5-Fluorouracil (5-FU) is one of the most commonly used chemotherapeutic agents in colon cancer treatment, but has a narrow therapeutic index limited by its toxicity. Melatonin exerts antitumor activity in various cancers, but it has never been combined with 5-FU as an anticolon cancer treatment to improve the chemotherapeutic effect of 5-FU. In this study, we assessed such combinational use in colon cancer and investigated whether melatonin could synergize the antitumor effect of 5-FU. We found that melatonin significantly enhanced the 5-FU-mediated inhibition of cell proliferation, colony formation, cell migration and invasion in colon cancer cells. We also found that melatonin synergized with 5-FU to promote the activation of the caspase/PARP-dependent apoptosis pathway and induce cell cycle arrest. Further mechanism study demonstrated that melatonin synergized the antitumor effect of 5-FU by targeting the PI3K/AKT and NF-κB/inducible nitric oxide synthase (iNOS) signaling. Melatonin in combination with 5-FU markedly suppressed the phosphorylation of PI3K, AKT, IKKα, IκBα, and p65 proteins, promoted the translocation of NF-κB p50/p65 from the nuclei to cytoplasm, abrogated their binding to the iNOS promoter, and thereby enhanced the inhibition of iNOS signaling. In addition, pretreatment with a PI3K- or iNOS-specific inhibitor synergized the antitumor effects of 5-FU and melatonin. Finally, we verified in a xenograft mouse model that melatonin and 5-FU exerted synergistic antitumor effect by inhibiting the AKT and iNOS signaling pathways. Collectively, our study demonstrated that melatonin synergized the chemotherapeutic effect of 5-FU in colon cancer through simultaneous suppression of multiple signaling pathways.

A polarized macrophage response is presumed to have a pivotal role in a variety of immunological pathophysiology. However, the molecular mechanism underlying macrophage functional shaping remains largely unknown. In this study, we reveal a pivotal role of miR-127 in macrophage development and thereby the pathogenesis of inflammation and lung injury. In particular, miR-127 was demonstrated to be prominently induced upon TLR engagement and repressed by the M2-prone cytokines. Enforced expression of miR-127 in macrophages resulted in significantly increased production of proinflammatory cytokines, whereas deletion of miR-127 impaired M1 gene expression and led to a M2-biased response. Accordingly, intratracheal administration of miR-127 resulted in an exaggerated pulmonary inflammation and injury. Conversely, antagonizing of miR-127 suppressed production of the proinflammatory cytokines and rendered the mice more refractory to the inflammation-associated pathology. Mechanistically, miR-127 demonstrated to target B cell lymphoma 6 (Bcl6) and remarkably downregulated its expression and subsequently dual specificity phosphatase 1 (Dusp1), which in turn enhanced the activation of JNK kinase and hence the development of proinflammatory macrophages. Thereby, reconstitution with the expression of Bcl6 or Dusp1 or inhibition of JNK activity impaired miR-127–mediated skewing of M1 proinflammatory macrophages, whereas interference of Bcl6 or Dusp1 expression abrogated the anti-inflammatory property of anti–miR-127. Together, these data establish miR-127 as a molecular switch during macrophage development and as a potential target for treatment of inflammatory diseases.

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Damage-associated molecular patterns (DAMP) trigger innate immune response and exacerbate inflammation to combat infection and cellular damage. Identifying DAMPs and revealing their functions are thus of crucial importance. Here we report that two molecules, N-myc and STAT interactor (NMI) and interferon-induced protein 35 (IFP35) act as DAMPs and are released by activated macrophages during lipopolysaccharide-induced septic shock or acetaminophen-induced liver injury. We show that extracellular NMI and IFP35 activate macrophages to release proinflammatory cytokines by activating nuclear factor-κB through the Toll-like receptor 4 pathway. In addition, the serum levels of NMI are increased in patients who succumbed to severe inflammation. NMI deficiency reduces inflammatory responses and mortality in mouse models of sepsis and liver injury. We therefore propose that extracellular NMI and IFP35 exacerbate inflammation as DAMPs, making them potential therapeutic targets for clinical intervention.

Aim of this study was to develop a new simpler and more effective severity score for community-acquired pneumonia (CAP) patients. A total of 1640 consecutive hospitalized CAP patients in Second Affiliated Hospital of Zhejiang University were included. The effectiveness of different pneumonia severity scores to predict mortality was compared, and the performance of the new score was validated on an external cohort of 1164 patients with pneumonia admitted to a teaching hospital in Italy. Using age ≥ 65 years, LDH > 230 u/L, albumin < 3.5 g/dL, platelet count < 100 × 109/L, confusion, urea > 7 mmol/L, respiratory rate ≥ 30/min, low blood pressure, we assembled a new severity score named as expanded-CURB-65. The 30-day mortality and length of stay were increased along with increased risk score. The AUCs in the prediction of 30-day mortality in the main cohort were 0.826 (95% CI, 0.807–0.844), 0.801 (95% CI, 0.781–0.820), 0.756 (95% CI, 0.735–0.777), 0.793 (95% CI, 0.773–0.813) and 0.759 (95% CI, 0.737–0.779) for the expanded-CURB-65, PSI, CURB-65, SMART-COP and A-DROP, respectively. The performance of this bedside score was confirmed in CAP patients of the validation cohort although calibration was not successful in patients with health care-associated pneumonia (HCAP). The expanded CURB-65 is objective, simpler and more accurate scoring system for evaluation of CAP severity, and the predictive efficiency was better than other score systems.

Macrophage polarization is critical for dictating host defense against pathogens and injurious agents. Dysregulation of macrophage differentiation has been implicated in infectious and inflammatory diseases. Here, we show that protein kinase B/Akt1 signaling induced by Staphylococcus aureus is essential in shifting macrophages from an antimicrobial phenotype (M1) to a functionally inert signature. Akt1(-/-)mice consistently had enhanced bacterial clearance and greater survival, compared with their wild-type littermates. The blunted M1 macrophage reaction driven by Akt1 was associated with decreased RelA/nuclear factor κB activity. Furthermore, by repression of the expression of suppressor of cytokine signaling 1 (SOCS1), microRNA 155 revealed to promote the transcription of M1 signature genes in macrophages from Akt1(-/-) mice. Accordingly, blocking of microRNA 155 in macrophages from Akt1(-/-)mice or knockdown of SOCS1 in cells from wild-type mice disabled or enabled, respectively, an M1 macrophage shift and antibacterial response. These results thus establish an Akt1-mediated, microRNA-involved circuit that regulates pathogen-driven macrophage polarization and, subsequently, the host response to infection.

Secondary bacterial pneumonias are a frequent complication of influenza and other respiratory viral infections, but the mechanisms underlying viral-induced susceptibility to bacterial infections are poorly understood. In particular, it is unclear whether the host's response against the viral infection, independent of the injury caused by the virus, results in impairment of antibacterial host defense. Here, we sought to determine whether the induction of an “antiviral” immune state using various viral recognition receptor ligands was sufficient to result in decreased ability to combat common bacterial pathogens of the lung. Using a mouse model, animals were administered polyinosine-polycytidylic acid (poly I:C) or Toll-like 7 ligand (imiquimod or gardiquimod) intranasally, followed by intratracheal challenge with Streptococcus pneumoniae. We found that animals pre-exposed to poly I:C displayed impaired bacterial clearance and increased mortality. Poly I:C-exposed animals also had decreased ability to clear methicillin-resistant Staphylococcus aureus. Furthermore, we showed that activation of Toll-like receptor (TLR)3 and Retinoic acid inducible gene (RIG-I)/Cardif pathways, which recognize viral nucleic acids in the form of dsRNA, both contribute to poly I:C mediated impairment of bacterial clearance. Finally, we determined that poly I:C administration resulted in significant induction of type I interferons (IFNs), whereas the elimination of type I IFN signaling improved clearance and survival following secondary bacterial pneumonia. Collectively, these results indicate that in the lung, poly I:C administration is sufficient to impair pulmonary host defense against clinically important gram-positive bacterial pathogens, which appears to be mediated by type I IFNs.

We have previously demonstrated that exosomes from dendritic cells (DCs) secreting TGF-β1 (sTGF-β1-EXOs) delay the development of murine inflammatory bowel disease (IBD). In this study, we isolated exosomes from DCs expressing membrane-associated TGF-β1 (mTGF-β1-EXOs) and found mTGF-β1-EXOs had more potent immunosuppressive activity than sTGF-β1-EXOs in vitro. Treatment of mice with mTGF-β1-EXOs inhibited the development and progression of myelin oligodendrocyte glycoprotein (MOG)peptide-induced EAE even after disease onset. Treatment of mice with mTGF-β1-EXOs also impaired Ag-specific Th1 and IL-17 responses, but promoted IL-10 responses ex vivo. Treatment with mTGF-β1-EXOs decreased the frequency of Th17 cells in EAE mice, which might be associated with the down-regulation of the p38, ERK, Stat3, and NF-κB activation and IL-6 expression in DCs. Treatment with mTGF-β1-EXOs maintained the regulatory capacity of Treg cells, and adoptive transfer of CD4 + Foxp3 + Treg cells from mTGF-β1-EXO-treated EAE mice dramatically prevented the development of EAE in the recipients. Moreover, treatment with mTGF-β1-EXOs from C57BL/6 mice effectively prevented and inhibited proteolipid protein (PLP) peptide-induced EAE in BALB/c mice. These results indicate that mTGF-β1-EXOs possess powerful immunosuppressive ability and can effectively inhibit the development and progression of EAE in different strains of mice.Keywords: Autoimmune diseases r Exosomes r TGF-β1 r Th17 r Treg cells IntroductionDCs are unique professional APCs and important for the initiation and regulation of immune response [1,2]. Immature DCs have lower levels of co-stimulatory molecules and usually Correspondence: Dr. Zhijian Cai e-mail: caizj@zju.edu.cn; jlwang@zju.edu.cn induce Ag-specific T-cell anergy [3]. TGF-β1 is a negative regulator of pro-inflammatory immune responses. We previously found that systemic administration of TGF-β1 gene-modified immature DCs delayed the development of dextran sulfate sodiuminduced murine inflammatory bowel disease (IBD) [4]. Therefore, * These authors contributed equally to this work. * * These authors share senior co-authorship.C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 2462 Lei Yu et al. Eur. J. Immunol. 2013. 43: 2461-2472 TGF-β1 may modify DC development toward a regulatory phenotype, inhibiting inflammation. Exosomes, released by almost all types of cells, are small lipid bilayer vesicles with a size of 50-100 nm. They are formed by membrane budding into the lumen of an endocytic compartment, leading to the formation of multivesicular bodies. Fusion of multivesicular bodies to plasma membrane leads to the extracellular release of exosomes [5,6]. Exosomes from DCs transfected with vIL-10, FasL, or IL-4 gene inhibit delayed-type hypersensitivity and collagen-induced murine arthritis [7,8]. These results indicate that exosomes can be effective vehicles to carry immunoregulatory molecules for the treatment of autoimmune diseases.Ag-specific Th1 and Th17 cells are crucial for the development of...