27 Sep 2013: (2013) Correction: Changes in Mouse Thymus and Spleen after Return from the STS-135 Mission in Space. PLoS ONE 8(9): 10.1371/annotation/e66bdc4e-2409-4582-b163-7bc182db275e. doi: 10.1371/annotation/e66bdc4e-2409-4582-b163-7bc182db275e | View correction
Our previous results with flight (FLT) mice showed abnormalities in thymuses and spleens that have potential to compromise immune defense mechanisms. In this study, the organs were further evaluated in C57BL/6 mice after Space Shuttle Atlantis returned from a 13-day mission. Thymuses and spleens were harvested from FLT mice and ground controls housed in similar animal enclosure modules (AEM). Organ and body mass, DNA fragmentation and expression of genes related to T cells and cancer were determined. Although significance was not obtained for thymus mass, DNA fragmentation was greater in the FLT group (P<0.01). Spleen mass alone and relative to body mass was significantly decreased in FLT mice (P<0.05). In FLT thymuses, 6/84 T cell-related genes were affected versus the AEM control group (P<0.05; up: IL10, Il18bp, Il18r1, Spp1; down: Ccl7, IL6); 15/84 cancer-related genes had altered expression (P<0.05; up: Casp8, FGFR2, Figf, Hgf, IGF1, Itga4, Ncam1, Pdgfa, Pik3r1, Serpinb2, Sykb; down: Cdc25a, E2F1, Mmp9, Myc). In the spleen, 8/84 cancer-related genes were affected in FLT mice compared to AEM controls (P<0.05; up: Cdkn2a; down: Birc5, Casp8, Ctnnb1, Map2k1, Mdm2, NFkB1, Pdgfa). Pathway analysis (apoptosis signaling and checkpoint regulation) was used to map relationships among the cancer–related genes. The results showed that a relatively short mission in space had a significant impact on both organs. The findings also indicate that immune system aberrations due to stressors associated with space travel should be included when estimating risk for pathologies such as cancer and infection and in designing appropriate countermeasures. Although this was the historic last flight of NASA’s Space Shuttle Program, exploration of space will undoubtedly continue.
Citation: Gridley DS, Mao XW, Stodieck LS, Ferguson VL, Bateman TA, et al. (2013) Changes in Mouse Thymus and Spleen after Return from the STS-135 Mission in Space. PLoS ONE 8(9): e75097. doi:10.1371/journal.pone.0075097
Editor: Kamaleshwar Singh, Texas Tech University, United States of America
Received: June 12, 2013; Accepted: August 8, 2013; Published: September 19, 2013
Copyright: © 2013 Gridley et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work was supported by NASA grant NNX09AQ76G and the LLUMC Department of Radiation Medicine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Astronaut health and safety are of key importance to the success of long-term missions in space. The immune system consists of a complex network of organs, tissues and cells that are essential for both maintenance of homeostasis in the body and for defense against infectious microbes and aberrant cell types with potential to progress to cancer. Additional pathologies associated with immune dysfunction include autoimmunity, hypersensitivity and poor wound healing. Although understanding of the underlying mechanisms remains limited, there are numerous reports that stressors in the space flight environment, e.g., microgravity, radiation and psychological stress of confinement, can have a profound impact on immune system status [1-16]. A better understanding of space flight-related health issues is needed, particularly when missions proceed beyond Earth’s protective geomagnetic field. Even more importantly, knowledge gained on the biological effects of factors in the space flight environment is relevant also to the general public as high altitude commercial airline flights and space tourism will undoubtedly continue to increase.
Current predictions of health consequences for space flight personnel, including risk for cancer, remain based on minimal data with large uncertainties [17,18]. If flight-related stressors result in higher mutation rates, as has been reported for bacteria , and/or modulated gene expression patterns [20,21], the possibility for malignant transformation could certainly be increased. This possibility together with radiation-induced immune depression or dysfunction due to a solar particle event (SPE) and reactivation of endogenous viruses with oncogenic potential, e.g. Epstein-Barr virus [22,23], could further increase the risk for cancer. In addition, it has been recently reported that microgravity was the cause for impaired human T cell activation during space flight due to inhibition of immediate early gene transcription .
Induction of thymic lymphoma in mice is a classical model for studying radiation-induced carcinogenesis . In addition, the thymus serves as a source of T cell reconstitution after great loss of this leukocyte type , which could certainly result from radiation exposure during an SPE. Our ground-based mouse studies using space-relevant radiation, including simulated SPE, have shown dramatic loss of leukocytes and effects on other parameters in various body compartments [27-34]. Proliferation of sub-lethally damaged cells during reconstitution, regardless of specific site of origin, increases the risk for transformation to a malignant phenotype. In addition, dysregulation of T cell maturation would have a negative impact on cell functions and interactions after their transit to other body sites, thus compromising capacity to destroy malignant or potentially malignant cells throughout the body. Our interest in the spleen comes from the fact that it is a major source of immune cells that function continuously via humoral and cell-mediated pathways. It is also a major site at which antigen presentation and production of opsonins that facilitate phagocytosis of bacteria and other particulate materials occurs.
We have previously evaluated thymus, spleen and other tissue responses obtained from mice flown to and from the International Space Station (ISS) onboard Space Shuttle Endeavour, i.e. Space Transportation System 77 (STS-77), STS-108 and STS-118 [20,35-42]. Numerous differences were noted in the flight (FLT) mice compared to control animals on ground. An intriguing finding especially relevant to the present study was that the expression of 30/84 genes related to cancer was significantly modulated in the thymus from FLT mice flown on STS-118 compared to ground controls housed in similar animal enclosure modules (AEM).
For the present study, thymus and spleen samples were collected from mice on July 21, 2011 at the Kennedy Space Center (KSC) after a 13-day flight of Space Shuttle Atlantis (STS-135). This was the historic final flight of NASA’s Space Shuttle Program. Our overall hypothesis was that important organs of the immune system in FLT mice would have excessive DNA fragmentation and altered expression of genes related to T helper (Th) cells and cancer. This study was part of the NASA Ames Research Center’s Biospecimen Sharing Program of Commercial Biomedical Test Module-3 (CBTM-3) and collaboration among investigators at the NASA Ames Research Center, BioServe Space Technologies and Amgen, Inc.
Materials and Methods
Animals and Housing
Growing (9 weeks old at start of experiment) female C57BL/6 mice (Charles River Laboratories, Inc., Wilmington, MA) were used in this study. FLT mice were housed in AEM, enclosed habitats with a raised mesh floor that provides ventilation, lighting and waste collection, as well as food and water for the rodents. The ground controls were also housed in flight hardware (AEM group). The environment (e.g., temperature, humidity, CO2 level) for the AEM mice was kept equivalent to that of the FLT mice based on 48 h delayed telemetry data. For example, temperature was 26-28°C and a 12-h light:dark cycle was maintained. Both groups of animals were fed a reformulated rodent food bar diet approved by NASA. Consumption of food and water was monitored daily throughout this study.
The LLU IACUC was consulted for our portion of the study and no protocol was required, since only tissues obtained after euthanasia (no live animals) were processed at our institution. All NASA activities involving vertebrate animals are carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH). The animal studies performed were all approved by multiple ACUC review boards in accordance with NASA’s standard operating procedures including the NASA Ames Research Center ACUC, the NASA, Kennedy Space Center ACUC, the University of Colorado at Boulder IACUC (this is the home institution for the Principal Investigator of the entire study – Dr. V. L. Ferguson - where tissues were collected for analysis as presented in this paper). These three ACUCs reviewed and approved all of our protocols as listed above: "in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the NIH.”
All dissections occurred at the Space Life Sciences Laboratory (SLSL) at KSC within 3-5 h after return of the Space Shuttle Atlantis from a 13-day mission to the ISS. Mice, approximately 11 weeks of age, were euthanized using 4% isoflurane followed by cardiac puncture and exsanguination. Tissues were distributed across a large team of investigators under the guidance/organization of the NASA’s Biospecimen Sharing Program. Thymuses and spleens were extracted and prepared in 4% paraformaldehyde (thymus) or snap frozen in liquid nitrogen (thymus and spleen). The number of samples shipped overnight to Loma Linda University (LLU) for analyses were as follows: thymus (AEM n = 7, FLT n = 4) and spleen (AEM n = 8, FLT n = 5).
Thymus and Spleen Mass, Alone and Relative to Body Mass (RTM, RSM)
Mice were first weighed by personnel at the SLSL shortly after the Space Shuttle landed. The animals were then euthanized and their thymuses and spleens were excised and weighed shortly thereafter. The following formula was used to calculate organ mass relative to body mass: RTM (or RSM) = organ mass (mg)/body mass (g). Means for thymus mass and RTM represent 7 AEM and 4 FLT mice; for spleen mass and RSM, values represent 8 AEM and 5 FLT mice.
TUNEL Assay on Thymus
The thymus was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay according to standard procedures. Data were obtained for 6 AEM and 3 FLT mice. The assay detects DNA fragmentation by incorporating fluorescein-12-dUTP at 3’-OH DNA ends using the recombinant terminal deoxynucleotidyl transferase enzyme (rTdT). Briefly, 6 µm paraffin embedded thymus sections were processed using an in situ cell death detection kit (DeadEnd Fluorometric TUNEL system, catalog no. TB235; Promega, Madison, WI) according to the manufacturer’s instructions. After deparaffinization, rehydration, fixation and permeabilization steps, slides were incubated with a mixture of fluorescein-labeled nucleotides and rTdT at 37°C for 1 h. rTdT catalyzes the incorporation of fluorescein nucleotides to free 3’-OH terminals of DNA fragments. Slides incubated with fluorescein-labeled nucleotide mixture without rTdT served as negative control. Cells treated with DNAse I (10 units/ml; Sigma Aldrich, St. Louis, MO) to induce breaks in DNA strands served as positive control. Fluorescence microscopy was performed with a microscope (BX61; Olympus, Central Valley, PA). For quantitative analysis, numbers of apoptotic cells were counted in nine sections from each animal. The surface of each section was measured on digital microphotographs using ImageJ v. 1.4 (available as freeware from http://rsbweb.nih.gov/ij/). Density profiles were expressed as mean number of apoptotic cells per square millimeter. Similar procedures have been described in the literature [43,44].
Gene Expression in Thymus and Spleen
Frozen thymus and spleen samples were thawed before quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. For the thymus, expression of 84 genes using the Mouse Th1-Th2-Th3 RT2 ProfilerTM PCR Array (PAMM-034A) and 84 genes using the Mouse Cancer PathwayFinder RT2 ProfilerTM PCR Array (PAMM-033A) was determined. For genes in the spleen, the same cancer array was used as for the thymus. Both arrays were obtained from SABiosciences/Qiagen Corp., Frederick, MD and the RT-PCR was performed at the SABiosciences Technical Core. The details of the procedures have been previously reported . Briefly, the extracted RNA was run on a bioanalyzer (Agilent Technologies, Santa Ana, CA) and its integrity was confirmed by assessing 18s and 28s rRNA peaks and by RNA integrity number (RIN). Spectrophotometrical measurements showed that 260/280 and 260/230 ratios for all samples were above 2.0 and 1.7, respectively. PCR reactions were performed on a Biorad cycler (Bio-Rad Laboratories, Hercules, CA) using RT2 Real-TimeTM SYBR Green PCR Master Mix PA-011 (SABiosciences/Qiagen) and relative changes were calculated using the Ct (threshold cycle) method; five housekeeping genes, RT controls and positive PCR controls were included. Comparison was made between the FLT versus AEM ground controls.
Ingenuity Pathway Analysis (IPA) (Ingenuity® Systems, Redwood City, CA; www.ingenuity.com) was used to map some of the more important relationships among the characterized cancer-related genes. The assessments included both the thymus and spleen. Due to the nature of the genes assessed, the limited number of significant changes and the tissue sources, we relaxed the statistical constraints for the pathway analysis and focused on two specific canonical pathways: Myc Mediated Apoptosis Signaling and Cell Cycle: G1/S Checkpoint Regulation. Comprehensive description of the legend in the IPA figures can be found here: http://ingenuity.force.com/ipa/articles/Feature_Description/Legend.
Gene expression data were analyzed using Student’s t-test at the SABiosciences/Qiagen Technical Core. This statistical test is widely accepted as valid when used for results after relative quantification with RT-PCR, as was done in the present study. Other data were analyzed using Student’s T test (Sigmaplot 12.3; Systat Software, Inc., Chicago, IL). Means and standard error of means (SEM) are reported. P values less than 0.05 were selected to indicate significance.
Food and Water Consumption
Intake of food and water was monitored daily. Food consumed per mouse per day was virtually identical for the AEM and FLT groups, i.e., 4.08 g and 4.09 g, respectively. Water consumption, however, between the two groups was somewhat different: 3.38 ml/mouse/day for the AEM group and 2.73 ml/mouse/day for the FLT group.
Thymus and Spleen Mass Alone and Relative Organ Mass (RTM and RSM)
Prior to take-off, body mass was very similar for the two groups; the FLT and AEM mice weighed 20.3 ± 1.2 g and 20.7 ± 1.2 g, respectively. Body mass shortly after landing was also not significantly different between the two groups: 18.1 ± 0.5 g (FLT) and 19.3 ± 0.5 g (AEM). The mass of both organs alone and relative to body mass are shown in Figure 1. Although the thymus mass and RTM values were relatively low for the FLT group compared to the AEM group, statistical significance was not obtained possibly due to low sample size. For the FLT group, the decrease in thymus mass alone approached significance (P=0.101 vs. AEM). However, Figure 1 also shows that the FLT animals had significantly lower spleen mass and RSM values compared to the AEM ground controls (P<0.05).
Figure 1. Thymus and spleen mass alone and relative to body mass (RTM, RSM).
Bars represent mean ± SEM. For thymus mass and RTM, n = 7 (AEM group) and n = 4 (FLT group). For spleen mass and RSM, n = 8 (AEM group) and n = 5 (FLT group). AEM, animal enclosure module (control mice housed on ground); FLT, flight mice. *P<0.05 vs. AEM. For FLT vs. AEM thymus mass, P=0.101.doi:10.1371/journal.pone.0075097.g001
DNA Fragmentation in Thymus
Representative images of DNA fragmentation in thymuses obtained from two mice in each of the groups are presented in the top four panels of Figure 2. Quantification of fluorescence, based on the TUNEL assay, indicated that there was a greater impact on the mice flown in space (Figure 2, bottom panel). The density of TUNEL-positive cells was significantly higher for FLT group compared to the AEM controls (P<0.01).
Figure 2. DNA fragmentation in thymus.
The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used. The top panel shows two representative examples from each of the groups. AEM (A34 and A42): control mice housed on ground in animal enclosure modules; FLT (F64 and F74): flight mice. The bottom panel shows the results after quantification; bars represent mean ± SEM for 6 AEM and 3 FLT mice. *P<0.01 vs. AEM.doi:10.1371/journal.pone.0075097.g002
Th Cell Subset-related Genes in Thymus
Genes related to the T cells with significant expression differences in FLT thymuses compared to the AEM counterparts housed on ground (P<0.05) are shown in Table 1. Of the 6 genes that were affected in the FLT group out of a total 84 evaluated, four were up-regulated (Il10, Spp1, Il18r1, Il18bp) and two were down-regulated (Il6, Ccl7). Fold-change was greater than 2.0 for all of these genes except Il18bp. Of the remaining genes, the expression of only Il15 approached statistical significance (fold-change was 1.5; P<0.1 vs. AEM group).
|Spp1||2.77||Secreted phosphoprotein 1; also known as osteopontin|
|Il18r1||2.01||Interleukin 18 receptor 1|
|Il18bp||1.75||Interleukin 18 binding protein|
|Ccl7||-2.37||Chemokine (C-C motif) ligand 7|
Cancer-related Genes in Thymus
Table 2 shows that 15 cancer-related genes were significantly modulated in FLT thymuses compared to the AEM ground controls (P<0.05) out of a total of 84 evaluated. Of these genes, 11 were up-regulated (Figf, Ncam1, Serpinb2, Itga4, Hgf, Fgfr2, Pik3r1, Sykb, Pdgfa, Casp8, Igf1) and four were down-regulated (Myc, E2f1, Mmp9, Cdc25a). Fold-change was greater than 2.0 in three of these genes and all were up-regulated. Table 2 also presents the genes with a trend toward significant modulation (P = 0.05 to <0.1); nine were up- and one was down-regulated. The placement of highlighted genes in the Myc and G1/S pathways are shown in Figures 3 and 4, respectively.
|P<0.05||Figf||2.75||C-fos induced growth factor|
|Ncam1||2.49||Neural cell adhesion molecule 1|
|Serpinb2||2.47||Serine (or cysteine) peptidase inhibitor, clade B, member 2|
|Itga4||1.82||Integrin alpha 4|
|Hgf||1.80||Hepatocyte growth factor|
|Fgfr2||1.68||Fibroblast growth factor receptor 2|
|Pik3r1||1.56||Phosphatidylinositol 3-kinase, regulatory subunit, polypeptide 1|
|Sykb||1.55||Spleen tyrosine kinase|
|Pdgfa||1.54||Platelet derived growth factor, alpha|
|Igf1||1.39||Insulin-like growth factor 1|
|E2f1||-1.82||E2F transcription factor 1|
|Mmp9||-1.78||Matrix metallopeptidase 9|
|Cdc25a||-1.27||Cell division cycle 25 homolog A (S. pombe)|
|P = 0.05 to <0.1||Serpine1||3.08||Serine (or cysteine) peptidase inhibitor, clade B, member 2|
|Plaur||1.92||Plasminogen activator, urokinase receptor|
|Itga3||1.89||Integrin alpha 3|
|Col18a1||1.71||Collagen, type XVIII, alpha 1|
|Itga2||1.69||Integrin alpha 2|
|Egfr||1.56||Epidermal growth factor receptor|
|Nfkb1||1.21||Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1|
|Cdk2||-1.35||Cyclin-dependent kinase 2|
Figure 3. Impact of space flight on Myc-mediated apoptosis signaling in the thymus.
Pathways are modified versions of the canonical pathways generated with IPA software. The up-down-regulated genes are from the cancer gene array. Red = Up-regulated. Green = Down-regulated. White = Not measured. Further description of the legend can be found at the IPA website indicated in the Materials and Methods. This analysis was based on gene expression levels in thymuses obtained from 4 AEM and 3 FLT mice.doi:10.1371/journal.pone.0075097.g003
Figure 4. Impact of space flight on G1/S checkpoint regulation in the thymus.
Pathways are modified versions of the canonical pathways generated with IPA software. The up-down-regulated genes are from the cancer gene array. Red = Up-regulated. Green = Down-regulated. White = Not measured. Further description of the legend can be found at the IPA website indicated in the Materials and Methods. This analysis was based on gene expression levels in thymuses obtained from 4 AEM and 3 FLT mice.doi:10.1371/journal.pone.0075097.g004
Cancer-related Genes in Spleen
Table 3 presents the 8/84 significantly modulated genes in spleens from FLT mice when compared to the AEM ground controls (P<0.05). Of these genes, only one was up-regulated (Cdkn2a), whereas seven were down-regulated (Map2k1, Casp8, Birc5, Mdm2, Pdgfa, Nfkb1, Ctnnb1). Also shown in Table 3 are the genes with near-significant modulation (P=0.05 to <0.1); five were up- and six were down-regulated. The placement of highlighted genes in the Myc and G1/S pathways are presented in Figures 5 and 6, respectively.
|P<0.05||Cdkn2a||1.47||Cyclin-dependent kinase inhibitor 2A|
|Map2k1||-1.67||Mitogen-activated protein kinase kinase 1|
|Birc5||-1.48||Baculoviral IAP repeat-containing 5|
|Mdm2||-1.45||Transformed mouse 3T3 cell double minute 2|
|Pdgfa||-1.42||Platelet derived growth factor, alpha|
|Nfkb1||-1.31||Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1|
|Ctnnb1||-1.26||Catenin (cadherin associated protein), beta 1|
|P = 0.05 to <0.1||Plau||1.62||Plasminogen activator, urokinase|
|Itga3||1.48||Integrin alpha 3|
|Figf||1.40||C-fos induced growth factor; Vegfd|
|Mmp2||1.32||Matrix metallopeptidase 2|
|Plaur||1.30||Plasminogen activator, urokinase receptor|
|Tert||-1.65||Telomerase reverse transcriptase|
|Itgb3||-1.31||Integrin beta 3|
|Nme4||-1.28||Non-metastatic cells 4, protein expressed in|
|Trp53||-1.26||Transformation related protein 53|
|Grb2||-1.24||Growth factor receptor bound protein 2|
|Bax||-1.23||Bcl2-associated X protein|
Figure 5. Impact of space flight on Myc-mediated apoptosis signaling in the spleen.
Pathways are modified versions of the canonical pathways generated with IPA software. The up-down-regulated genes are from the cancer gene array. Red = Up-regulated. Green = Down-regulated. White = Not measured. Further description of the legend can be found at the IPA website indicated in the Materials and Methods. This analysis was based on gene expression levels in spleens obtained from 5 AEM and 4 FLT mice.doi:10.1371/journal.pone.0075097.g005
Figure 6. Impact of space flight on G1/S checkpoint regulation in the spleen.
Pathways are modified versions of the canonical pathways generated with IPA software. The up-down-regulated genes are from the cancer gene array. Red = Up-regulated. Green = Down-regulated. White = Not measured. Further description of the legend can be found at the IPA website indicated in the Materials and Methods. This analysis was based on gene expression levels in spleens obtained from 5 AEM and 4 FLT mice.doi:10.1371/journal.pone.0075097.g006
The data show that there was some degree of atrophy in both organs collected from the FLT mice shortly after landing compared to AEM controls, although statistical significance was obtained only for the spleen. Overall, this is consistent with our previous findings on rodents flown in space and is likely due to a combination of stressors that lead to loss of sensitive cells in these body sites. We have previously found decreases in leukocyte populations in rodent models after three space shuttle flights (STS-77, STS-108 and STS-118) that had duration times similar to the STS-135 flight [20,37,40,41]. A possible contributing factor is the relatively low volume of water consumed by the FLT mice, i.e., approximately 19% less than the amount consumed by the AEM controls. Hyperosmolarity, which occurs during dehydration, has been reported to decrease cellularity in spleens and thymuses of mice . However, the degree of dehydration did not appear to be severe; personnel inspecting the animals after landing found no obvious signs of ill health.
Evaluation of thymus samples using the TUNEL assay revealed that the FLT group had significantly more DNA fragmentation compared to the AEM group. Damage to DNA has long been associated with apoptotic cell death. Research indicates, however, that necrosis, autophagy and mitotic catastrophe are also possibilities [46,47]. A decrease in leukocytes, regardless of specific mechanism, could certainly contribute to low mass of immune-related organs. It seems likely that the increased DNA fragmentation in FLT thymuses includes the combination of microgravity and increased radiation while in space plus the hypergravity experienced during take-off and landing (maximum of approximately 3-G). A study of brains from rats kept under simulated weightlessness followed by hypergravity found that the highest level of apoptotic cells based on TUNEL-positivity occurred in animals subjected to both conditions . In addition, studies with cells (including lymphocytes) subjected to simulated microgravity on Earth have demonstrated numerous abnormalities including mitochondrial disruption and apoptosis .
Because we were part of NASA’s large BSP-organized team, the tissue fixation process for the TUNEL assay was not ideal. In terms of section thickness, 6 µm was chosen as the standard for all organ samples scheduled for histology, when slices <4 µm would have been better for this particular assay. The thymus has high cell density, thus making it a challenge for us to count TUNEL-positive cells based on total cell nuclei. In order to normalize the quantification process and minimize stochastic error, we therefore adapted our quantification methods and counting criteria using ImageJ software on digital microphotographs. Cell density profiles were expressed as mean number of positive cells per square millimeter for the TUNEL assay. Similar procedures have been described in the literature for various tissues that include the thymus [43,44,50].
Our results do show increased DNA fragmentation in the thymuses from FLT mice, thus supporting our hypothesis. This information increases the importance to study immune/cellular mechanisms triggered by stress stimuli, especially in the context of space flight.
The T cell-related genes evaluated in the thymus included those associated with Th1 and Th2 cytokines, CD4+ T cell markers, the T regulatory (Treg) cell network, T and B cell activation, relevant transcription factors and transcription regulators. The data showed that the expression of six genes was significantly different in the FLT group compared to the AEM group. The fact that relatively few of these genes displayed significant modulation may be at least partly due to the relative immaturity of thymocytes compared to Th cells in the periphery. Another contributing factor may be related to thymic atrophy in the FLT mice which likely triggered regenerative mechanisms that may have further increased the proportion of immature cells. The most up-regulated gene was Il10 that encodes interleukin-10 (IL-10), a cytokine produced by several leukocyte types including T lymphocytes. IL-10 is a potent immunosuppressor that is crucial for limitation and ultimate termination of inflammatory responses . However, it is a pleiotropic cytokine that can also enhance B cell survival, antibody production and phagocyte function . In our previous study of STS-118 mice, IL-10 level was high in supernatants of spleen cells from the FLT animals after activation with anti-CD3 monoclonal antibody , indicating that the capacity to produce this cytokine was enhanced after landing. In blood from astronauts who experienced reactivation of latent herpes virus infection during short-duration spaceflights, elevated IL-10 has been reported shortly after landing .
Genes encoding IL-18 receptor 1 (Il18r1) and IL-18 binding protein (Il18bp) were also up-regulated in FLT thymuses compared to the AEM group. IL-18, together with several other cytokines, induces phenotypic and functional changes in immature cells within the thymus that can facilitate T cell development . Expression of the Il18 gene itself in the FLT and AEM thymuses, however, was similar at the time of assessment. The other up-regulated gene was Spp1 that encodes secreted phosphoprotein 1 (perhaps better known as osteopontin, OPN). OPN is a multifunctional protein involved in adhesion, signaling and survival of cells, as well as tissue remodeling and immune regulation [55,56]. Its level is increased in response to various stressors . In a space flight simulation with hind limb unloaded mice (wild type and OPN knockouts), it was demonstrated that OPN was required for thymus and spleen atrophy and loss in body mass . Hence, it seems possible that enhanced OPN production (up-regulated Spp1) may have been involved in the relatively low thymus mass noted in the present study.
Il6, encoding IL-6, was one of the two down-regulated genes in FLT compared to AEM thymuses. Since this cytokine is known to cause thymic involution , the decreased expression of Il6 in our FLT mice is consistent with the need for regeneration. Furthermore, since IL-6 stimulates immune responses when needed, e.g. during infection , susceptibility to microbes may be increased when Il6 is down-regulated. However, after a 7-day mission in space (STS-54), mitogen-induced production of IL-6 by rat thymocytes was enhanced compared to control rats housed on ground . Ccl7 was the other down-regulated gene in the FLT versus AEM comparison. This gene encodes chemokine (C-C motif) ligand 7 (CCL7; originally called monocyte-specific chemokine 3, MCP-3), a small protein that attracts monocytes and regulates macrophage function . Thus, the decreased expression of Ccl7 in the present study suggests an anti-inflammatory response in FLT thymuses.
Status of genes relevant to cancer was determined in both thymus and spleen. The array included genes important in cell cycle control, DNA damage repair, apoptosis, cell senescence, signal transduction, transcription, adhesion, angiogenesis, invasion and metastasis. Of the genes we assessed, more were affected in the thymus than in the spleen when the FLT group was compared with the AEM controls. Of the 84 genes evaluated in each organ, the expression of only Figf, Ncam1 and Serpinb2 was modulated by more than 2-fold. All three of these genes were in the thymus and all three were up-regulated. Of these genes, the expression of Figf, encoding c-fos induced growth factor, was the most enhanced. This protein belongs in the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family and is also known as VEGF-D. It promotes angiogenesis and endothelial cell growth, thus facilitating tumor progression [63,64]. Enhanced expression of Figf was also evident in our previous study of mouse thymuses after return from the STS-118 flight . Ncam1 encodes neural cell adhesion molecule (NCAM, often referred to as cluster of differentiation 56 or CD56). Expression of CD56 on a variety of malignant and pre-malignant cells has been reported in patients, including those with precursor T-cell lymphoblastic leukemia/lymphoma (T-ALL/LBL) cells [65-68]. The Serpinb2 (serine peptidase inhibitor, clade B member 2; also known as plasminogen activator inhibitor 2 or PAI-2) is a member of the large serpin family. Although the functions of this molecule are not entirely clear, there is evidence that it may decrease risk for tumor metastasis, inhibit apoptosis and protect against cytotoxic effects of viruses [69-71]. Casp8 and Pdgfa were the only genes significantly affected in both the thymus and spleen. However, these two genes were up-regulated in the thymus, but down-regulated in the spleen, indicating that body compartment can make a great difference in the results.
In our previous study using the same cancer gene array on thymus samples from mice flown on STS-118, a very different profile was obtained . The number of cancer-related genes affected in the thymus from FLT mice in the present study (15/84) was less than what we found in FLT mice flown on STS-118 (30/84). Previously, there were four up- and nine down-regulated genes in the FLT group versus the AEM group, but only the enhanced expression of Figf was noted after both flights. The most likely explanation for this discrepancy is that the STS-118 mice were subjected to muscle strength testing and nuclear magnetic resonance body composition measurements by investigators prior to euthanasia and collection of samples for further testing at our institution. The extent of these procedures, and the resulting stress generated by involving the mice in these assays, on gene expression profiles is not known. Possible additional contributing factors to the discrepancy between the two flights are differences in the method of euthanasia and the commercial source of the mice . The impact of these latter factors, if present, seems likely to be minor. We believe that the results from the present study more accurately reflect the modulatory effects of stressors associated with space flight, since mice flown on STS-118 underwent additional procedures (as noted above) prior to sacrifice and organ collection.
The cancer gene expression profiles were, indeed, quite different for the thymus and spleen. However, this is not entirely surprising, since gene expression tends to reflect organ-specific cell composition and function at the molecular level. Organ variability in gene expression has been previously reported in animal models, as well as in humans [73,74]. Furthermore, lack of correlation in gene regulation between tissue types is common under microgravity conditions. In fact, most studies have described anomalies in the typical gene expression patterns in well-known pathways. For example, Singh and colleagues have detected epigenetic changes under microgravity conditions , thereby suggesting that these changes may be the result of alternative folding/conformation state of the DNA which then can give rise to alternative gene expression.
To determine the effects of space flight on relevant processes, we relaxed the statistical constraints to focus on fold changes and limited the analysis to cancer-related genes in two specific canonical pathways: Myc Mediated Apoptosis Signaling and Cell Cycle: G1/S Checkpoint Regulation. The importance of these pathways in carcinogenesis has been well documented [76,77]. Although pathway analysis works best with large numbers of genes and we included only the cancer-related genes, we believe this analysis is justified for three reasons: 1) the generated data are unique; 2) the availability of the shared tissues was very limited and repetition is unlikely in the near future as this was the last flight of NASA’s Space Shuttle Program; and 3) the sample size for this study was somewhat limited, particularly as transformation to an abnormal phenotype and immune function were not primary goals for this space shuttle experiment.
In the pathway analysis, Myc and p53 appear to be down-regulated in both the thymus and spleen suggesting that DNA damage-induced apoptosis is not a major factor after space flight (Figures 3 and 5). Similarly, mitochondria-dependent apoptosis also appears to be down-regulated in both organs. In the spleen, this is indicated by the down-regulation of caspase 8 and BAX, both known to induce mitochondrial membrane permeabilization. While these genes are actually up-regulated in the thymus, along with a similar increase in the expression of BAD, their impact on membrane permeability are minimized by a corresponding up-regulation of BCL2 in the mitochondria and the activation of IFG-1/Ras pathways in the cytoplasm. The latter results in the activation of AKT and the subsequent phosphorylation of both BAD and caspase 8. When phosphorylated, BAD cannot bind to BCL2 and caspase 8 cannot be activated, thereby minimizing mitochondrial-dependent apoptosis. Although there is a similar up-regulation of IGF-1 and AKT in the spleen, the down-regulation of BAX in the mitochondria makes a full activation of the IFG-1/Ras pathway unnecessary.
Surprisingly, despite the down-regulation of apoptosis signaling and space flight-induced decreases in virtually all immune populations (often reported previously), cell cycle progression appears to be arrested in both the spleen and thymus based on pathway analysis (Figures 4 and 6). This is indicated by a space flight-induced down-regulation of virtually all G1/S checkpoint genes (including Cdc25A, CDK2, CDK4/6, Rb and E2F) with a corresponding up-regulation of cell cycle progression inhibitors (including both p16INK4 and p21/Cip1). This suggests that immunocyte recovery, if occurring at all immediately after flight, is not a primary function of the thymus or spleen.
Because p21 is associated also with cell differentiation, the up-regulation of this gene in the thymus, combined with the apparent cell cycle arrest, suggests that T cell maturation may be enhanced. Although the expression of Il18 was not affected, the increased expression of IL18bp and IL18r1 tends to support this possibility [54,78]. However, genes encoding cytokines known to be involved in thymic regeneration, e.g., Il7, Il15, Il17, Il22 and Il23 [78-81] were not significantly up-regulated by space flight. Indeed, the cytokine-related genes that were altered in the thymus (Ccl7, Il6, Il10 and Il18bp) are all involved in the down-regulation of cell-mediated immunity, including T cell differentiation. Similarly, with the exception of Il18r, there were no significant space flight-induced changes in many of the surface receptor genes commonly associated with Th cell differentiation that were included in the array (Cd28, Cd40, Cd40lg, Cd80, Cd86, IL2Rα, Il4ra and Il12rb2). A previous study of granulocytic lineage cells in the bone marrow from mice shortly after Space Shuttle Endeavour (STS-118) landed found a higher level of differentiated and lower level of less differentiated cells compared to AEM-housed ground controls, and there was no radical disruption in the distribution of subpopulations .
In conclusion, the data clearly show that shortly after a 12.8 or ~13-day flight in space there was a significant impact on the assessed parameters. Spleen mass was especially reduced, increased DNA fragmentation was noted in the thymus and a number of important genes associated with T cells and cancer were modulated by flight compared to the AEM ground controls. Although gene expression in FLT mice was not always greater than 2-fold different compared to the AEM controls, we believe it is important to present all significantly modified genes due to the uniqueness of the study and because there is no compelling evidence that a greater than 2-fold change always has a greater biological impact than a less than 2-fold change. In future space flight studies, it would be important to evaluate protein levels associated with these genes. Pathway analysis suggested down-regulation of apoptosis and inhibition of cell cycling. Since the FLT group had an abundance of apoptotic/dead cells, the down-regulation of apoptosis signaling and cell cycling pathways in the remaining viable cells (those still capable of gene expression) may reflect a great need for survival. Multiple factors could certainly be responsible for the changes, including altered gravity, increased radiation and stress of handling, as well as the body’s attempt to regain homeostasis after return to Earth. It remains to be determined whether the aberrations are persistent and translate into increased risk for pathologies such as leukemia/lymphoma. These malignancies have been identified as a dominant risk to astronauts exposed to an SPE (as indicated at the NASA Technical Reports Server). Finally, in September 2011, NASA announced that it will proceed with a new Space Launch System that will allow human travel into space further than ever before. Thus, a better understanding of the precise mechanisms that result in immunological changes and possible health consequences associated with space travel should remain a high research priority so that appropriate countermeasures can be developed.
We would like to thank Amgen, Inc. and their support team for the opportunity to participate in this study and BioServe Space Technologies at the University of Colorado at Boulder for organizing and overseeing this project. We would also like to thank all team members of the Ferguson, Bateman and Bouxsein labs who participated in the tissue processing at KSC. We would also like to thank Paula Dumars and Vera Vizir of the BSP and Ames Research Center, as well as the KSC SLSL support staff. Finally, the strong support of Dr. James M. Slater at LLUMC for space-related research is greatly appreciated.
Conceived and designed the experiments: DSG LSS VLF TAB MJP. Performed the experiments: DSG XWM LSS VLF TAB MM CEC TAM JMS MJP. Analyzed the data: DSG XWM LSS VLF TAB MM CEC TAM JMS MJP. Wrote the manuscript: DSG XWM MJP.
- 1. Allebban Z, Ichiki AT, Gibson LA, Jones JB, Congdon CC et al. (1994) Effects of space flight on the number of rat peripheral blood leukocytes and lymphocyte subsets. J Leukoc Biol 55: 209-213. PubMed: 8301218.
- 2. Boonyaratanakornkit JB, Cogoli A, Li CF, Schopper T, Pippia P et al. (2005) Key gravity-sensitive signaling pathways drive T cell activation. FASEB J 19: 2020-2022. PubMed: 16210397.
- 3. Chapes SK (2004) Lessons from Immune 1-3: what did we learn and what do we need to do in the future? J Gravit Physiol 11: 45-48.
- 4. Chapes SK, Morrison DR, Guikema JA, Lewis ML (1992) Spooner BS. Cytokine secretion by immune cells in space. J Leukoc Biol 52: 104-110. PubMed: 1640164.
- 5. Congdon CC, Allebban Z, Gibson LA, Kaplansky A, Strickland KM et al. (1996) Lymphatic tissue changes in rats flown on Spacelab Life Sciences-2. J Appl Physiol 81: 172-177. PubMed: 8828660.
- 6. Fedorenko BS, Snigireva GP, Ivanov AA (2010) Cytogenetic analysis of cosmonaut’s blood lymphocytes exposed to low space radiation doses and its place in clinical-physiological health monitoring. Aviakosm Ekol Med 44: 9-12.
- 7. George K, Chappell LJ, Cucinotta FA (2010) Persistence of space radiation induced cytogenetic damage in the blood lymphocytes of astronauts. Mutat Res 701: 75-79. doi:10.1016/j.mrgentox.2010.02.007. PubMed: 20176126.
- 8. Guéguinou N, Huin-Schohn C, Bascove M, Bueb JL, Tschirhart E et al. (2009) Could spaceflight-associated immune system weakening preclude the expansion of human presence beyond Earth’s orbit? J Leukoc Biol 86: 1027-1038. doi:10.1189/jlb.0309167. PubMed: 19690292.
- 9. Hatton JP, Gaubert F, Lewis ML, Darsel Y, Ohlmann P et al. (1999) The kinetics of translocation and cellular quantity of protein kinase C in human leukocytes are modified during spaceflight. FASEB J 13 Suppl: S23-33. PubMed: 10352142.
- 10. Ichiki AT, Gibson LA, Jago TL, Strickland KM, Johnson DL et al. (1996) Effects of space flight on rat peripheral blood leukocytes and bone marrow progenitor cells. J Leukoc Biol 60: 37-43. PubMed: 8699121.
- 11. Kaur I, Simons ER, Kapadia AS, Ott CM, Pierson DL (2008) Effect of space flight on ability of monocytes to respond to endotoxins of gram-negative bacteria. Clin Vaccine Immunol 15: 1523-1528. doi:10.1128/CVI.00065-08. PubMed: 18768671.
- 12. Mehta SK, Kaur I, Grimm EA, Smid C, Feeback DL et al. (2001) Decreased non-MHC restricted (CD56+) killer cell cytotoxicity after spaceflight. J Appl Physiol 91: 1814-1818. PubMed: 11568167.
- 13. Misurova E, Tigranyan RA, Praslicka M (1981) Changes of deoxyribonucleoprotein in the spleen, thymus and liver of rats exposed to weightlessness and artificial gravity aboard the Cosmos biosatellites. Adv Space Res 1: 225-230. doi:10.1016/0273-1177(81)90111-3. PubMed: 11541714.
- 14. Sonnenfeld G, Mandel AD, Konstantinova IV, Berry WD, Taylor GR et al. (1992) Space flight alters immune cell function and distribution. J Appl Physiol 73: 191S-195S. PubMed: 1526951.
- 15. Sonnenfeld G, Miller ES (1993) The role of cytokines in immune changes induced by spaceflight. J Leukoc Biol 54: 253-258. PubMed: 8371055.
- 16. Stowe RP, Sams CF, Pierson DL (2003) Effects of mission duration on neuroimmune responses in astronauts. Aviat Space Environ Med 74: 1281-1284. PubMed: 14692473.
- 17. Cucinotta FA, Durante M (2006) Cancer risk from exposure to galactic cosmic rays: implications for space exploration by human beings. Lancet Oncol 7: 431-435. doi:10.1016/S1470-2045(06)70695-7. PubMed: 16648048.
- 18. Durante M, Cucinotta FA (2008) Heavy ion carcinogenesis and human space exploration. Nat Rev Cancer 8: 465-472. doi:10.1038/nrc2391. PubMed: 18451812.
- 19. Moeller R, Reitz G, Nicholson The Protect Team WL, Horneck G (2012) Mutagenesis in bacterial spores exposed to space and simulated martian conditions: data from the EXPOSE-E space flight experiment PROTECT. Astrobiology 12: 457-468. doi:10.1089/ast.2011.0739. PubMed: 22680692.
- 20. Baqai FP, Gridley DS, Slater JM, Luo-Owen X, Stodieck LS et al. (2009) Effects of space flight on innate immune function and antioxidant gene expression. J Appl Physiol 106: 1935-1942. doi:10.1152/japplphysiol.91361.2008. PubMed: 19342437.
- 21. Guo F, Li Y, Liu Y, Huang J, Zhang Z et al. (2012) Identification of genes associated with tumor development in CaSki cells in the cosmic space. Mol Biol Rep 39: 6923-6931. doi:10.1007/s11033-012-1519-x. PubMed: 22302396.
- 22. Kaneda A, Matsusaka K, Aburatani H, Fukayama M (2012) Epstein-Barr virus infection as an epigenetic driver of tumorigenesis. Cancer Res 72: 3445-3450. doi:10.1158/1538-7445.AM2012-3445. PubMed: 22761333.
- 23. Stowe RP, Kozlova EV, Sams CF, Pierson DL, Walling DM (2011) Latent and lytic Epstein-Barr virus gene expression in the peripheral blood of astronauts. J Med Virol 83: 1071-1077. doi:10.1002/jmv.22079. PubMed: 21503923.
- 24. Chang TT, Walther I, Li CF, Boonyaratanakornkit J, Galleri G et al. (2012) The Rel/NF-κB pathway and transcription of immediate early genes in T cell activation are inhibited by microgravity. J Leukoc Biol 92: 1133-1145. doi:10.1189/jlb.0312157. PubMed: 22750545.
- 25. Kominami R, Niwa O (2006) Radiation carcinogenesis in mouse thymic lymphomas. Cancer Sci 97: 575-581. doi:10.1111/j.1349-7006.2006.00218.x. PubMed: 16827796.
- 26. Mackall CL, Fleisher TA, Brown MR, Andrich MP, Chen CC et al. (1995) Age, thymopoiesis, and CD4+ T-lymphocyte regeneration after intensive chemotherapy. N Engl J Med 332: 143-149. doi:10.1056/NEJM199501193320303. PubMed: 7800006.
- 27. Gridley DS, Coutrakon GB, Rizvi A, Bayeta EJM, Luo-Owen X et al. (2008) Low dose photons modify liver response to simulated solar particle event protons. Radiat Res 169: 280-287. doi:10.1667/RR1155.1. PubMed: 18302490.
- 28. Gridley DS, Luo-Owen X, Rizvi A, Makinde AY, Pecaut MJ et al. (2010) Low-dose photon and simulated solar particle event proton effects on Foxp3+ T regulatory cells and other leukocytes. Technol Cancer Res Treat 9: 637-649. PubMed: 21070086.
- 29. Gridley DS, Rizvi A, Luo-Owen X, Makinde AY, Coutrakon GB et al. (2008) Variable hematopoietic responses to acute photons, protons and simulated solar particle event protons. In Vivo 22: 159-170. PubMed: 18468399.
- 30. Gridley DS, Rizvi A, Makinde AY, Luo-Owen X, Mao XW et al. (2013) Space-relevant radiation modifies cytokine profiles, signaling pathways and Foxp3+ T cells. Int J Radiat Biol 89: 26-35. doi:10.3109/09553002.2012.715792. PubMed: 22852758.
- 31. Mao XW, Crapo JD, Gridley DS (2012) Mitochondrial oxidative stress-induced apoptosis and radioprotection in proton-irradiated rat retina. Radiat Res 178: 118-125. doi:10.1667/RR2821.1. PubMed: 22780102.
- 32. Rizvi A, Pecaut MJ, Gridley DS (2011) Low-dose gamma-rays and simulated solar particle event protons modify splenocyte gene and cytokine expression patterns. J Radiat Res 52: 701-711. doi:10.1269/jrr.10107. PubMed: 21971034.
- 33. Rizvi A, Pecaut MJ, Slater JM, Subramaniam S, Gridley DS (2011) Low-dose γ-rays modify CD4+ T cell signalling response to simulated solar particle event protons in a mouse model. Int J Radiat Biol 87: 24-35. doi:10.3109/09553002.2010.518206. PubMed: 21142612.
- 34. Tian J, Pecaut MJ, Coutrakon GB, Slater JM, Gridley DS (2009) Response of extracellular matrix regulators in mouse lung after exposure to photons, protons and simulated solar particle event protons. Radiat Res 172: 30-41. doi:10.1667/RR1670.1. PubMed: 19580505.
- 35. Gridley DS, Nelson GA, Peters LL, Kostenuik PJ, Bateman TA et al. (2003) Genetic models in applied physiology: selected contribution: effects of space flight on immunity in the C57BL/6 mouse. II. Activation, cytokines, erythrocytes, and platelets. J Appl Physiol 94: 2095-2103.. doi: 10.1152/japplphysiol.01053.2002
- 36. Gridley DS, Pecaut MJ, Green LM, Herrmann EC, Bianski B et al. (2012) Effects of space flight on the expression of liver proteins in the mouse. J Proteomics Bioinform 5: 256-261.
- 37. Gridley DS, Slater JM, Luo-Owen X, Rizvi A, Chapes SK et al. (2009) Spaceflight effects on T lymphocyte distribution, function and gene expression. J Appl Physiol 106: 194-202. PubMed: 18988762.
- 38. Lebsack TW, Fa V, Woods CC, Gruener R, Manziello AM et al. (2010) Microarray analysis of spaceflown murine thymus tissue reveals changes in gene expression regulating stress and glucocorticoid receptors. J Cell Biochem 110: 372-381. PubMed: 20213684.
- 39. Ortega MT, Pecaut MJ, Gridley DS, Stodieck LS, Ferguson VL et al. (2009) Shifts in bone marrow cell phenotypes caused by space flight. J Appl Physiol 106: 548-555. PubMed: 19056998.
- 40. Pecaut MJ, Nelson GA, Peters LL, Kostenuik PJ, Bateman TA et al. (2003) Genetic models in applied physiology: selected contribution: Effects of space flight on immunity in the C57BL/6 mouse, Part I: Immune population distributions. J Appl Physiol 94: 2085-2094. PubMed: 12514166.
- 41. Pecaut MJ, Simske SJ, Fleshner M (2000) Space flight induces changes in splenocyte subpopulations: effectiveness of ground-based models. Am J Physiol Regul Integr Comp Physiol 279: R2072-R2078. PubMed: 11080071.
- 42. Tian J, Pecaut MJ, Slater JM, Gridley DS (2010) Space flight modulates expression of extracellular matrix, adhesion and profibrotic molecules in mouse lung. J Appl Physiol 108: 162-171. doi:10.1152/japplphysiol.00730.2009. PubMed: 19850731.
- 43. Bejarano-Escobar R, Blasco M, Martín-Partido G, Francisco-Morcillo J (2012) Light-induced degeneration and microglial response in the retina of an epibenthonic pigmented teleost: age-dependent photoreceptor susceptibility to cell death. J Exp Biol 215: 3799-3812. doi:10.1242/jeb.072124. PubMed: 22811246.
- 44. Santos AM, Martín-Oliva D, Ferrer-Martín RM, Tassi M, Calvente R et al. (2010) Microglial response to light-induced photoreceptor degeneration in the mouse retina. J Comp Neurol 518: 477-492. doi:10.1002/cne.22227. PubMed: 20020538.
- 45. Go WY, Liu X, Roti MA, Liu F, Ho SN (2004) NFAT5/TonEBP mutant mice define osmotic stress as a critical feature of the lymphoid microenvironment. Proc Natl Acad Sci U S A 101: 10673-10678. doi:10.1073/pnas.0403139101. PubMed: 15247420.
- 46. Grasl-Kraupp B, Ruttkay-Nedecky B, Koudelka H, Bukowska K, Bursch W et al. (1995) In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note. Hepatology 21: 1465-1468. doi:10.1002/hep.1840210534. PubMed: 7737654.
- 47. Surova O, Zhivotovsky B (2012 Dec 3) Various modes of cell death induced by DNA damage. Oncogene. [Epub ahead of print]. PubMed: 23208502.
- 48. Sun XQ, Xu ZP, Zhang S, Cao XS, Liu TS (2009) Simulated weightlessness aggravates hypergravity-induced impairment of learning and memory and neuronal apoptosis in rats. Behav Brain Res 199: 197-202. doi:10.1016/j.bbr.2008.11.035. PubMed: 19100783.
- 49. Uva BM, Strollo F, Ricci F, Pastorino M, Mason JI et al. (2005) Morpho-functional alterations in testicular and nervous cells submitted to modelled microgravity. J Endocrinol Invest 28 Suppl Proceedings(11: 84-91. PubMed: 16760632.
- 50. Garcia-Suarez O, Blanco-Gelaz MA, Lopez ML, Germana A, Cabo R et al. (2002) Massive lymphocyte apoptosis in the thymus of functionally deficient TrkB mice. J Neuroimmunol 129: 25-34. doi:10.1016/S0165-5728(02)00166-2. PubMed: 12161017.
- 51. Moore KW, de Waal Malefyt R, Coffman RL, O’Garra A (2001) Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 19: 683–765. doi:10.1146/annurev.immunol.19.1.683. PubMed: 11244051.
- 52. Sabat R, Grütz G, Warszawska K, Kirsch S, Witte E et al. (2010) Biology of interleukin-10. Cytokine Growth Factor Rev 21: 331-344. doi:10.1016/j.cytogfr.2010.09.002. PubMed: 21115385.
- 53. Mehta SK, Crucian BE, Stowe RP, Simpson RJ, Ott CM et al. (2013) Reactivation of latent viruses is associated with increased plasma cytokines in astronauts. Cytokine 61: 205-209. doi:10.1016/j.cyto.2012.09.019. PubMed: 23107825.
- 54. Rodriguez-Galán MC, Bream JH, Farr A, Young HA (2005) Synergistic effect of IL-2, IL-12, and IL-18 on thymocyte apoptosis and Th1/Th2 cytokine expression. J Immunol 174: 2796-2804. PubMed: 15728489.
- 55. Denhardt DT, Noda M, O’Regan AW, Pavlin D, Berman JS (2001) Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival. J Clin Invest 107: 1055-1061. doi:10.1172/JCI12980. PubMed: 11342566.
- 56. Diao H, Iwabuchi K, Li L, Onoe K, Van Kaer L et al. (2008) Osteopontin regulates development and function of invariant natural killer T cells. Proc Natl Acad Sci U S A 105: 15884-15889. doi:10.1073/pnas.0806089105. PubMed: 18836077.
- 57. Wang KX, Denhardt DT (2008) Osteopontin: role in immune regulation and stress responses. Cytokine Growth Factor Rev 19: 333-345. doi:10.1016/j.cytogfr.2008.08.001. PubMed: 18952487.
- 58. Wang KX, Shi Y, Denhardt DT (2007) Osteopontin regulates hindlimb-unloading-induced lymphoid organ atrophy and weight loss by modulating corticosteroid production. Proc Natl Acad Sci U S A 104: 14777-14782. doi:10.1073/pnas.0703236104. PubMed: 17785423.
- 59. Gruver AL, Sempowski GD (2008) Cytokines, leptin, and stress-induced thymic atrophy. J Leukoc Biol 84: 915-923. doi:10.1189/jlb.0108025. PubMed: 18495786.
- 60. van der Poll T, Keogh CV, Guirao X, Buurman WA, Kopf M et al. (1997) Interleukin-6 gene deficient mice show impaired defense against pneumococcal pneumonia. J Infect Dis 176: 439–444. doi:10.1086/514062. PubMed: 9237710.
- 61. Miller ES, Koebel DA, Sonnenfeld G (1995) Influence of space flight on the production of interleukin-3 and interleukin-6 by rat spleen and thymus cells. J Appl Physiol 78: 810-813. PubMed: 7775323.
- 62. Opdenakker G, Froyen G, Fiten P, Proost P, Van Damme J (1993) Human monocyte chemotactic protein-3 (MCP-3): molecular cloning of the cDNA and comparison with other chemokines. Biochem Biophys Res Commun 191: 535–542. doi:10.1006/bbrc.1993.1251. PubMed: 8461011.
- 63. Qiu X, Yao S, Zhang S (2010) Advances in the research on lymphangiogenesis in carcinoma tissues (Review ). Oncol Lett 1: 579-582 PubMed: 22966346.
- 64. Stacker SA, Caesar C, Baldwin ME, Thornton GE, Williams RA et al. (2001) VEGF-D promotes the metastatic spread of tumor cells via the lymphatics. Nat Med 7: 186-191. doi:10.1038/84635. PubMed: 11175849.
- 65. Onishi Y, Matsuno Y, Tateishi U, Maeshima AM, Kusumoto M et al. (2004) Two entities of precursor T-cell lymphoblastic leukemia/lymphoma based on radiologic and immunophenotypic findings. Int J Hematol 80: 43-51. doi:10.1532/IJH97.04061. PubMed: 15293567.
- 66. Schieke SM, Sharaf MA, Lerner A, Rünger TM, Mahalingam M (2012) Primary cutaneous CD56 positive lymphoma: a diagnostic conundrum in an unusual case of lymphoma. J Cutan Pathol 39: 540-544. doi:10.1111/j.1600-0560.2012.01874.x. PubMed: 22519525.
- 67. Stacchini A, Barreca A, Demurtas A, Aliberti S, di Celle PF et al. (2012) Flow cytometric detection and quantification of CD56 (neural cell adhesion molecule, NCAM) expression in diffuse large B cell lymphomas and review of the literature. Histopathology 60: 452-459. doi:10.1111/j.1365-2559.2011.04098.x. PubMed: 22276608.
- 68. Terada T, Maruo H (2012) Esophageal combined carcinomas: Immunohistochemical and molecular genetic studies. World J Gastroenterol 18: 1545-1551. doi:10.3748/wjg.v18.i13.1545. PubMed: 22509088.
- 69. Allen BJ, Tian Z, Rizvi SM, Li Y, Ranson M (2003) Preclinical studies of targeted alpha therapy for breast cancer using 213Bi-labelled-plasminogen activator inhibitor type 2. Br J Cancer 88: 944–950. doi:10.1038/sj.bjc.6600838. PubMed: 12644835.
- 70. Antalis TM, La Linn M, Donnan K, Mateo L, Gardner J et al. (1998) The serine proteinase inhibitor (serpin) plasminogen activation inhibitor type 2 protects against viral cytopathic effects by constitutive interferon α/β priming. J Exp Med 187: 1799–1811. doi:10.1084/jem.187.11.1799. PubMed: 9607921.
- 71. Medcalf RL, Stasinopoulis SJ (2005) The undecided serpin. The ins and outs of plasminogen activator inhibitor type 2. FEBS J 272: 4858-4867. doi: 10.1111/j.1742-4658.2005.04879.x
- 72. Kajioka EH, Andres ML, Nelson GA, Gridley DS (2000) Immunological variables in male and female C57BL/6 mice from two sources. Comp Med 50: 288-291. PubMed: 10894494.
- 73. Cowley MJ, Cotsapas CJ, Williams RB, Chan EK, Pulvers JN et al. (2009) Intra- and inter-individual genetic differences in gene expession. Mamm Genome 20: 281-295. doi:10.1007/s00335-009-9181-x. PubMed: 19424753.
- 74. Son CG, Bilke S, Davis S, Greer BT, Wei JS et al. (2005) Database of mRNA gene expression profiles of multiple human organs. Genome Res 15: 443-450. doi:10.1101/gr.3124505. PubMed: 15741514.
- 75. Singh KP, Kumari R, Dumond JW (2010) Simulated microgravity-induced epigenetic changes in human lymphocytes. J Cell Biochem 111: 123-129. doi:10.1002/jcb.22674. PubMed: 20506542.
- 76. Deckbar D, Jeggo PA, Löbrich M (2011) Understanding the limitations of radiation-induced cell cycle checkpoints. Crit Rev Biochem Mol Biol 46: 271-283. doi:10.3109/10409238.2011.575764. PubMed: 21524151.
- 77. Hoffman B, Liebermann DA (2008) Apoptotic signaling by c-MYC. Oncogene 27: 6462-6472. doi:10.1038/onc.2008.312. PubMed: 18955973.
- 78. Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H (2001) Interleukin-18 regulates both Th1 and Th2 responses. Annu Rev Immunol 19: 423-474. doi:10.1146/annurev.immunol.19.1.423. PubMed: 11244043.
- 79. Porter BO, Malek TR (2000) Thymic and intestinal intraepithelial T lymphocyte development are each regulated by the gammac-dependent cytokines IL-2, IL-7, and IL-15. Semin Immunol 12: 465-474. doi:10.1006/smim.2000.0264. PubMed: 11085179.
- 80. Sekai M, Tani-Ichi S, Yoneyama M, Fujita T, Kina T et al. (2013) Lymphocyte-stromal cell interaction induces IL-7 expression by interferon regulatory factors. Mol Immunol 54: 378-385. doi:10.1016/j.molimm.2013.01.002. PubMed: 23376291.
- 81. Zúñiga LA, Jain R, Haines C, Cua DJ (2013) Th17 cell development: from the cradle to the grave. Immunol Rev 252: 78-88. doi:10.1111/imr.12036. PubMed: 23405896.