Post-Spaceflight (STS-135) Mouse Splenocytes Demonstrate Altered Activation Properties and Surface Molecule Expression

Abstract

Alterations in immune function have been documented during or post-spaceflight and in ground based models of microgravity. Identification of immune parameters that are dysregulated during spaceflight is an important step in mitigating crew health risks during deep space missions. The in vitro analysis of leukocyte activity post-spaceflight in both human and animal species is primarily focused on lymphocytic function. This report completes a broader spectrum analysis of mouse lymphocyte and monocyte changes post 13 days orbital flight (mission STS-135). Analysis includes an examination in surface markers for cell activation, and antigen presentation and co-stimulatory molecules. Cytokine production was measured after stimulation with T-cell mitogen or TLR-2, TLR-4, or TLR-5 agonists. Splenocyte surface marker analysis immediate post-spaceflight and after in vitro culture demonstrated unique changes in phenotypic populations between the flight mice and matched treatment ground controls. Post-spaceflight splenocytes (flight splenocytes) had lower expression intensity of CD4+CD25+ and CD8+CD25+ cells, lower percentage of CD11c+MHC II+ cells, and higher percentage of CD11c+MHC I+ populations compared to ground controls. The flight splenocytes demonstrated an increase in phagocytic activity. Stimulation with ConA led to decrease in CD4+ population but increased CD4+CD25+ cells compared to ground controls. Culturing with TLR agonists led to a decrease in CD11c+ population in splenocytes isolated from flight mice compared to ground controls. Consequently, flight splenocytes with or without TLR-agonist stimulation showed a decrease in CD11c+MHC I+, CD11c+MHC II+, and CD11c+CD86+ cells compared to ground controls. Production of IFN-γ was decreased and IL-2 was increased from ConA stimulated flight splenocytes. This study demonstrated that expression of surface molecules can be affected by conditions of spaceflight and impaired responsiveness persists under culture conditions in vitro.

Fig 1. Cell population differences in total splenocytes collected from flight mice.

Splenocytes collected immediately post-spaceflight were stained with CD4, CD8, MHC I, MHC II, and CD11c. Analysis was conducted by flow cytometry. (1a) Example of gating strategy between flight mice and ground (AEM; animal enclosure module controls). (1b) Example of positive population composition of each gate identified in 1a from flight mice. (1c) Graph of percent positive and MFI from each marker of splenocytes from flight mice.

Fig 2. Cell population differences in total splenocytes collected from ground control mice.

Splenocytes collected immediately post-sacrifice of ground control mice were stained with CD4, CD8, MHC I, MHC II, and CD11c. Analysis was conducted by flow cytometry. (2a) Example of gating strategy between flight mice and ground controls. (2b) Example of positive population composition of each gate identified in 2a from ground AEM control mice. (2c) Graph of percent positive and MFI from each marker of splenocytes from AEM control mice.

Fig 3. Bead uptake by splenocytes from flight mice and ground controls.

Splenocytes were incubate with fluorescent beads and collected for analysis by flow cytometry. Percent positive events were separated by peak mean florescent intensity and labeled as low, med, and high. Data was plotted for each individual mouse. Mean indicated by dotted line (ground AEM control) or solid line (flight mice). *** = p<0.001; **p = <0.01

Fig 4. Surface markers expression of CD4⁺ splenocytes.

Flight (solid square) or ground AEM control (open circle) splenocytes were cultured in DMEM with 10% FBS for 24 hours with only media (control), anti-CD3/CD28, or ConA. Cells were collected and stained for expression of CD4, CD28, and CD25. (A) CD4 positive cells in total splenocyte population. (B) CD4⁺CD28⁺ cells. (C) CD4⁺CD25⁺ cells. (D) CD4⁺ cells that are also CD28⁺CD25⁺. *** = p<0.001 **p = <0.01; * = p<0.05

Fig 5. Expression of CD8 on non-activated and ConA activated splenocytes.

Flight (solid square) or ground AEM control (open circle) splenocytes were cultured in DMEM with 10% FBS for 24 hours with or without ConA (2μg/mL). Cells were collected and stained for expression of CD8. Representative flow dot graphs of CD8 positive populations after 24 hours culture in media only (left) or in the presence of ConA (right).

Fig 6. Expression of surface markers of CD8^lo and CD8^hi splenocytes.

Flight (solid square) or ground AEM control (open circle) splenocytes were cultured in DMEM with 10% FBS for 24 hours with only media (control), anti-CD3/CD28, or ConA. Cells were collected and stained for expression of CD8, CD28, and CD25. *** = p<0.001 **p = <0.01; * = p<0.05

Fig 7. Antigen presentation molecule expression under TLR agonist stimulation.

Flight (solid square) or ground AEM control (open circle) splenocytes were cultured in DMEM with 10% FBS with or without TLR agonists for 24 hours. Cells were collected and stained for expression of MHC I (H-2k^b) and MHC II (I-A^b). (7a) Representative dot plot of MHC I⁺ populations. (7b) Percent positive and MFI of MHC I expression from G2 and G3 populations. *** = p<0.001.

Fig 8. Dendritic cell populations changes after TLR stimulation in post-spaceflight mice.

Flight (solid square) or ground AEM control (open circle) splenocytes were cultured in DMEM with 10% FBS with or without TLR agonists for 24 hours. Cells were collected and stained for expression of CD11c, MHC I, MHC II, CD86, and CD80. (8a) Total percent of dendritic cells (CD11c) of post-spaceflight or ground AEM control mice under various TLR stimulation conditions. (8b) Percent and MFI of dendritic cells expressing MHC I. (8c) Percent and MFI of dendritic cells expressing MHC II. 8d) Percent and MFI of dendritic cells expressing CD86. *** = p<0.001 **p = <0.01 * = p<0.05.