LRRK1 regulation of actin assembly in osteoclasts involves serine 5 phosphorylation of L-plastin.

Mice with disruption of Lrrk1 and patients with nonfunctional mutant Lrrk1 exhibit severe osteopetrosis phenotypes because of osteoclast cytoskeletal dysfunction. To understand how Lrrk1 regulates osteoclast function by modulating cytoskeleton rearrangement, we examined the proteins that are differentially phosphorylated in wild-type mice and Lrrk1-deficient osteoclasts by metal affinity purification coupled liquid chromatography/mass spectrometry (LC/MS) analyses. One of the candidates that we identified by LC/MS is L-plastin, an actin bundling protein. We found that phosphorylation of L-plastin at serine (Ser) residues 5 was present in wild-type osteoclasts but not in Lrrk1-deficient cells. Western blot analyses with antibodies specific for Ser5 phosphorylated L-plastin confirmed the reduced L-plastin Ser5 phosphorylation in Lrrk1 knockout (KO) osteoclasts. micro computed tomography (Micro-CT) analyses revealed that the trabecular bone volume of the distal femur was increased by 27% in the 16 to 21-week-old L-plastin KO females as compared with the wild-type control mice. The ratio of bone volume to tissue volume and connectivity density were increased by 44% and 47% (both P < 0.05), respectively, in L-plastin KO mice. Our data suggest that targeted disruption of L-plastin increases trabecular bone volume, and phosphorylation of Ser5 in L-plastin in the Lrrk1 signaling pathway may in part contribute to actin assembly in mature osteoclasts.

Alveolar macrophage development in mice requires L-plastin for cellular localization in alveoli.

Alveolar macrophages are lung-resident sentinel cells that develop perinatally and protect against pulmonary infection. Molecular mechanisms controlling alveolar macrophage generation have not been fully defined. Here, we show that the actin-bundling protein L-plastin (LPL) is required for the perinatal development of alveolar macrophages. Mice expressing a conditional allele of LPL (CD11c.Crepos-LPLfl/fl) exhibited significant reductions in alveolar macrophages and failed to effectively clear pulmonary pneumococcal infection, showing that immunodeficiency results from reduced alveolar macrophage numbers. We next identified the phase of alveolar macrophage development requiring LPL. In mice, fetal monocytes arrive in the lungs during a late fetal stage, maturing to alveolar macrophages through a prealveolar macrophage intermediate. LPL was required for the transition from prealveolar macrophages to mature alveolar macrophages. The transition from prealveolar macrophage to alveolar macrophage requires the upregulation of the transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ), which is induced by exposure to granulocyte-macrophage colony-stimulating factor (GM-CSF). Despite abundant lung GM-CSF and intact GM-CSF receptor signaling, PPAR-γ was not sufficiently upregulated in developing alveolar macrophages in LPL-/- pups, suggesting that precursor cells were not correctly localized to the alveoli, where GM-CSF is produced. We found that LPL supports 2 actin-based processes essential for correct localization of alveolar macrophage precursors: (1) transmigration into the alveoli, and (2) engraftment in the alveoli. We thus identify a molecular pathway governing neonatal alveolar macrophage development and show that genetic disruption of alveolar macrophage development results in immunodeficiency.

Mst1 Kinase Regulates the Actin-Bundling Protein L-Plastin To Promote T Cell Migration.

Exploring the mechanisms controlling lymphocyte trafficking is essential for understanding the function of the immune system and the pathophysiology of immunodeficiencies. The mammalian Ste20-like kinase 1 (Mst1) has been identified as a critical signaling mediator of T cell migration, and loss of Mst1 results in immunodeficiency disease. Although Mst1 is known to support T cell migration through induction of cell polarization and lamellipodial formation, the downstream effectors of Mst1 are incompletely defined. Mice deficient for the actin-bundling protein L-plastin (LPL) have phenotypes similar to mice lacking Mst1, including decreased T cell polarization, lamellipodial formation, and cell migration. We therefore asked whether LPL functions downstream of Mst1. The regulatory N-terminal domain of LPL contains a consensus Mst1 phosphorylation site at Thr(89) We found that Mst1 can phosphorylate LPL in vitro and that Mst1 can interact with LPL in cells. Removal of the Mst1 phosphorylation site by mutating Thr(89) to Ala impaired localization of LPL to the actin-rich lamellipodia of T cells. Expression of the T89A LPL mutant failed to restore migration of LPL-deficient T cells in vitro. Furthermore, expression of T89A LPL in LPL-deficient hematopoietic cells, using bone marrow chimeras, failed to rescue the phenotype of decreased thymic egress. These results identify LPL as a key effector of Mst1 and establish a novel mechanism linking a signaling intermediate to an actin-binding protein critical to T cell migration.

HIV-1 Nef drives macrophages into hiding.

In this issue of Blood, Vérollet et al show that expression of the HIV-1­derived protein Nef alters the migratory mode adopted by macrophages, enhancing macrophage tissue infiltration and explaining the observed accumulation of tissue-resident macrophages in some HIV-infected patients.

Taking a toll road to better vaccines.

Effective subunit vaccines must elicit strong CD4(+) T cell responses. In this issue of Immunity, Malherbe et al. (2008) find that the ability of adjuvants to stimulate high-avidity T cell responses correlates with Toll-like-receptor engagement.

The actin-bundling protein L-plastin supports T-cell motility and activation.

Tight regulation of actin dynamics is essential for T-cell trafficking and activation. Recent studies in human and murine T cells reveal that T-cell motility and full T-cell activation require the hematopoietic-specific, actin-bundling protein L-plastin (LPL). T cells lacking LPL do not form fully mature synapses and thus demonstrate reduced cytokine production and proliferation. Reduction or loss of LPL expression also reduces the velocity of T cells and impairs thymic egress and intranodal motility. Whereas dispensable for proximal T-cell receptor and chemokine receptor signaling, LPL is critical to the later stages of synapse maturation and cellular polarization. Serine phosphorylation, calcium, and calmodulin binding regulate the bundling activity and localization of LPL following T-cell receptor and chemokine receptor engagement. However, the interaction between these regulatory domains and resulting changes in local control of actin cytoskeletal structures has not been fully elucidated. Circumstantial evidence suggests a function for LPL in either the formation or maintenance of integrin-associated adhesion structures. As LPL may be a target of the commonly used immunosuppressive agent dexamethasone, full elucidation of the regulation and function of LPL in T-cell biology may illuminate new pathways for clinically useful immunotherapeutics.

L-plastin is essential for alveolar macrophage production and control of pulmonary pneumococcal infection.

We report that mice deficient for the hematopoietic-specific, actin-bundling protein L-plastin (LPL) succumb rapidly to intratracheal pneumococcal infection. The increased susceptibility of LPL(-/-) mice to pulmonary pneumococcal challenge correlated with reduced numbers of alveolar macrophages, consistent with a critical role for this cell type in the immediate response to pneumococcal infection. LPL(-/-) mice demonstrated a very early clearance defect, with an almost 10-fold-higher bacterial burden in the bronchoalveolar lavage fluid 3 h following infection. Clearance of pneumococci from the alveolar space in LPL(-/-) mice was defective compared to that in Rag1(-/-) mice, which lack all B and T lymphocytes, indicating that innate immunity is defective in LPL(-/-) mice. We did not identify defects in neutrophil or monocyte recruitment or in the production of inflammatory cytokines or chemokines that would explain the early clearance defect. However, efficient alveolar macrophage regeneration following irradiation required LPL. We thus identify LPL as being key to alveolar macrophage development and essential to an effective antipneumococcal response. Further analysis of LPL(-/-) mice will illuminate critical regulators of the generation of alveolar macrophages and, thus, effective pulmonary innate immunity.

Intrinsic T- and B-cell defects impair T-cell-dependent antibody responses in mice lacking the actin-bundling protein L-plastin.

Germinal center development, critical for long-term humoral immunity, requires the trafficking of T and B lymphocytes to defined tissues and locations after antigenic challenge. The molecular mechanisms that support lymphocyte trafficking through the linkage of extracellular chemotactic and adhesive cues to the actin cytoskeleton are not yet fully defined. We have previously identified the actin-bundling protein L-plastin (LPL) as a requisite intermediary in both naive B and T lymphocyte migration and in T-cell activation. We tested the hypothesis that humoral immunity would require LPL. We show that mice lacking LPL demonstrated defective germinal center formation and reduced production of T-cell-dependent antibodies. T cells from LPL(-/-) mice exhibited defective expansion of the follicular helper T population. Reduced expansion of LPL(-/-) follicular helper T cells correlated with impaired trafficking to or retention of cells in the spleen following challenge, highlighting the importance of initial lymphocyte recruitment to the eventual success of the immune response. Furthermore, LPL(-/-) B cells demonstrated cell-intrinsic defects in population expansion and in differentiation into germinal center B cells. LPL thus modulates both T- and B-cell function during the germinal center reaction and the production of T-cell-dependent antibody responses.

The actin-bundling protein L-plastin is essential for marginal zone B cell development.

B cell development is exquisitely sensitive to location within specialized niches in the bone marrow and spleen. Location within these niches is carefully orchestrated through chemotactic and adhesive cues. In this article, we demonstrate the requirement for the actin-bundling protein L-plastin (LPL) in B cell motility toward the chemokines CXCL12 and CXCL13 and the lipid chemoattractant sphingosine-1-phosphate, which guide normal B cell development. Impaired motility of B cells in LPL(-/-) mice correlated with diminished splenic maturation of B cells, with a moderate (40%) loss of follicular B cells and a profound (>80%) loss of marginal zone B cells. Entry of LPL(-/-) B cells into the lymph nodes and bone marrow of mice was also impaired. Furthermore, LPL was required for the integrin-mediated enhancement of Transwell migration but was dispensable for integrin-mediated lymphocyte adhesion. These results suggest that LPL may participate in signaling that enables lymphocyte transmigration. In support of this hypothesis, the phosphorylation of Pyk-2, a tyrosine kinase that integrates chemotactic and adhesive cues, is diminished in LPL(-/-) B cells stimulated with chemokine. Finally, a well-characterized role of marginal zone B cells is the generation of a rapid humoral response to polysaccharide Ags. LPL(-/-) mice exhibited a defective Ab response to Streptococcus pneumoniae, indicating a functional consequence of defective marginal zone B cell development in LPL(-/-) mice.

Expression of non-phosphorylatable S5A-L-plastin exerts phenotypes distinct from L-plastin deficiency during podosome formation and phagocytosis.

Introduction: The actin cytoskeleton remodels to enable diverse processes essential to immunity, such as cell adhesion, migration and phagocytosis. A panoply of actin-binding proteins regulate these rapid rearrangements to induce actin-based shape changes and to generate force. L-plastin (LPL) is a leukocyte-specific, actin-bundling protein that is regulated in part by phosphorylation of the Ser-5 residue. LPL deficiency in macrophages impairs motility, but not phagocytosis; we recently found that expression of LPL in which the S5 residue is converted to a non-phosphorylatable alanine (S5A-LPL) resulted in diminished phagocytosis, but unimpaired motility. Methods: To provide mechanistic insight into these findings, we now compare the formation of podosomes (an adhesive structure) and phagosomes in alveolar macrophages derived from wild-type (WT), LPL-deficient, or S5A-LPL mice. Both podosomes and phagosomes require rapid remodeling of actin, and both are force-transmitting. Actin rearrangement, force generation, and signaling rely upon recruitment of many actin-binding proteins, including the adaptor protein vinculin and the integrin-associated kinase Pyk2. Prior work suggested that vinculin localization to podosomes was independent of LPL, while Pyk2 was displaced by LPL deficiency. We therefore chose to compare vinculin and Pyk2 co-localization with F-actin at sites of adhesion of phagocytosis in AMs derived from WT, S5A-LPL or LPL-/- mice, using Airyscan confocal microscopy. Results: As described previously, podosome stability was significantly disrupted by LPL deficiency. In contrast, LPL was dispensable for phagocytosis and was not recruited to phagosomes. Recruitment of vinculin to sites of phagocytosis was significantly enhanced in cells lacking LPL. Expression of S5A-LPL impeded phagocytosis, with reduced appearance of ingested bacteria-vinculin aggregates. Discussion: Our systematic analysis of the regulation of LPL during podosome vs. phagosome formation illuminates essential remodeling of actin during key immune processes.

The actin-bundling protein L-plastin dissociates CCR7 proximal signaling from CCR7-induced motility.

Chemokines promote lymphocyte motility by triggering F-actin rearrangements and inducing cellular polarization. Chemokines can also enhance cell-cell adhesion and costimulate T cells. In this study, we establish a requirement for the actin-bundling protein L-plastin (LPL) in CCR7- and sphingosine-1-phosphate-mediated T cell chemotaxis using LPL(-/-) mice. Disrupted motility of mature LPL(-/-) thymocytes manifested in vivo as diminished thymic egress. Two-photon microscopy of LPL(-/-) lymphocytes revealed reduced velocity and motility in lymph nodes. Defective migration resulted from defective cellular polarization following CCR7 ligation, as CCR7 did not polarize to the leading edge in chemokine-stimulated LPL(-/-) T cells. However, CCR7 signaling to F-actin polymerization and CCR7-mediated costimulation was intact in LPL(-/-) lymphocytes. The differential requirement for LPL in CCR7-induced cellular adhesion and CCR7-induced motility allowed assessment of the contribution of CCR7-mediated motility to positive selection of thymocytes and lineage commitment. Results suggest that normal motility is not required for CCR7 to function in positive selection and lineage commitment. We thus identify LPL as a molecule critical for CCR7-mediated motility but dispensable for early CCR7 signaling. The requirement for actin bundling by LPL for polarization reveals a novel mechanism of regulating actin dynamics during T cell motility.

Actin-bundling protein L-plastin regulates T cell activation.

Engagement of TCRs induces actin rearrangements, which are critical for T cell activation. T cell responses require new actin polymerization, but the significance of higher-order actin structures, such as microfilament bundles, is unknown. To determine the role of the actin-bundling protein leukocyte-plastin (L-plastin; LPL) in this process, T cells from LPL(-/-) mice were studied. LPL(-/-) T cells were markedly defective in TCR-mediated cytokine production and proliferation. LPL(-/-) T cells also spread inefficiently on surfaces with immobilized TCR ligands and formed smaller immunological synapses with APCs, likely due to defective formation of lamellipodia. LPL(-/-) mice showed delayed rejection of skin allografts after release from immunosuppression. Moreover, LPL(-/-) mice developed much less severe neurologic symptoms in experimental autoimmune encephalomyelitis, which correlated with impaired T cell responses to Ag, manifested by reduced proliferation and production of IFN-γ and IL-17. Thus, LPL-dependent actin bundling facilitates the formation of lamellipodia and normal immunological synapses and thereby enables T cell activation.

Efficient T Cell Migration and Activation Require L-Plastin.

Rapid re-organization of the actin cytoskeleton supports T-cell trafficking towards immune sites and interaction with antigen presenting cells (APCs). F-actin rearrangement enables T-cell trafficking by stabilizing adhesion to vascular endothelial cells and promoting transendothelial migration. T-cell/APC immune synapse (IS) maturation also relies upon f-actin-anchored LFA-1:ICAM-1 ligation. Therefore, efficient T-cell responses require tight regulation of f-actin dynamics. In this review, we summarize how the actin-bundling protein L-plastin (LPL) regulates T-cell activation and migration. LPL enhances f-actin polymerization and also directly binds to the ß2 chain of the integrin LFA-1 to support intercellular adhesion and IS formation in human and murine T cells. LPL- deficient T cells migrate slowly in response to chemo-attractants such as CXCL12, CCL19, and poorly polarize towards ICAM-1. Loss of LPL impairs thymic egress and intranodal motility. LPL is also required for T-cell IS maturation with APCs, and therefore for efficient cytokine production and proliferation. LPL-/- mice are less susceptible to T-cell mediated pathologies, such as allograft rejection and experimental autoimmune encephalomyelitis (EAE). LPL activity is regulated by its N-terminal "headpiece", which contains serine and threonine phosphorylation and calcium- and calmodulin-binding sites. LPL phosphorylation is required for lamellipodia formation during adhesion and migration, and also for LFA-1 clustering during IS formation. However, the precise molecular interactions by which LPL supports T-cell functional responses remain unclear. Future studies elucidating LPL-mediated regulation of T-cell migration and/or activation may illuminate pathways for therapeutic targeting in T-cell-mediated diseases.

High-resolution imaging of protein secretion at the single-cell level using plasmon-enhanced FluoroDOT assay.

Secreted proteins mediate essential physiological processes. With conventional assays, it is challenging to map the spatial distribution of proteins secreted by single cells, to study cell-to-cell heterogeneity in secretion, or to detect proteins of low abundance or incipient secretion. Here, we introduce the "FluoroDOT assay," which uses an ultrabright nanoparticle plasmonic-fluor that enables high-resolution imaging of protein secretion. We find that plasmonic-fluors are 16,000-fold brighter, with nearly 30-fold higher signal-to-noise compared with conventional fluorescence labels. We demonstrate high-resolution imaging of different secreted cytokines in the single-plexed and spectrally multiplexed FluoroDOT assay that revealed cellular heterogeneity in secretion of multiple proteins simultaneously. Using diverse biochemical stimuli, including Mycobacterium tuberculosis infection, and a variety of immune cells such as macrophages, dendritic cells (DCs), and DC-T cell co-culture, we demonstrate that the assay is versatile, facile, and widely adaptable for enhancing biological understanding of spatial and temporal dynamics of single-cell secretome.

Dietary lipids inhibit mitochondria transfer to macrophages to divert adipocyte-derived mitochondria into the blood.

Adipocytes transfer mitochondria to macrophages in white and brown adipose tissues to maintain metabolic homeostasis. In obesity, adipocyte-to-macrophage mitochondria transfer is impaired, and instead, adipocytes release mitochondria into the blood to induce a protective antioxidant response in the heart. We found that adipocyte-to-macrophage mitochondria transfer in white adipose tissue is inhibited in murine obesity elicited by a lard-based high-fat diet, but not a hydrogenated-coconut-oil-based high-fat diet, aging, or a corn-starch diet. The long-chain fatty acids enriched in lard suppress mitochondria capture by macrophages, diverting adipocyte-derived mitochondria into the blood for delivery to other organs, such as the heart. The depletion of macrophages rapidly increased the number of adipocyte-derived mitochondria in the blood. These findings suggest that dietary lipids regulate mitochondria uptake by macrophages locally in white adipose tissue to determine whether adipocyte-derived mitochondria are released into systemic circulation to support the metabolic adaptation of distant organs in response to nutrient stress.

Protein kinase C-theta is required for efficient positive selection.

Protein kinase C-theta (PKCtheta) is critical for TCR-initiated signaling in mature T cells, but initial reports found no requirement for PKCtheta in thymocyte development. Thymocytes and peripheral T cells utilize many of the same signaling components and, given the significant role of PKCtheta in peripheral T cells, it was surprising that it was not involved at all in TCR signaling in thymocytes. We decided to re-evaluate the role of PKCtheta in thymocyte development using the well-characterized class II-restricted n3.L2 TCR-transgenic TCR model. Analysis of n3.L2 PKCtheta(-/-) mice revealed a defect in thymocyte-positive selection, resulting in a 50% reduction in the generation of n3.L2 CD4 single-positive thymocytes and n3.L2 CD4 mature T cells. Competition between n3.L2 WT and n3.L2 PKCtheta(-/-) thymocytes in bone marrow chimeras revealed a more dramatic defect, with a >80% reduction in generation of n3.L2 CD4 single-positive thymocytes derived from PKCtheta(-/-) mice. Inefficient positive selection of n3.L2 PKCtheta(-/-) CD4 single-positive cells resulted from "weaker" signaling through the TCR and correlated with diminished ERK activation. The defect in positive selection was not complete in the PKCtheta(-/-) mice, most likely accounted for by compensation by other PKC isoforms not evident in peripheral cells. Similar decreased positive selection of both CD4 and CD8 single-positive thymocytes was also seen in nontransgenic PKCtheta(-/-) mice. These findings now place PKCtheta as a key signaling molecule in the positive selection of thymocytes as well as in the activation of mature T cells.

Neutrophil L-Plastin Controls Ocular Paucibacteriality and Susceptibility to Keratitis.

Why ocular mucosa is paucibacterial is unknown. Many different mechanisms have been suggested but the comprehensive experimental studies are sparse. We found that a deficiency in L-plastin (LCP1), an actin bundling protein, resulted in an ocular commensal overgrowth, characterized with increased presence of conjunctival Streptococcal spp. The commensal overgrowth correlated with susceptibility to P. aeruginosa-induced keratitis. L-plastin knock-out (KO) mice displayed elevated bacterial burden in the P. aeruginosa-infected corneas, altered inflammatory responses, and compromised bactericidal activity. Mice with ablation of LPL under the LysM Cre (LysM. CreposLPLfl/fl ) and S100A8 Cre (S100A8.CreposLPLfl/fl ) promoters had a similar phenotype to the LPL KOs mice. In contrast, infected CD11c.CreposLPLfl/fl mice did not display elevated susceptibility to infection, implicating the myeloid L-plastin-sufficient cells (e.g., macrophages and neutrophils) in maintaining ocular homeostasis. Mechanistically, the elevated commensal burden and the susceptibility to infection were linked to defects in neutrophil frequencies at steady state and during infection and compromised bactericidal activities upon priming. Macrophage exposure to commensal organisms primed neutrophil responses to P. aeruginosa, augmenting PMN bactericidal capacity in an L-plastin dependent manner. Cumulatively, our data highlight the importance of neutrophils in controlling ocular paucibacteriality, reveal molecular and cellular events involved in the process, and suggest a link between commensal exposure and resistance to infection.

Cells under stress: The mechanical environment shapes inflammasome responses to danger signals.

Many intracellular signals, such as host danger-associated molecules and bacterial toxins during infection, elicit inflammasome activation. However, the mechanical environment in tissues may also influence the sensitivity of various inflammasomes to activation. The cellular mechanical environment is determined by the extracellular tissue stiffness, or its inverse, tissue compliance. Tissue stiffness is sensed by the intracellular cytoskeleton through a process termed mechanotransduction. Thus, extracellular compliance and the intracellular cytoskeleton may regulate the sensitivity of inflammasome activation. Control of proinflammatory signaling by tissue compliance may contribute to the pathogenesis of diseases such as ventilator-induced lung injury during bacterial pneumonia and tissue fibrosis in inflammatory disorders. The responsible signaling cascades in inflammasome activation pathways and mechanotransduction crosstalk are not yet fully understood. This rather different immunomodulatory perspective will be reviewed and open questions discussed here.

Technical Advance: New in vitro method for assaying the migration of primary B cells using an endothelial monolayer as substrate.

Migration of B cells supports their development and recruitment into functional niches. Therefore, defining factors that control B cell migration will lead to a better understanding of adaptive immunity. In vitro cell migration assays with B cells have been limited by poor adhesion of cells to glass coated with adhesion molecules. We have developed a technique using monolayers of endothelial cells as the substrate for B cell migration and used this technique to establish a robust in vitro assay for B cell migration. We use TNF-α to up-regulate surface expression of the adhesion molecule VCAM-1 on endothelial cells. The ligand VLA-4 is expressed on B cells, allowing them to interact with the endothelial monolayer and migrate on its surface. We tested our new method by examining the role of L-plastin (LPL), an F-actin-bundling protein, in B cell migration. LPL-deficient (LPL-/-) B cells displayed decreased speed and increased arrest coefficient compared with wild-type (WT) B cells, following chemokine stimulation. However, the confinement ratios for WT and LPL-/- B cells were similar. Thus, we demonstrate how the use of endothelial monolayers as a substrate will support future interrogation of molecular pathways essential to B cell migration.