Down-regulation of GATA1-dependent erythrocyte-related genes in the spleens of mice exposed to a space travel

Secondary lymphoid organs are critical for regulating acquired immune responses. The aim of this study was to characterize the impact of spaceflight on secondary lymphoid organs at the molecular level. We analysed the spleens and lymph nodes from mice flown aboard the International Space Station (ISS) in orbit for 35 days, as part of a Japan Aerospace Exploration Agency mission. During flight, half of the mice were exposed to 1 g by centrifuging in the ISS, to provide information regarding the effect of microgravity and 1 g exposure during spaceflight. Whole-transcript cDNA sequencing (RNA-Seq) analysis of the spleen suggested that erythrocyte-related genes regulated by the transcription factor GATA1 were significantly down-regulated in ISS-flown vs. ground control mice. GATA1 and Tal1 (regulators of erythropoiesis) mRNA expression was consistently reduced by approximately half. These reductions were not completely alleviated by 1 g exposure in the ISS, suggesting that the combined effect of space environments aside from microgravity could down-regulate gene expression in the spleen. Additionally, plasma immunoglobulin concentrations were slightly altered in ISS-flown mice. Overall, our data suggest that spaceflight might disturb the homeostatic gene expression of the spleen through a combination of microgravity and other environmental changes

Hypergravity Provokes a Temporary Reduction in CD4+CD8+ Thymocyte Number and a Persistent Decrease in Medullary Thymic Epithelial Cell Frequency in Mice.

Gravity change affects many immunological systems. We investigated the effects of hypergravity (2G) on murine thymic cells. Exposure of mice to 2G for three days reduced the frequency of CD4+CD8+ thymocytes (DP) and mature medullary thymic epithelial cells (mTECs), accompanied by an increment of keratin-5 and keratin-8 double-positive (K5+K8+) TECs that reportedly contain TEC progenitors. Whereas the reduction of DP was recovered by a 14-day exposure to 2G, the reduction of mature mTECs and the increment of K5+K8+ TEC persisted. Interestingly, a surgical lesion of the inner ear's vestibular apparatus inhibited these hypergravity effects. Quantitative PCR analysis revealed that the gene expression of Aire and RANK that are critical for mTEC function and development were up-regulated by the 3-day exposure and subsequently down-regulated by the 14-day exposure to 2G. Unexpectedly, this dynamic change in mTEC gene expression was independent of the vestibular apparatus. Overall, data suggest that 2G causes a temporary reduction of DP and a persistent reduction of mature mTECs in a vestibular system-dependent manner, and also dysregulates mTEC gene expression without involving the vestibular system. These data might provide insight on the impact of gravity change on thymic functions during spaceflight and living

The short-term hypergravity exposure causes a reduction of CD4⁺CD8⁺ double positive thymocytes in a vestibular apparatus-dependent manner.

<p>(A) Ratio of thymic weight to body weight (left) and total thymic cell number (right) of mice exposed to 2 <i>g</i> gravity (labeled as 2G) for 3 day or normal gravity control (labeled as C). Vestibular apparatus were surgically disrupted in some groups of mice (labeled as VL). N = 10 for C and 2G, N = 8 for C with VL, and N = 9 for 2G with VL. The asterisks indicate statistical significance at ***P < 0.001 and **P < 0.01 (Student’s <i>t</i>-test). NS indicates that the difference is not significant (Student’s <i>t</i>-test). (B) Flow cytometric analysis of thymocytes in mice exposed to 2G for 3 days. Thymocytes were analyzed by staining with CD4 and CD8α antibodies (left figures). Numbers in panels indicates percentage of each fraction. Percentages of CD4- and CD8-double positive thymocytes (CD4⁺CD8⁺DP) in total thymocytes are summarized in right figures. Mice were exposed to 2 <i>g</i> gravity (2G) for 3 days. “C” indicates 1G control. Vestibular apparatus are surgically disrupted in some groups of mice (VL). N = 10 for C and 2G, N = 8 for C with VL, and N = 9 for 2G with VL. The asterisks indicate statistical significance at ***P < 0.001 and *P < 0.05 (Student’s <i>t</i>-test). NS indicates that the difference is not significant (Student’s <i>t</i>-test). (C) Cell numbers of CD4⁺CD8⁺ (CD4⁺CD8⁺DP), CD4⁺CD8^- (CD4SP), CD4^-CD8⁺ (CD8SP), and CD4^-CD8^- (DN) fractions in mice exposed to 2G for 3days. N = 10 for C and 2G, N = 8 for C with VL, and N = 9 for 2G with VL. The asterisks indicate statistical significance at ***P < 0.001 and *P < 0.05 (Student’s <i>t</i>-test). NS indicates that the difference is not significant (Student’s <i>t</i>-test).</p

The short-term hypergravity exposure causes a reduction in frequency of mature medullary thymic epithelial cells in a vestibular apparatus-dependent manner.

<p>(A) Flow cytometric analysis of thymic epithelial cells (TECs) in mice exposed to 2G for 3 days. Mice were exposed to 2 <i>g</i> gravity (2G) for 3 days or left under 1G (Control). Vestibular apparatus are surgically disrupted in some mice (labeled as VL). TECs (CD45^–TER119^– EpCAM⁺) in total thymic cells were analyzed by staining with UEA-1-lectin, an mTEC marker, and CD80 antibody. Numbers in panels indicates percentage of each fraction. The percentages of UEA-1^– CD80^lo (containing cTECs), UEA-1⁺CD80^high (mTEC^hi), and UEA-1⁺CD80^low (mTEC^lo) cells among thymic stroma cells in the thymus are summarized in right figures. “C” in graphs indicates 1G control. N = 5 each C, 2G, C with VL, and 2G with VL groups. The asterisks indicate statistical significance at **P < 0.01 (Student’s <i>t</i>-test). (B) Cell numbers of total TECs (CD45^–TER119^– EpCAM⁺), UEA-1^– CD80^lo TECs (containing cTECs), mTECs (UEA-1⁺), mTEC^hi (UEA-1⁺CD80^high), and mTEC^lo (UEA-1⁺CD80^low) in the thymus are summarized in figures. N = 5 each C, 2G, C with VL, and 2G with VL groups.</p

Expressions of mTEC-related molecules in the thymus and plasma of mice exposed to 2G.

<p>(A) Expressions of RANK, Aire, and RANKL in the whole thymus of mice exposed to 2G (2G) for 3 days. “C” indicates 1G control. Vestibular apparatus are surgically disrupted in some groups of mice (labeled as VL). Expression of RANK (<i>Rank</i>), Aire (<i>Aire</i>), and RANKL (<i>Rankl</i>) mRNAs was evaluated by qPCR analysis. N = 5 each C, 2G, C with VL, and 2G with VL groups. The asterisks indicate statistical significance at **P < 0.01 and *P < 0.05 (Student’s <i>t</i>-test). (B) Expressions of RANK and Aire in the whole thymus of mice exposed to 2G for 14 days. Expression of RANK and Aire mRNAs was evaluated by qPCR analysis. N = 6 each C, C with VL, and 2G with VL groups. N = 5 for 2G. The asterisks indicate statistical significance at *P < 0.05, *P < 0.01, and ***P < 0.001 (Student’s <i>t</i>-test). NS indicates that the difference is not significant (Student’s <i>t</i>-test). (C) RANKL and OPG protein levels in plasma of mice exposed to 2G for 3 days. Plasma concentration of RANKL (left) and OPG (middle) protein was determined by ELISA. Ratio of RANKL to OPG (RANKL/OPG) was exhibited in the right figure. N = 5 each C, C with VL, and 2G with VL groups. N = 4 for 2G. The asterisks indicate statistical significance at **P < 0.01 (Student’s <i>t</i>-test). (D) Corticosterone level in plasma of mice exposed to 2G for 3 days. Concentration of corticosterone was determined by ELISA. N = 6 each C, G, C with VL, and 2G with VL groups. NS indicates that the difference is not significant (Student’s <i>t</i>-test).</p