The Receptor Slamf1 on the Surface of Myeloid Lineage Cells Controls Susceptibility to Infection by Trypanosoma cruzi

Trypanosoma cruzi, the protozoan parasite responsible for Chagas'' disease, causes severe myocarditis often resulting in death. Here, we report that Slamf1−/− mice, which lack the hematopoietic cell surface receptor Slamf1, are completely protected from an acute lethal parasite challenge. Cardiac damage was reduced in Slamf1−/− mice compared to wild type mice, infected with the same doses of parasites, as a result of a decrease of the number of parasites in the heart even the parasitemia was only marginally less. Both in vivo and in vitro experiments reveal that Slamf1-defIcient myeloid cells are impaired in their ability to replicate the parasite and show altered production of cytokines. Importantly, IFN-γ production in the heart of Slamf1 deficient mice was much lower than in the heart of wt mice even though the number of infiltrating dendritic cells, macrophages, CD4 and CD8 T lymphocytes were comparable. Administration of an anti-Slamf1 monoclonal antibody also reduced the number of parasites and IFN-γ in the heart. These observations not only explain the reduced susceptibility to in vivo infection by the parasite, but they also suggest human Slamf1 as a potential target for therapeutic target against T. cruzi infection.     

Cell Migration Is Regulated by Platelet-Derived Growth Factor Receptor Endocytosis

Cell migration requires spatial and temporal processes that detect and transfer extracellular stimuli into intracellular signals. The platelet-derived growth factor (PDGF) receptor is a cell surface receptor on fibroblasts that regulates proliferation and chemotaxis in response to PDGF. How the PDGF signal is transmitted accurately through the receptor into cells is an unresolved question. Here, we report a new intracellular signaling pathway by which DOCK4, a Rac1 guanine exchange factor, and Dynamin regulate cell migration by PDGF receptor endocytosis. We showed by a series of biochemical and microscopy techniques that Grb2 serves as an adaptor protein in the formation of a ternary complex between the PDGF receptor, DOCK4, and Dynamin, which is formed at the leading edge of cells. We found that this ternary complex regulates PDGF-dependent cell migration by promoting PDGF receptor endocytosis and Rac1 activation at the cell membrane. This study revealed a new mechanism by which cell migration is regulated by PDGF receptor endocytosis.Chemoattractants bind to cell surface receptors, resulting in the cytoskeletal reorganization that permits the migration of cells toward a stimulus. In fibroblasts, the platelet-derived growth factor receptor β (PDGFRβ) is a cell surface receptor tyrosine kinase (RTK) that regulates cell proliferation and chemotaxis in response to PDGF. PDGF binding activates PDGF receptor autophosphorylation, which in turn mediates a series of intracellular signaling cascades initiated by the association of SH2 domain-containing adaptor proteins (25). The adaptor protein Grb2 at the plasma membrane binds to Ras exchange factor Sos1, activating mitogen-activated protein kinase (MAPK) and cell proliferation signals (19). Grb2 also plays a critical role in receptor internalization via its interaction with dynamin, an exchange factor that facilitates receptor entry into endocytic vesicles (32). Grb2 regulates ubiquitination and the degradation of the receptor via its interaction with Cbl, an E3 ubiquitin ligase (33). While the role of Grb2 in modulating receptor levels and facilitating growth factor-dependent mitogenic signals is defined, its role in coordinating receptor-dependent chemotaxis has not been elucidated.The small GTPase Rac1 plays a crucial role in PDGF-mediated chemotaxis by regulating cortical actin at the leading edge of cells. PDGF receptor activation promotes GTP loading and the translocation of Rac1 to the cell membrane via guanine exchange factors (GEFs). The DOCK family of Rac1 GEFs, also called CDM proteins (for Caenorhabditis elegans ced-5, vertebrate DOCK180, and Drosophila myoblast city), are regulators of cell migration and have been implicated in various biological processes, such as lymphocyte migration, phagocytosis, and cancer progression (6, 10, 30, 35). In migrating fibroblasts, DOCK proteins localize to the cell''s leading edge via their interaction with the phospholipid PIP3, but a direct molecular link to PDGF has not been established (5). Biochemical studies show that Rac activation requires the DHR2/docker domain of DOCK proteins and the expression of the PH domain-containing protein Ced-12/ELMO. Previously we identified DOCK4 in a screen for novel tumor suppressor genes using representational difference analysis on mouse tumor cell lines (35). DOCK4, like other CDM proteins, binds ELMO and exerts its biochemical effects on the small GTPases Rac and Rap1 (30, 35). An interesting observation is that the amino acid sequence toward the C terminus is not conserved among individual DOCK family members. The alternate splicing of the DOCK4 gene has been reported, but how amino acid sequence variation alters the signaling properties of DOCK4 for the regulation of cell migration is unknown.Members of the Nck family of adaptor proteins, CrkII and Nck, have been reported to bind to the C terminus of DOCK180 (12, 29). Here, we show that the third member of the family of Nck adaptors, namely Grb2, binds to wild-type DOCK4. We found that a ternary complex formed by Grb2-DOCK4-Dynamin2 interacts with PDGF-activated PDGFβ receptor and promotes growth factor-dependent migration without altering cell proliferation. PDGF-dependent migration requires receptor endocytosis and is regulated by the formation of a DOCK4-Grb2-Dynamin2-PDGFRβ complex at the cell''s leading edge. These studies provide novel mechanistic insights into PDGFRβ regulation and cell migration.     

Cell Migration Is Regulated by AGE-RAGE Interaction in Human Oral Cancer Cells In Vitro

Advanced glycation end products (AGEs) are produced in an irreversible non-enzymatic reaction of carbohydrates and proteins. Patients with diabetes mellitus (DM) are known to have elevated AGE levels, which is viewed as a risk factor of diabetes-related complications. In a clinical setting, it has been shown that patients with oral cancer in conjunction with DM have a higher likelihood of cancer metastasis and lower cancer survival rates. AGE-RAGE (a receptor of AGEs) is also correlated with metastasis and angiogenesis. Recent studies have suggested that the malignancy of cancer may be enhanced by glyceraldehyde-derived AGEs; however, the underlying mechanism remains unclear. This study examined the apparently close correlation between AGE-RAGE and the malignancy of SAS oral cancer cell line. In this study, AGEs increased ERK phosphorylation, enhanced cell migration, and promoted the expression of RAGE, MMP2, and MMP9. Using PD98059, RAGE antibody, and RAGE RNAi to block RAGE pathway resulted in the inhibition of ERK phosphorylation. Cell migration, MMP2 and MMP9 expression were also reduced by this treatment. Our findings demonstrate the importance of AGE-RAGE with regard to the malignancy of oral cancer, and help to explain the poor prognosis of DM subjects with oral cancer.     

The Group Migration of Dictyostelium Cells Is Regulated by Extracellular Chemoattractant Degradation

Starvation of Dictyostelium induces a developmental program in which cells form an aggregate that eventually differentiates into a multicellular structure. The aggregate formation is mediated by directional migration of individual cells that quickly transition to group migration in which cells align in a head-to-tail manner to form streams. Cyclic AMP acts as a chemoattractant and its production, secretion, and degradation are highly regulated. A key protein is the extracellular phosphodiesterase PdsA. In this study we examine the role and localization of PdsA during chemotaxis and streaming. We find that pdsA⁻ cells respond chemotactically to a narrower range of chemoattractant concentrations compared with wild-type (WT) cells. Moreover, unlike WT cells, pdsA⁻ cells do not form streams at low cell densities and form unusual thick and transient streams at high cell densities. We find that the intracellular pool of PdsA is localized to the endoplasmic reticulum, which may provide a compartment for storage and secretion of PdsA. Because we find that cAMP synthesis is normal in cells lacking PdsA, we conclude that signal degradation regulates the external cAMP gradient field generation and that the group migration behavior of these cells is compromised even though their signaling machinery is intact.     

细胞膜表面糖链结构与肿瘤转移的关系

连接在天冬酰胺上的N-糖链,连接在丝氨酸和苏氨酸上的O-糖链,连接在丝氨酸上的糖胺聚糖,连接在脂类物质上的糖脂等糖基化修饰在真核细胞表面普遍存在,并调节了细胞的各种功能,它们不仅参与生命活动中正常的生理生化活动,而且于疾病的发生发展密不可分。肿瘤细胞表面糖基异常化,如糖链在表达水平上的差异以及特殊糖链结构的出现,均与肿瘤细胞的侵袭和转移有密切关系。本综述主要介绍了肿瘤细胞糖基化的改变:一些主要的糖基化结构如β1,6分支和唾液酸的表达的增加会使某些肿瘤细胞的迁移能力增强。同时对引起这种变化的作用机制进行了介绍,提出一些潜在的抗肿瘤研究靶点,为肿瘤糖生物学的深入研究提供参考。     

Maintenance of Leukemia-Initiating Cells Is Regulated by the CDK Inhibitor Inca1

Functional differences between healthy progenitor and cancer initiating cells may provide unique opportunities for targeted therapy approaches. Hematopoietic stem cells are tightly controlled by a network of CDK inhibitors that govern proliferation and prevent stem cell exhaustion. Loss of Inca1 led to an increased number of short-term hematopoietic stem cells in older mice, but Inca1 seems largely dispensable for normal hematopoiesis. On the other hand, Inca1-deficiency enhanced cell cycling upon cytotoxic stress and accelerated bone marrow exhaustion. Moreover, AML1-ETO9a-induced proliferation was not sustained in Inca1-deficient cells in vivo. As a consequence, leukemia induction and leukemia maintenance were severely impaired in Inca1^−/− bone marrow cells. The re-initiation of leukemia was also significantly inhibited in absence of Inca1^−/− in MLL—AF9- and c-myc/BCL2-positive leukemia mouse models. These findings indicate distinct functional properties of Inca1 in normal hematopoietic cells compared to leukemia initiating cells. Such functional differences might be used to design specific therapy approaches in leukemia.     

Hematopoietic Stem/Progenitor Cell Proliferation and Differentiation Is Differentially Regulated by High-Density and Low-Density Lipoproteins in Mice

Rationale

Hematopoietic stem/progenitor cells (HSPC) are responsible for maintaining the blood system as a result of their self-renewal and multilineage differentiation capacity. Recently, studies have suggested that HDL cholesterol may inhibit and impaired cholesterol efflux may increase HSPC proliferation and differentiation.

Objectives

We hypothesized that LDL may enhance HSPC proliferation and differentiation while HDL might have the opposing effect which might influence the size of the pool of inflammatory cells.

Methods and Results

HSPC number and function were studied in hypercholesterolemic LDL receptor knockout (LDLr^−/−) mice on high fat diet. Hypercholesterolemia was associated with increased frequency of HSPC, monocytes and granulocytes in the peripheral blood (PB). In addition, an increased proportion of BM HSPC was in G₂M of the cell cycle, and the percentage of HSPC and granulocyte-macrophage progenitors (GMP) increased in BM of LDLr^−/− mice. When BM Lin-Sca-1+cKit+ (i.e. “LSK”) cells were cultured in the presence of LDL in vitro we also found enhanced differentiation towards monocytes and granulocytes. Furthermore, LDL promoted lineage negative (Lin−) cells motility. The modulation by LDL on HSPC differentiation into granulocytes and motility was inhibited by inhibiting ERK phosphorylation. By contrast, when mice were infused with human apoA-I (the major apolipoprotein of HDL) or reconstituted HDL (rHDL), the frequency and proliferation of HSPC was reduced in BM in vivo. HDL also reversed the LDL-induced monocyte and granulocyte differentiation in vitro.

Conclusion

Our data suggest that LDL and HDL have opposing effects on HSPC proliferation and differentiation. It will be of interest to determine if breakdown of HSPC homeostasis by hypercholesterolemia contributes to inflammation and atherosclerosis progression.     

Group 2 Innate Lymphoid Cell Production of IL-5 Is Regulated by NKT Cells during Influenza Virus Infection

Respiratory virus infections, such as influenza, typically induce a robust type I (pro-inflammatory cytokine) immune response, however, the production of type 2 cytokines has been observed. Type 2 cytokine production during respiratory virus infection is linked to asthma exacerbation; however, type 2 cytokines may also be tissue protective. Interleukin (IL)-5 is a prototypical type 2 cytokine that is essential for eosinophil maturation and egress out of the bone marrow. However, little is known about the cellular source and underlying cellular and molecular basis for the regulation of IL-5 production during respiratory virus infection. Using a mouse model of influenza virus infection, we found a robust transient release of IL-5 into infected airways along with a significant and progressive accumulation of eosinophils into the lungs, particularly during the recovery phase of infection, i.e. following virus clearance. The cellular source of the IL-5 was group 2 innate lymphoid cells (ILC2) infiltrating the infected lungs. Interestingly, the progressive accumulation of eosinophils following virus clearance is reflected in the rapid expansion of c-kit⁺ IL-5 producing ILC2. We further demonstrate that the enhanced capacity for IL-5 production by ILC2 during recovery is concomitant with the enhanced expression of the IL-33 receptor subunit, ST2, by ILC2. Lastly, we show that NKT cells, as well as alveolar macrophages (AM), are endogenous sources of IL-33 that enhance IL-5 production from ILC2. Collectively, these results reveal that c-kit⁺ ILC2 interaction with IL-33 producing NKT and AM leads to abundant production of IL-5 by ILC2 and accounts for the accumulation of eosinophils observed during the recovery phase of influenza infection.     

Pulmonary Inflammation Is Regulated by the Levels of the Vesicular Acetylcholine Transporter

Acetylcholine (ACh) plays a crucial role in physiological responses of both the central and the peripheral nervous system. Moreover, ACh was described as an anti-inflammatory mediator involved in the suppression of exacerbated innate response and cytokine release in various organs. However, the specific contributions of endogenous release ACh for inflammatory responses in the lung are not well understood. To address this question we have used mice with reduced levels of the vesicular acetylcholine transporter (VAChT), a protein required for ACh storage in secretory vesicles. VAChT deficiency induced airway inflammation with enhanced TNF-α and IL-4 content, but not IL-6, IL-13 and IL-10 quantified by ELISA. Mice with decreased levels of VAChT presented increased collagen and elastic fibers deposition in airway walls which was consistent with an increase in inflammatory cells positive to MMP-9 and TIMP-1 in the lung. In vivo lung function evaluation showed airway hyperresponsiveness to methacholine in mutant mice. The expression of nuclear factor-kappa B (p65-NF-kB) in lung of VAChT-deficient mice were higher than in wild-type mice, whereas a decreased expression of janus-kinase 2 (JAK2) was observed in the lung of mutant animals. Our findings show the first evidence that cholinergic deficiency impaired lung function and produce local inflammation. Our data supports the notion that cholinergic system modulates airway inflammation by modulation of JAK2 and NF-kB pathway. We proposed that intact cholinergic pathway is necessary to maintain the lung homeostasis.     

The Epithelial Calcium Channel TRPV5 Is Regulated Differentially by Klotho and Sialidase

The transient receptor potential vanilloid type 5 (TRPV5) Ca²⁺ channel facilitates transcellular Ca²⁺ transport in the distal convoluted tubule (DCT) of the kidney. The channel is glycosylated with a complex type N-glycan and it has been postulated that hydrolysis of the terminal sialic acid(s) stimulate TRPV5 activity. The present study delineates the role of the N-glycan in TRPV5 activity using biochemical assays in Human Embryonic Kidney 293 cells expressing TRPV5, isoelectric focusing and total internal reflection fluorescent microscopy. The anti-aging hormone klotho and other glycosidases stimulate TRPV5-dependent Ca²⁺ uptake. Klotho was found to increase the plasma membrane stability of TRPV5, via the TRPV5 N-glycan. Sialidase mimicked this stimulatory action. However, this effect was independent of the N-glycosylation state of TRPV5, since the N-glycosylation mutant (TRPV5^N358Q) was activated to the same extent. We showed that the increased TRPV5 activity after sialidase treatment is caused by inhibition of lipid raft-mediated internalization. In addition, sialidase modified the N-glycan of transferrin, a model glycoprotein, differently from klotho. Previous studies showed that after klotho treatment, galectin-1 binds the TRPV5 N-glycan and thereby increases TRPV5 activity. However, galectin-3, but not galectin-1, was expressed in the DCT. Furthermore, an increase in TRPV5-mediated Ca²⁺ uptake was detected after galectin-3 treatment. In conclusion, two distinct TRPV5 stimulatory mechanisms were demonstrated; a klotho-mediated effect that is dependent on the N-glycan of TRPV5 and a sialidase-mediated stimulation that is lipid raft-dependent and independent of the N-glycan of TRPV5.     

Heterogeneous CD8+ T Cell Migration in the Lymph Node in the Absence of Inflammation Revealed by Quantitative Migration Analysis

The three-dimensional positions of immune cells can be tracked in live tissues precisely as a function of time using two-photon microscopy. However, standard methods of analysis used in the field and experimental artifacts can bias interpretations and obscure important aspects of cell migration such as directional migration and non-Brownian walk statistics. Therefore, methods were developed for minimizing drift artifacts, identifying directional and anisotropic (asymmetric) migration, and classifying cell migration statistics. These methods were applied to describe the migration statistics of CD8+ T cells in uninflamed lymph nodes. Contrary to current models, CD8+ T cell statistics are not well described by a straightforward persistent random walk model. Instead, a model in which one population of cells moves via Brownian-like motion and another population follows variable persistent random walks with noise reproduces multiple statistical measures of CD8+ T cell migration in the lymph node in the absence of inflammation.     

DAAM1 Is a Formin Required for Centrosome Re-Orientation during Cell Migration

Background

Disheveled-associated activator of morphogenesis 1 (DAAM1) is a formin acting downstream of Wnt signaling that is important for planar cell polarity. It has been shown to promote proper cell polarization during embryonic development in both Xenopus and Drosophila. Importantly, DAAM1 binds to Disheveled (Dvl) and thus functions downstream of the Frizzled receptors. Little is known of how DAAM1 is localized and functions in mammalian cells. We investigate here how DAAM1 affects migration and polarization of cultured cells and conclude that it plays a key role in centrosome polarity.

Methodology/Principal Findings

Using a specific antibody to DAAM1, we find that the protein localizes to the acto-myosin system and co-localizes with ventral myosin IIB-containing actin stress fibers. These fibers are particularly evident in the sub-nuclear region. An N-terminal region of DAAM1 is responsible for this targeting and the DAAM1(1-440) protein can interact with myosin IIB fibers independently of either F-actin or RhoA binding. We also demonstrate that DAAM1 depletion inhibits Golgi reorientation in wound healing assays. Wound-edge cells exhibit multiple protrusions characteristic of unpolarized cell migration. Finally, in U2OS cells lines stably expressing DAAM1, we observe an enhanced myosin IIB stress fiber network which opposes cell migration.

Conclusions/Significance

This work highlights the importance of DAAM1 in processes underlying cell polarity and suggests that it acts in part by affecting the function of acto-myosin IIB system. It also emphasizes the importance of the N-terminal half of DAAM1. DAAM1 depletion strongly blocks centrosomal re-polarization, supporting the concept that DAAM1 signaling cooperates with the established Cdc42 associated polarity complex. These findings are also consistent with the observation that ablation of myosin IIB but not myosin IIA results in polarity defects downstream of Wnt signaling. The structure-function analysis of DAAM1 in cultured cells parallels more complex morphological events in the developing embryo.     

Elongation Factor 2 Kinase Is Regulated by Proline Hydroxylation and Protects Cells during Hypoxia

Protein synthesis, especially translation elongation, requires large amounts of energy, which is often generated by oxidative metabolism. Elongation is controlled by phosphorylation of eukaryotic elongation factor 2 (eEF2), which inhibits its activity and is catalyzed by eEF2 kinase (eEF2K), a calcium/calmodulin-dependent α-kinase. Hypoxia causes the activation of eEF2K and induces eEF2 phosphorylation independently of previously known inputs into eEF2K. Here, we show that eEF2K is subject to hydroxylation on proline-98. Proline hydroxylation is catalyzed by proline hydroxylases, oxygen-dependent enzymes which are inactivated during hypoxia. Pharmacological inhibition of proline hydroxylases also stimulates eEF2 phosphorylation. Pro98 lies in a universally conserved linker between the calmodulin-binding and catalytic domains of eEF2K. Its hydroxylation partially impairs the binding of calmodulin to eEF2K and markedly limits the calmodulin-stimulated activity of eEF2K. Neuronal cells depend on oxygen, and eEF2K helps to protect them from hypoxia. eEF2K is the first example of a protein directly involved in a major energy-consuming process to be regulated by proline hydroxylation. Since eEF2K is cytoprotective during hypoxia and other conditions of nutrient insufficiency, it may be a valuable target for therapy of poorly vascularized solid tumors.     

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen^1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response³.DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment⁴. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens⁵. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma^6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.     

MicroRNAs and Their Targets Are Differentially Regulated in Adult and Neonatal Mouse CD8+ T Cells

Immunological memory, which protects organisms from re-infection, is a hallmark of the mammalian adaptive immune system and the underlying principle of vaccination. In early life, however, mice and other mammals are deficient at generating memory CD8+ T cells, which protect organisms from intracellular pathogens. The molecular basis that differentiates adult and neonatal CD8+ T cells is unknown. MicroRNAs (miRNAs) are both developmentally regulated and required for normal adult CD8+ T cell functions. We used next-generation sequencing to identify mouse miRNAs that are differentially regulated in adult and neonatal CD8+ T cells, which may contribute to the impaired development of neonatal memory cells. The miRNA profiles of adult and neonatal cells were surprisingly similar during infection; however, we observed large differences prior to infection. In particular, miR-29 and miR-130 have significant differential expression between adult and neonatal cells before infection. Importantly, using RNA-Seq, we detected reciprocal changes in expression of messenger RNA targets for both miR-29 and miR-130. Moreover, targets that we validated include Eomes and Tbx21, key genes that regulate the formation of memory CD8+ T cells. Notably, age-dependent changes in miR-29 and miR-130 are conserved in human CD8+ T cells, further suggesting that these developmental differences are biologically relevant. Together, these results demonstrate that miR-29 and miR-130 are likely important regulators of memory CD8+ T cell formation and suggest that neonatal cells are committed to a short-lived effector cell fate prior to infection.     

Role of Arginase 1 from Myeloid Cells in Th2-Dominated Lung Inflammation

Th2-driven lung inflammation increases Arginase 1 (Arg1) expression in alternatively-activated macrophages (AAMs). AAMs modulate T cell and wound healing responses and Arg1 might contribute to asthma pathogenesis by inhibiting nitric oxide production, regulating fibrosis, modulating arginine metabolism and restricting T cell proliferation. We used mice lacking Arg1 in myeloid cells to investigate the contribution of Arg1 to lung inflammation and pathophysiology. In six model systems encompassing acute and chronic Th2-mediated lung inflammation we observed neither a pathogenic nor protective role for myeloid-expressed Arg1. The number and composition of inflammatory cells in the airways and lungs, mucus secretion, collagen deposition, airway hyper-responsiveness, and T cell cytokine production were not altered if AAMs were deficient in Arg1 or simultaneously in both Arg1 and NOS2. Our results argue that Arg1 is a general feature of alternative activation but only selectively regulates Th2 responses. Therefore, attempts to experimentally or therapeutically inhibit arginase activity in the lung should be examined with caution.