Microarray analysis of spaceflown murine thymus tissue reveals changes in gene expression regulating stress and glucocorticoid receptors.

The detrimental effects of spaceflight and simulated microgravity on the immune system have been extensively documented. We report here microarray gene expression analysis, in concert with quantitative RT-PCR, in young adult C57BL/6NTac mice at 8 weeks of age after exposure to spaceflight aboard the space shuttle (STS-118) for a period of 13 days. Upon conclusion of the mission, thymus lobes were extracted from space flown mice (FLT) as well as age- and sex-matched ground control mice similarly housed in animal enclosure modules (AEM). mRNA was extracted and an automated array analysis for gene expression was performed. Examination of the microarray data revealed 970 individual probes that had a 1.5-fold or greater change. When these data were averaged (n = 4), we identified 12 genes that were significantly up- or down-regulated by at least 1.5-fold after spaceflight (P < or = 0.05). The genes that significantly differed from the AEM controls and that were also confirmed via QRT-PCR were as follows: Rbm3 (up-regulated) and Hsph110, Hsp90aa1, Cxcl10, Stip1, Fkbp4 (down-regulated). QRT-PCR confirmed the microarray results and demonstrated additional gene expression alteration in other T cell related genes, including: Ctla-4, IFN-alpha2a (up-regulated) and CD44 (down-regulated). Together, these data demonstrate that spaceflight induces significant changes in the thymic mRNA expression of genes that regulate stress, glucocorticoid receptor metabolism, and T cell signaling activity. These data explain, in part, the reported systemic compromise of the immune system after exposure to the microgravity of space.

Tissue inhibitor of metalloproteinase-2 inhibits T-cell infiltration and preserves pancreatic beta-cell function in an in vitro type 1 diabetes mellitus model.

Type 1 diabetes mellitus (T1DM) results from autoreactive T-cells that attack and destroy insulin producing pancreatic beta-cells. This knowledge has provided a framework for numerous efforts to prevent or mitigate T1DM at various stages of the disease. In this study, we utilized an organ culture model of type 1 diabetes to determine whether tissue inhibitors of metalloproteinases (TIMPs) could block T-cell migration into the pancreas and ultimately preserve beta-cell function. We measured T-cell repertoires, insulin secretion, and performed immunohistochemistry and confocal laser microscopy in order to evaluate the effect of TIMP-1, TIMP-2, and TIMP-3 on our in vitro T1DM organ culture model. TIMP-2 decreased T-cell transmigration and preserved insulin production in our T1DM organ culture model. Moreover, TIMP-2 inhibited transmigration of diabetogenic T-cells across an islet microvascular endothelial cell layer. Our findings suggest that TIMP-2 is effective at blocking infiltration of autoreactive T-cells into target pancreas tissue thereby preserving pancreatic beta-cell mass.

Production of human B cells from CD34+CD38- T- B- progenitors in organ culture by sequential cytokine stimulation.

We investigated sequential cytokine addition on human hematopoietic stem cell (HSC) differentiation in murine fetal liver (FL), fetal spleen (FS) and bone marrow (BM) organ cultures (OC). Tissues were colonized with unpurified or FACS sorted CD34+CD38-CD10-CD19-CD3-CD8-CD4-(T- B-) cells from human cord blood (HUCB). CD19+ cell production and kinetics differed in each tissue. Fetal liver organ cultures (FLOC) inoculated with CD34+CD38-T-B- cells produced fewer CD19+ cells than fetal liver organ culture (FLOC) cultured with unpurified HUCB. CD19+ cell production was restored in the CD34+CD38-T-B- organ cultures by treating with SCF, LIF and IL-6 followed by IL-7 and removing all cytokines for the last 3 days of culture (a six-fold increase). FLOC also produced CD34+CD38-T-B- cells and monocyte-lineage CD33+CD14- cells, both of which increased after cytokine treatment. Re-colonization of secondary FLOC with CD34+CD38-T-B- cells generated in primary FLOC produced additional B-cells, monocytes and CD34+CD38- cells suggesting that the primary cells retained HSC activity. Expansion and differentiation of HSCs depended on the microenvironment of the recipient tissue as well as addition of cytokines in the appropriate order.

Use of a microgravity organ culture dish system to demonstrate the signal dampening effects of modeled microgravity during T cell development.

Recently, we have shown that exposure of fetal thymus organ cultures (FTOC) to modeled microgravity (MMG) using a clinostat with a microgravity organ culture dish system (MOCDS) blocks T cell development in a manner independent of steroid stress hormones present in vivo. In this study, we describe the development of the MOCDS system, as well as its use in attempting to understand the mechanism by which T cell development is inhibited in MMG. We show that after MMG exposure FTOC exhibited a significant reduction in CD4+CD8+ double positive (DP) cell production, but those DP cells which remained expressed higher levels of the T cell receptor (TCR) associated molecule, CD3. Interestingly, CD4-CD8- double negative (DN) cells expressed lower levels of CD3 on their surface. DN, as well as immature single positive (ISP) cells, also expressed reduced levels of the IL-7 receptor alpha chain (CD127). These changes in CD3 and CD127 expression were concomitantly associated with an increased production of tumor necrosis factor (TNF)-alpha. We were also able to show that addition of an exogenous signal (anti-CD3epsilon monoclonal antibody) to these cultures effectively mitigated the MMG-induced effects, suggesting that MMG-exposure causes a signal dampening effect on developing thymocytes.

Substance P effects on developing thymocytes in hindlimb unloaded rats.

INTRODUCTION: The purpose of this study was to examine the effects of substance P (SP) on the immune system in a condition similar to microgravity. We analyzed immune disturbances caused by subjecting Fischer 344 rats to a 45 degrees antiorthostatic suspension technique, otherwise known as the hindlimb unloading (HU) model. METHODS: Four groups of rats were assigned to either the prone control non-substance P group (P-NSP), prone control substance P group (P-SP), hindlimb unloaded non-substance P (HU-NSP) or the hindlimb unloaded substance P group (HU-SP). SP was administered at 10 ml of a 1 micromol x L(-1) concentration for 15 min x d(-1). HU and SP exposure for all groups lasted 16 d. After 16 d, 500 microl of blood was obtained to assay for both T-cell phenotype and corticosterone (CS) levels. Thymus lobes were excised in order to examine T-cell phenotype. Thymocytes were counted and stained for lymphocyte markers (CD4, CD8, and CD3). An analysis of variance (ANOVA) test was used to determine significance between groups (p < or = 0.05). RESULTS: HU-NSP rats showed a decrease in thymic CD4+CD8 +/- cells from 85.51 +/- 1.9% to 62.06 +/- 1.9% when compared with P-NSP rats. SP reversed these effects and returned CD4+CD8+ cells to control levels (76.60 +/- 1.9%). DISCUSSION: Daily SP treatment was found to reverse the deleterious effects caused by HU and corticosterone in rat thymic immune cells. SP could prove to be an effective means for keeping the immune system functioning at normal levels in microgravity, allowing astronauts to stay in space longer and maintain a more productive immune system.