A properly functioning immune system is essential to crew health and the success of long-term missions. Evidence indicates that astronauts have significantly increased rates of infection after flight. Through an animal study, Dr. Yufang Shi is simulating spaceflight conditions to study the effect on the immune system. He has found that infection-fighting white blood cells inappropriately die-off leading to immunosuppression. The overall goals of this study are to determine the mechanisms underlying immune system changes that occur in space and to aid in the development of countermeasures to overcome the problem.
Overview
Apoptosis and Immune Homeostasis During Hindlimb Unloading
Principal Investigator:
Yufang Shi, D.V.M., Ph.D.
Organization:
University of Medicine and Dentistry of New Jersey Robert Wood Johnson Medical School
Technical Summary
Spaceflight impacts the immune system, often resulting in lymphopenia and abnormally low responses to antigenic stimulation. These changes are believed to be due to factors such as microgravity, lack of load-bearing, psychological and physical stress, and exposure to radiation. But, the exact mechanisms are unknown. Hindlimb unloading (HU) in rodents, a ground-based animal model, has been used to simulate some of the factors associated with spaceflight. Interestingly, HU induces similar changes in the immune system of rodents as those resulting from spaceflight in humans. Based on our previous studies, we hypothesize that some of these changes are modulated through the induction of apoptosis in lymphocytes.
Specific Aims
- Elucidate the role of Fas and endogenous opioids in the modulation of the immune system by HU and radiation
- Determine the effect of HU and radiation on lymphocyte dynamics and immune responses
- Determine whether HU and radiation promote autoimmunity because autoimmune disease paradoxically is often associated with stress and radiation exposure
Progress
In a study at the NASA Space Radiation Laboratory at Brookhaven National Laboratory (BNL), we examined the acute effects of whole body HZE-particle irradiation. Mice were subjected to high-energy Fe-56 ions, and the effects on various lymphocyte populations were determined. We found that lymphocyte numbers in the spleen and thymus were reduced in a dose-dependent manner (using doses of 200, 100 and 50 cGy), with thymocytes being more sensitive than splenocytes.
Subpopulation analysis revealed that all subsets declined in number, except for CD4+CD25+ regulatory T cells. To examine the mechanism, mice deficient in apoptosis-inducing genes were tested. Deficiency in Fas ligand (gld mutation) or TRAIL (TNF-related apoptosis-inducing ligand) did not prevent lymphocyte losses, indicating that these apoptosis pathways are not involved in particle radiation-induced lymphocyte losses. In contrast, gamma-radiation-induced cell depletion does require FasL and TRAIL. Thus, there are divergent pathways of apoptosis induction by particle radiation vs. gamma-radiation. Using specific inhibitors in vitro, we also found that particle radiation-induced lymphocyte apoptosis is caspase-dependent but does not utilize nitric oxide or oxygen free radicals.
We have reported that endogenous granzyme B (GzmB) is a critical regulator of the balance between Th1/Th2 helper T cells. Highly expressed in Th2 cells, GzmB is required to induce apoptosis in these cells. We have found that GzmB-deficient mice develop stronger Th2-type immune responses. Since GzmB expression can be inhibited by steroid hormones and corticosteroid levels are often elevated during stress, these findings may explain the increased levels of Th2 cytokines found in astronauts post-flight. Mice subjected to HU also have increased Th2 responses and serum glucocorticoids. We are still examining the role of GzmB in immune responses of mice subjected to HU.
Stress, microgravity and space radiation are well-known to induce apoptosis in lymphoid and other cells. Although apoptotic cells can induce immunosuppression, the molecular mechanisms are unknown. We found that exposure to apoptotic cells rendered dendritic cells (DC) capable of producing much greater amounts of nitric oxide in response to exogenous IFN-gamma compared to normal DC. We believe that these results have important ramifications for understanding spaceflight-induced immunosuppression.
Mesenchymal stem cells (MSCs) are a unique population of multi-potent stem cells that are derived from adult bone marrow. These cells are immunosuppressive and also seem to protect splenic and thymic lymphocytes from the deleterious effects of microgravity and radiation. We found that transfusing mice with MSCs immediately after gamma-irradiation or before HU significantly reduced splenocyte losses. In vitro, irradiated lymphocytes were protected from apoptosis when co-cultured with MSCs. These results indicate that MSCs provide survival signals that prevent lymphocyte apoptosis. In fact, we have found several such MSC-produced cytokines, which may allow for development of pharmaceutical interventions to prevent lymphocyte loss during spaceflight.
We also examined the interaction between the immune and skeletal systems. Osteopontin (OPN), a bone matrix protein, has also been shown affect the immune system, as a cytokine and possibly as a transcription factor. We examined the role of OPN in HU-induced lymphocyte depletion using OPN-knockout mice, collaborating with David Denhardt and Kathryn Wang. We have reported that OPN deficiency confers resistance to HU-induced thymocyte loss. This protective effect derives from decreased corticosteroid levels in OPN-deficient mice, indicating that OPN is important in regulating the steroid response during HU. This suggests a similar role for OPN in the response to spaceflight conditions. Importantly, injection of a specific anti-OPN antibody (2C5) into wild-type mice ameliorates organ atrophy induced by chronic restraint; changes in corticosterone levels were also partially reversed. These studies reveal that circulating OPN plays a significant role in the regulation of the hypothalamus-pituitary-adrenal axis hormones, and that it augments chronic restraint-induced organ atrophy.
Future Plans
Continue ongoing studies of the molecular mechanisms that contribute to the cell survival/radiation-protective effects of MSCs.
Examine the effects on the immune parameters of mice after three months of spaceflight, using control and osf-1 transgenic mice. Compare these results to our HU studies.
Earth Applications
We hope to gain an understanding of the mechanisms by which stress affects immune homeostasis. With this information, it may be possible to devise dietary and lifestyle changes that would reduce the deleterious effects of stress on the immune system. Such changes may lead to an improvement in public health. We also hope to identify food supplements, such as vitamin C and seaweed extracts, that may reduce the health impact of stress, bringing benefit to the general population, especially those who suffer from high levels of occupational or life stress.
By determining the role of cellular apoptosis in mediating the deleterious effects of stress on the immune system, we may be able to aid in the development of therapies for the maintenance of immune tolerance for clinical use in organ transplant or autoimmune disease.
We are studying the role of a specific subset of T lymphocytes called CD4+CD25+ regulatory T cells, which seem to be involved in the deleterious effect of stress on the immune system. Learning how these cells exert their effects may lead to potential interventions to prevent the negative health impacts of autoimmunity, cancer and other diseases that arise from an improperly regulated immune system.
It is difficult to measure the level of stress that an individual experiences for the purpose of allowing appropriate intervention. Identification of particular serum proteins whose concentration is related to the stress level of an individual could serve as a biomarker for the assessment of stress levels for medical or occupational purposes.
By determining the role of cellular apoptosis in mediating the deleterious effects of stress on the immune system, we may be able to aid in the development of therapies for the maintenance of immune tolerance for clinical use in organ transplant or autoimmune disease.
We are studying the role of a specific subset of T lymphocytes called CD4+CD25+ regulatory T cells, which seem to be involved in the deleterious effect of stress on the immune system. Learning how these cells exert their effects may lead to potential interventions to prevent the negative health impacts of autoimmunity, cancer and other diseases that arise from an improperly regulated immune system.
It is difficult to measure the level of stress that an individual experiences for the purpose of allowing appropriate intervention. Identification of particular serum proteins whose concentration is related to the stress level of an individual could serve as a biomarker for the assessment of stress levels for medical or occupational purposes.
This project's funding ended in 2009