Podosomes, But Not the Maturation Status, Determine the Protease-Dependent 3D Migration in Human Dendritic Cells.
ABSTRACT: Dendritic cells (DC) are professional Antigen-Presenting Cells scattered throughout antigen-exposed tissues and draining lymph nodes, and survey the body for pathogens. Their ability to migrate through tissues, a 3D environment, is essential for an effective immune response. Upon infection, recognition of Pathogen-Associated Molecular Patterns (PAMP) by Toll-like receptors (TLR) triggers DC maturation. Mature DC (mDC) essentially use the protease-independent, ROCK-dependent amoeboid mode in vivo, or in collagen matrices in vitro. However, the mechanisms of 3D migration used by human immature DC (iDC) are still poorly characterized. Here, we reveal that human monocyte-derived DC are able to use two migration modes in 3D. In porous matrices of fibrillar collagen I, iDC adopted the amoeboid migration mode. In dense matrices of gelled collagen I or Matrigel, iDC used the protease-dependent, ROCK-independent mesenchymal migration mode. Upon TLR4 activation by LPS, mDC-LPS lose the capacity to form podosomes and degrade the matrix along with impaired mesenchymal migration. TLR2 activation by Pam3CSK4 resulted in DC maturation, podosome maintenance, and efficient mesenchymal migration. Under all these conditions, when DC used the mesenchymal mode in dense matrices, they formed 3D podosomes at the tip of cell protrusions. Using PGE2, known to disrupt podosomes in DC, we observed that the cells remained in an immature status and the mesenchymal migration mode was abolished. We also observed that, while CCL5 (attractant of iDC) enhanced both amoeboid and mesenchymal migration of iDC, CCL19 and CCL21 (attractants of mDC) only enhanced mDC-LPS amoeboid migration without triggering mesenchymal migration. Finally, we examined the migration of iDC in tumor cell spheroids, a tissue-like 3D environment. We observed that iDC infiltrated spheroids of tumor cells using both migration modes. Altogether, these results demonstrate that human DC adopt the mesenchymal mode to migrate in 3D dense environments, which relies on their capacity to form podosomes independent of their maturation status, paving the way of further investigations on in vivo DC migration in dense tissues and its regulation during infections.
Project description:Filamin A (FLNa) is a cross-linker of actin filaments and serves as a scaffold protein mostly involved in the regulation of actin polymerization. It is distributed ubiquitously, and null mutations have strong consequences on embryonic development in humans, with organ defects which suggest deficiencies in cell migration. We have reported previously that macrophages, the archetypal migratory cells, use the protease- and podosome-dependent mesenchymal migration mode in dense three-dimensional environments, whereas they use the protease- and podosome-independent amoeboid mode in more porous matrices. Because FLNa has been shown to localize to podosomes, we hypothesized that the defects seen in patients carrying FLNa mutations could be related to the capacity of certain cell types to form podosomes. Using strategies based on FLNa knock-out, knockdown, and rescue, we show that FLNa (i) is involved in podosome stability and their organization as rosettes and three-dimensional podosomes, (ii) regulates the proteolysis of the matrix mediated by podosomes in macrophages, (iii) is required for podosome rosette formation triggered by Hck, and (iv) is necessary for mesenchymal migration but dispensable for amoeboid migration. These new functions assigned to FLNa, particularly its role in mesenchymal migration, could be directly related to the defects in cell migration described during the embryonic development in FLNa-defective patients.
Project description:Transmission of measles virus (MV) from dendritic to airway epithelial cells is considered as crucial to viral spread late in infection. Therefore, pathways and effectors governing this process are promising targets for intervention. To identify these, we established a 3D respiratory tract model where MV transmission by infected dendritic cells (DCs) relied on the presence of nectin-4 on H358 lung epithelial cells. Access to recipient cells is an important prerequisite for transmission, and we therefore analyzed migration of MV-exposed DC cultures within the model. Surprisingly, enhanced motility toward the epithelial layer was observed for MV-infected DCs as compared to their uninfected siblings. This occurred independently of factors released from H358 cells indicating that MV infection triggered cytoskeletal remodeling associated with DC polarization enforced velocity. Accordingly, the latter was also observed for MV-infected DCs in collagen matrices and was particularly sensitive to ROCK inhibition indicating infected DCs preferentially employed the amoeboid migration mode. This was also implicated by loss of podosomes and reduced filopodial activity both of which were retained in MV-exposed uninfected DCs. Evidently, sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) as produced in response to virus-infection in DCs contributed to enhanced velocity because this was abrogated upon inhibition of sphingosine kinase activity. These findings indicate that MV infection promotes a push-and-squeeze fast amoeboid migration mode via the SphK/S1P system characterized by loss of filopodia and podosome dissolution. Consequently, this enables rapid trafficking of virus toward epithelial cells during viral exit.
Project description:M2-polarized macrophages have been shown to adapt their 3D migration mode to physical properties of surrounding extracellular matrix. They migrate in the integrin-mediated adhesion and proteolytic activity-dependent "mesenchymal" mode in stiff matrices and in the integrin and protease-independent "amoeboid" mode in low density, porous environments. To find out what impact the switching between the migration modes has on expression of both protein-coding and non-coding genes we employed RNA sequencing of total RNA depleted of ribosomal RNA isolated from macrophages migrating in either mode in 3D collagens. Differentially expressed genes from both categories have been detected and the changes in expression of selected genes were further validated with RT-qPCR. The acquired data will facilitate better understanding of how mechanical properties of tissue microenvironment reflect in macrophage immune function and how the transitions between mesenchymal and amoeboid migratory modes are regulated at the gene expression level.
Project description:Tumor cells exhibit two interconvertible modes of cell motility referred to as mesenchymal and amoeboid migration. Mesenchymal mode is characterized by elongated morphology that requires high GTPase Rac activation, whereas amoeboid mode is dependent on actomyosin contractility induced by Rho/Rho-associated protein kinase (ROCK) signaling. While elongated morphology is driven by Rac-induced protrusion at the leading edge, how Rho/ROCK signaling controls amoeboid movement is not well understood. We identified FilGAP, a Rac GTPase-activating protein (GAP), as a mediator of Rho/ROCK-dependent amoeboid movement of carcinoma cells. We show that depletion of endogenous FilGAP in carcinoma cells induced highly elongated mesenchymal morphology. Conversely, forced expression of FilGAP induced a round/amoeboid morphology that requires Rho/ROCK-dependent phosphorylation of FilGAP. Moreover, depletion of FilGAP impaired breast cancer cell invasion through extracellular matrices and reduced tumor cell extravasation in vivo. Thus phosphorylation of FilGAP by ROCK appears to promote amoeboid morphology of carcinoma cells, and FilGAP contributes to tumor invasion.
Project description:The focal adhesion proteins Hic-5 and paxillin have been previously identified as key regulators of MDA-MB-231 breast cancer cell migration and morphologic mesenchymal-amoeboid plasticity in three-dimensional (3D) extracellular matrices (ECMs). However, their respective roles in other cancer cell types have not been evaluated. Herein, utilizing 3D cell-derived matrices and fibronectin-coated one-dimensional substrates, we show that across a variety of cancer cell lines, the level of Hic-5 expression serves as the major indicator of the cells primary morphology, plasticity, and in vitro invasiveness. Domain mapping studies reveal sites critical to the functions of both Hic-5 and paxillin in regulating phenotype, while ectopic expression of Hic-5 in cell lines with low endogenous levels of the protein is sufficient to induce a Rac1-dependent mesenchymal phenotype and, in turn, increase amoeboid-mesenchymal plasticity and invasion. We show that the activity of vinculin, when coupled to the expression of Hic-5 is required for the mesenchymal morphology in the 3D ECM. Taken together, our results identify Hic-5 as a critical modulator of tumor cell phenotype that could be utilized in predicting tumor cell migratory and invasive behavior in vivo.
Project description:M2-polarized macrophages have been shown to adapt their migration mode to physical properties of the surrounding extracellular matrix. They migrate in the integrin-mediated adhesion and proteolytic activity-dependent mesenchymal mode in stiff matrices, and in the integrin- and protease-independent amoeboid mode in low density, porous environments. To find out what impact has the switching between the migration modes on expression of both protein-coding and non-coding genes we employed RNA sequencing of total RNA depleted of ribosomal RNA isolated from M2 macrophages migrating in three-dimensional collagen gels of high or low concentration for 48 hours.
Project description:p27(kip1) (p27) is an inhibitor of cyclin/cyclin-dependent kinase complexes, whose nuclear loss indicates a poor prognosis in various solid tumors. When located in the cytoplasm, p27 binds Op18/stathmin (stathmin), a microtubule (MT)-destabilizing protein, and restrains its activity. This leads to MT stabilization, which negatively affects cell migration. Here, we demonstrate that this p27 function also influences morphology and motility of cells immersed in three-dimensional (3D)matrices. Cells lacking p27 display a decrease in MT stability, a rounded shape when immersed in 3D environments, and a mesenchymal-amoeboid conversion in their motility mode. Upon cell contact to extracellular matrix, the decreased MT stability observed in p27 null cells results in accelerated lipid raft trafficking and increased RhoA activity. Importantly, cell morphology, motility, MT network composition, and distribution of p27 null cells were rescued by the concomitant genetic ablation of Stathmin, implicating that the balanced expression of p27 and stathmin represents a crucial determinant for cytoskeletal organization and cellular behavior in 3D contexts.
Project description:As dendritic cells (DCs) are the most potent professional antigen-presenting cells, they are being tested as cancer vaccines for immunotherapy of established cancers. Although numerous studies have characterized DCs by their phenotype and function, few have identified potential molecular markers of antigen presentation prior to vaccination of host. In this study we generated pre-immature DC (piDC), immature DC (iDC), and mature DC (mDC) from human peripheral blood monocytes (PBMC) obtained from HLA-A2 healthy donors, and pulsed them with human papillomavirus E7 peptide (p11-20), a class I HLA-A2 binding antigen. We then characterized DCs for cell surface phenotype and gene expression profile by microarray technology. We identified a set of 59 genes that distinguished three differentiation stages of DCs (piDC, iDC and mDC). When piDC, iDC and mDC were pulsed with E7 peptide for 2 hrs, the surface phenotype did not change, however, iDCs rather than mDCs showed transcriptional response by up-regulation of a set of genes. A total of 52 genes were modulated in iDC upon antigen pulsing. Elongation of pulse time for iDCs to 10 and 24 hrs did not significantly bring further changes in gene expression. The E7 peptide up-modulated immune response (KPNA7, IGSF6, NCR3, TREM2, TUBAL3, IL8, NFKBIA), pro-apoptosis (BTG1, SEMA6A, IGFBP3 and SRGN), anti-apoptosis (NFKBIA), DNA repair (MRPS11, RAD21, TXNRD1), and cell adhesion and cell migration genes (EPHA1, PGF, IL8 and CYR61) in iDCs. We confirmed our results by Q-PCR analysis. The E7 peptide but not control peptide (PADRE) induced up-regulation of NFKB1A gene only in HLA-A2 positive iDCs and not in HLA-A2 negative iDCs. These results suggest that E7 up-regulation of genes is specific and HLA restricted and that these genes may represent markers of antigen presentation and help rapidly assess the quality of dendritic cells prior to administration to the host.
Project description:The definitions of tolerogenic vs immunogenic dendritic cells (DC) remain controversial. Immature DC have been shown to induce T regulatory cells (Treg) specific for foreign and allogeneic Ags. However, we have previously reported that mature DC (mDC) prevented the onset of autoimmune diabetes, whereas immature DC (iDC) were therapeutically ineffective. In this study, islet-specific CD4(+) T cells from BDC2.5 TCR-transgenic mice were stimulated in the absence of exogenous cytokine with iDC or mDC pulsed with high- or low-affinity antigenic peptides and examined for Treg induction. Both iDC and mDC presenting low peptide doses induced weak TCR signaling via the Akt/mammalian target of rapamycin (mTOR) pathway, resulting in significant expansion of Foxp3(+) Treg. Furthermore, unpulsed mDC, but not iDC, also induced Treg. High peptide doses induced strong Akt/mTOR signaling and favored the expansion of Foxp3(neg) Th cells. The inverse correlation of Foxp3 and Akt/mTOR signaling was also observed in DO11.10 and OT-II TCR-transgenic T cells and was recapitulated with anti-CD3/CD28 stimulation in the absence of DC. IL-6 production in these cultures correlated positively with Ag dose and inversely with Treg expansion. Studies with T cells or DC from IL-6(-/-) mice revealed that IL-6 production by T cells was more important in the inhibition of Treg induction at low Ag doses. These studies indicate that the strength of Akt/mTOR signaling, a critical T cell-intrinsic determinant for Treg vs Th induction, can be controlled by adjusting the dose of antigenic peptide. Furthermore, this operates in a dominant fashion over DC phenotype and cytokine production.
Project description:In the present study, 3D biomimetic platforms were fabricated with guiding grating to mimic extracellular matrix topography, porous membrane to resemble the epithelial porous interface and trenches below to represent blood vessels as an in vitro tissue microenvironment. Fabrication technologies were developed to integrate the transparent biocompatible polydimethylsiloxane platforms with preciously controlled dimensions. Cell migration behaviors of an immortalized nasopharyngeal epithelial cell line (NP460) and a nasopharyngeal carcinoma cell line (NPC43) were studied on the 2D and 3D platforms. The NP460 and NPC43 cells traversing through the porous membrane and migrating in the trenches below were studied by time-lapse imaging. Before traversing through the pores, NP460 and NPC43 cells migrated around the pores but NPC43 cells had a lower migration speed with less lamellipodia spreading. After traversing to trenches below, NPC43 cells moved faster with an alternated elongated morphology (mesenchymal migration mode) and round morphology (amoeboid migration mode) compared with only mesenchymal migration mode for NP460 cells. The cell traversing probability through porous membrane on platforms with 30 ?m wide trenches below was found to be the highest when the guiding grating was perpendicular to the trenches below and the lowest when the guiding grating was parallel to the trenches below. The present study shows important information on cell migration in complex 3D microenvironment with various dimensions and could provide insight for pathology and treatment of nasopharyngeal carcinoma.