The Natural Combination Medicine Traumeel (Tr14) Improves Resolution of Inflammation by Promoting the Biosynthesis of Specialized Pro-Resolving Mediators.

The resolution of inflammation is an integral part of the acute inflammatory response and eventually leads to the return to homeostasis. It is supported by specialized pro-resolving mediators (SPMs) that act as immunoresolvents via specific G-protein-coupled receptors. In contrast to classical non-steroidal anti-inflammatory drugs (NSAIDs) that suppress the formation of pro-inflammatory lipid mediators such as prostaglandins, novel pharmacotherapeutic concepts propose to foster the biosynthesis of beneficial SPMs. Here, we demonstrate that the natural combination medicine Traumeel (Tr14) improves resolution of inflammation by promoting SPM formation. Tr14 enhanced the biosynthesis of 12-/15-lipoxygenase (LOX) products and of SPMs in zymosan-induced mouse peritonitis as well as in human monocyte-derived macrophages challenged with Staphylococcus aureus. Importantly, in the peritonitis model, Tr14 supported the recruitment of innate leukocytes and the efferocytotic capacity of macrophages, and positively influenced the inflammation resolution index. Taken together, we suggest that based on these properties Tr14 may possess therapeutic potential as an enhancer for the resolution of inflammatory processes.

Rho/ROCK pathway inhibition by the CDK inhibitor p27(kip1) participates in the onset of macrophage 3D-mesenchymal migration.

Infiltration of macrophages into tissue can promote tumour development. Depending on the extracellular matrix architecture, macrophages can adopt two migration modes: amoeboid migration--common to all leukocytes, and mesenchymal migration--restricted to macrophages and certain tumour cells. Here, we investigate the initiating mechanisms involved in macrophage mesenchymal migration. We show that a single macrophage is able to use both migration modes. Macrophage mesenchymal migration is correlated with decreased activity of Rho/Rho-associated protein kinase (ROCK) and is potentiated when ROCK is inhibited, suggesting that amoeboid inhibition participates in mechanisms that initiate mesenchymal migration. We identify the cyclin-dependent kinase (CDK) inhibitor p27(kip1) (also known as CDKN1B) as a new effector of macrophage 3D-migration. By using p27(kip1) mutant mice and small interfering RNA targeting p27(kip1), we show that p27(kip1) promotes mesenchymal migration and hinders amoeboid migration upstream of the Rho/ROCK pathway, a process associated with a relocation of the protein from the nucleus to the cytoplasm. Finally, we observe that cytoplasmic p27(kip1) is required for in vivo infiltration of macrophages within induced tumours in mice. This study provides the first evidence that silencing of amoeboid migration through inhibition of the Rho/ROCK pathway by p27(kip1) participates in the onset of macrophage mesenchymal migration.

Blood leukocytes and macrophages of various phenotypes have distinct abilities to form podosomes and to migrate in 3D environments.

Leukocytes migrate through most tissues in the body, a process which takes place in 3D environments. We have previously shown that macrophages use the amoeboid migration mode in porous matrices such as fibrillar collagen I and the mesenchymal mode involving podosomes and matrix proteolysis in dense matrices such as Matrigel. Whether such a plasticity may apply to other leukocytes and to all subsets of macrophages is unknown. Here, we therefore provide a comparative analysis of the in vitro 3D migration modes adopted by primary human leukocytes. Blood-derived monocytes, neutrophils and T lymphocytes were found to use the amoeboid mode in a porous fibrillar collagen I matrix but were unable to infiltrate dense Matrigel and to form podosomes. M2-polarized macrophages and elicited peritoneal macrophages formed podosome rosettes, degraded the ECM and infiltrated both matrices. In contrast, M1 macrophages were motionless in 2D and 3D environments, whilst resident macrophages, devoid of podosomes, were only able to use the amoeboid mode. Thus, we conclude that whereas all leukocytes use the amoeboid mode to migrate through porous matrices, it is only certain macrophages that can adopt the mesenchymal mode that permits migration through dense matrices. Interestingly, the acquisition of mesenchymal migration capacity by macrophages correlates with the presence of podosomes and with their capacity to organize those as rosettes, which appears to be modulated by their differentiation and polarization states. As a perspective, specific control of the mesenchymal migration would be a potential target for therapeutic approaches aiming at decreasing macrophage tissue infiltration.

The Drosophila TRPP cation channel, PKD2 and Dmel/Ced-12 act in genetically distinct pathways during apoptotic cell clearance.

Apoptosis, a genetically programmed cell death, allows for homeostasis and tissue remodelling during development of all multi-cellular organisms. Phagocytes swiftly recognize, engulf and digest apoptotic cells. Yet, to date the molecular mechanisms underlying this phagocytic process are still poorly understood. To delineate the molecular mechanisms of apoptotic cell clearance in Drosophila, we have carried out a deficiency screen and have identified three overlapping phagocytosis-defective mutants, which all delete the fly homologue of the ced-12 gene, known as Dmel\ced12. As anticipated, we have found that Dmel\ced-12 is required for apoptotic cell clearance, as for its C. elegans and mammalian homologues, ced-12 and elmo, respectively. However, the loss of Dmel\ced-12 did not solely account for the phenotypes of all three deficiencies, as zygotic mutations and germ line clones of Dmel\ced-12 exhibited weaker phenotypes. Using a nearby genetically interacting deficiency, we have found that the polycystic kidney disease 2 gene, pkd2, which encodes a member of the TRPP channel family, is also required for phagocytosis of apoptotic cells, thereby demonstrating a novel role for PKD2 in this process. We have also observed genetic interactions between pkd2, simu, drpr, rya-r44F, and retinophilin (rtp), also known as undertaker (uta), a gene encoding a MORN-repeat containing molecule, which we have recently found to be implicated in calcium homeostasis during phagocytosis. However, we have not found any genetic interaction between Dmel\ced-12 and simu. Based on these genetic interactions and recent reports demonstrating a role for the mammalian pkd-2 gene product in ER calcium release during store-operated calcium entry, we propose that PKD2 functions in the DRPR/RTP pathway to regulate calcium homeostasis during this process. Similarly to its C. elegans homologue, Dmel\Ced-12 appears to function in a genetically distinct pathway.

[Phagocyte migration: an overview]. / La migration des phagocytes: tour d'horizon.

Phagocytes are the first line of host defense thanks to their capacity to infiltrate infected and wounded tissues, where they exert their bactericidal and tissue repair functions. However, tissue infiltration of phagocytes also stimulates the progression of pathologies such as cancer and chronic inflammatory diseases. It is therefore necessary to identify the molecular and cellular mechanisms that control this process to identify new therapeutic targets. Phagocytes leave the blood stream by crossing the vascular wall and infiltrate interstitial tissues, a three-dimensional environment. A state-of-the-art of the different steps of phagocyte tissue recruitment in vivo and of the different in vitro models is developed in this synthesis. We focus on recent data concerning the migration of phagocytes in three-dimensional environments. The use of two different migration modes, amoeboid and mesenchymal, by macrophages and the role of podosomes and proteases in the mesenchymal migration are discussed.

The process of macrophage migration promotes matrix metalloproteinase-independent invasion by tumor cells.

Tumor-associated macrophages are known to amplify the malignant potential of tumors by secreting a variety of cytokines and proteases involved in tumor cell invasion and metastasis, but how these macrophages infiltrate tumors and whether the macrophage migration process facilitates tumor cell invasion remain poorly documented. To address these questions, we used cell spheroids of breast carcinoma SUM159PT cells as an in vitro model of solid tumors. We found that macrophages used both the mesenchymal mode requiring matrix metalloproteinases (MMPs) and the amoeboid migration mode to infiltrate tumor cell spheroids. Whereas individual SUM159PT cells invaded Matrigel using an MMP-dependent mesenchymal mode, when they were grown as spheroids, tumor cells were unable to invade the Matrigel surrounding spheroids. When spheroids were infiltrated or in contact with macrophages, tumor cell invasiveness was restored. It was dependent on the capacity of macrophages to remodel the matrix and migrate in an MMP-independent mesenchymal mode. This effect of macrophages was much reduced when spheroids were infiltrated by Matrigel migration-defective Hck(-/-) macrophages. In the presence of macrophages, SUM159PT migrated into Matrigel in the proximity of macrophages and switched from an MMP-dependent mesenchymal migration to an amoeboid mode resistant to protease inhibitors.Thus, in addition to the well-described paracrine loop between macrophages and tumor cells, macrophages can also contribute to the invasiveness of tumor cells by remodeling the extracellular matrix and by opening the way to exit the tumor and colonize the surrounding tissues in an MMP-dispensable manner.

Macrophage podosomes go 3D.

Macrophage tissue infiltration is a critical step in the immune response against microorganisms and is also associated with disease progression in chronic inflammation and cancer. Macrophages are constitutively equipped with specialized structures called podosomes dedicated to extracellular matrix (ECM) degradation. We recently reported that these structures play a critical role in trans-matrix mesenchymal migration mode, a protease-dependent mechanism. Podosome molecular components and their ECM-degrading activity have been extensively studied in two dimensions (2D), but yet very little is known about their fate in three-dimensional (3D) environments. Therefore, localization of podosome markers and proteolytic activity were carefully examined in human macrophages performing mesenchymal migration. Using our gelled collagen I 3D matrix model to obligate human macrophages to perform mesenchymal migration, classical podosome markers including talin, paxillin, vinculin, gelsolin, cortactin were found to accumulate at the tip of F-actin-rich cell protrusions together with ß1 integrin and CD44 but not ß2 integrin. Macrophage proteolytic activity was observed at podosome-like protrusion sites using confocal fluorescence microscopy and electron microscopy. The formation of migration tunnels by macrophages inside the matrix was accomplished by degradation, engulfment and mechanic compaction of the matrix. In addition, videomicroscopy revealed that 3D F-actin-rich protrusions of migrating macrophages were as dynamic as their 2D counterparts. Overall, the specifications of 3D podosomes resembled those of 2D podosome rosettes rather than those of individual podosomes. This observation was further supported by the aspect of 3D podosomes in fibroblasts expressing Hck, a master regulator of podosome rosettes in macrophages. In conclusion, human macrophage podosomes go 3D and take the shape of spherical podosome rosettes when the cells perform mesenchymal migration. This work sets the scene for future studies of molecular and cellular processes regulating macrophage trans-migration.

Matrix architecture dictates three-dimensional migration modes of human macrophages: differential involvement of proteases and podosome-like structures.

Tissue infiltration of macrophages, although critical for innate immunity, is also involved in pathologies, such as chronic inflammation and cancer. In vivo, macrophages migrate mostly in a constrained three-dimensional (3D) environment. However, in vitro studies, mainly focused on two dimensions, do not provide meaningful clues about the mechanisms involved in 3D macrophage migration. In contrast, tumor cell 3D migration is well documented. It comprises a protease-independent and Rho kinase (ROCK)-dependent amoeboid migration mode and a protease-dependent and ROCK-independent mesenchymal migration mode. In this study, we examined the influence of extracellular matrix (composition, architecture, and stiffness) on 3D migration of human macrophages derived from blood monocytes (MDMs). We show that: 1) MDMs use either the amoeboid migration mode in fibrillar collagen I or the mesenchymal migration mode in Matrigel and gelled collagen I, whereas HT1080 tumor cells only perform mesenchymal migration; 2) when MDMs use the mesenchymal migratory mode, they form 3D collagenolytic structures at the tips of cell protrusions that share several markers with podosomes as described in two dimensions; 3) in contrast to tumor cells, matrix metalloproteinase inhibitors do not impair protease-dependent macrophage 3D migration, suggesting the involvement of other proteolytic systems; and 4) MDMs infiltrating matrices of similar composition but with variable stiffness adapt their migration mode primarily to the matrix architecture. In conclusion, although it is admitted that leukocytes 3D migration is restricted to the amoeboid mode, we show that human macrophages also perform the mesenchymal mode but in a distinct manner than tumor cells, and they naturally adapt their migration mode to the environmental constraints.

Undertaker, a Drosophila Junctophilin, links Draper-mediated phagocytosis and calcium homeostasis.

Phagocytosis is important during development and in the immune response for the removal of apoptotic cells and pathogens, yet its molecular mechanisms are poorly understood. In Caenorhabditis elegans, the CED2/5/10/12 pathway regulates actin during phagocytosis of apoptotic cells, whereas the role of the CED1/6/7 pathway in phagocytosis is unclear. We report that Undertaker (UTA), a Drosophila Junctophilin protein, is required for Draper (CED-1 homolog)-mediated phagocytosis. Junctophilins couple Ca2+ channels at the plasma membrane to those of the endoplasmic reticulum (ER), the Ryanodine receptors. We place Draper, its adaptor drCed-6, UTA, the Ryanodine receptor Rya-r44F, the ER Ca2+ sensor dSTIM, and the Ca2+-release-activated Ca2+ channel dOrai in the same pathway that promotes calcium homeostasis and phagocytosis. Thus, our results implicate a Junctophilin in phagocytosis and link Draper-mediated phagocytosis to Ca2+ homeostasis, highlighting a previously uncharacterized role for the CED1/6/7 pathway.

Requirement for a Drosophila E3-ubiquitin ligase in phagocytosis of apoptotic cells.

Many cells die by apoptosis during animal development. Apoptotic cells are rapidly removed through phagocytosis by their neighbors or by macrophages. To genetically dissect this process, we performed an in vivo screen for genes required for efficient phagocytosis of apoptotic cells by Drosophila macrophages and identified pallbearer (pall), which encodes an F box protein. F box proteins generally provide substrate specificity to Skp Cullin F box (SCF) complexes, acting as E3 ligases that target phosphorylated proteins to ubiquitylation and degradation via the 26S proteasome. We showed that Pallbearer functions in an SCF-dependent manner and provided direct evidence of a role for ubiquitylation and proteasomal degradation in phagocytosis of apoptotic corpses in vivo. This work might further our understanding of the regulation of apoptotic cell engulfment and thus our understanding of innate immunity as a whole.