Acta Astronautica 60 (2007) 247 – 253 www.elsevier.com/locate/actaastro

Predictors of immune function in space flight夡 William T. Shearera,∗ , Shaojie Zhangb , James M. Reubenc , Bang-Ning Leec , Janet S. Butelb a Department of Pediatrics, Section of Allergy and Immunology, Texas Children’s Hospital, 6621 Fannin Street

(MC: FC330.01), Houston, TX 77030, USA b Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA c Department of Hematopathology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA

Available online 19 October 2006

Abstract Of all of the environmental conditions of space flight that might have an adverse effect upon human immunity and the incidence of infection, space radiation stands out as the single-most important threat. As important as this would be on humans engaged in long and deep space flight, it obviously is not possible to plan Earth-bound radiation and infection studies in humans. Therefore, we propose to develop a murine model that could predict the adverse effects of space flight radiation and reactivation of latent virus infection for humans. Recent observations on the effects of gamma and latent virus infection demonstrate latent virus reactivation and loss of T cell mediated immune responses in a murine model. We conclude that using this small animal method of quantitating the amounts of radiation and latent virus infection and resulting alterations in immune responses, it may be possible to predict the degree of immunosuppression in interplanetary space travel for humans. Moreover, this model could be extended to include other space flight conditions, such as microgravity, sleep deprivation, and isolation, to obtain a more complete assessment of space flight risks for humans. © 2006 Elsevier Ltd. All rights reserved.

Keywords: Space flight; Radiation; Polyoma virus infection; T lymphocytes; Interferon-gamma; Latent virus reactivation

1. Radiation and latent viruses: risk factors for space travel

夡

Supported by National Aeronautics and Space Administration Cooperative Agreement NCC 9-58 (project numbers IIH00202, IIH00204, and IIH00403) through the National Space Biomedical Research Institute. The subject of this article was presented in part at the 15th International Academy of Aeronautics Humans in Space Symposium (Helmut H. Hinghofer-Szalkay, Rupert Gerzer, Organizers): The Immune System, (G. Sonnenfeld, Chair), May 24, 2005, in Graz, Austria. ∗ Corresponding author. Tel.: +1 832 824 1274; fax: +1 832 825 7131. E-mail address: [email protected] (W.T. Shearer). 0094-5765/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.actaastro.2006.08.005

Predicting immune function in space flight must be one of the most important long-term goals of preparing for interplanetary travel, because of the immune system’s unique capability of preserving life free of infection and cancer [1]. Because of the numerous restraints of previous and present modes of space flight, it has been important to utilize several models of space flight using both animal and human subjects, each designed to test hypotheses of the effects of space travel on immune responses [2–5]. Because of these studies and information derived from non-space medical research,

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it is well known that the space flight conditions of radiation, microgravity, microbial contamination, isolation, stress, and sleep deprivation all have adverse health consequences, often leading to immune dysregulation and immunosuppression [6–16]. Depending upon the degree of alteration of normal immune function, acute, chronic, and reactivated infections, particularly latent viral infections, occur and further set the stage for loss of containment of malignant cell growths. Although many of the space flight models can utilize human subjects, certain conditions, such as space radiation [17] and latent virus reactivation [18] can only be explored in animal subjects [19–23]. Nevertheless, the quantitative exposure to different types of space radiation and specific latent viruses make it possible to predict the danger of immune compromise that will result in latent virus reactivation and tumorigenesis [24–28]. Moreover, with certain animal models it is now possible to test the combined effects of radiation, microgravity, latent virus infections, and other space conditions upon immune responses—innate and acquired, humoral and cellular, regulatory and surveillance. Thus, by using new and combined models of space flight with quantitative and specific exposure to conditions encountered in space travel, it will be possible to better prepare humans in space for adverse health consequences and the plan for immune mitigations of those challenges [29]. 2. Model to assess effects of space radiation and latent virus 2.1. Design of model The model system consisted of inoculating BALB/c female mice (Harlan Sprague Dawley, Indianapolis, IN) with murine polyoma virus (PyV) (T Benjamin, Harvard Medical School) and giving 3 Gy of whole body gamma radiation (137 Cesium, gamma irradiator, Atomic Energy of Canada Ltd., Ottawa, Ont., Canada) prior to and after intraperitoneal inoculation of viruses [30] (75 hemagglutination units [HAU]). Periodically, the appearance of PyV in tissues was measured using a quantitative polymerase chain reaction technique. In addition, spleen weight, spleen cell counts, and the proliferation of spleen cells was measured in response to a T cell mitogen, concanavalin A (Con A) and the secretion of interferon-gamma (IFN-) by spleen cells stimulated by lipopolysaccharide (LPS). 2.2. Observations with model In a group of 40 animals that had been irradiated at day 0 and injected with PyV on day 1, the

percentage of tissue specimens that contained PyV virus was higher from irradiated (3 Gy) mice as compared to control, non-irradiated mice (Table 1). Splenic tissue demonstrated significant differences between PyV mice (n = 2) and the PyV + radiation-treated mice (n = 6) at days 3, 7 and 12. At day 12, for example observed differences were as follows: (1) spleen weight 140±29 mg (mean±SEM) for PyV versus 110±10 mg for PyV + 3 Gy (P = .004); (2) lymphoproliferation to Con A 59, 139 ± 10, 373 cpm for PyV versus 13, 539±1369 cpm for PyV±3 Gy (P =.001); (3) lymphoproliferation to LPS 12, 470 ± 1034 cpm for PyV versus 2601 ± 894 cpm for PyV ± 3 Gy (P < .001); (4) Con A stimulated IFN- production 1891 ± 466 pg/mL for PyV versus 789 ± 493 pg/mL for PyV + 3 Gy (P = −.033); and (5) LPS stimulated IFN- production 513 ± 122 pg/mL for PyV versus 88.3 ± 40.1 pg/mL for PyV + 3 Gy (P = .001). In a group of 168 animals a second dose of radiation was employed to look for reactivation of latent viruses (Table 2). By day 49 both control and irradiated animals had cleared the virus. Ten days later, viral replication was detected in animals given radiation on day 49. Spleens of animals were analyzed for weight, cell count, proliferative response to Con A, and IFN- production to Con A for days 0–49 (Fig. 1) and days 49–69 (Fig. 2). PyV and 3 Gy separately and collectively exerted profound changes in each of these measurements made in primary injection (Fig. 1) and reactivated infection (Fig. 2). In every instance 3 Gy reduced the value of the item measured in PyV infected or reactivated cells compared to just PyV treated cells. 2.3. Implications of model The model demonstrates acute changes in the ability of host defense mechanisms to contain latent virus infection that can be quantitated with regard to several immune responses, principally T cell mediated immune responses. Immune intact animals infected with PyV demonstrated detectable virus replication rate through 10 days after inoculation, with the virus being undetectable at 20 days. Radiation given one day prior to PyV inoculation raised the viral replication incidence in animals at day 10; virus remained evident at day 20, but was cleared by day 49. Subsequent exposure to radiation on day 49 in PyV-infected animals resulted in virus reactivation and replication to detectable levels by day 59. One of the two functional consequences of radiation was a decrease in the ability of splenic cells to respond to activation by the T-cell agonist, Con A, which had not recovered 7 weeks after gamma radiation. There

W.T. Shearer et al. / Acta Astronautica 60 (2007) 247 – 253

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Table 1 Effect of radiation on clearance of murine PyV after acute infection of BALB/c mice Days postinoculation

Number (%) tissue samples PyVa,b Total tissuesc

3 7 12 24 30

High virus tissues (kidney, liver, bone)d

Low virus tissues (salivary gland, lung, mammary gland, skin)

Not irradiated

Irradiatede

Not irradiated

Irradiated

Not irradiated

Irradiated

7 7 1 2 0

32 26 30 23 9

5 6 1 2 0

17 18 15 14 7

2 1 0 0 0

15 8 15 9 2

(50) (50) (7) (14) (0)

(76) (62) (71) (55) (21)

(83) (100) (17) (33) (0)

(94) (100) (83) (78) (39)

(25) (12) (0) (0) (0)

(62) (33) (62) (38) (8)

Note: Taken with permission from [30]. a Mice were inoculated with 75 HAU PyV intraperitoneally on day 1 of experiment. b Detection of PyV was performed with a PCR assay (see Methods). c Tissues (n = 7) analyzed per mouse: kidney, liver, bone, salivary gland, lung, mammary gland, and skin. The spleen was harvested for immunological studies. Number of mice/time point: not irradiated = 2; irradiated = 6. d High virus tissues were defined as those for which  50% of specimens from control (non-irradiated) mice collected over the period of the first 7 days postinoculation were virus-positive; low virus tissues, < 50% of specimens from controls were virus-positive. e Mice were irradiated with -rays (3 Gy) with a 137 Cs source on day 0.

Table 2 Effect of -irradiation on replication of PyV in mouse spleen cells harvested from BALB/c mice Day

Experimental group Controlsa

PyV inoculated (day 1)

0 3 10 20 49 54 59 69

A1

A2

B1

B2

C

D

Irradiated ND 5/6 3/6 0/6 Irradiated 0/6 2/6 1/6

Irradiated ND ND ND ND Not irradiated 0/6 0/6 0/6

Not irradiated 5/6 3/6 0/6 0/6 Irradiated 0/6 5/6 1/6

Not irradiated ND ND ND ND Not irradiated 0/6 0/6 0/6

Irradiated ND 0/2 0/2 0/2

Not irradiated ND 0/2 ND 0/2

ND ND ND

ND ND ND

Groups of mice (n = 6) were irradiated with -rays (3 Gy) with a 137 Cs source on days 0∗ and 49† and were infected with 75 HAU of PyV on day 1‡ (see key below). Detection of PyV replication and reactivation was made with a PCR assay (see Methods). The number of animals per group testing positive for viral DNA in spleen cells is indicated as a ratio for each time point. ND, Not done. A1, 3 Gy∗ /PyV‡ /3 Gy† ; A2, 3 Gy∗ /PyV‡ /0 Gy; B1, 0 Gy/PyV‡ /3 Gy† ; B2, 0 Gy/PyV‡ /0 Gy; C, 3 Gy∗ /PBS/0 Gy† ; D, 0 Gy/PBS/0 Gy. Note: Taken with permission from [30]. a In uninfected control groups C and D, six mice per time point were harvested for immunological studies, but only two animals at selected time points were tested for PyV.

was also an impairment of the ability of these stimulated splenic cells to secrete IFN-, a cytokine that is essential for the elimination of PyV infection. In the context of PyV infection, radiation prolonged primary virus replication in animals and radiation of latently infected animals reactivated virus replication. Radiation reduced splenic cell proliferation in animals showing active PyV replication. The increased production of

IFN- by splenic cells in PyV-infected animals, a normal immune response, was impaired by radiation. 3. Lessons from human medicine It is known that humans will be subject to continuous and intermittent doses of radiation in deep space—protons, gamma rays, and heavy metal ions

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A

B

Spleen Weight

Spleen Cell Count 150

250

150

100 A1 B1 C D

50

Mean (x106) ± SEM

Mean (mg) ± SEM

200

0

100

50

A1 B1 C D

0 01 3

10

20

49

01 3

10

day (d) P values A1 vs D B1 vs D C vs D A1 vs B1 A1 vs C

C

d3 <0.001 <0.001 <0.001 <0.001 NS

d10 <0.001 <0.001 <0.001 <0.001 NS

49

day (d) d20 0.055 0.011 0.033 <0.001 NS

d49 <0.001 <0.001 NS 0.017 <0.001

Proliferative Response to Con-A

P values A1 vs D B1 vs D C vs D A1 vs B1 A1 vs C

D

100000

d3 <0.001 <0.001 <0.001 <0.001 NS

d10 <0.001 NS <0.002 <0.001 NS

d20 NS <0.001 NS <0.001 0.003

10000

1000

A1 B1 C D

100

d49 0.005 NS NS 0.006 <0.001

IFN-γ Production to Con-A 500

Mean (pg/mg) ± SEM

Mean (cpm) ± SEM

20

A1 B1 C D

400

300

200

100

0 01 3

10

20

49

01 3

10

day (d) P values A1 vs D B1 vs D C vs D A1 vs B1 A1 vs C

d3 <0.001 <0.001 <0.001 0.003 NS

d10 0.001 <0.006 <0.001 <0.001 NS

20

49

day (d) d20 <0.001 NS <0.001 <0.001 NS

d49 <0.001 <0.001 <0.001 0.004 NS

P values A1 vs D B1 vs D C vs D A1 vs B1 A1 vs C

d3 0.001 NS 0.012 <0.001 NS

d10 0.003 <0.001 NS NS 0.012

d20 0.002 <0.001 NS 0.008 0.007

d49 NS NS 0.023 NS 0.004

Fig. 1. Effects of -irradiation (day 0, arrow) and primary PyV infection (75 HAU; day 1, open box) on spleen weights (A), spleen cell counts (B), proliferative responses to concanavalin A (Con A) stimulation (C), and IFN- production in response to Con A stimulation (D). Significance factors for differences between groups of mice (key in box in each panel) analyzed by ANOVA-LSD are given in a grid below each panel. The treatments for each group of mice (A1, B1, C, D) are described in Table 2. There were six animals/group/time point. Note: Taken with permission from [30].

W.T. Shearer et al. / Acta Astronautica 60 (2007) 247 – 253

A

Spleen Weight

B

200

251

Spleen Cell Count

A1 A2

200

150

100 A1

50

A2

Mean (x106) ± SEM

Mean (mg) ± SEM

B1 B2

100

B1 B2

0

0 49

54

59

49

69

54

day (d) P values A1 vs B2 A2 vs B2 B1 vs B2 A1 vs A2 A1 vs B1

C

d49 0.029 0.018 NS NS 0.026

d54 <0.001 <0.001 <0.001 <0.001 NS

59

69

day (d) d59 <0.001 NS 0.001 <0.001 <0.001

d69 NS NS NS NS NS

Proliferative Response to Con-A 100000

P values A1 vs B2 A2 vs B2 B1 vs B2 A1 vs A2 A1 vs B1

d49 0.017 0.005 NS NS 0.017

d54 <0.001 NS <0.001 <0.001 NS

d59 <0.001 NS <0.001 <0.001 NS

IFN-γ Production to Con-A

D

A1

200

A1

A2

10000

1000

100

Mean (pg/mg) ± SEM

Mean (cpm) ± SEM

A2 B1 B2

d69 <0.001 <0.001 <0.001 NS NS

B1 B2

150

100

50

0 49

54

69

59

49

54

day (d) P values A1 vs B2 A2 vs B2 B1 vs B2 A1 vs A2 A1 vs B1

d49 <0.001 0.001 NS NS <0.001

d54 NS 0.002 NS <0.001 0.051

69

59 day (d)

d59 NS 0.006 NS 0.007 NS

d69 NS 0.040 NS NS NS

P values A1 vs B2 A2 vs B2 B1 vs B2 A1 vs A2 A1 vs B1

d49 NS NS NS NS NS

d54 NS NS 0.013 0.002 <0.001

d59 0.031 0.009 NS NS 0.047

d69 0.004 NS 0.018 0.011 NS

Fig. 2. Effects of -irradiation (day 49, arrow) and reactivated PyV infection on mouse spleen weights (A), spleen cell counts (B), proliferative responses to concanavalin A (Con A) stimulation (C), and IFN- production in response to Con A stimulation (D). Significance factors for differences between groups of mice (key in box in each panel) analyzed by ANOVA-LSD are given in a grid below each panel. The treatments for each group of mice (A1, A2, B1, B2) are described in Table 2. There were six animals/group/time point.

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[31]. Since virtually all humans carry latent viruses, such as Epstein-Barr and PyV, weakening of immunity by radiation is very likely to lead to reactivation of these latent viruses, chronic viral infection, and viral transformation of normal cells into malignant clones. These preliminary studies must be followed by similar experiments of longer duration to assess the incidence of tumorigenesis. Recently, Thorley-Lawson has described how Epstein-Barr virus takes up residence in human B cells and occasionally produces B cell lymphomas in patients who are immunosuppressed [32]. Virtually all of these patients are those undergoing bone marrow and organ transplantation and those with congenital immunodeficiency [33]. Once Epstein-Barr virus enters B cells, it remains latent and its viral genes latent membrane protein 1 (LMP1) and LMP2 inhibit surface expression of viral antigens that could be recognized by cytotoxic T cells. However, with further disruption of the immune system, such as would happen with radiation, the virus begins to replicate and produce clones of B cells that, unless they are killed off by cytotoxic T cells or undergo apoptosis, can develop into B cell lymphomas [34,35]. Fortunately for humans suffering from B cell lymphomas, the golden age of antigen specific (e.g., EBV) monoclonal antibodies has arrived and remarkable cures are now being observed [36–38]. For space travelers, too, the development of these biological therapies that can be taken into space offer a powerful yet practical rescue from radiation and latent virus induced malignancies. 4. Summary In summary, use of this animal model of radiation and latent virus infection will very likely be able to predict some of the long-term health risks of humans exposed to space travel condition. The addition of other conditions of long-term travel into deep space (i.e., microgravity, sleep deprivation, isolation) is likely to add to the value of this model system. Acknowledgments We thank Maria Shlyapobersky and De-Yu Shen for technical assistance, and Carolyn Jackson for assistance with manuscript preparation. References [1] G. Sonnenfeld, J.S. Butel, W.T. Shearer, Effects of the space flight environment on the immune system, Reviews on Environmental Health 18 (2003) 1–17.

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