The reactivation of latent herpes viruses poses a significant risk to the health and safety of the crew during exploration class space flight missions. Of particular concern is cytomegalovirus (CMV), which infects 50-80% of the US population and is known to cause significant morbidity and mortality in immunocompromised patients. CMV DNA is frequently found in the body fluids of astronauts during space travel indicating that the space flight environment can initiate active CMV infections. As space flight also causes huge alterations in the normal functioning of the immune system, CMV reactivation in a potentially immunosuppressed crewmember could have disastrous consequences during long-duration exploration class missions. The effects of microgravity on CMV replication and its potential to evade the host immune system are unknown. In this study, investigators will utilize the rotating wall vessel (RWV) cell culture system to simulate microgravity on Earth allowing them to study the virulent and infectivity properties of CMV and how it interacts with host immune cells in the absence of gravity. The investigators hypothesized that modeled microgravity wiould evoke changes in cellular gene expression that will underpin CMV reactivation and increase virus replication and infectivity. They further postulated that modeled microgravity would impair the anti-CMV responses of host T-cells and NK-cells that will allow viral persistence and replication to occur.
This study had the following specific aims:
1. Determine the effects of modeled microgravity and radiation on CMV replication and the expression of genes associated with latency and lytic activity.
2. Determine the impact of modeled microgravity and radiation on the anti-CMV activity of host Tcells and NK-cells.
3. Determine the impact of modeled microgravity on the anti-CMV activity of host T-cells and NK-cells.
The methodology to address Specific Aim 1: Investigators determined the effects of simulated microgravity and/or radiation on: (i) CMV infectivity and viral yield using human MRC5 cells and low-passage clinical and laboratory CMV strains; (ii) reactivation of CMV during monocyte to dendritic cell differentiation; and (iii) the molecular profile of CMV-infected cells before and after differentiation by measuring viral transcripts of the latency-control locus and of select lytic and latent genes using a human CMV gene array.
The methodology to address Specific Aim 2: Investigators determined the effects of simulated microgravity and/or acute radiation exposure on: (i) CMV--specific T-cell expansion and their recognition and functional responses to CMV viral antigens; (ii) the cytokine secretion profile of expanded CMV-specific CTLs; (iii) NK-cell phenotypic shifts and cytotoxic activity against standard and CMV-specific target cells in vitro; (iv) NK-cell expansion in response to CMV and CMV infection analogs; and (v) the ability of CMV-specific NK-cells and secretory products from CMV-specific T-cells to curtail CMV reactivation, replication and infectivity.
Findings from Specific Aim 1
The researchers established a positive infection model for Kasumi-3 myeloid progenitor cells using a low fibroblast passaged, BAC-derived TB40/E clinical strain of CMV. The virus is cell associated as confirmed by both real-time PCR and confocal imaging. Cultures are separated into cell pellet and supernatant fractions via high sped centrifugation, 14,000 x g, and the viral copy number is highest in the cell pellet fraction; confocal imaging confirms internalization of the virus. Lytic infection has been confirmed by direct single color antibody staining using mouse anti-human mab810X-AlexaFluor-488(IE-1) and flow cytometry. Additionally, Kasumi-3 cells pre-treated to 12 days of simulated microgravity (SMG) are seemingly resistant to initial infection at the early 12-24 hour time point, with viral copy numbers being consistently lower than non-treated controls.
Findings from Specific Aim 2
All Kasumi-3 cells from the CMV infection experiments (with and without SMG) have been cryopreserved for future DNA extraction and gene expression assays by RT-PCR. The researchers expect that any increase in CMV reactivation due to SMG or otherwise will be preceded by an expression of lytic genes and a downregulation of latent genes. They also expect to see latent gene expression increase following primary infection to indicate that the virus has become latent. Downregulated IE-1 expression should occur concomitantly with this.
Findings from Specific Aim 3
In the process of understanding the mechanistic underpinnings of suppression in NK cell function seen in the International Space Station (ISS) study, the researchers have established an in vitro model using a rotary cell culture system to simulate microgravity in human peripheral blood mononuclear cells. Using this model, they were able to show that microgravity adversely affects immune cell viability (<90% viable) after a 12-hour exposure to microgravity. Researchers have also replicated the suppression in NK cell function seen in crewmembers. NK cells exposed to microgravity (SMG) killed fewer cancer cells compared to NK cells exposed to both 1g (static) and a rotational control (RC) (for shear stress). This suppression in function was evident despite absence of any phenotypic differences among the three groups (SMG, static, and RC). Microgravity reduced NK cell perforin expression but did not affect their conjugation rate to target cells. Furthermore, the researchers used cytometry-by-time-of-flight to look at various functional and maturation markers, which hinted at reduced degranulation and cytokine production in NK cells exposed to microgravity. They confirmed this using flow cytometry, discovering that microgravity lowered degranulation of cytotoxic granules (reduced CD107a+ expression) and suppressed TNF-a and IFN-gamma expression in NK cells that were exposed to cancer cells.
No datasets exist for this study. A final report was archived.
|Mission||Launch/Start Date||Landing/End Date||Duration|