Regulation of miRNA expression by α4ß1 integrin-dependent multiple myeloma cell adhesion.

The α4ß1 integrin regulates the trafficking of multiple myeloma (MM) cells and contributes to MM disease progression. MicroRNAs (miRNAs) can have both tumor suppressor and oncogenic roles and thus are key controllers of tumor evolution, and have been associated with different phases of MM pathogenesis. Using small RNAseq analysis, we show here that α4ß1-dependent MM cell adhesion regulates the expression of forty different miRNAs, therefore expanding our current view of the α4ß1 involvement in MM cell biology. Specific upregulation of miR-324-5p and miR-331-3p in cells attached to α4ß1 ligands was confirmed upon silencing the α4 integrin subunit, and their increased levels found to be dependent on Erk1/2- and PI3K-Akt-, but not Src-dependent signaling. Enhanced miR-324-5p expression upon α4ß1-mediated MM cell adhesion aimed the hedgehog (Hh) component SMO, revealing that the miR-324-5p-SMO module represents a α4ß1-regulated pathway that could control Hh-dependent cellular responses in myeloma. Our results open new therapy research avenues around the α4ß1 contribution to MM progression that deserve to be investigated.

Early p38 Activation Regulated by MKP-1 Is Determinant for High Levels of IL-10 Expression Through TLR2 Activation.

Toll-like receptors (TLRs) play a crucial role in the recognition of pathogen-derived components as a first line of defense against infections. It has been suggested that depending on the nature of the pathogens, TLRs activation induce a distinct cytokine profile that may contribute to the polarization of the acquired immune response. Here, we investigated the early MAPK signaling activation via TLR4 and TLR2 receptors and its impact in differential cytokine profile by macrophages. We found that TLR2 ligands activated MAPKs p38 and ERK earlier compared to the TLR4 ligand LPS in macrophages. Higher IL-10/IL-12 and IL-10/TNF-α ratios were also observed at later time points in response to TLR2 ligands compared to LPS. The results also indicate an earlier activation of the phosphatase MKP-1 and that MKP-1 KO macrophages show a prolongation in p38 phosphorylation in response to TLR2 stimulation. Furthermore, p38 is critical for IL-10 expression in response to TLR2 ligands, which triggers the macrophage change to a M2 and regulatory phenotype in contrast to the M1 phenotype induced by TLR4 activation. Therefore, the early TLR2-mediated p38 induction contributes for the high IL-10 production, likely as a virulence strategy to suppress host Th1 response against certain types of pathogens.

Induction of TLR4/TLR2 Interaction and Heterodimer Formation by Low Endotoxic Atypical LPS.

The Toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2) complex is considered the major receptor of the innate immune system to recognize lipopolysaccharides (LPSs). However, some atypical LPSs with different lipid A and core saccharide moiety structures and compositions than the well-studied enterobacterial LPSs can induce a TLR2-dependent response in innate immune cells. Ochrobactrum intermedium, an opportunistic pathogen, presents an atypical LPS. In this study, we found that O. intermedium LPS exhibits a weak inflammatory activity compared to Escherichia coli LPS and, more importantly, is a specific TLR4/TLR2 agonist, able to signal through both receptors. Molecular docking analysis of O. intermedium LPS predicts a favorable formation of a TLR2/TLR4/MD-2 heterodimer complex, which was experimentally confirmed by fluorescence resonance energy transfer (FRET) in cells. Interestingly, the core saccharide plays an important role in this interaction. This study reveals for the first time TLR4/TLR2 heterodimerization that is induced by atypical LPS and may help to escape from recognition by the innate immune system.

Cathepsin L3 From Fasciola hepatica Induces NLRP3 Inflammasome Alternative Activation in Murine Dendritic Cells.

The production of IL-1-family cytokines such as IL-1ß and IL-18 is finely regulated by inflammasome activation after the recognition of pathogens associated molecular pattern (PAMPs) and danger associated molecular patterns (DAMPs). However, little is known about the helminth-derived molecules capable of activating the inflammasome. In the case of the helminth trematode Fasciola hepatica, the secretion of different cathepsin L cysteine peptidases (FhCL) is crucial for the parasite survival. Among these enzymes, cathepsin L3 (FhCL3) is expressed mainly in the juvenile or invasive stage. The ability of FhCL3 to digest collagen has demonstrated to be critical for intestinal tissue invasion during juvenile larvae migration. However, there is no information about the interaction of FhCL3 with the immune system. It has been shown here that FhCL3 induces a non-canonical inflammasome activation in dendritic cells (DCs), leading to IL-1ß and IL-18 production without a previous microbial priming. Interestingly, this activation was depending on the cysteine protease activity of FhCL3 and the NLRP3 receptor, but independent of caspase activation. We also show that FhCL3 is internalized by DCs, promoting pro-IL-1ß cleavage to its mature and biologically active form IL-1ß, which is released to the extracellular environment. The FhCL3-induced NLRP3 inflammasome activation conditions DCs to promote a singular adaptive immune response, characterized by increased production of IFN-γ and IL-13. These data reveal an unexpected ability of FhCL3, a helminth-derived molecule, to activate the NLRP3 inflammasome, which is independent of the classical mechanism involving caspase activation.

Stripe artifact elimination based on nonsubsampled contourlet transform for light sheet fluorescence microscopy.

Stripe artifacts, caused by high-absorption or high-scattering structures in the illumination light path, are a common drawback in both unidirectional and multidirectional light sheet fluorescence microscopy (LSFM), significantly deteriorating image quality. To circumvent this problem, we present an effective multidirectional stripe remover (MDSR) method based on nonsubsampled contourlet transform (NSCT), which can be used for both unidirectional and multidirectional LSFM. In MDSR, a fast Fourier transform (FFT) filter is designed in the NSCT domain to shrink the stripe components and eliminate the noise. Benefiting from the properties of being multiscale and multidirectional, MDSR succeeds in eliminating stripe artifacts in both unidirectional and multidirectional LSFM. To validate the method, MDSR has been tested on images from a custom-made unidirectional LSFM system and a commercial multidirectional LSFM system, clearly demonstrating that MDSR effectively removes most of the stripe artifacts. Moreover, we performed a comparative experiment with the variational stationary noise remover and the wavelet-FFT methods and quantitatively analyzed the results with a peak signal-to-noise ratio, showing an improved noise removal when using the MDSR method.

Modulation of endothelial function by Toll like receptors.

Endothelial cells (EC) are able to actively control vascular permeability, coagulation, blood pressure and angiogenesis. Most recently, a role for endothelial cells in the immune response has been described. Therefore, the endothelium has a dual role controlling homeostasis but also being the first line for host defence and tissue damage repair thanks to its ability to mount an inflammatory response. Endothelial cells have been shown to express pattern-recognition receptors (PRR) including Toll-like receptors (TLR) that are activated in response to stimuli within the bloodstream including pathogens and damage signals. TLRs are strategic mediators of the immune response in endothelial cells but they also regulate the angiogenic process critical for tissue repair. Nevertheless, endothelial activation and angiogenesis can contribute to some pathologies. Thus, inappropriate endothelial activation, also known as endothelial dysfunction, through TLRs contributes to tissue damage during autoimmune and inflammatory diseases such as atherosclerosis, hypertension, ischemia and diabetes associated cardiovascular diseases. Also TLR induced angiogenesis is required for the growth of some tumors, atherosclerosis and rheumatoid arthritis, among others. In this review we discuss the importance of various TLRs in modulating the activation of endothelial cells and their importance in immunity to infection and vascular disease as well as their potential as therapeutic targets.

Polarization-sensitive optical projection tomography for muscle fiber imaging.

Optical projection tomography (OPT) is a tool used for three-dimensional imaging of millimeter-scale biological samples, with the advantage of exhibiting isotropic resolution typically in the micron range. OPT can be divided into two types: transmission OPT (tOPT) and emission OPT (eOPT). Compared with eOPT, tOPT discriminates different tissues based on their absorption coefficient, either intrinsic or after specific staining. However, it fails to distinguish muscle fibers whose absorption coefficients are similar to surrounding tissues. To circumvent this problem, in this article we demonstrate a polarization sensitive OPT system which improves the detection and 3D imaging of muscle fibers by using polarized light. We also developed image acquisition and processing protocols that, together with the system, enable the clear visualization of muscles. Experimental results show that the muscle fibers of diaphragm and stomach, difficult to be distinguished in regular tOPT, were clearly displayed in our system, proving its potential use. Moreover, polarization sensitive OPT was fused with tOPT to investigate the stomach tissue comprehensively. Future applications of polarization sensitive OPT could be imaging other fiber-like structures such as myocardium or other tissues presenting high optical anisotropy.

Advances in optical imaging for pharmacological studies.

Imaging approaches are an essential tool for following up over time representative parameters of in vivo models, providing useful information in pharmacological studies. Main advantages of optical imaging approaches compared to other imaging methods are their safety, straight-forward use and cost-effectiveness. A main drawback, however, is having to deal with the presence of high scattering and high absorption in living tissues. Depending on how these issues are addressed, three different modalities can be differentiated: planar imaging (including fluorescence and bioluminescence in vivo imaging), optical tomography, and optoacoustic approaches. In this review we describe the latest advances in optical in vivo imaging with pharmacological applications, with special focus on the development of new optical imaging probes in order to overcome the strong absorption introduced by different tissue components, especially hemoglobin, and the development of multimodal imaging systems in order to overcome the resolution limitations imposed by scattering.

Fluorescent Molecular Tomography for In Vivo Imaging of Mouse Atherosclerosis.

Optical imaging technologies such as fluorescence molecular tomography (FMT) are gaining great relevance in cardiovascular research. The main reason is the increased number of available fluorescent agents, especially those termed "activatable probes," which remain quenched under baseline conditions and are fluorescent when a specific enzymatic activity is present. A major characteristic of FMT is the possibility of obtaining quantitative data of fluorescence signal distribution in a noninvasive fashion and using nonionizing radiation, making FMT an invaluable tool for longitudinal studies with biomedical applications. Here, we describe a standard procedure to perform FMT experiments in atherosclerosis mouse models, from the handling of the animals to the reconstruction of the 3D images.

In-vivo optical tomography of small scattering specimens: time-lapse 3D imaging of the head eversion process in Drosophila melanogaster.

Even though in vivo imaging approaches have witnessed several new and important developments, specimens that exhibit high light scattering properties such as Drosophila melanogaster pupae are still not easily accessible with current optical imaging techniques, obtaining images only from subsurface features. This means that in order to obtain 3D volumetric information these specimens need to be studied either after fixation and a chemical clearing process, through an imaging window--thus perturbing physiological development -, or during early stages of development when the scattering contribution is negligible. In this paper we showcase how Optical Projection Tomography may be used to obtain volumetric images of the head eversion process in vivo in Drosophila melanogaster pupae, both in control and headless mutant specimens. Additionally, we demonstrate the use of Helical Optical Projection Tomography (hOPT) as a tool for high throughput 4D-imaging of several specimens simultaneously.

Vertically scanned laser sheet microscopy.

Laser sheet microscopy is a widely used imaging technique for imaging the three-dimensional distribution of a fluorescence signal in fixed tissue or small organisms. In laser sheet microscopy, the stripe artifacts caused by high absorption or high scattering structures are very common, greatly affecting image quality. To solve this problem, we report here a two-step procedure which consists of continuously acquiring laser sheet images while vertically displacing the sample, and then using the variational stationary noise remover (VSNR) method to further reduce the remaining stripes. Images from a cleared murine colon acquired with a vertical scan are compared with common stitching procedures demonstrating that vertically scanned light sheet microscopy greatly improves the performance of current light sheet microscopy approaches without the need for complex changes to the imaging setup and allows imaging of elongated samples, extending the field of view in the vertical direction.

Helical optical projection tomography.

A new technique termed Helical Optical Projection Tomography (hOPT) has been developed with the aim to overcome some of the limitations of current 3D optical imaging techniques. hOPT is based on Optical Projection Tomography (OPT) with the major difference that there is a translation of the sample in the vertical direction during the image acquisition process, requiring a new approach to image reconstruction. Contrary to OPT, hOPT makes possible to obtain 3D-optical images of intact long samples without imposing limits on the sample length. This has been tested using hOPT to image long murine tissue samples such as spinal cords and large intestines. Moreover, 3D-reconstructed images of the colon of DSS-treated mice, a model for Inflammatory Bowel Disease, allowed the identification of the structural alterations. Finally, the geometry of the hOPT device facilitates the addition of a Selective Plane Illumination Microscopy (SPIM) arm, providing the possibility of delivering high resolution images of selected areas together with complete volumetric information.

VIP in inflammatory bowel disease: state of the art.

The pathogenesis of inflammatory bowel syndrome (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC) is poorly understood. However, an inflammatory component is a common hallmark. It has been suggested that CD principally involves Th1 and/or Th17 cells, while UC is considered to be more Th2 driven. Because vasoactive intestinal peptide (VIP) has emerged in the last decade as a putative candidate for the treatment of inflammatory diseases with a Th1 component, it may as well serve as a therapeutic target in CD. In addition, experiments using mice deficient in VIP or its receptors have revealed that the endogenously-produced VIP may participate in the regulation of immunity. The aim of the present review is to summarize the quite considerable array of data which suggests that the VIP-receptor system plays a key role in modulating multiple molecular and cellular players involved in IBD.

Akt1 and Akt2 protein kinases differentially contribute to macrophage polarization.

Activated macrophages are described as classically activated or M1 type and alternatively activated or M2 type, depending on their response to proinflammatory stimuli and the expression of genetic markers including iNOS, arginase1, Ym1, and Fizz1. Here we report that Akt kinases differentially contribute to macrophage polarization, with Akt1 ablation giving rise to an M1 and Akt2 ablation resulting in an M2 phenotype. Accordingly, Akt2(-/-) mice were more resistant to LPS-induced endotoxin shock and to dextran sulfate sodium (DSS)-induced colitis than wild-type mice, whereas Akt1(-/-) mice were more sensitive. Cell depletion and reconstitution experiments in a DSS-induced colitis model confirmed that the effect was macrophage-dependent. Gene-silencing studies showed that the M2 phenotype of Akt2(-/-) macrophages was cell autonomous. The microRNA miR-155, whose expression was repressed in naive and in LPS-stimulated Akt2(-/-) macrophages, and its target C/EBPß appear to play a key role in this process. C/EBPß, a hallmark of M2 macrophages that regulates Arg1, was up-regulated upon Akt2 ablation or silencing. Overexpression or silencing of miR-155 confirmed its central role in Akt isoform-dependent M1/M2 polarization of macrophages.

Intravital spectral imaging as a tool for accurate measurement of vascularization in mice.

BACKGROUND: Quantitative determination of the development of new blood vessels is crucial for our understanding of the progression of several diseases, including cancer. However, in most cases a high throughput technique that is simple, accurate, user-independent and cost-effective for small animal imaging is not available. METHODS: In this work we present a simple approach based on spectral imaging to increase the contrast between vessels and surrounding tissue, enabling accurate determination of the blood vessel area. This approach is put to test with a 4T1 breast cancer murine in vivo model and validated with histological and microvessel density analysis. RESULTS: We found that one can accurately measure the vascularization area by using excitation/emission filter pairs which enhance the surrounding tissue's autofluorescence, significantly increasing the contrast between surrounding tissue and blood vessels. Additionally, we found excellent correlation between this technique and histological and microvessel density analysis. CONCLUSIONS: Making use of spectral imaging techniques we have shown that it is possible to accurately determine blood vessel volume intra-vitally. We believe that due to the low cost, accuracy, user-independence and simplicity of this technique, it will be of great value in those cases where in vivo quantitative information is necessary.

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

INTRODUCTION: 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. METHODS: 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. RESULTS: Array analysis revealed among other genes that CRF induced the expression of SMAD2 and ß-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- ß-catenin signaling. CRF induced 4T1 cell proliferation and augmented the TGF-ß action on proliferation confirming its impact on TGFß/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. CONCLUSION: 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 ß-catenin expression.

The kinase Akt1 controls macrophage response to lipopolysaccharide by regulating microRNAs.

MicroRNAs regulated by lipopolysaccharide (LPS) target genes that contribute to the inflammatory phenotype. Here, we showed that the protein kinase Akt1, which is activated by LPS, positively regulated miRNAs let-7e and miR-181c but negatively regulated miR-155 and miR-125b. In silico analyses and transfection studies revealed that let-7e repressed Toll-like receptor 4 (TLR4), whereas miR-155 repressed SOCS1, two proteins critical for LPS-driven TLR signaling, which regulate endotoxin sensitivity and tolerance. As a result, Akt1(-/-) macrophages exhibited increased responsiveness to LPS in culture and Akt1(-/-) mice did not develop endotoxin tolerance in vivo. Overexpression of let-7e and suppression of miR-155 in Akt1(-/-) macrophages restored sensitivity and tolerance to LPS in culture and in animals. These results indicate that Akt1 regulates the response of macrophages to LPS by controlling miRNA expression.

Adiponectin promotes endotoxin tolerance in macrophages by inducing IRAK-M expression.

High levels of plasma adiponectin are associated with low levels of inflammatory markers and cardioprotection. The mechanism via which adiponectin exerts its anti-inflammatory effect is yet unknown. In the present study, we demonstrate that globular adiponectin (gAd) induces the expression of the inactive isoform of IL-1R-associated kinases (IRAK), IRAK-M. Homologous deletion of IRAK-M in IRAK-M(-/-) mice abolished the tolerogenic properties of gAd because pretreatment of IRAK-M(-/-) macrophages with gAd did not suppress LPS-induced proinflammatory cytokine production. GAd activated the MAPKs MEK1/2 and ERK1/2 in macrophages via their upstream regulator Tpl2. Activation of ERK1/2 via Tpl2 appeared necessary for the induction of IRAK-M because gAd did not induce IRAK-M in Tpl2(-/-) macrophages or in macrophages pretreated with the MEK1/2 inhibitor UO126. In addition, activation of PI3K and Akt1 also appeared necessary for the induction of IRAK-M by gAd, because treatment of Akt1(-/-) macrophages or pretreatment of macrophages with the PI3K inhibitor wortmannin abolished gAd-induced IRAK-M expression. Analysis of IRAK-M expression in human peripheral blood cells confirmed that serum adiponectin was negatively associated with IRAK-M and responsiveness to LPS. In conclusion, our data demonstrate that IRAK-M is a major mediator of gAd-induced endotoxin tolerance in primary macrophages, expression of which depends on the activation of Tpl2/ERK and PI3K/Akt1 signaling pathways.

VIP reverses the expression profiling of TLR4-stimulated signaling pathway in rheumatoid arthritis synovial fibroblasts.

Since recent evidences point out the potential involvement of Toll-like receptors (TLRs) in the therapeutic effect of vasoactive intestinal peptide (VIP), the purpose of this study is to elucidate the role of VIP as a negative regulator of TLR-signaling. To this aim, we analyzed in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) or osteoarthritis (OA), the expression profile of TLR-pathway related molecules, as well as the alterations induced by LPS stimulation in RA-FLS and the effect of VIP treatment. Cultured FLS were obtained from patients with RA or OA. RA-FLS were next stimulated with lipopolysaccharide (LPS) in presence or absence of VIP. The gene expression profiling of molecules involved in LPS-mediated TLR4-signaling was studied by cRNA microarray analysis. Twenty three molecules involved in TLR signaling resulted over-expressed at mRNA level in basal RA-FLS compared to OA-FLS. Moreover, in RA-FLS, 23 of the analyzed genes were found to be up-regulated by LPS stimulation whereas 30 were not affected. VIP down-regulated the LPS-induced RNA expression of molecules involved in TLR signaling pathway. Up-regulation of RNA expression of CD14, MD2, TRAM, TRIF, IRAK4, TAB2, TRAF6 and TBK1 was corroborated by RT-PCR as well as the VIP regulatory effect. Increased protein levels of TRAF6, TBK1 and pIRAK1 after exposure to LPS, and the inhibitory effect of VIP, were described by Western blotting. As functional consequences, it was observed the VIP-induced impaired production of IL-6 and RANTES/CCL5 after LPS stimulation. In conclusion, VIP acts as a negative modulator of the TLR4-signaling by overturning the production of several checkpoints molecules of the cascade and thus, widening its potential therapeutic effects.

Vasoactive intestinal peptide suppresses toll-like receptor 4 expression in macrophages via Akt1 reducing their responsiveness to lipopolysaccharide.

Toll-like receptor 4 (TLR4) recognizes and initiates signals from Gram-negative bacterial lipopolysaccharide (LPS) triggering the inflammatory response. Expression levels of TLR4 on macrophages partly regulate the magnitude of the response to LPS. Vasoactive Intestinal Peptide (VIP) is known to block inflammatory responses by inhibiting pro-inflammatory cytokine production from activated macrophages. In the present report we demonstrate that VIP directly suppressed TLR4 expression on naïve primary mouse macrophages utilizing signalling cascades that control TLR4 transcription. VIP-induced suppression of TLR4 occurred at the transcriptional level by decreasing PU.1 DNA binding. Mutation of the proximal PU.1 but not the AP-1-binding site on the TLR4 promoter abrogated VIP-induced suppression of TLR4 transcription. Moreover, inhibition of PI3K by wortmannin or homologous deletion of the Akt1 isoform, a pathway known to act as a negative regulator of macrophage activation, alleviated the suppressive action of VIP on TLR4 expression. To evaluate the biological significance of VIP effect on TLR4 expression, Raw264.7 macrophages were pre-treated with VIP for 24h and then exposed to LPS. Pre-treatment with VIP rendered macrophages hypo-responsive to LPS resulting in reduced pro-inflammatory cytokine production. Moreover, in vivo administration of VIP in C57BL/6 mice resulted in lower IL-6 production upon treatment with LPS. Overall, the results indicate that VIP promotes endotoxin tolerance by downregulating TLR4 expression via Akt1.