B-Cell Homeostasis Is Maintained During Two Months of Head-Down Tilt Bed Rest With or Without Antioxidant Supplementation.

Alterations of the immune system could seriously impair the ability to combat infections during future long-duration space missions. However, little is known about the effects of spaceflight on the B-cell compartment. Given the limited access to astronaut samples, we addressed this question using blood samples collected from 20 healthy male volunteers subjected to long-duration bed rest, an Earth-based analog of spaceflight. Hematopoietic progenitors, white blood cells, total lymphocytes and B-cells, four B-cell subsets, immunoglobulin isotypes, six cytokines involved in inflammation, cortisone and cortisol were quantified at five time points. Tibia microarchitecture was also studied. Moreover, we investigated the efficiency of antioxidant supplementation with a cocktail including polyphenols, omega 3, vitamin E and selenium. Our results show that circulating hematopoietic progenitors, white blood cells, total lymphocytes and B-cells, and B-cell subsets were not affected by bed rest. Cytokine quantification suggested a lower systemic inflammatory status, supported by an increase in serum cortisone, during bed rest. These data confirm the in vivo hormonal dysregulation of immunity observed in astronauts and show that bed rest does not alter B-cell homeostasis. This lack of an impact of long-term bed rest on B-cell homeostasis can, at least partially, be explained by limited bone remodeling. None of the evaluated parameters were affected by the administration of the antioxidant supplement. The non-effectiveness of the supplement may be because the diet provided to the non-supplemented and supplemented volunteers already contained sufficient antioxidants. Given the limitations of this model, further studies will be required to determine whether B-cell homeostasis is affected, especially during future deep-space exploration missions that will be of unprecedented durations.

Analysis of femurs from mice embarked on board BION-M1 biosatellite reveals a decrease in immune cell development, including B cells, after 1 wk of recovery on Earth.

Bone loss and immune dysregulation are among the main adverse outcomes of spaceflight challenging astronauts' health and safety. However, consequences on B-cell development and responses are still under-investigated. To fill this gap, we used advanced proteomics analysis of femur bone and marrow to compare mice flown for 1 mo on board the BION-M1 biosatellite, followed or not by 1 wk of recovery on Earth, to control mice kept on Earth. Our data revealed an adverse effect on B lymphopoiesis 1 wk after landing. This phenomenon was associated with a 41% reduction of B cells in the spleen. These reductions may contribute to explain increased susceptibility to infection even if our data suggest that flown animals can mount a humoral immune response. Future studies should investigate the quality/efficiency of produced antibodies and whether longer missions worsen these immune alterations.-Tascher, G., Gerbaix, M., Maes, P., Chazarin, B., Ghislin, S., Antropova, E., Vassilieva, G., Ouzren-Zarhloul, N., Gauquelin-Koch, G., Vico, L., Frippiat, J.-P., Bertile, F. Analysis of femurs from mice embarked on board BION-M1 biosatellite reveals a decrease in immune cell development, including B cells, after 1 wk of recovery on Earth.

The urodele amphibian Pleurodeles waltl has a diverse repertoire of immunoglobulin heavy chains with polyreactive and species-specific features.

Urodele amphibians are an interesting model because although they possess the cardinal elements of the vertebrate immune system, their immune response is apparently subdued. This phenomenon, sometimes regarded as a state of immunodeficiency, has been attributed by some authors to limited antibody diversity. We reinvestigated this issue in Pleurodeles waltl, a metamorphosing urodele, and noted that upsilon transcripts of its IgY repertoire were as diverse as alpha transcripts of the mammalian IgA repertoire. Mu transcripts encoding the IgM repertoire were less diverse, but could confer more plasticity. Both isotypes present potential polyreactive features that may confer urodele antibodies with the ability to bind to a variety of antigens. Finally, we observed additional cysteines in CDR1 and 2 of the IGHV5 and IGHV6 domains, some of which specific to urodeles, that could allow the establishment of a disulfide bond between these CDRs. Together, these data suggest that urodele antibody diversity is not as low as previously thought.

LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro.

BACKGROUND: Patients with metastatic melanoma have a poor median rate of survival. It is therefore necessary to increase our knowledge about melanoma cell dissemination which includes extravasation, where cancer cells cross the endothelial barrier. Extravasation is well understood during travelling of white blood cells, and involves integrins such as LFA-1 (composed of two chains, CD11a and CD18) expressed by T cells, while ICAM-1 is induced during inflammation by endothelial cells. Although melanoma cell lines cross endothelial cell barriers, they do not express LFA-1. We therefore hypothesized that melanoma-endothelial cell co-culture might induce the LFA-1/ICAM ligand/receptor couple during melanoma transmigration. METHODS: A transwell approach has been used as well as blocking antibodies against CD11a, CD18 and ICAM-1. Data were analyzed with an epifluorescence microscope. Fluorescence intensity was quantified with the ImageJ software. RESULTS: We show here that HUVEC-conditioned medium induce cell-surface expression of LFA-1 on melanoma cell lines. Similarly melanoma-conditioned medium activates ICAM-1 expression in endothelial cells. Accordingly blocking antibodies of ICAM-1, CD11a or CD18 strongly decrease melanoma transmigration. We therefore demonstrate that melanoma cells can cross endothelial monolayers in vitro due to the induction of ICAM-1 and LFA-1 occurring during the co-culture of melanoma and endothelial cells. Our data further suggest a role of LFA-1 and ICAM-1 in the formation of melanoma cell clumps enhancing tumor cell transmigration. CONCLUSION: Melanoma-endothelial cell co-culture induces LFA-1 and ICAM-1 expression, thereby favoring in vitro melanoma trans-migration.

PHF19 and Akt control the switch between proliferative and invasive states in melanoma.

Melanoma tumor cells shift between proliferative and invasive states based on their plasticity and microenvironmental conditions. Our team has shown that melanoma cells, grown as spheroids in a neural cell crest medium, polarize toward an invasive phenotype, characterized by a higher motility, a poor proliferation rate and a gain of pluripotency gene expression (Nanog and Oct4) when compared with cells grown in two dimensions in a serum-contaning medium. In agreement with the phenotypic switching hypothesis, most of these features are reversible. Microarray studies comparing two- vs. three-dimensional cultures revealed the downregulation of a polycomb-like protein, PHF19 (PHD finger protein 19), in the spheroids. As Polycomb proteins are involved in the epigenetic control of gene expression, we hypothesized that PHF19 might play a role in the switch between proliferative and invasive phenotypes. In this report, we show that PHF19 silencing reduces the cell proliferation rate and increases the transendothelial migration capacities of melanoma cell lines. However, PHF19 does not modulate the transcription level of Oct4 and Nanog. In the search of an upstream transcriptional regulator of the above genes, we identified the Akt signaling cascade as an inhibitor of Oct4 and Nanog expression and an activator for PHF19 expression. Through chromatin immunoprecipitation, we further provide evidence that phospho-Akt is part of the transcriptional complex associated to the promoters of all three genes. Our data therefore indicate the role of PHF19 and its upstream regulator, Akt, in the phenotype switch of melanoma cells from proliferative to invasive states.

Plasticity of melanoma cells induced by neural cell crest conditions and three-dimensional growth.

Melanoma tumors have been shown to comprise both invasive and proliferative cell subpopulations. These populations are highly plastic, thus hampering full characterization and therapeutic targeting of dormant and partially dedifferentiated invasive cells. We have reported, previously, that melanoma cells grown in a serum-free neural crest medium, in which they propagate as spheroids, show higher invasiveness and increased immune escape. In addition, in spheroids, we showed the increased expression of several genes which are involved in pluripotency, differentiation, and invasion. We therefore proposed that these culture conditions favor the polarization of proliferative melanoma cells toward an invasive state. As plasticity may suggest a reversible polarization, the aim of this report is to assess the transient phenotype of invasive cells generated through this procedure. We provide evidence that spheroid cells mimic dormant populations, and that this phenotype is fully reversible when cells are reintroduced into culture media that contain serum in which they grow as a monolayer. We also show that most transcriptional deregulations can be reversed. To further explain this plasticity in melanoma cells, we explored the epigenetic status of four gene promoters, assuming changes in acetylation or dimethylation on histone 3. We show reversible modifications on lysine 9 and lysine 4. We propose that spheroids allow the transient polarization of melanoma cells toward enhanced dormancy, loss of differentiation, and invasiveness, thereby reproducing the properties and plasticity of invasive subpopulations in melanoma tumors. This in-vitro model will allow further characterization and targeting of melanoma invasive cell populations.

Nanog and Oct4 overexpression increases motility and transmigration of melanoma cells.

PURPOSE: Melanoma tumors are highly heterogeneous and can undergo phenotypic modifications depending on their plasticity and the microenvironment, with shifts between proliferative and invasive states. We have shown that melanoma cells, grown as spheroids in a neural crest cell medium, polarize toward an invasive and motile phenotype, in agreement with transcriptomic modulations, including the up-regulation of Nanog and Oct4. Overexpression of these genes was shown to be associated with poor prognosis and metastatic forms of some cancers. We thus investigated implication of Nanog and Oct4, two embryonic transcription factors, in melanoma motility. METHODS: Our team used stable transfection of Nanog or Oct4 in A375 melanoma cell line to investigate motility in a wound healing assay and a transendothelial migration assay. Using semiquantitative RT-PCR, expression of two gene panels involved either in mesenchymal motility or in amoeboid migration was studied. RESULTS: Strongly enhanced capacities of motility and extravasation were observed with cells overexpressing Oct4 and Nanog. The A375 cell line has been described as having a mesenchymal migration type. However, in the Oct4 and Nanog transfectants, several amoeboid migration markers are strongly induced. Accordingly, amoeboid migration inhibitors decrease significantly the transmigration of Oct4- and Nanog-expressing cells through endothelial cells. CONCLUSIONS: We propose here that Nanog and Oct4 pluripotency marker expression in melanoma cells increases the transmigration capacity of these cells through the gain of amoeboid motility, leading to higher invasiveness and aggressiveness.

Junctional adhesion molecules are required for melanoma cell lines transendothelial migration in vitro.

One of the main steps of metastasis is extravasation, a phenomenon well described in lymphocytes but remaining to be fully uncovered for melanoma. Junctional adhesion molecules (JAMs) control the transendothelial migration of leukocytes. To date, the role of the JAM proteins, notably JAM-A and JAM-C, has not been examined in melanoma. Here, we compared two melanoma tumor cell lines, A375 and SLM8 cells, the A375 cell line being four times more efficient than the SLM8 cells in the crossing of the endothelial monolayer. We show evidence of the differential expression of JAM-A and JAM-C in these cell lines with JAM-C mainly expressed in the A375 cell line, and JAM-A detected preferentially in the SLM8 cells. To further dissect the respective roles of these proteins, we used both siRNA and blocking antibodies to decrease JAM-A and JAM-C expression.

Coexpression of major histocompatibility complex class II with chemokines and nuclear NFkappaB p50 in melanoma: a rational for their association with poor prognosis.

The constitutive expression of major histocompatibility complex class II (MHC II) molecules in melanoma is highly unusual and has been associated with unfavorable clinical outcome and higher metastatic dissemination. This association remains poorly understood and therefore, in this study we looked to whether it is caused by intracellular events that promote tumor progression. We previously reported that MHC II expression in melanoma cells requires active mitogen-activated protein kinase/extracellular signal-related kinase. However, our comparative and molecular analyses of a panel of melanoma cell lines herein provide clear evidence that mitogen-activated protein kinase/extracellular signal-related kinase is not sufficient for HLA-DR expression. We found that the expression of HLA-DR in these tumors rather coincides with the expression of CXCL-1 and CXCL-8 chemokines, both known to be expressed in tumors that invade early and are related to invasive stages of melanoma. The expression of HLA-DR also nicely paralleled that of the nuclear NFkappaB p50 subunit, regulating the expression of these chemokines in melanoma and previously correlated with poor prognosis of melanoma patients, although we provide evidence that NFkappaB is not directly regulating MHC II expression level. The molecular basis for class II transactivator and HLA-DR expression in melanoma therefore remains unsolved, but our findings linking together the expression of HLA-DR, of chemokines involved in invasiveness, and of nuclear NFkappaB p50 strongly support the content that MHC II may be a marker of invasive primary melanoma, and could explain the long-standing association of MHC II expression with overall poor prognosis and unfavorable clinical outcome.