A meta-analysis indicates that the regulation of cell motility is a non-intrinsic function of chemoattractant receptors that is governed independently of directional sensing.

Chemoattraction, defined as the migration of a cell toward a source of a chemical gradient, is controlled by chemoattractant receptors. Chemoattraction involves two basic activities, namely, directional sensing, a molecular mechanism that detects the direction of a source of chemoattractant, and actin-based motility, which allows the migration of a cell towards it. Current models assume first, that chemoattractant receptors govern both directional sensing and motility (most commonly inducing an increase in the migratory speed of the cells, i.e. chemokinesis), and, second, that the signaling pathways controlling both activities are intertwined. We performed a meta-analysis to reassess these two points. From this study emerge two main findings. First, although many chemoattractant receptors govern directional sensing, there are also receptors that do not regulate cell motility, suggesting that is the ability to control directional sensing, not motility, that best defines a chemoattractant receptor. Second, multiple experimental data suggest that receptor-controlled directional sensing and motility can be controlled independently. We hypothesize that this independence may be based on the existence of separated signalling modules that selectively govern directional sensing and motility in chemotactic cells. Together, the information gathered can be useful to update current models representing the signalling from chemoattractant receptors. The new models may facilitate the development of strategies for a more effective pharmacological modulation of chemoattractant receptor-controlled chemoattraction in health and disease.

The Actin Cytoskeleton at the Immunological Synapse of Dendritic Cells.

Dendritic cells (DCs) are considered the most potent antigen-presenting cells. DCs control the activation of T cells (TCs) in the lymph nodes. This process involves forming a specialized superstructure at the DC-TC contact zone called the immunological synapse (IS). For the sake of clarity, we call IS(DC) and IS(TC) the DC and TC sides of the IS, respectively. The IS(DC) and IS(TC) seem to organize as multicentric signaling hubs consisting of surface proteins, including adhesion and costimulatory molecules, associated with cytoplasmic components, which comprise cytoskeletal proteins and signaling molecules. Most of the studies on the IS have focused on the IS(TC), and the information on the IS(DC) is still sparse. However, the data available suggest that both IS sides are involved in the control of TC activation. The IS(DC) may govern activities of DCs that confer them the ability to activate the TCs. One key component of the IS(DC) is the actin cytoskeleton. Herein, we discuss experimental data that support the concept that actin polarized at the IS(DC) is essential to maintaining IS stability necessary to induce TC activation.

The Chemokine Receptor CCR7 Uses Distinct Signaling Modules With Biased Functionality to Regulate Dendritic Cells.

Chemotaxis is a molecular mechanism that confers leukocytes the ability to detect gradients of chemoattractants. Chemokine receptors are well-known regulators of chemotaxis in leukocytes; however, they can regulate several other activities in these cells. This information has been often neglected, probably due to the paramount role of chemotaxis in the immune system and in biology. Therefore, the experimental data available on the mechanisms used by chemokine receptors to regulate other functions of leukocytes is sparse. The results obtained in the study of the chemokine receptor CCR7 in dendritic cells (DCs) provide interesting information on this issue. CCR7 guides the DCs from the peripheral tissues to the lymph nodes, where these cells control T cell activation. CCR7 can regulate DC chemotaxis, survival, migratory speed, cytoarchitecture, and endocytosis. Biochemical and functional analyses show: first, that CCR7 uses in DCs the PI3K/Akt pathway to control survival, the MAPK pathway to control chemotaxis, and the RhoA pathways to regulate actin dynamics, which in turn controls migratory speed, cytoarchitecture, and endocytosis; second, that these three signaling pathways behave as modules with a high degree of independence; and third, that although each one of these routes can regulate several functions in different settings, CCR7 promotes in DCs a functional bias in each pathway. The data uncover an interesting mechanism used by CCR7 to regulate the DCs, entailing multifunctional signaling pathways organized in modules with biased functionality. A similar mechanism could be used by other chemoattractant receptors to regulate the functions of leukocytes.

Fully endoscopic transoral resection of high cervical osteophyte. How I do it?

BACKGROUND: Fully endoscopic transoral approaches (FETOA) constitute a reasonable option for the treatment of middling compressive pathology that involve the craniocervical junction and higher cervical levels. METHODS: We describe, step by step, the FETOA for the treatment of upper cervical lesions. More specifically, the ones that are located between C1 and C3. A giant anterior C1-C2 osteophyte resection will be used as an illustration of these approaches. CONCLUSIONS: This technique represents a minimally invasive treatment option for these kinds of high cervical lesions. It offers optimal visualization, maximizing the resection of these lesions and decreasing the morbidity and mortality.

Immunological Synapse Formation Induces Mitochondrial Clustering and Mitophagy in Dendritic Cells.

The immunological synapse (IS) is a superstructure formed during T cell activation at the zone of contact between T cells and dendritic cells (DCs). The IS includes specific molecular components in the T cell and DCs sides that may result in different functionality. Most of the studies on the IS have focused on the T cell side of this structure and, in contrast, the information available on the IS of DCs is sparse. Autophagy is a cellular process involved in the clearance of damaged proteins and organelles via lysosomal degradation. Mitophagy is the selective autophagy of damaged mitochondria. In this study, it is shown that IS formation induces clustering of mitochondria in the IS of DCs and partial depolarization of these organelles. At the IS of the DCs also accumulate autophagy and mitophagy markers, even when the kinase complex mTORC1, an inhibitor of the autophagy, is active. Together the results presented indicate that IS formation induces local clustering of mitochondria and mitophagy, which could be a homeostatic mechanism to control the quality of mitochondria in this region. The data underline the complexity of the regulatory mechanisms operating in the IS of DCs.

Macrophage-specific MHCII expression is regulated by a remote Ciita enhancer controlled by NFAT5.

MHCII in antigen-presenting cells (APCs) is a key regulator of adaptive immune responses. Expression of MHCII genes is controlled by the transcription coactivator CIITA, itself regulated through cell type-specific promoters. Here we show that the transcription factor NFAT5 is needed for expression of Ciita and MHCII in macrophages, but not in dendritic cells and other APCs. NFAT5-deficient macrophages showed defective activation of MHCII-dependent responses in CD4+ T lymphocytes and attenuated capacity to elicit graft rejection in vivo. Ultrasequencing analysis of NFAT5-immunoprecipitated chromatin uncovered an NFAT5-regulated region distally upstream of Ciita This region was required for CIITA and hence MHCII expression, exhibited NFAT5-dependent characteristics of active enhancers such as H3K27 acetylation marks, and required NFAT5 to interact with Ciita myeloid promoter I. Our results uncover an NFAT5-regulated mechanism that maintains CIITA and MHCII expression in macrophages and thus modulates their T lymphocyte priming capacity.

Remodeling our concept of chemokine receptor function: From monomers to oligomers.

The chemokines direct leukocyte recruitment in both homeostatic and inflammatory conditions, and are therefore critical for immune reactions. By binding to members of the class A G protein-coupled receptors, the chemokines play an essential role in numerous physiological and pathological processes. In the last quarter century, the field has accumulated much information regarding the implications of these molecules in different immune processes, as well as mechanistic insight into the signaling events activated through their binding to their receptors. Here, we will focus on chemokine receptors and how new methodological approaches have underscored the role of their conformations in chemokine functions. Advances in biophysical-based techniques show that chemokines and their receptors act in very complex networks and therefore should not be considered isolated entities. In this regard, the chemokine receptors can form homo- and heterodimers as well as oligomers at the cell surface. These findings are changing our view as to how chemokines influence cell biology, identify partners that regulate chemokine function, and open new avenues for therapeutic intervention.

Liver X Receptor Nuclear Receptors Are Transcriptional Regulators of Dendritic Cell Chemotaxis.

The liver X receptors (LXRs) are ligand-activated nuclear receptors with established roles in the maintenance of lipid homeostasis in multiple tissues. LXRs exert additional biological functions as negative regulators of inflammation, particularly in macrophages. However, the transcriptional responses controlled by LXRs in other myeloid cells, such as dendritic cells (DCs), are still poorly understood. Here we used gain- and loss-of-function models to characterize the impact of LXR deficiency on DC activation programs. Our results identified an LXR-dependent pathway that is important for DC chemotaxis. LXR-deficient mature DCs are defective in stimulus-induced migration in vitro and in vivo Mechanistically, we show that LXRs facilitate DC chemotactic signaling by regulating the expression of CD38, an ectoenzyme important for leukocyte trafficking. Pharmacological or genetic inactivation of CD38 activity abolished the LXR-dependent induction of DC chemotaxis. Using the low-density lipoprotein receptor-deficient (LDLR-/-) LDLR-/- mouse model of atherosclerosis, we also demonstrated that hematopoietic CD38 expression is important for the accumulation of lipid-laden myeloid cells in lesions, suggesting that CD38 is a key factor in leukocyte migration during atherogenesis. Collectively, our results demonstrate that LXRs are required for the efficient emigration of DCs in response to chemotactic signals during inflammation.

Beyond Chemoattraction: Multifunctionality of Chemokine Receptors in Leukocytes.

The word chemokine is a combination of the words chemotactic and cytokine, in other words cytokines that promote chemotaxis. Hence, the term chemokine receptor refers largely to the ability to regulate chemoattraction. However, these receptors can modulate additional leukocyte functions, as exemplified by the case of CCR7 which, apart from chemotaxis, regulates survival, migratory speed, endocytosis, differentiation and cytoarchitecture. We present evidence highlighting that multifunctionality is a common feature of chemokine receptors. Based on the activities that they regulate, we suggest that chemokine receptors can be classified into inflammatory (which control both inflammatory and homeostatic functions) and homeostatic families. The information accrued also suggests that the non-chemotactic functions controlled by chemokine receptors may contribute to optimizing leukocyte functioning under normal physiological conditions and during inflammation.

The Mammalian Sterile 20-like 1 Kinase Controls Selective CCR7-Dependent Functions in Human Dendritic Cells.

The chemokine receptor CCR7 directs mature dendritic cells (mDCs) to the lymph nodes where these cells control the initiation of the immune response. CCR7 regulates chemotaxis, endocytosis, survival, migratory speed, and cytoarchitecture in mDCs. The molecular mechanisms used by CCR7 to regulate these functions in mDCs are not completely understood. The mammalian sterile 20-like 1 kinase (Mst1) plays a proapoptotic role under stress conditions; however, recently, it has been shown that Mst1 can also control homeostatic cell functions under normal conditions. In this study, we show that stimulation of CCR7 in mDCs induces Gαi-dependent activation of Mst1, suggesting the involvement of this kinase in the control of CCR7-dependent functions. Analysis of the mDCs in which Mst1 expression levels were reduced with small interfering RNA shows that this kinase mediates CCR7-dependent effects on cytoarchitecture, endocytosis and migratory speed but not on chemotaxis or survival. In line with these results, biochemical analysis indicates that Mst1 does not control key signaling regulators of CCR7-dependent chemotaxis or survival. In contrast, Mst1 regulates downstream of CCR7 and, of note, independently of Gα13, the RhoA pathway. Reduction of Mst1 inhibits CCR7-dependent phosphorylation of downstream targets of RhoA, including cofilin, myosin L chain, and myosin L chain phosphatase. Consistent with the role of the latter molecules as modulators of the actin cytoskeleton, mDCs with reduced Mst1 also displayed a dramatic reduction in actin barbed-end formation that could not be recovered by stimulating CCR7. The results indicate that the kinase Mst1 controls selective CCR7-dependent functions in human mDCs.

Plasma membrane-associated superstructure: Have we overlooked a new type of organelle in eukaryotic cells?

A variety of intriguing plasma membrane-associated regions, including focal adhesions, adherens junctions, tight junctions, immunological synapses, neuromuscular junctions and the primary cilia, among many others, have been described in eukaryotic cells. Emphasizing their importance, alteration in their molecular structures induces or correlates with different pathologies. These regions display surface proteins connected to intracellular molecules, including cytoskeletal component, which maintain their cytoarchitecture, and signalling proteins, which regulate their organization and functions. Based on the molecular similarities and other common features observed, we suggest that, despite differences in external appearances, all these regions are just the same superstructure that appears in different locations and cells. We hypothesize that this superstructure represents an overlooked new type of organelle that we call plasma membrane-associated superstructure (PMAS). Therefore, we suggest that eukaryotic cells include classical organelles (e.g. mitochondria, Golgi and others) and also PMAS. We speculate that this new type of organelle might be an innovation associated to the emergence of eukaryotes. Finally we discuss the implications of the hypothesis proposed.

CXCL12 Regulates through JAK1 and JAK2 Formation of Productive Immunological Synapses.

The adaptive immune response requires interaction between T cells and APC to form a specialized structure termed the immune synapse (IS). Although the TCR is essential for IS organization, other factors such as chemokines participate in this process. In this study, we show that the chemokine CXCL12-mediated signaling contributes to correct IS organization and therefore influences T cell activation. CXCR4 downregulation or blockade on T cells caused defective actin polymerization at the contact site with APC, altered microtubule-organizing center polarization and the IS structure, and reduced T cell/APC contact duration. T cell activation was thus inhibited, as shown by reduced expression of CD25 and CD69 markers and of IL-2 mRNA levels. The results indicate that, through Gi and JAK1 and 2 kinases activation, CXCL12 signaling cooperates to build the IS and to maintain adhesive contacts between APC and T cells, required for continuous TCR signaling.

A novel MEK-ERK-AMPK signaling axis controls chemokine receptor CCR7-dependent survival in human mature dendritic cells.

Chemokine receptor CCR7 directs mature dendritic cells (mDCs) to secondary lymph nodes where these cells regulate the activation of T cells. CCR7 also promotes survival in mDCs, which is believed to take place largely through Akt-dependent signaling mechanisms. We have analyzed the involvement of the AMP-dependent kinase (AMPK) in the control of CCR7-dependent survival. A pro-apoptotic role for AMPK is suggested by the finding that pharmacological activators induce apoptosis, whereas knocking down of AMPK with siRNA extends mDC survival. Pharmacological activation of AMPK also induces apoptosis of mDCs in the lymph nodes. Stimulation of CCR7 leads to inhibition of AMPK, through phosphorylation of Ser-485, which was mediated by G(i)/Gßγ, but not by Akt or S6K, two kinases that control the phosphorylation of AMPK on Ser-485 in other settings. Using selective pharmacological inhibitors, we show that CCR7-induced phosphorylation of AMPK on Ser-485 is mediated by MEK and ERK. Coimmunoprecipitation analysis and proximity ligation assays indicate that AMPK associates with ERK, but not with MEK. These results suggest that in addition to Akt-dependent signaling mechanisms, CCR7 can also promote survival of mDCs through a novel MEK1/2-ERK1/2-AMPK signaling axis. The data also suggest that AMPK may be a potential target to modulate mDC lifespan and the immune response.

Detecting apoptosis of leukocytes in mouse lymph nodes.

Although there are multiple methods for analyzing apoptosis in cultured cells, methodologies for analyzing apoptosis in vivo are sparse. In this protocol, we describe how to detect apoptosis of leukocytes in mouse lymph nodes (LNs) via the detection of apoptotic caspases. We have previously used this protocol to study factors that modulate dendritic cell (DC) survival in LNs; however, it can also be used to analyze other leukocytes that migrate to the LNs. DCs labeled with a fluorescent cell tracker are subcutaneously injected in the posterior footpads of mice. Once the labeled DCs reach the popliteal LN (PLN), the animals are intravenously injected with FLIVO, a permeant fluorescent reagent that selectively marks active caspases and consequently apoptotic cells. Explanted PLNs are then examined under a two-photon microscope to look for the presence of apoptotic cells among the DCs injected. The protocol requires 6-6.5 h for preparation and analysis plus an additional 34-40 h to allow apoptosis of the injected DCs in the PLN.

Antigen presentation by dendritic cells in rheumatoid arthritis.

Rheumatoid Arthritis (RA) is a chronic autoimmune inflammatory disease that affects largely synovial joints. It has been postulated that activated autoreactive CD4 T cells play a key role in triggering and/or maintaining the chronic inflammatory process in RA. Dendritic cells (DCs) are antigen-presenting cells that activate cognate clonal CD4 T cells in the lymph nodes. The activation process involves the formation of a molecular structure at the DC-CD4 T cell contact zone called immunological synapse (IS). In RA, the synovium, a thin layer of tissue below the capsule in the joints, shows a massive infiltration of DCs and CD4 T cells. Subjects bearing HLA-DRB1 alleles of the Major Histocompatibility Complex II gene displaying a motif called RA "shared epitope (SE)", have an enhanced susceptibility to suffer RA. Interestingly, the SE-containing HLA-DRB1 molecules display a pocket with a high affinity for citrullinated antigens, which are found at higher levels in subjects prone to develop RA. Thus, it is possible that the DCs of susceptible individuals may form IS with particular features that may present citrullinated peptides to autorreactive naïve CD4 T clones that, after being activated, contribute to the initiation or development of the disease. Herein I put forward a model of RA initiation based on current information on the immune response and RA.

Aryl hydrocarbon receptor contributes to the MEK/ERK-dependent maintenance of the immature state of human dendritic cells.

Dendritic cells (DCs) promote tolerance or immunity depending on their maturation state, which is enhanced or accelerated upon MEK-ERK signaling pathway inhibition. We have determined the contribution of MEK-ERK activation to the profile of gene expression of human immature monocyte-derived dendritic cells (MDDCs) and peripheral blood myeloid DCs. ERK inhibition altered the expression of genes that mediate Chemokine (C-C motif) ligand 19 (CCL19)-directed migration (CCR7) and low-density lipoprotein (LDL) binding (CD36, SCARB1, OLR1, CXCL16) by immature DCs. In addition, ERK upregulated CCL2 expression while impairing the expression of DC maturation markers (RUNX3, ITGB7, IDO1). MEK-ERK-regulated genes exhibited an overrepresentation of cognate sequences for the aryl hydrocarbon receptor (AhR) transcription factor, whose transcriptional and DNA-binding activities increased in MDDCs upon exposure to the MEK1/2 inhibitor U0126. Therefore, the MEK-ERK signaling pathway regulates antigen capture, lymph node homing, and acquisition of maturation-associated genes, and its contribution to the maintenance of the immature state of MDDCs and myeloid DCs is partly dependent on the activity of AhR. Since pharmacologic modulation of the MEK-ERK signaling pathway has been proposed as a potential therapeutic strategy for cancer, our findings indicate that ERK inhibitors might influence antitumor responses through regulation of critical DC effector functions.

Chemokine CXCL12 uses CXCR4 and a signaling core formed by bifunctional Akt, extracellular signal-regulated kinase (ERK)1/2, and mammalian target of rapamycin complex 1 (mTORC1) proteins to control chemotaxis and survival simultaneously in mature dendritic cells.

Chemokines control several cell functions in addition to chemotaxis. Although much information is available on the involvement of specific signaling molecules in the control of single functions controlled by chemokines, especially chemotaxis, the mechanisms used by these ligands to regulate several cell functions simultaneously are completely unknown. Mature dendritic cells (maDCs) migrate through the afferent lymphatic vessels to the lymph nodes, where they regulate the initiation of the immune response. As maDCs are exposed to chemokine CXCL12 (receptors CXCR4 and CXCR7) during their migration, its functions are amenable to be regulated by this ligand. We have used maDCs as a model system to analyze the mechanisms whereby CXCL12 simultaneously controls chemotaxis and survival in maDCs. We show that CXCL12 uses CXCR4, but not CXCR7, and the components of a signaling core that includes G(i)/Gßγ, PI3K-α/-δ/-γ, Akt, ERK1/2 and mammalian target of rapamycin complex 1 (mTORC1), which organize hierarchically to control both functions. Downstream of Akt, Forkhead box class O (FOXO) regulates CXCL12-dependent survival, but not chemotaxis, suggesting that downstream of the aforementioned signaling core, additional signaling molecules may control more selectively CXCL12-dependent chemotaxis or survival. Finally, the data obtained also show that CXCR4 uses a signaling signature that is different from that used by CCR7 to control similar functions.

The neuronal protein Kidins220/ARMS associates with ICAM-3 and other uropod components and regulates T-cell motility.

Kinase D interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is a protein that is mainly expressed in brain and neural cells where its function is only starting to be characterized. Here, we show that Kidins220/ARMS is also expressed in T lymphocytes where it is highly concentrated at the uropod of polarized T cells. In this cellular model, Kidins220/ARMS colocalizes with typical uropod T-cell molecules and coimmunoprecipitates with ICAM-3. Furthermore, Kidins220/ARMS associates with raft domains at the uropod and coimmunoprecipitates with caveolin-1, a molecule we show here to be also expressed in T cells. Importantly, induction of morphological polarization in primary T lymphocytes and Jurkat cells enhances Kidins220/ARMS colocalization with ICAM-3. Conversely, disruption of cell polarity provokes Kidins220/ARMS redistribution from the uropod to other cellular regions and drastically impairs its association with ICAM-3 in a protein kinase C-dependent manner. Finally, Kidins220/ARMS knockdown in human polarized T-cell lines promotes both basal and stromal cell-derived factor-1α-induced directed migration, identifying a novel function for this molecule. Altogether, our findings show that Kidins220/ARMS is a novel component of the uropod involved in the regulation of T-cell motility, an essential process for the immune response.

CD69 modulates sphingosine-1-phosphate-induced migration of skin dendritic cells.

In this study, we have investigated the role of CD69, an early inducible leukocyte activation receptor, in murine dendritic cell (DC) differentiation, maturation, and migration. Skin DCs and DC subsets present in mouse lymphoid organs express CD69 in response to maturation stimuli. Using a contact sensitization model, we show that skin DCs migrated more efficiently to draining lymph nodes (LNs) in the absence of CD69. This was confirmed by subcutaneous transfer of CD69-/- DCs, which presented an increased migration to peripheral LNs. Two-photon microscopy analysis showed that once DCs reached the LNs, CD69 deficiency did not alter DC interstitial motility in the LNs. Chemotaxis to sphingosine-1-phosphate (S1P) was enhanced in CD69-/- DCs compared with wild-type DCs. Accordingly, we detected a higher expression of S1P receptor type-1 (S1P(1)) by CD69-/- DCs, whereas S1P(3) expression levels were similar in wild-type and CD69-/- DCs. Moreover, in vivo treatment with S1P analogs SEW2871 and FTY720 during skin sensitization reduced skin DC migration to peripheral LNs. These results suggest that CD69 regulates S1P-induced skin DC migration by modulating S1P(1) function. Together, our findings increase our knowledge on DC trafficking patterns in the skin, enabling the development of new directed therapies using DCs for antigen (Ag) delivery.