate that L. monocytogenes promotes significant changes in the miRNA expression profile in macrophages, and reveal a vacuolar-dependent miRNA signature, listeriolysin-independent and MyD88-dependent. These miRNAs are predicted to target immune genes and are therefore most likely involved in regulation of the macrophage innate immune response against infection at post-transcriptional levels

The impact of stress on tumor growth: peripheral CRF mediates tumor-promoting effects of stress

<p>Abstract</p> <p>Introduction</p> <p>Stress has been shown to be a tumor promoting factor. Both clinical and laboratory studies have shown that chronic stress is associated with tumor growth in several types of cancer. Corticotropin Releasing Factor (CRF) is the major hypothalamic mediator of stress, but is also expressed in peripheral tissues. Earlier studies have shown that peripheral CRF affects breast cancer cell proliferation and motility. The aim of the present study was to assess the significance of peripheral CRF on tumor growth as a mediator of the response to stress in vivo.</p> <p>Methods</p> <p>For this purpose we used the 4T1 breast cancer cell line in cell culture and in vivo. Cells were treated with CRF in culture and gene specific arrays were performed to identify genes directly affected by CRF and involved in breast cancer cell growth. To assess the impact of peripheral CRF as a stress mediator in tumor growth, Balb/c mice were orthotopically injected with 4T1 cells in the mammary fat pad to induce breast tumors. Mice were subjected to repetitive immobilization stress as a model of chronic stress. To inhibit the action of CRF, the CRF antagonist antalarmin was injected intraperitoneally. Breast tissue samples were histologically analyzed and assessed for neoangiogenesis.</p> <p>Results</p> <p>Array analysis revealed among other genes that CRF induced the expression of SMAD2 and β-catenin, genes involved in breast cancer cell proliferation and cytoskeletal changes associated with metastasis. Cell transfection and luciferase assays confirmed the role of CRF in WNT- β-catenin signaling. CRF induced 4T1 cell proliferation and augmented the TGF-β action on proliferation confirming its impact on TGFβ/SMAD2 signaling. In addition, CRF promoted actin reorganization and cell migration, suggesting a direct tumor-promoting action. Chronic stress augmented tumor growth in 4T1 breast tumor bearing mice and peripheral administration of the CRF antagonist antalarmin suppressed this effect. Moreover, antalarmin suppressed neoangiogenesis in 4T1 tumors in vivo.</p> <p>Conclusion</p> <p>This is the first report demonstrating that peripheral CRF, at least in part, mediates the tumor-promoting effects of stress and implicates CRF in SMAD2 and β-catenin expression.</p

Ο ρόλος των νευροπεπτιδίων του στρες στη φλεγμονή

O παράγοντας έκλυσης της κορτικοτροπίνης (CRF) είναι ο κύριος ρυθμιστής της απόκρισης στο στρες. Εκτός από τον υποθάλαμο, ο CRF και τα ομόλογα πεπτίδια UCN1 και UCN2, ανιχνεύονται μεταξύ άλλων σε ιστούς και κύτταρα του ανοσοποιητικού συστήματος. Ενώ η έκκριση του υποθαλαμικού CRF οδηγεί σε μερική ανοσοκαταστολή μέσω αύξησης των επιπέδων των γλυκοκορτικοειδών και ενεργοποίησης του συμπαθητικού νευρικού συστήματος, στην περιφέρεια τα πεπτίδια CRF εμφανίζουν θετική δράση στη φλεγμονή. Σε ορισμένες περιπτώσεις έχουν επιπλέον παρατηρηθεί και αντιφλεγμονώδεις δράσεις από τα πεπτίδια αυτά. Τα μακροφάγα παίζουν πρωταρχικό και κυρίαρχο ρόλο στην φλεγμονώδη ανoσοαπάντηση. Ο ρόλος του τοπικά παραγόμενου CRF αλλά και των UCNs στην δράση των μακροφάγων παραμένει αδιευκρίνιστος. Στην παρούσα διδακτορική διατριβή μελετήθηκε ο ρόλος των πεπτιδίων CRF αλλά και των υποδοχέων τους, CRF1 και CRF2, στα μακροφάγα. Πιο συγκεκριμένα, διερευνήθηκε η δράση των πεπτιδίων CRF στην ενεργοποίηση και απόπτωση των μακροφάγων, ενώ ταυτόχρονα εξετάστηκε και ο μηχανισμός που εμπλέκεται σε κάθε δράση. Βρέθηκε ότι, σε μακροφάγα Raw264.7 αλλά και σε πρωτογενή περιτοναϊκά μακροφάγα ποντικού, τα πεπτίδια CRF, UCN1 και UCN2 μέσω του υποδοχέα CRF2 ενεργοποιούν τους μεταγραφικούς παράγοντες PU.1 και AP-1, οι οποίοι προσδένονται στον υποκινητή του TLR4 και επάγουν την μεταγραφή του. Μέσω του TLR4 διαμεσολαβείται η δράση του LPS στα μακροφάγα και συνεπώς η παραγωγή προ-φλεγμονωδών κυτοκινών. Το σήμα του CRF2 έχει ως αποτέλεσμα να αναστέλλεται η κατασταλτική επίδραση του LPS στην έκφραση του υποδοχέα TLR4, γεγονός παρέχει έναν πιθανό μηχανισμό για την προ-φλεγμονώδη δράση των νευροπεπτιδίων της οικογένειας CRF στα μακροφάγα. Τα σήματα των υποδοχέων CRF ευοδώνουν και την παραγωγή PGE2 σε ενεργοποιημένα με LPS μακροφάγα, μέσω επαγωγής της έκφρασης των ενζύμων Cox-1 και Cox-2. Η επίδραση των πεπτιδίων CRF στην PGE2 έμμεσα οδηγεί σε σύντομη και παροδική καταστολή του TNF-α. Μάλιστα, η ενεργοποίηση της PI3K από τα πεπτίδια CRF είναι απαραίτητη για την καταστολή του TNF-α. Προτείνεται λοιπόν ένας επιπλέον μηχανισμός δράσης των πεπτιδίων CRF στα μακροφάγα, στον οποίο συμμετέχουν οι προσταγλανδίνες και πιθανά σχετίζεται με κάποιες από τις παρατηρούμενες αντί-φλεγμονώδεις ιδιότητες των πεπτιδίων. Η παροδική καταστολή του παραγόμενου TNF-α από ενεργοποιημένα με LPS μακροφάγα έχει επίπτωση στην απόπτωσή τους, καθώς παρατηρήθηκε σημαντική μείωση της απόπτωσης σε σύντομο χρονικό διάστημα που συμπίπτει με την καταστολή του TNF-α από τα πεπτίδια. Αντίθετα, σε μεγαλύτερα χρονικά διαστήματα ο CRF επάγει απόπτωση σε ενεργοποιημένα μακροφάγα. Τα πεπτίδια UCN1 και UCN2 βρέθηκε να επάγουν απόπτωση και σε μη ενεργοποιημένα μακροφάγα μέσω του υποδοχέα CRF2 υποδοχέα και την επαγωγή των προ-αποπτωτικών πρωτεϊνών Βax και Βad. Το γεγονός αυτό αποκαλύπτει έναν αντί-φλεγμονώδη ρόλο των συγκεκριμένων πεπτιδίων, ο οποίος λαμβάνει χώρα πριν την ενεργοποίηση των μακροφάγων. Οι υποδοχείς CRF1 και CRF2 ανιχνεύτηκαν τόσο σε πρωτογενή μακροφάγα όσο και στις κυτταρικές σειρές που χρησιμοποιήθηκαν. Με βάση τα παραπάνω προτείνεται ότι ο υποδοχέας CRF2 έχει τον κυρίαρχο ρόλο στην φυσιολογία τους, γεγονός που επιβεβαιώθηκε από τις μελέτες που πραγματοποιήθηκαν σε πρωτογενή περιτοναϊκά μακροφάγα από CRF1-/- και CRF2-/- ποντικούς. Η απουσία του υποδοχέα CRF2 είχε ως αποτέλεσμα σημαντικά ελαττωμένη παραγωγή προ- φλεγμονωδών κυτοκινών (IL-6 και TNF-α) μετά από ενεργοποίηση με LPS. Αντίθετα, τα CRF1-/- μακροφάγα βρέθηκε να παράγουν σημαντικά υψηλότερα επίπεδα TNF-α και IL-6, αλλά και να εκφράζουν υψηλότερα επίπεδα Cox-2 μετά από ενεργοποίηση με LPS. Το αποτέλεσμα αυτό πιθανά να σχετίζεται με τα χαμηλά επίπεδα κορτικοστερόνης των CRF1-/- ζώων. Ωστόσο, τα υψηλά επίπεδα έκφρασης της Cox-1 (η έκφραση της οποίας δεν επηρεάζεται από τα γλυκοκορτικοειδή) στα CRF1-/- και στα CRF2-/- μακροφάγα, αποτέλεσε ένα ιδιαίτερα ενδιαφέρον αποτέλεσμα, το οποίο χρίζει περαιτέρω διερεύνησης. Συμπερασματικά, η παρούσα διατριβή παρέχει νέα δεδομένα στην αλληλεπίδραση του νευροενδοκρινικού με το ανοσοποιητικό σύστημα συνηγορώντας στο χαρακτηρισμό μιας νέας οικογένειας ανοσορυθμιστών, αυτής των πεπτιδίων της οικογένειας του CRF.Corticotropin- releasing factor (CRF) is the principal regulator of the stress response. Hypothalamic CRF acts indirectly as an anti- inflammatory agent through the production of glucocorticoids. CRF as well as the structurally related peptides Urocortin 1 and Urocortin 2 (UCN1 and UCN2) are also secreted locally at the inflammatory sites from peripheral nerves or inflammatory cells themselves directly affecting the immune system. In vivo and in vitro studies describe peripheral CRF as a pro- inflammatory factor while anti- inflammatory effects of CRF and UCN have also been observed. Among inflammatory cells, macrophages are important players in innate immunity because of their non selective response to almost all infectious microorganisms. Activated macrophages produce cytokines such as TNF-α, IL-6 and NO, which are crucial mediators of the inflammatory response. We have previously shown that CRF augments LPS- induced pro-inflammatory cytokine secretion from Raw264.7 macrophages. In this thesis we demonstrate that CRF, as well as its related peptides UCN1 and UCN2, can induce the transcription and surface expression of TLR4 in Raw264.7 and murine primary peritoneal macrophages through the activation of PU.1 and AP-1 transcription factors. The up-regulation of TLR4 by the peptides provides a mechanism for the increased sensitivity to LPS observed in macrophages treated with CRF. Using antagonists of CRF1 and CRF2 receptors we found that CRF2 mediates the effect of CRF peptides on TLR4. Thus, the CRF2 ligands UCN1 and UCN2 also augmented LPS- induced pro- inflammatory cytokine secretion in primary macrophages. Moreover, using primary macrophages from wild type or CRF2 -/- mice, we demonstrated that CRF2 is involved in the induction of pro-inflammatory cytokine in macrophages following LPS activation. In the absence of CRF2 macrophages produced significantly reduced levels of IL-6 and TNF-α. On the contrary, CRF1-/- macrophages produced higher levels of IL-6 and TNF-α in response to LPS, possibly due to glucocorticoid insufficiency of CRF1-/- mice. Interestingly, both CRF1-/- and CRF2-/- macrophages expressed significantly higher levels of Cox-1 than wild type. CRF peptides induced Cox-1 and Cox-2 expression and PGE2 production from macrophages, transiently inhibiting TNF-α production. PI3K mediated the inhibitory effect of CRF peptides on TNF-α. These data suggest an additional mechanism for the pro-inflammatory effect of CRF peptides, as well as a mechanism of a transient anti-inflammatory effect on macrophages. Along with the effect of CRF peptides on pro-inflammatory cytokine secretion we demonstrated that CRF peptides affect apoptosis of LPS- activated macrophages in a time dependent manner. More specifically, prolonged exposure to CRF augments apoptosis of activated macrophages, while shortly after stimulation the LPS-induced apoptosis is transiently inhibited by CRF. CRF augments NO production from activated macrophages simultaneous with the induction of apoptosis. The early inhibitory effect of CRF on apoptosis on the other hand is consistent with the transient inhibitory effect of CRF on TNF-α. UCN1 and UCN2, via CRF2, induced apoptosis in naïve, non- activated macrophages, suggesting another anti- inflammatory effect of CRF peptides. Moreover, macrophages expressed high levels of UCN1 which was reduced upon LPS simulation. In contrast to the mechanism of LPS- induced apoptosis in macrophages, we found that UCNs induce apoptosis via Bax and Bad up-regulation. In conclusion, these data provide supporting evidence on the peripheral role of CRF peptides as modulators of the immune response acting directly on macrophages, the primary and principal coordinators of the inflammatory cascade

Differential role of MyD88 and TRIF signaling in myeloid cells in the pathogenesis of autoimmune diabetes

Type 1 diabetes (T1D) is caused by the autoimmune destruction of the insulin-producing pancreatic beta cells. While the role of adaptive immunity has been extensively studied, the role of innate immune responses and particularly of Toll-like Receptor (TLR) signaling in T1D remains poorly understood. Here we show that myeloid cell-specific MyD88 deficiency considerably protected mice from the development of streptozotocin (STZ)-induced diabetes. The protective effect of MyD88 deficiency correlated with increased expression of the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) in pancreatic lymph nodes from STZ-treated mice and in bone marrow-derived dendritic cells (BMDC) stimulated with apoptotic cells. Mice with myeloid cell specific TIR-domain-containing adapter-inducing interferon-beta (TRIF) knockout showed a trend towards accelerated onset of STZ-induced diabetes, while TRIF deficiency resulted in reduced IDO expression in vivo and in vitro. Moreover, myeloid cell specific MyD88 deficiency delayed the onset of diabetes in Non-Obese Diabetic (NOD) mice, whereas TRIF deficiency had no effect. Taken together, these results identify MyD88 signaling in myeloid cells as a critical pathogenic factor in autoimmune diabetes, which is antagonized by TRIF-dependent responses. This differential function of MyD88 and TRIF depends at least in part on their opposite effects in regulating IDO expression in phagocytes exposed to apoptotic cells

Differential effect of MyD88 and TRIF deficiency on <i>Ido</i> expression and Treg induction in PLNs of STZ-treated mice.

<p>(A) The mRNA expression of the indicated genes was measured by qRT-PCR in the PLNs of MyD88^CD11c-KO (n = 5), TRIF^CD11c-KO (n = 9) and their respective littermate control <i>Myd88</i>^FL (n = 4) and <i>Trif</i>^FL (n = 8) mice one week after the completion of STZ injections. Results are depicted as fold increase compared to untreated mice (buffer-only) of each genotype. (B) FACS analysis for Tregs (CD4⁺CD25⁺Foxp3⁺) in the PLNs of STZ-treated mice one week after the completion of the STZ injections. Representative plots of gated live, CD4⁺ PLN cells stained with CD25 and intracellular Foxp3. Bar graph shows quantification of CD4⁺CD25⁺Foxp3⁺ cells in STZ-treated <i>Myd88</i>^FL (n = 4), MyD88^CD111c-KO (n = 6), <i>Trif</i>^FL (n = 5) and TRIF^CD11c-KO (n = 6), as well as untreated <i>Myd88</i>^FL (n = 3), MyD88^CD111c-KO (n = 2), <i>Trif</i>^FL (n = 2) and TRIF^CD11c-KO (n = 2) mice.</p

Differential response of MyD88- or TRIF-deficient DCs to apoptotic cell phagocytosis.

<p>(A) WT, <i>Myd88</i>^-/- and <i>Trif</i>^-/- BMDCs were stimulated with apoptotic splenocytes for 6 or 24 hours and the expression of <i>Tnf</i>, <i>Il10</i> and <i>Ido</i> mRNAs was measured by qRT-PCR. Data shown are representative of three independent experiments. (B) The expression of NF-κB proteins was assessed by immunoblotting with specific antibodies in cytoplasmic and nuclear extracts from BMDCs stimulated with apoptotic splenocytes for 4 hours. Tubulin and HDAC1 were used as loading controls. Blots are representative of three independent experiments. (C) The mRNA expression of the indicated genes was measured by qRT-PCR in peritoneal cells collected from MyD88^CD11c-KO, TRIF^CD11c-KO and Cre negative littermate control mice (n = 4 per genotype) 18 hours after i.p. injection of 10⁷ apoptotic thymocytes or medium-only. Data are representative of two independent experiments. (D) The mRNA and protein expression of IDO was analyzed by qRT-PCR and immunoblotting respectively in WT, <i>Myd88</i>^-/- and <i>Trif</i>^-/- BMDCs that were stimulated with primary apoptotic islets (immunoblot was performed on protein extracts prepared from islets stimulated for 12 hours with STZ). Data are representative of two independent experiments.</p

Development of autoimmune diabetes in NOD mice with myeloid- specific MyD88 or TRIF deficiency.

<p>(A and C) Graphs depicting the incidence of diabetes in mice with the indicated genotypes. (B and D) Representative images and quantification of H&E stained paraffin sections of pancreatic tissue from 10-week-old mice with the indicated genotypes. Graph depicts the percentage of mice with a given histology score per genotype: 0 or I, no islet infiltrates or only small peri-islet infiltrates; II, invasive insulitis (<50% of islet area); III, severe invasive insulitis (>50% of islet area). 20–30 islets per mouse were counted. NOD.MyD88^CD11c-KO (n = 8), NOD.MyD88^LysM-KO (n = 9), NOD.TRIF^CD11c-KO (n = 7) and NOD.TRIF^LysM-KO (n = 9), <i>NOD</i>.<i>Myd88</i>^FL (n = 13), and <i>NOD</i>.<i>Trif</i>^FL (n = 12).</p

Immune cell infiltration in pancreatic islets of STZ-treated mice.

<p>Paraffin sections of pancreatic islets collected from mice one week after completion of the STZ treatment were stained for CD3 (A) or F4/80 (B). CD3⁺ or F4/80⁺ cells were counted on 30–50 islets per genotype of mice scored with insulitis. <i>Myd88</i>^FL (n = 4), MyD88^CD11c-KO (n = 3), <i>Trif</i>^FL (n = 5) and TRIF^CD11c-KO (n = 6).</p

STZ-induced diabetes development in mice with myeloid cell-specific MyD88 or TRIF deficiency.

<p>(A and C) Graphs depicting the incidence of diabetes in mice with the indicated genotypes after treatment with 50 mg/kg STZ for five consecutive days. (B and D) Representative images and quantification of H&E stained paraffin sections of pancreatic tissue from mice with the indicated genotypes one week after completion of the STZ treatment. Graph depicts the percentage of mice with a given histology score per genotype: 0, no islet infiltrates, I, peri-insulitis, II, invasive insulitis. 30–50 islets per mouse were examined. MyD88^CD11c-KO (n = 8), MyD88^LysM-KO (n = 6), <i>Myd88</i>^FL (n = 9), TRIF^CD11c-KO (n = 6), TRIF^LysM-KO (n = 7) and <i>Trif</i>^FL (n = 11).</p