Visualization of Lipid Droplets in the Alveolar Macrophage Cell Line MH-S with Live-cell Imaging by 3D Holotomographic Microscopy (Nanolive)

Lipid droplets (LD), triglycerides and sterol esters among them, are well known for their capacity as lipid storage organelles. Recently, they have emerged as critical cytoplasmic structures involved in numerous biological functions. LD storage is generated de novo by the cell and provides an energy reserve, lipid precursors, and cell protection. Moreover, LD accumulation can be observed in some pathologies as obesity, atherosclerosis, or lung diseases. Fluorescence imaging techniques are the most widely used techniques to visualize cellular compartments in live cells, including LD. Nevertheless, presence of fluorophores can damage subcellular components and induce cytotoxicity, or even alter the dynamics of the organelles. As an alternative to fluorescence microscopy, label-free techniques such as stimulated Raman scattering and coherent anti-stokes Raman scattering microscopy offer a solution to avoid the undesirable effects caused by dyes and fluorescent proteins, but are expensive and complex. Here, we describe a label-free method using live-cell imaging by 3D holotomographic microscopy (Nanolive) to visualize LD accumulation in the MH-S alveolar macrophage cell line after treatment with oleic acid, a monounsaturated fatty acid that promotes lipid accumulation.We are grateful to Instituto de Salud Carlos III for financial support to S.H.(PI20CIII/00018). A. Pérez-Montero is supported by Comunidad de Madrid (PEJ-2020-AI/BMD-17651).S

Current status of terpenoids as inflammasome inhibitors

Increasing evidence supports NLRP3 inflammasome as a new target to control inflammation. Dysregulation of NLRP3 inflammasome has been reported to be involved in the pathogenesis of several human inflammatory diseases. However, no NLRP3 inflammasome inhibitors are available in clinic. Terpenoids are natural products with multi-target activities against inflammation. Recent studies have revealed that these compounds are capable of inhibiting the activation of NLRP3 inflammasome in several mouse models of NLRP3 inflammasome-related pathogenesis. Thus, terpenoids represent an interesting pharmacological approach for the treatment of inflammatory diseases as they are endowed with a dual mechanism of inhibition of NF-KB transcription factor and inflammasome activation, both critically involved in their anti-inflammatory effects. This work provides an overview of the current knowledge on the therapeutic potential of terpenoids as NLRP3 inflammasome inhibitors.This work was supported by grant PI11/00036, PI14/00055, and PI17/00012 from the FIS, MPY 1410/09 from ISCIII and Spanish Ministry of Health (Instituto de Salud Carlos III; RD12/0036/0059) to SH and grant RTI2018-094356-B-C21 from Ministerio de Economía y Competitividad to LGC, IC and BH. LGC received a predoctoral fellowship award from the Spanish Ministry of Education, Culture and Sports (FPU17/03519).S

Tumor suppressor ARF regulates tissue microenvironment and tumor growth through modulation of macrophage polarization

Tumor microenvironment has been described to play a key role in tumor growth, progression, and metastasis. Macrophages are a major cellular constituent of the tumor stroma, and particularly tumor associated macrophages (TAMs or M2-like macrophages) exert important immunosuppressive activity and a pro-tumoral role within the tumor microenvironment. Alternative-reading frame (ARF) gene is widely inactivated in human cancer. We have previously demonstrated that ARF deficiency severely impairs inflammatory response establishing a new role for ARF in the regulation of innate immunity. On the basis of these observations, we hypothesized that ARF may also regulates tumor growth through recruitment and modulation of the macrophage phenotype in the tumor microenvironment. Xenograft assays of B16F10 melanoma cells into ARF-deficient mice resulted in increased tumor growth compared to those implanted in WT control mice. Tumors from ARF-deficient mice exhibited significantly increased number of TAMs as well as microvascular density. Transwell assays showed crosstalk between tumor cells and macrophages. On the one hand, ARF-deficient macrophages modulate migratory ability of the tumor cells. And on the other, tumor cells promote the skewing of ARF-/- macrophages toward a M2-type polarization. In conclusion, these results demonstrate that ARF deficiency facilitates the infiltration of macrophages into the tumor mass and favors their polarization towards a M2 phenotype, thus promoting tumor angiogenesis and tumor growth. This work provides novel information about the critical role of ARF in the modulation of tumor microenvironment.This study was supported by grant PI11.0036 and PI14.0055 from the FIS, MPY 1410/09 from ISCIII and Spanish Ministry of Health (Instituto de Salud Carlos III; RD12/0036/0059) to SH, and by grant TPY-M-1068/13 and IERPY 1149/16 to AL. L J-G was supported by FIS (FI12/00340). AL was supported by FIS (CP12/03087). S Herranz was supported by TPY-M-1068/13 from ISCIII. We thank Fernando González Camacho and Silvia Hernández Esteban for Confocal Microscopy assistance.S

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.This work was supported by the Ministerio de Economía y Competitividad (MINECO) and the Instituto de Salud Carlos III (ISCIII) (grant number IERPY 1149/16 to A.L.; MPY 1410/09 to S. Hortelano); by the MINECO through the Fondo de Investigación en Salud (FIS) (grants numbers PI11.0036 and PI14.0055 to S. Hortelano). S. Herranz was supported by IERPY 1149/16 from ISCIII.S

ILK mediates LPS-induced vascular adhesion receptor expression and subsequent leucocyte trans-endothelial migration†

Aims The inflammatory response to injurious agents is tightly regulated to avoid adverse consequences of inappropriate leucocyte accumulation or failed resolution. Lipopolysaccharide (LPS)-activated endothelium recruits leucocytes to the inflamed tissue through controlled expression of membrane-associated adhesion molecules. LPS responses in macrophages are known to be regulated by integrin-linked kinase (ILK); in this study, we investigated the role of ILK in the regulation of the LPS-elicited inflammatory response in endothelium. Methods and results This study was performed on immortalized mouse endothelial cells (EC) isolated from lung and coronary vasculature. Cells were thoroughly characterized and the role of ILK in the regulation of the LPS response was investigated by suppressing ILK expression using siRNA and shRNA technologies. Phenotypic and functional analyses confirmed that the immortalized cells behaved as true EC. LPS induced the expression of the inflammatory genes E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). ILK knockdown impaired LPS-mediated endothelial activation by preventing the induction of ICAM-1 and VCAM-1. Blockade of the LPS-induced response inhibited the inflammatory-related processes of firm adhesion and trans-endothelial migration of leucocytes. Conclusion ILK is involved in the expression of cell adhesion molecules by EC activated with the inflammatory stimulus LPS. This reduced expression modulates leucocyte adhesion to the endothelium and the extravasation process. This finding suggests ILK as a potential anti-inflammatory target for the development of vascular-specific treatments for inflammation-related disease

IL-10 released by a new inflammation-regulated lentiviral system efficiently attenuates zymosan-induced arthritis

We thank Dr Filip Lim for critical reading of the manuscript, and Dr S. Bartlett for English editing and helpful discussions. We also thank Drs. David Sancho and M. A. del Pozo for providing us with DCs and immortalized MEFs, respectively. AR is supported by Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+I) and Instituto de Salud Carlos III (FIS; PI060122), the Spanish Ministry of Science and Innovation (MICINN;SAF2009-10691) and the Comunidad Autónoma de Madrid (S2006/BIO-0236 and S2010/BMD-2312). JMR is supported by MICINN (RECAVA RD06/0014/005) and by from Fundació La Marató de TV3 (Grant 080731

Labdane conjugates protect cardiomyocytes from doxorubicin-induced cardiotoxicity

The cardiovascular side effects associated with doxorubicin (DOX), a wide spectrum anticancer drug, have limited its clinical application. Therefore, to explore novel strategies with cardioprotective effects, a series of new labdane conjugates were prepared (6a-6c and 8a-8d) from the natural diterpene labdanodiol (1). These hybrid compounds contain anti-inflammatory privileged structures such as naphthalimide, naphthoquinone, and furanonaphthoquinone. Biological activity of these conjugates against DOX-induced cardiotoxicity was tested in vitro and the potential molecular mechanisms of protective effects were explored in H9c2 cardiomyocytes. Three compounds 6c, 8a, and 8b significantly improved cardiomyocyte survival, via inhibition of reactive oxygen species-mediated mitogen-activated protein kinase signaling pathways (extracellular signal-regulated kinase and c-Jun N-terminal kinase) and autophagy mediated by Akt activation. Some structure-activity relationships were outlined, and the best activity was achieved with the labdane-furonaphthoquinone conjugate 8a having an N-cyclohexyl substituent. The findings of this study pave the way for further investigations to obtain more compounds with potential cardioprotective activity.This study was supported by Grant RTI2018‐094356‐BC21 from the Ministerio de Ciencia, Innovación y Universidades (MICIU) to A. E.‐B., I. C., L. G.‐C., and B. H.; Grant PI17/00012 and PI20/00018 from the Instituto de Salud Carlos III to S. H. These projects are also cofunded by the European Regional Development Fund (FEDER). A. A. and S. O.‐R. thank the Cabildo de Tenerife (Agustín de Betancourt Program).S

Synthesis of Quinoline and Dihydroquinoline Embelin Derivatives as Cardioprotective Agents

A set of new dihydroquinoline embelin derivatives was obtained from the reaction of the natural benzoquinone embelin (1) with anilines and aromatic aldehydes in the presence of AgOTf. The synthesis of these compounds involves the formation of a Knoevenagel adduct, followed by nucleophilic addition of aniline and subsequent electrocyclic ring closure. The scope of the reaction regarding the aldehydes and anilines was determined. Quinoline derivatives were also obtained from the corresponding dihydroquinolines under oxidation with DDQ. The cardioprotective activity of the synthesized compounds was screened using a doxorubicin-induced cardiotoxicity model in H9c2 cardiomyocytes. Some structure-activity relationships were outlined, and the best activities were achieved with quinoline-embelin derivatives having a 4-nitrophenyl group attached at the pyridine ring. The obtained results indicated that embelin derivatives 4i, 6a, 6d, 6k, and 6m could have potential as cardioprotective agents, as they attenuated a DOX-induced cardiotoxicity effect acting on oxidative stress and apoptosis.We gratefully acknowledge the financial support from the Spanish MICIU RTI2018-094356-B-C21 to A.E.B., I.C., and B.H. and Agencia Canaria de Investigación, Innovación y Sociedad de la Información Pro ID 2021010037 to A.E.B. These projects are also cofunded by the European Regional Development Fund (FEDER). We thank Dr. A. Tapia and G. Feresin for providing the natural embeline. We are grateful to Instituto de Salud Carlos III for financial support to S.H.S

Semisynthesis and Inhibitory Effects of Solidagenone Derivatives on TLR-Mediated Inflammatory Responses

A series of nine derivatives (2⁻10) were prepared from the diterpene solidagenone (1) and their structures were elucidated by means of spectroscopic studies. Their ability to inhibit inflammatory responses elicited in peritoneal macrophages by TLR ligands was investigated. Compounds 5 and 6 showed significant anti-inflammatory effects, as they inhibited the protein expression of nitric oxide synthase (NOS-2), cyclooxygenase-2 (COX-2), and cytokine production (TNF-α, IL-6, and IL-12) induced by the ligand of TLR4, lipopolysaccharide (LPS), acting at the transcriptional level. Some structure⁻activity relationships were outlined. Compound 5 was selected as a representative compound and molecular mechanisms involved in its biological activity were investigated. Inhibition of NF-κB and p38 signaling seems to be involved in the mechanism of action of compound 5. In addition, this compound also inhibited inflammatory responses mediated by ligands of TLR2 and TLR3 receptors. To rationalize the obtained results, molecular docking and molecular dynamic studies were carried out on TLR4. All these data indicate that solidagenone derivative 5 might be used for the design of new anti-inflammatory agents.S

α-Hispanolol Induces Apoptosis and Suppresses Migration and Invasion of Glioblastoma Cells Likely via Downregulation of MMP-2/9 Expression and p38MAPK Attenuation

α-Hispanolol (α-H) is a labdane diterpenoid that has been shown to induce apoptosis in several human cancer cells. However, the effect of α-H in human glioblastoma cells has not been described. In the present work, we have investigated the effects of α-H on apoptosis, migration, and invasion of human glioblastoma cells with the aim of identifying the molecular targets underlying its mechanism of action. The results revealed that α-H showed significant cytotoxicity against human glioma cancer cell lines U87 and U373 in a concentration- and time-dependent manner. This effect was higher in U87 cells and linked to apoptosis, as revealed the increased percentage of sub-G1 population by cell cycle analysis and acquisition of typical features of apoptotic cell morphology. Apoptosis was also confirmed by significant presence of annexin V-positive cells and caspase activation. Pretreatment with caspase inhibitors diminishes the activities of caspase 8, 9, and 3 and maintains the percentage of viable glioblastoma cells, indicating that α-H induced cell apoptosis through both the extrinsic and the intrinsic pathways. Moreover, we also found that α-H downregulated the anti-apoptotic Bcl-2 and Bcl-xL proteins and activated the pro-apoptotic Bid and Bax proteins. On the other hand, α-H exhibited inhibitory effects on the migration and invasion of U87 cells in a concentration-dependent manner. Furthermore, additional experiments showed that α-H treatment reduced the enzymatic activities and protein levels of matrix metalloproteinase MMP-2 and MMP-9 and increased the expression of TIMP-1 inhibitor, probably via p38MAPK regulation. Finally, xenograft assays confirmed the anti-glioma efficacy of α-H. Taken together, these findings suggest that α-H may exert anti-tumoral effects in vitro and in vivo through the inhibition of cell proliferation and invasion as well as by the induction of apoptosis in human glioblastoma cells. This research describes α-H as a new drug that may improve the therapeutic efficacy against glioblastoma tumors.This study was supported by grant PI11/00036, PI14/00055, and PI17/00012 from the FIS, MPY 1410/09 from ISCIII and Spanish Ministry of Health (Instituto de Salud Carlos III; RD12/0036/0059) to SoH and by grants IERPY 1149/16 and IERPY-M 389/18 to AL. L JG was supported by FIS (FI12/00340). SaH was supported by IERPY 1149/16 from ISCIII.S