Cardiotrophin-1 is an anti-inflammatory cytokine and promotes IL-4-induced M2 macrophage polarization.

Macrophages play a central role in tissue remodeling, repair, and resolution of inflammation. Macrophage polarization to M1 or M2 activation status may determine the progression or resolution of the inflammatory response. We have previously reported that cardiotrophin-1 (CT-1) displays both cytoprotective and metabolic activities. The role of CT-1 in inflammation remains poorly understood. Here, we employed recombinant CT-1 (rCT-1) and used CT-1-null mice and myeloid-specific CT-1 transgenic mice to investigate whether CT-1 might play a role in the modulation of the inflammatory response. We observed that CT-1 deficiency was associated with enhanced release of inflammatory mediators and with stronger activation of NF-κB in response to LPS, whereas the inflammatory response was attenuated in CT-1 transgenic mice or by administering rCT-1 to wild-type animals prior to LPS challenge. We found that CT-1 promoted IL-6 expression only by nonhematopoietic cells, whereas LPS up-regulated IL-6 expression in both hematopoietic and nonhematopoietic cells. Notably, rCT-1 inhibited LPS-mediated soluble IL-6R induction. Using IL-6-/- mice, we showed that rCT-1 inhibited LPS-induced TNF-α and IFN-γ response in an IL-6-independent manner. Importantly, we demonstrated that CT-1 primes macrophages for IL-4-dependent M2 polarization by inducing IL-4 receptor expression. Mechanistic analyses showed that the signal transducer and activator of transcription 3-suppressor of cytokine signaling 3 axis mediates this effect. Our findings support the notion that CT-1 is a critical regulator of inflammation and suggest that rCT-1 could be a molecule with potential therapeutic application in inflammatory conditions.-Carneros, D., Santamaría, E. M., Larequi, E., Vélez-Ortiz, J. M., Reboredo, M., Mancheño, U., Perugorria, M. J., Navas, P., Romero-Gómez, M., Prieto, J., Hervás-Stubbs, S., Bustos, M. Cardiotrophin-1 is an anti-inflammatory cytokine and promotes IL-4-induced M2 macrophage polarization.

Strict requirement for vector-induced type I interferon in efficacious antitumor responses to virally encoded IL12.

Host responses are increasingly considered important for the efficacious response to experimental cancer therapies that employ viral vectors, but little is known about the specific nature of host responses required. In this study, we investigated the role of host type I interferons (IFN-I) in the efficacy of virally delivered therapeutic genes. Specifically, we used a Semliki Forest virus encoding IL12 (SFV-IL12) based on its promise as an RNA viral vector for cancer treatment. Intratumoral injection of SFV-IL12 induced production of IFN-I as detected in serum. IFN-I production was abolished in mice deficient for the IFNß transcriptional regulator IPS-1 and partially attenuated in mice deficient for the IFNß signaling protein TRIF. Use of bone marrow chimeric hosts established that both hematopoietic and stromal cells were involved in IFN-I production. Macrophages, plasmacytoid, and conventional dendritic cells were each implicated based on cell depletion experiments. Further, mice deficient in the IFN-I receptor (IFNAR) abolished the therapeutic activity of SFV-IL12, as did a specific antibody-mediated blockade of IFNAR signaling. Reduced efficacy was not caused by an impairment in IL12 expression, because IFNAR-deficient mice expressed the viral IL12 transgene even more strongly than wild-type (WT) hosts. Chimeric host analysis for the IFNAR involvement established a strict requirement in hematopoietic cells. Notably, although tumor-specific CD8 T lymphocytes expanded robustly after intratumoral injection of WT mice with SFV-IL12, this did not occur in mice where IFNAR was inactivated genetically or pharmacologically. Overall, our results argued that the antitumor efficacy of a virally based transgene therapeutic relied strongly on a vector-induced IFN-I response, revealing an unexpected mechanism of action that is relevant to a broad array of current translational products in cancer research.

Zolmitriptan: a novel portal hypotensive agent which synergizes with propranolol in lowering portal pressure.

OBJECTIVE: Only a limited proportion of patients needing pharmacological control of portal hypertension are hemodynamic responders to propranolol. Here we analyzed the effects of zolmitriptan on portal pressure and its potential interaction with propranolol. METHODS: ZOLMITRIPTAN, PROPRANOLOL OR BOTH WERE TESTED IN TWO RAT MODELS OF PORTAL HYPERTENSION: common bile duct ligation (CBDL) and CCl4-induced cirrhosis. In these animals we measured different hemodynamic parameters including portal venous pressure, arterial renal flow, portal blood flow and cardiac output. We also studied the changes in superior mesenteric artery perfusion pressure and in arterial wall cAMP levels induced by zolmitriptan, propranolol or both. Moreover, we determined the effect of splanchnic sympathectomy on the response of PVP to zolmitriptan. RESULTS: In both models of portal hypertension zolmitriptan induced a dose-dependent transient descent of portal pressure accompanied by reduction of portal flow with only slight decrease in renal flow. In cirrhotic rats, splanchnic sympathectomy intensified and prolonged zolmitriptan-induced portal pressure descent. Also, propranolol caused more intense and durable portal pressure fall when combined with zolmitriptan. Mesenteric artery perfusion pressure peaked for about 1 min upon zolmitriptan administration but showed no change with propranolol. However propranolol enhanced and prolonged the elevation in mesenteric artery perfusion pressure induced by zolmitriptan. In vitro studies showed that propranolol prevented the inhibitory effects of ß2-agonists on zolmitriptan-induced vasoconstriction and the combination of propranolol and zolmitriptan significantly reduced the elevation of cAMP caused by ß2-agonists. CONCLUSION: Zolmitriptan reduces portal hypertension and non-selective beta-blockers can improve this effect. Combination therapy deserves consideration for patients with portal hypertension failing to respond to non-selective beta-blockers.

Transcriptomic effects of Tet-on and mifepristone-inducible systems in mouse liver.

Control of transgene expression from long-term expression vectors can be achieved with inducible and regulated promoters. The two most commonly used inducible systems employ doxycycline or mifepristone as the drug activating a silent trans-activator, which is expressed from a constitutive promoter. We evaluated the alterations provoked by constitutive expression in the liver of rtTA2(S)-M2 (rtTA2; second-generation reverse tetracycline-controlled trans-activator) and GLp65, which are the trans-activators of the doxycyline- and mifepristone-inducible systems, respectively. To this end we performed transcriptomic analysis of mice expressing these trans-activators in the liver over 1 month. rtTA2 expression induced alterations in a few genes (69 gene probesets; false discovery rate [FDR], approximately 0.05), whereas GLp65 caused more numerous changes (1059 gene probe-sets, an FDR of approximately 0.05). However, only 20 and 53 of the genes from the rtTA2 and GLp65 groups, respectively, showed changes (R-fold >or= 3). Functional assignments indicate that alterations were mild and of little general significance. Few additional transcriptomic changes were observed when expressing trans-activators in the presence of inducer drugs; most were due to the drugs themselves. These results and the absence of toxicity observed in treated animals indicate that the two inducible systems are well tolerated and have little impact on the liver transcriptome profile. The milder alterations found with the use of rtTA2 suggest that this system is possibly safer for gene therapy applications.

Human cytomegalovirus proteins encoded by UL37 exon 1 protect infected fibroblasts against virus-induced apoptosis and are required for efficient virus replication.

Human cytomegalovirus (HCMV) strain AD169 mutants carrying transposon insertions or large deletions in UL37 exon 1 (UL37x1) were recovered from modified bacterial artificial chromosomes by reconstitution in human fibroblasts expressing the adenovirus anti-apoptotic protein E1B19K. UL37x1 mutant growth was severely compromised in normal fibroblasts, with minimal release of infectious progeny. Growth in E1B19K-expressing cells was restored, but did not reach wild-type levels. Normal fibroblasts infected by UL37x1 mutants underwent apoptosis spontaneously between 48 and 96 h after infection. Apoptosis was inhibited by treatment of cells with the broad-spectrum caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone, resulting in substantially increased release of virus. Inhibition of viral DNA replication by phosphonoformate or ganciclovir also inhibited apoptosis, implying that death was triggered by late viral functions or by replication and packaging of the viral genome. Immunofluorescent staining showed that although viral proteins accumulated normally during delayed-early phase and viral DNA replication compartments formed, viral late proteins were detected only rarely, suggesting that spontaneous apoptosis occurs early in late phase. These results demonstrate that anti-apoptotic proteins encoded by HCMV UL37x1 [pUL37x1 (vMIA), gpUL37 and gpUL37(M)] prevent apoptosis that would otherwise be initiated by the replication programme of the virus and are required for efficient and sustainable virus replication.