Alfonso Luque

CCR7 Is Mainly Expressed in Teleost Gills, Where It Defines an IgD+IgM− B Lymphocyte Subset

Chemokine receptor CCR7, the receptor for both CCL19 and CCL21 chemokines, regulates the recruitment and clustering of circulating leukocytes to secondary lymphoid tissues, such as lymph nodes and Peyers patches. Even though teleost fish do not have either of these secondary lymphoid structures, we have recently reported a homolog to CCR7 in rainbow trout (Oncorhynchus mykiss). In the present work, we have studied the distribution of leukocytes bearing extracellular CCR7 in naive adult tissues by flow cytometry, observing that among the different leukocyte populations, the highest numbers of cells with membrane (mem)CCR7 were recorded in the gill (7.5 ± 2% CCR7+ cells). In comparison, head kidney, spleen, thymus, intestine, and peripheral blood possessed <5% CCR7+ cells. When CCR7 was studied at early developmental stages, we detected a progressive increase in gene expression and protein CCR7 levels in the gills throughout development. Surprisingly, the majority of the CCR7+ cells in the gills were not myeloid cells and did not express membrane CD8, IgM, nor IgT, but expressed IgD on the cell surface. In fact, most IgD+ cells in the gills expressed CCR7. Intriguingly, the IgD+CCR7+ population did not coexpress memIgM. Finally, when trout were bath challenged with viral hemorrhagic septicemia virus, the number of CCR7+ cells significantly decreased in the gills while significantly increased in head kidney. These results provide evidence of the presence of a novel memIgD+memIgM− B lymphocyte subset in trout that expresses memCCR7 and responds to viral infections. Similarities with IgD+IgM− subsets in mammals are discussed.

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 diseases.

Macrophages, Inflammation, and Tumor Suppressors: ARF, a New Player in the Game

The interaction between tumor progression and innate immune system has been well established in the last years. Indeed, several lines of clinical evidence indicate that immune cells such as tumor-associated macrophages (TAMs) interact with tumor cells, favoring growth, angiogenesis, and metastasis of a variety of cancers. In most tumors, TAMs show properties of an alternative polarization phenotype (M2) characterized by the expression of a series of chemokines, cytokines, and proteases that promote immunosuppression, tumor proliferation, and spreading of the cancer cells. Tumor suppressor genes have been traditionally linked to the regulation of cancer progression; however, a growing body of evidence indicates that these genes also play essential roles in the regulation of innate immunity pathways through molecular mechanisms that are still poorly understood. In this paper, we provide an overview of the immunobiology of TAMs as well as what is known about tumor suppressors in the context of immune responses. Recent advances regarding the role of the tumor suppressor ARF as a regulator of inflammation and macrophage polarization are also reviewed.

Transcriptional heterogeneity of IgM+ cells in rainbow trout (Oncorhynchus mykiss) tissues.

Two major classes of B lymphocytes have been described to date in rainbow trout: IgM+ and IgT+ cells. IgM+ cells are mainly localized in the spleen, peripheral blood and kidney but are also found in other tissues. However, differences among IgM+ cell populations attending to its location are poorly defined in fish. Thus, the aim of this work was to characterize the expression of different immune molecules such as chemokine receptors, Toll-like receptors (TLRs) and transcription factors on sorted IgM+ lymphocytes from different rainbow trout tissues. IgM+ populations from blood, spleen, kidney, gills, intestine and liver were isolated by cell sorting and the constitutive levels of transcription of these genes evaluated by real-time PCR. To further characterize B cells, we identified an MS4A sequence. In humans, the MS4A family includes several genes with immune functions, such as the B cell marker CD20 or FcRβ. Subsequently, we have also evaluated the mRNA levels of this MS4A gene in the different IgM+ populations. The relevant differences in transcriptional patterns observed for each of these IgM+ populations analyzed, point to the presence of functionally different tissue-specific B cell populations in rainbow trout. The data shown provides a pattern of genes transcribed in IgM+ B cells not previously revealed in teleost fish. Furthermore, the constitutive expression of all the TLR genes analyzed in IgM+ cells suggests an important role for these cells in innate immunity.

Molecular characterization of three novel chemokine receptors in rainbow trout (Oncorhynchus mykiss).

Chemokines signal through a family of seven-transmembrane domain G-coupled receptors in order to regulate both leukocyte mobilization and activate the recruited cells. Although many chemokines have been identified in rainbow trout (Oncorhynchus mykiss), only a few chemokine receptors have been reported to date. In this work, we have cloned three novel chemokine receptors in rainbow trout. One of these receptors seems to be a clear orthologue of CCR6, while the second one constitutes a novel CCR9 gene different from the previous CCR9 reported in this species. This gene, which we have designated as CCR9B, represents another lineage of fish CCR9 genes, not previously identified. Finally, a deeper phylogenetic analysis of the third novel chemokine receptor gene, which had been identified on the basis of sequence similarity to CCR3, constitutes a novel lineage of CCR receptors which has no equivalent in humans and that may be teleost-specific. We have designated this novel gene as CCR13, to avoid any possible ascription to mammalian genes. Further transcriptional studies revealed that CCR6 was constitutively transcribed in thymus, gills, hindgut and peripheral blood leukocytes (PBLs), while CCR9B was strongly transcribed in thymus and PBLs but also in spleen, gills, hindgut and brain at lower levels. CCR13, on the other hand, was strongly detected in spleen, head kidney and PBLs and faintly in thymus, gills, brain and gonad. The data provided constitutes a step forward the identification of novel chemokine receptors that may contribute to a future understanding of chemokine signalling in fish.

The Tumor Suppressor ARF Regulates Innate Immune Responses in Mice

The innate immune system is the first line of defense against invading organisms, and TLRs are the main sensors of microbial components, initiating signaling pathways that induce the production of proinflammatory cytokines and type I IFNs. An antiviral action for the tumor suppressor alternative reading frame (ARF) has been reported; however, the precise role of ARF in innate immunity is unknown. In this study, we show that ARF plays an important role in regulation of inflammatory responses. In peritoneal macrophages and bone marrow-derived macrophages from ARF-deficient animals, the induction of proinflammatory cytokines and chemokines by TLR ligands was severely impaired. The altered responses of ARF−/− cells to TLR ligands result from aberrant activation of intracellular signaling molecules including MAPKs, IκBα degradation, and NF-κB activation. Additionally, animals lacking ARF were resistant to LPS-induced endotoxic shock. This impaired activation of inflammation in ARF−/− mice was not restricted to TLRs, as it was also shown in response to non-TLR signaling pathways. Thus, ARF−/− mice were also unable to trigger a proper inflammatory response in experimental peritonitis or in 12-O-tetradecanoylphorbol-13-acetate–induced edema. Overexpression of ARF, but not its downstream target p53, rescued the ARF-deficient phenotype, increasing TLR4 levels and restoring inflammatory reaction. An increase in the E2F1 protein levels observed in ARF−/− macrophages at basal condition and after LPS stimulation may be involved in the impaired response in this system, as E2F1 has been described as an inflammatory suppressor. These results indicate that tumor suppressor ARF is a new regulator of inflammatory cell signaling.

Selective Impairment of P2Y Signaling by Prostaglandin E2 in Macrophages: Implications for Ca2+-Dependent Responses

Extracellular nucleotides have been recognized as important modulators of inflammation via their action on specific pyrimidine receptors (P2). This regulation coexists with the temporal framework of proinflammatory and proresolution mediators released by the cells involved in the inflammatory response, including macrophages. Under proinflammatory conditions, the expression of cyclooxygenase-2 leads to the release of large amounts of PGs, such as PGE2, that exert their effects through EP receptors and other intracellular targets. The effect of these PGs on P2 receptors expressed in murine and human macrophages was investigated. In thioglycollate-elicited and alternatively activated macrophages, PGE2 selectively impairs P2Y but not P2X7 Ca2+ mobilization. This effect is absent in LPS-activated cells and is specific for PGE2 because it cannot be reproduced by other PGs with cyclopentenone structure. The inhibition of P2Y responses by PGE2 involves the activation of nPKCs (PKCε) and PKD that can be abrogated by selective inhibitors or by expression of dominant-negative forms of PKD. The inhibition of P2Y signaling by PGE2 has an impact on the cell migration elicited by P2Y agonists in thioglycollate-elicited and alternatively activated macrophages, which provide new clues to understand the resolution phase of inflammation, when accumulation of PGE2, anti-inflammatory and proresolving mediators occurs.

Regulation of endothelial dynamics by PGC-1α relies on ROS control of VEGF-A signaling

UNLABELLED Peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α) is a regulator of mitochondrial metabolism and reactive oxygen species (ROS) that is known to play a relevant role in angiogenesis. AIMS This study aims to investigate the role of ROS on the regulation by PGC-1α of angiogenesis. METHODS AND RESULTS We found that endothelial cells (ECs) from mice deleted for PGC-1α display attenuated adhesion to the extracellular matrix, together with slower and reversible spreading. Structural analysis demonstrates unstable formation of focal adhesions, defective cytoskeleton reorganization in response to cellular matrix adhesion, cell migration and cell-cell adhesion. Confluent cultures showed also a reduction of membrane bound VE-cadherin, suggesting defective inter-cellular junction formation. Functional consequences included impaired directional migration, and enhanced tip phenotype in aortic explants sprouting assays. At the molecular level, PGC-1α-deleted ECs exhibit a constitutive activation of the vascular endothelial growth factor-A (VEGF-A) signaling pathway and a defective response to VEGF-A. All these alterations are partially reversed by administration of the antioxidant EUK-189. The contribution of mitochondrial ROS and NOX activation was confirmed using a mitochondrial targeted antioxidant (MitoTEMPO) and a NOX inhibitor (VAS-2870). These results indicate that elevated production of ROS in the absence of PGC-1α is a key factor in the alteration of the VEGF-A signaling pathway and the capacity of endothelial cells to form stable interactions with other endothelial cells and with the extracellular matrix. Our findings show that PGC-1α control of ROS homeostasis plays an important role in the control of endothelial response to VEGF-A.

Chemokines and relapses in childhood acute lymphoblastic leukemia: A role in migration and in resistance to antileukemic drugs

We studied whether chemokines may have a role in relapses in childhood acute lymphoblastic leukemia (ALL). We compared the levels of chemokine receptors in marrow samples from 82 children with ALL at diagnosis versus 15 at relapses, and quantified the levels of chemokines in central system fluid (CSF) samples. The functional role of specific chemokines was studied in vitro and in vivo. The expression of some chemokine receptors was upregulated upon leukemic relapse, both in B- and in T-ALL, and in cases of medullary and extramedullary involvement. CXCL10 induced chemotaxis in leukemic cell lines and in primary leukemic cells, depending upon the levels of CXCR3 expression. CXCL10 specifically diminished chemotherapy-induced apoptosis on ALL cells expressing CXCR3, partially inhibiting caspase activation and maintaining the levels of the antiapoptotic protein Bcl-2. Finally, immunodeficient mice engrafted with CXCR3-expressing human leukemic cells showed decreased infiltration of marrow, spleen, and CNS after receiving a CXCR3-antagonist molecule. CXCR3 signaling in ALL may have a dual function: chemotactic for the localisation of leukemic blasts in specific niches, and it may also confer resistance to chemotherapy, enhancing the chances for relapses.

A Role for the CXCR3/CXCL10 Axis in Rasmussen Encephalitis

BACKGROUND Rasmussen encephalitis is a devastating pediatric syndrome of unknown etiology that is characterized by progressive loss of neurological function and intractable focal epilepsy. Cytotoxic T lymphocytes have an active role in the pathogenic process of Rasmussen encephalitis. We studied the implication of CXCL10-CXCR3, a chemotactic axis involved in the pathogenesis of several cases of immune encephalitis. METHODS We analyzed surgical specimens of children with Rasmussen encephalitis, and performed functional in vitro assays to test the implications of the pathological findings. RESULTS We found that cytotoxic T lymphocytes infiltrating the damaged areas of primary biopsies expressed CXCR3, whereas neurons and astrocytes in the same areas expressed CXCL10. The in vitro assays demonstrated we found that astrocytes upregulated the expression of CXCL10 messenger RNA and the release of CXCL10 to the supernatants on stimulation with polyinosinic-polycyticylic acid, a synthetic double-stranded RNA that mimics infections with either RNA or DNA viruses. Activated T lymphocytes responded to the production of CXCL10 by astrocytes by increasing their migration in a transwell assay. Finally, the chemotaxis induced by the stimulated astrocytes was completely abrogated in the presence of a small molecule antagonist of CXCR3. CONCLUSIONS Our results suggest that the CXCR3-CXCL10 axis has a role in recruiting pathogenic T lymphocytes into the brains of patients with Rasmussen encephalitis. This chemotactic mechanism may be targeted pharmacologically.