José María González-Granado

Miro-1 links mitochondria and microtubule dynein motors to control lymphocyte migration and polarity

ABSTRACT The recruitment of leukocytes to sites of inflammation is crucial for a functional immune response. In the present work, we explored the role of mitochondria in lymphocyte adhesion, polarity, and migration. We show that during adhesion to the activated endothelium under physiological flow conditions, lymphocyte mitochondria redistribute to the adhesion zone together with the microtubule-organizing center (MTOC) in an integrin-dependent manner. Mitochondrial redistribution and efficient lymphocyte adhesion to the endothelium require the function of Miro-1, an adaptor molecule that couples mitochondria to microtubules. Our data demonstrate that Miro-1 associates with the dynein complex. Moreover, mitochondria accumulate around the MTOC in response to the chemokine CXCL12/SDF-1α; this redistribution is regulated by Miro-1. CXCL12-dependent cell polarization and migration are reduced in Miro-1-silenced cells, due to impaired myosin II activation at the cell uropod and diminished actin polymerization. These data point to a key role of Miro-1 in the control of lymphocyte adhesion and migration through the regulation of mitochondrial redistribution.

High-Resolution Imaging of Intravascular Atherogenic Inflammation in Live Mice

Rationale: The inflammatory processes that initiate and propagate atherosclerosis remain poorly understood, largely because defining the intravascular behavior of immune cells has been technically challenging. Respiratory and pulsatile movements have hampered in vivo visualization of leukocyte accumulation in athero-prone arteries at resolutions achieved in other tissues. Objective: To establish and to validate a method that allows high-resolution imaging of inflammatory leukocytes and platelets within the carotid artery of atherosusceptible mice in vivo. Methods and Results: We have devised a procedure to stabilize the mouse carotid artery mechanically without altering blood dynamics, which dramatically enhances temporal and spatial resolutions using high-speed intravital microscopy in multiple channels of fluorescence. By applying this methodology at different stages of disease progression in atherosusceptible mice, we first validated our approach by assessing the recruitment kinetics of various leukocyte subsets and platelets in athero-prone segments of the carotid artery. The high temporal and spatial resolution allowed the dissection of both the dynamic polarization of and the formation of subcellular domains within adhered leukocytes. We further demonstrate that the secondary capture of activated platelets on the plaque is predominantly mediated by neutrophils. Finally, we couple this procedure with triggered 2-photon microscopy to visualize the 3-dimensional movement of leukocytes in intimate contact with the arterial lumen. Conclusions: The improved imaging of diseased arteries at subcellular resolution presented here should help resolve many outstanding questions in atherosclerosis and other arterial disorders.

CD81 Controls Sustained T Cell Activation Signaling and Defines the Maturation Stages of Cognate Immunological Synapses

ABSTRACT In this study, we investigated the dynamics of the molecular interactions of tetraspanin CD81 in T lymphocytes, and we show that CD81 controls the organization of the immune synapse (IS) and T cell activation. Using quantitative microscopy, including fluorescence recovery after photobleaching (FRAP), phasor fluorescence lifetime imaging microscopy-Föster resonance energy transfer (phasorFLIM-FRET), and total internal reflection fluorescence microscopy (TIRFM), we demonstrate that CD81 interacts with ICAM-1 and CD3 during conjugation between T cells and antigen-presenting cells (APCs). CD81 and ICAM-1 exhibit distinct mobilities in central and peripheral areas of early and late T cell-APC contacts. Moreover, CD81–ICAM-1 and CD81-CD3 dynamic interactions increase over the time course of IS formation, as these molecules redistribute throughout the contact area. Therefore, CD81 associations unexpectedly define novel sequential steps of IS maturation. Our results indicate that CD81 controls the temporal progression of the IS and the permanence of CD3 in the membrane contact area, contributing to sustained T cell receptor (TCR)-CD3-mediated signaling. Accordingly, we find that CD81 is required for proper T cell activation, regulating CD3ζ, ZAP-70, LAT, and extracellular signal-regulated kinase (ERK) phosphorylation; CD69 surface expression; and interleukin-2 (IL-2) secretion. Our data demonstrate the important role of CD81 in the molecular organization and dynamics of the IS architecture that sets the signaling threshold in T cell activation.

Embryological-Origin–Dependent Differences in Homeobox Expression in Adult Aorta Role in Regional Phenotypic Variability and Regulation of NF-κB Activity

Objective—Different vascular beds show differing susceptibility to the development of atherosclerosis, but the molecular mechanisms underlying these differences are incompletely understood. This study aims to identify factors that contribute to the phenotypic heterogeneity of distinct regions of the adult vasculature. Approach and Results—High-throughput mRNA profiling in adult mice reveals higher expression of the homeobox paralogous genes 6 to 10 (Hox6-10) in the athero-resistant thoracic aorta (TA) than in the athero-susceptible aortic arch (AA). Higher homeobox gene expression also occurs in rat and porcine TA, and is maintained in primary smooth muscle cells isolated from TA (TA–SMCs) compared with cells from AA (AA–SMCs). This region-specific homeobox gene expression pattern is also observed in human embryonic stem cells differentiated into neuroectoderm–SMCs and paraxial mesoderm–SMCs, which give rise to AA–SMCs and TA–SMCs, respectively. We also find that, compared with AA and AA–SMCs, TA and TA–SMCs have lower activity of the proinflammatory and proatherogenic nuclear factor-&kgr;B (NF-&kgr;B) and lower expression of NF-&kgr;B target genes, at least in part attributable to HOXA9-dependent inhibition. Conversely, NF-&kgr;B inhibits HOXA9 promoter activity and mRNA expression in SMCs. Conclusion—Our findings support a model of Hox6-10–specified positional identity in the adult vasculature that is established by embryonic cues independently of environmental factors and is conserved in different mammalian species. Differential homeobox gene expression contributes to maintaining phenotypic differences between SMCs from athero-resistant and athero-susceptible regions, at least in part through feedback regulatory mechanisms involving inflammatory mediators, for example, reciprocal inhibition between HOXA9 and NF-&kgr;B.Objective— Different vascular beds show differing susceptibility to the development of atherosclerosis, but the molecular mechanisms underlying these differences are incompletely understood. This study aims to identify factors that contribute to the phenotypic heterogeneity of distinct regions of the adult vasculature. Approach and Results— High-throughput mRNA profiling in adult mice reveals higher expression of the homeobox paralogous genes 6 to 10 ( Hox6-10 ) in the athero-resistant thoracic aorta (TA) than in the athero-susceptible aortic arch (AA). Higher homeobox gene expression also occurs in rat and porcine TA, and is maintained in primary smooth muscle cells isolated from TA (TA–SMCs) compared with cells from AA (AA–SMCs). This region-specific homeobox gene expression pattern is also observed in human embryonic stem cells differentiated into neuroectoderm–SMCs and paraxial mesoderm–SMCs, which give rise to AA–SMCs and TA–SMCs, respectively. We also find that, compared with AA and AA–SMCs, TA and TA–SMCs have lower activity of the proinflammatory and proatherogenic nuclear factor-κB (NF-κB) and lower expression of NF-κB target genes, at least in part attributable to HOXA9-dependent inhibition. Conversely, NF-κB inhibits HOXA9 promoter activity and mRNA expression in SMCs. Conclusion— Our findings support a model of Hox6-10–specified positional identity in the adult vasculature that is established by embryonic cues independently of environmental factors and is conserved in different mammalian species. Differential homeobox gene expression contributes to maintaining phenotypic differences between SMCs from athero-resistant and athero-susceptible regions, at least in part through feedback regulatory mechanisms involving inflammatory mediators, for example, reciprocal inhibition between HOXA9 and NF-κB. # Significance {#article-title-44}

Nuclear Envelope Lamin-A Couples Actin Dynamics with Immunological Synapse Architecture and T Cell Activation

Communication between the nuclear lamina protein lamin-A and the actin cytoskeleton is required for optimal T cell responses. Nuclear Support for T Cell Activation A-type lamins are filamentous proteins that form the nuclear lamina inside the inner nuclear membrane. As well as maintaining the mechanical integrity of the nucleus, A-type lamins physically connect the nucleus and the actin cytoskeleton by associating with the linker of nucleoskeleton and cytoskeleton (LINC) complex. González-Granado et al. found that the abundance of lamin-A was rapidly and transiently increased in mouse and human CD4+ T cells in response to stimulation of the T cell receptor. Lamin-A enhanced F-actin polymerization at the immunological synapse between the T cells and antigen-presenting cells, thereby boosting T cell activation. Disruption of the interaction between lamin-A and the LINC complex also decreased T cell activation in culture, and mice lacking lamin-A in immune cells exhibited reduced T cell activation in response to antigen. These results suggest that the nuclear skeleton communicates with the cytoskeleton to enable optimal T cell activation. In many cell types, nuclear A-type lamins regulate multiple cellular functions, including higher-order genome organization, DNA replication and repair, gene transcription, and signal transduction; however, their role in specialized immune cells remains largely unexplored. We showed that the abundance of A-type lamins was almost negligible in resting naïve T lymphocytes, but was increased upon activation of the T cell receptor (TCR). The increase in lamin-A was an early event that accelerated formation of the immunological synapse between T cells and antigen-presenting cells. Polymerization of F-actin in T cells is a critical step for immunological synapse formation, and lamin-A interacted with the linker of nucleoskeleton and cytoskeleton (LINC) complex to promote F-actin polymerization. We also showed that lamin-A expression accelerated TCR clustering and led to enhanced downstream signaling, including extracellular signal–regulated kinase 1/2 (ERK1/2) signaling, as well as increased target gene expression. Pharmacological inhibition of the ERK pathway reduced lamin-A–dependent T cell activation. Moreover, mice lacking lamin-A in immune cells exhibited impaired T cell responses in vivo. These findings underscore the importance of A-type lamins for TCR activation and identify lamin-A as a previously unappreciated regulator of the immune response.

Tetraspanins CD9 and CD151 at the immune synapse support T-cell integrin signaling

Understanding how the immune response is activated and amplified requires detailed knowledge of the stages in the formation of the immunological synapse (IS) between T lymphocytes and antigen‐presenting cells (APCs). We show that tetraspanins CD9 and CD151 congregate at the T‐cell side of the IS. Silencing of CD9 or CD151 blunts the IL‐2 secretion and expression of the activation marker CD69 by APC‐conjugated T lymphocytes, but does not affect the accumulation of CD3 or actin to the IS, or the translocation of the microtubule‐organizing center toward the T‐B contact area. CD9 or CD151 silencing diminishes the relocalization of α4β1 integrin to the IS and reduces the accumulation of high‐affinity β1 integrins at the cell–cell contact. These changes are accompanied by diminished phosphorylation of the integrin downstream targets FAK and ERK1/2. Our results suggest that CD9 and CD151 support integrin‐mediated signaling at the IS.

Nuclear envelope lamin-A as a coordinator of T cell activation

Nuclear lamins A/C control several critical cellular functions, e.g., chromatin organization, gene transcription, DNA replication, DNA damage responses, cell cycle progression, cell differentiation, and cell polarization during migration. However, few studies have addressed the role of lamins A/C in the control of the functions of immune cells. Recently, we have demonstrated that lamins A/C are induced in T cells upon antigen recognition. Lamins A/C enhance T cell responses by coupling the plasma membrane to the nucleus via the linker of nucleoskeleton and cytoskeleton (LINC) complex and the actin cytoskeleton. Here, we discuss the possible physiological relevance and functional context of lamin A/C in T cell activation and propose a model in which lamins A/C are key modulators of immune cell functions.

Sorting nexin 6 enhances lamin a synthesis and incorporation into the nuclear envelope

Nuclear lamins are important structural and functional proteins in mammalian cells, but little is known about the mechanisms and cofactors that regulate their traffic into the nucleus. Here, we demonstrate that trafficking of lamin A, but not lamin B1, and its assembly into the nuclear envelope are regulated by sorting nexin 6 (SNX6), a major component of the retromer that targets proteins and other molecules to specific subcellular locations. SNX6 interacts with lamin A in vitro and in vivo and links it to the outer surface of the endoplasmic reticulum in human and mouse cells. SNX6 transports its lamin A cargo to the nuclear envelope in a process that takes several hours. Lamin A protein levels in the nucleus augment or decrease, respectively, upon gain or loss of SNX6 function. We further show that SNX6-dependent lamin A nuclear import occurs across the nuclear pore complex via a RAN-GTP-dependent mechanism. These results identify SNX6 as a key regulator of lamin A synthesis and incorporation into the nuclear envelope.

Conventional CD4 + T cells present bacterial antigens to induce cytotoxic and memory CD8 + T cell responses

Bacterial phagocytosis and antigen cross-presentation to activate CD8+ T cells are principal functions of professional antigen presenting cells. However, conventional CD4+ T cells also capture and kill bacteria from infected dendritic cells in a process termed transphagocytosis (also known as transinfection). Here, we show that transphagocytic T cells present bacterial antigens to naive CD8+ T cells, which proliferate and become cytotoxic in response. CD4+ T-cell-mediated antigen presentation also occurs in vivo in the course of infection, and induces the generation of central memory CD8+ T cells with low PD-1 expression. Moreover, transphagocytic CD4+ T cells induce protective anti-tumour immune responses by priming CD8+ T cells, highlighting the potential of CD4+ T cells as a tool for cancer immunotherapy.Antigen presentation is generally considered the domain of innate immune cells, but CD4+ T cells can transphagocytose bacteria from infected dendritic cells. Here the authors show CD4+ T cells can transphagocytose bacterial and tumour antigens and present them to CD8+ T cells to activate memory and cytotoxic functions.

In Vitro Differentiation of Naïve CD4⁺ T Cells: A Tool for Understanding the Development of Atherosclerosis.

A complete knowledge of atherosclerosis requires a better understanding of how innate and adaptive immunity operate systemically and locally within the arterial wall. T-helper 1 (Th1) lymphocyte responses have proatherogenic effects in mice, contrasting with the responses of T regulatory cells (Tregs), which can suppress growth of atherosclerotic lesions. An imbalance in the differentiation of T-helper cells may therefore impact the development, size, and stability of atherosclerosis plaques. This chapter describes a method to isolate naïve CD4(+) T cells from atherosclerosis-prone mouse peripheral blood lymphocytes and to differentiate these CD4(+) T cells in vitro to various T helper cell lineages. These techniques allow the analysis of T lymphocytes in vitro, a necessary step in the study of molecular mechanisms involved in the inflammatory responses that trigger atherosclerosis.