Maria Antonia Meloni

Key gravity-sensitive signaling pathways drive T cell activation

Returning astronauts have experienced altered immune function and increased vulnerability to infection during spaceflights dating back to Apollo and Skylab. Lack of immune response in microgravity occurs at the cellular level. We analyzed differential gene expression to find gravity‐dependent genes and pathways. We found inhibited induction of 91 genes in the simulated freefall environment of the random positioning machine. Altered induction of 10 genes regulated by key signaling pathways was verified using real‐time RT‐PCR. We discovered that impaired induction of early genes regulated primarily by transcription factors NF‐κB, CREB, ELK, AP‐1, and STAT after crosslinking the T‐cell receptor contributes to T‐cell dysfunction in altered gravity environments. We have previously shown that PKA and PKC are key early regulators in T‐cell activation. Since the majority of the genes were regulated by NF‐κB, CREB, and AP‐1, we studied the pathways that regulated these transcription factors. We found that the PKA pathway was down‐regulated in vg. In contrast, PI3‐K, PKC, and its upstream regulator pLAT were not significantly down‐regulated by vectorless gravity. Since NF‐κB, AP‐1, and CREB are all regulated by PKA and are transcription factors predicted by microarray analysis to be involved in the altered gene expression in vectorless gravity, the data suggest that PKA is a key player in the loss of T‐cell activation in altered gravity.

Simulated microgravity inhibits the genetic expression of interleukin‐2 and its receptor in mitogen‐activated T lymphocytes

Experiments conducted in space in the last two decades have shown that T lymphocyte activation in vitro is remarkably reduced in microgravity. The data indicate that a failure of the expression of the interleukin‐2 receptor (measured as protein secreted in the supernatant) is responsible of the loss of activity. To test such hypothesis we have studied the genetic expression of interleukin‐2 and of its receptor in concanavalin A‐activated lymphocytes with the RT‐PCR technology. Microgravity conditions were simulated in the fast rotating clinostat and in the random positioning machine. The latter is an instrument introduced recently to study gravitational effects on single cells. Our data clearly show that the expression of both IL‐2 and IL‐2Rα genes is significantly inhibited in simulated 0×g. Thus full activation is prevented.

Activation signals of T lymphocytes in microgravity

Human peripheral blood lymphocytes and monocytes were activated with concanavalin A with or without exogenous recombinant interleukin 1 (IL-1) alone or IL-1 + interleukin 2 (IL-2) under microgravity conditions to test the hypothesis that lack of production of IL-1 by monocytes is the cause of the near total loss of activation observed earlier on several Spacelab flights. The 60 min failure of the on-board 1 x g reference centrifuge at the time of the addition of the activator renders the in-flight data at 1 x g unreliable. However, the data from a previous experiment on SLS-1 show that there is no difference between the results from the in-flight 1 x g centrifuge and 1 x g on ground. The comparison between the data of the cultures at 0 x g in space and of the synchronous control at 1 x g on ground show that exogenous IL-1 and IL-2 do not prevent the loss of activity (measured as the mitotic index) at 0 x g; production of interferon-gamma, however, is partially restored. In contrast to a previous experiment in space, the production of IL-1 is not inhibited.

Signal transduction in T lymphocytes--a comparison of the data from space, the free fall machine and the random positioning machine.

In this paper we discuss the effect of microgravity on T cells and we present the data of studies with two new machines for 0 g simulations. Several experiments in space show that mitogenic T cell activation is lost at 0 g. Immunocytochemistry indicates that such effect is associated with changes of the cytoskeleton. Biochemical studies suggest that the lack of expression of the interleukin-2 receptor is one of the major causes of the loss of activity. In fact, interleukin-2 is the third signal required for full activation. In order to deepen our investigations we are now working with the free-fall machine, FFM, invented by D. Mesland, and with the random positioning machine, RPM, or three-dimensional clinostat, developed by T. Hoson. The FFM produces periods of free-fall lasting approximately 800 ms followed by bounces of 15-30 g lasting 45-60 ms. The RPM eliminates the effect of gravity by rotating biological specimen randomly around two orthogonal axes. While the FFM failed to reproduce the results obtained with T lymphocytes in space, the data from the RPM are in good agreement with those in real microgravity. In fact, the inhibition of the mitotic index in the RPM is 89% compared to static controls. The RPM (as the FFM) can carry markedly larger specimen than the fast rotating clinostat and thus allows to conduct comprehensive studies to select suitable biological objects for further investigations in space.

The Rel/NF‐κB pathway and transcription of immediate early genes in T cell activation are inhibited by microgravity

This study tested the hypothesis that transcription of immediate early genes is inhibited in T cells activated in μg. Immunosuppression during spaceflight is a major barrier to safe, long‐term human space habitation and travel. The goals of these experiments were to prove that μg was the cause of impaired T cell activation during spaceflight, as well as understand the mechanisms controlling early T cell activation. T cells from four human donors were stimulated with Con A and anti‐CD28 on board the ISS. An on‐board centrifuge was used to generate a 1g simultaneous control to isolate the effects of μg from other variables of spaceflight. Microarray expression analysis after 1.5 h of activation demonstrated that μg‐ and 1g‐activated T cells had distinct patterns of global gene expression and identified 47 genes that were significantly, differentially down‐regulated in μg. Importantly, several key immediate early genes were inhibited in μg. In particular, transactivation of Rel/NF‐κB, CREB, and SRF gene targets were down‐regulated. Expression of cREL gene targets were significantly inhibited, and transcription of cREL itself was reduced significantly in μg and upon anti‐CD3/anti‐CD28 stimulation in simulated μg. Analysis of gene connectivity indicated that the TNF pathway is a major early downstream effector pathway inhibited in μg and may lead to ineffective proinflammatory host defenses against infectious pathogens during spaceflight. Results from these experiments indicate that μg was the causative factor for impaired T cell activation during spaceflight by inhibiting transactivation of key immediate early genes.

Cytoskeleton changes and impaired motility of monocytes at modelled low gravity

Summary.Investigations performed in space have shown that gravity changes affect important cellular mechanisms like proliferation, differentiation, genetic expression, cytoskeletal architecture, and motility in lymphocytes, monocytes, and other mammalian cells. In particular, a dramatic depression of the mitogenic in vitro activation of human peripheral blood lymphocytes was observed at low gravity. The hypothesis of the present work is that a reduced interaction between T lymphocytes and monocytes, essential for the second signalling pathway, might be one of the reasons for the observed depression of the in vitro activation of human lymphocytes. Cell motility and with it a continuous rearrangement of the cytoskeletal network within the cell is essential for cell-to-cell contacts. Whereas nonactivated lymphocytes in suspension are highly motile at low gravity, no data are available so far on the motility of adherent monocytes. It thus can be argued that impaired monocyte locomotion and cytoskeletal changes could be responsible for a reduced interaction of monocytes with T lymphocytes. In this study, the locomotion ability of J-111 cells, an adherent monocyte cell line, attached to colloidal gold particles on coverslips and exposed to modelled low gravity in the random positioning machine was found to be severely reduced compared with that of controls and the structures of actin, tubulin, and vinculin were affected.

Movements and interactions of leukocytes in microgravity.

The mitogenic activation of human lymphocytes resuspended in vitro is dramatically reduced in microgravity. As cell-cell contacts are one of the elements essential for activation, the behaviour of human leukocytes (mainly lymphocytes and monocytes as accessory cells) in the presence of the mitogen concanavalin A was studied in the centrifuge microscope NIZEMI at 0 x g. Aggregates (formed by intercellular bindings of membrane glycoproteins via the tetravalent alpha-glucoside ligand concanavalin A) were found at 0 x g as well as at 1 x g already 12 h after the addition of the mitogen. In general, the aggregates observed at 0 x g after an incubation time of 46 and 78 h were smaller than the corresponding aggregates in the ground control. The findings are of primary importance since they confirm the indirect evidence we had from earlier Spacelab experiments and demonstrate that cell-cell contacts are occurring also in microgravity. In addition, single cells in 0 x g show a significant higher locomotion velocity than the cells at 1 x g. The fact that the locomotion capability is not decreased during the 78-h incubation with concanavalin A provides further evidence that the cells are not proceeding through the cell cycle.

Space flight affects motility and cytoskeletal structures in human monocyte cell line J-111.

Certain functions of immune cells in returning astronauts are known to be altered. A dramatic depression of the mitogenic in vitro activation of human lymphocytes was observed in low gravity. T‐cell activation requires the interaction of different type of immune cells as T‐lymphocytes and monocytes. Cell motility based on a continuous rearrangement of the cytoskeletal network within the cell is essential for cell–cell contacts. In this investigation on the International Space Station we studied the influence of low gravity on different cytoskeletal structures in adherent monocytes and their ability to migrate. J‐111 monocytes were incubated on a colloid gold substrate attached to a cover slide. Migrating cells removed the colloid gold, leaving a track recording cell motility. A severe reduction of the motility of J‐111 cells was found in low gravity compared to 1g in‐flight and ground controls. Cell shape appeared more contracted, whereas the control cells showed the typical morphology of migrating monocytes, i.e., elongated and with pseudopodia. A qualitative and quantitative analysis of the structures of F‐actin, β‐tubulin and vinculin revealed that exposure of J‐111 cells to low gravity affected the distribution of the different filaments and significantly reduced the fluorescence intensity of F‐actin fibers. Cell motility relies on an intact structure of different cytoskeletal elements. The highly reduced motility of monocytes in low gravity must be attributed to the observed severe disruption of the cytoskeletal structures and may be one of the reasons for the dramatic depression of the in vitro activation of human lymphocytes.

5-Lipoxygenase-dependent apoptosis of human lymphocytes in the International Space Station: data from the ROALD experiment

The functional adaptation of the immune system to the surrounding environment is also a fundamental issue in space. It has been suggested that a decreased number of lymphocytes might be a cause of immunosuppression, possibly due to the induction of apoptosis. Early activation of 5‐lipoxygenase (5‐LOX) might play a central role in the initiation of the apoptotic program. The goal of the role of apoptosis in lymphocyte depression (ROALD) experiment, flown on the International Space Station as part of the BIO‐4 mission of the European Space Agency, was to ascertain the induction of apoptosis in human lymphocytes under authentic microgravity, and to elucidate the possible involvement of 5‐LOX. Our results demonstrate that exposure of human lymphocytes to microgravity for 48 h onboard the ISS remarkably increased apoptotic hallmarks such as DNA fragmentation (~3‐fold compared to ground‐based controls) and cleaved‐poly (ADP‐ribose) polymerase (PARP) protein expression (~3‐fold), as well as mRNA levels of apoptosis‐related markers such as p53 (~3‐fold) and calpain (~4‐fold); these changes were paralleled by an early increase of 5‐LOX activity (~2‐fold). Our findings provide a molecular background for the immune dysfunction observed in astronauts during space missions, and reveal potential new markers to monitor health status of ISS crew members.—Battista, N., Meloni, M. A., Bari, M., Mastrangelo, N., Galleri, G., Rapino, C., Dainese, E., Finazzi Agrò, A., Pippia, P., Maccarrone, M. 5‐Lipoxygenase‐dependent apoptosis of human lymphocytes in the International Space Station: data from the ROALD experiment. FASEB J. 26, 1791‐1798 (2012). www.fasebj.org

Channelling of deoxyribose moiety of exogenous DNA into carbohydrate metabolism: role of deoxyriboaldolase

In bacteria, the addition of (deoxy)nucleosides or (deoxy)ribose to the growth medium causes induction of enzymes involved in their catabolism, leading to the utilisation of the pentose moiety as carbon and energy source. In this respect, deoxyriboaldolase appears the key enzyme, allowing the utilisation of deoxyribose 5-P through glycolysis. We observed that not only deoxynucleosides, but also DNA added to the growth medium of Bacillus cereus induced deoxyriboaldolase; furthermore, the switch of the culture from aerobic to anaerobic conditions caused a further increase in enzyme activity, leading to a more efficient channelling of deoxyribose 5-P into glycolysis, probably as a response to the low energy yield of the sugar fermentation. In eukaryotes, the catabolism of (deoxy)nucleosides is well known. However, the research in this field has been mainly devoted to the salvage of the bases formed by the action of nucleoside phosphorylases, whereas the metabolic fate of the sugar moiety has been largely neglected. Our results indicate that the deoxyriboaldolase activity is present in the liver of several vertebrates and in a number of cell lines. We discuss our observations looking at the nucleic acids not only as informational molecules, but also as a not negligible source of readily usable phosphorylated sugar.