Normal Control Fas-Fc con Ig0

10000

20000

30000

40000 unstimanti-CD3-stim

HU treated

*

Pro

lifer

atio

n (c

pm)

Normal Immun Imm+Stress0

5000

10000

15000

20000

25000unstimOVA-stim

Pro

life

rati

on

(cp

m)

Apoptosis and Immune Homeostasis During Hindlimb UnloadingArthur Roberts, Erwei Sun, Lixin Wei, H.S. William Tan, Zengrong Yuan, Catherine Liu, Satish Devadas,

Duane Pierson, Satish Mehta, Kathryn Wang, David Denhardt, Yufang Shi UMDNJ-Robert Wood Johnson Medical School & Rutgers University, Piscataway, NJ and Johnson Space Center, Houston TX

ABSTRACTABSTRACTHindlimb unloading (HU) in rodent is a well-accepted ground-based model used to simulate some space flight conditions and reproduce the deleterious effects on the musculoskeletal, cardiovascular and immune systems.  In this model, mice are suspended by the tail so that only their forelegs contact the ground, while they are free to move about the cage, with food and water provided ad libitum. We have found that HU severely deplete various cell populations in the spleen and thymus. These changes are likely to be mediated by apoptosis, since DNA strand breaks indicative of apoptosis, were detected by TUNEL staining in spleno-cytes and thymocytes.  Administration of opioid antagonists or interference with the Fas-FasL interaction reduced the loss of splenocytes, but not thymocytes.  On the other hand, RU486, a steroid receptor antagon-ist, blocked the reduction of lymphocyte numbers in both spleen and thymus. Therefore, the effects of HU on the homeostasis of splenocytes and thymocytes must be exerted through distinct mechanisms.  HU exposure severely curtailed both non-specific TCR-induced and ovalbumin-specific splenocyte proliferation. The delayed-type hypersensitivity response measured by footpad swelling in response to secondary exposure to OVA was also decreased by HU. Similarly, the expansion of OVA-specific T cells was reduced. Interest-ingly, CD4+CD25+ regulatory T cells were found to be important in the reduction of lymphocyte number and immune suppression. Depletion of these cells by injecting monoclonal antibody against CD25 prevented lymphocyte reduction and improved the immune responses.  Thus, lymphocytes are drastically affected by HU in mice, and normal immune responses are severely blunted. These effects are mediated at least in part by apoptosis, and the Fas-FasL interaction is central in splenocyte reduction, but not so in the thymus.  In addition, CD4+CD25+ T cells play a critical role in modulating immune responses during HU. Further exploration into the mechanism of HU-induced lymphocyte apoptosis and immunosuppression will allow for the development of more effective countermeasures for use during space flight. We have begun to explore some of these: vitamin C to prevent HU-mediated lymphocyte loss and kelp extract as a protectant from radiation-induced lymphocyte depletion.

Fig. 2. Effect of blockade of opioid receptors and corticosteroid receptors on HU-induced lymphocyte reduction. Mice were treated with naltrexone (opioid receptor blocker) or RU486 (a steroid receptor antagonist) then subjected to HU for 2 days, and the effect on splenic and thymic cellularity measured by counting total lymphocytes. HU-induced splenocyte reduction was reversed by either naltrexone or RU486, while thymocytes were rescued only by RU486. *p<0.01 vs. control

Fig. 3. Blocking the Fas-FasL interaction prevents HU-induced splenocyte reduction. Male BALB/c mice were subjected to HU with or without administration of Fas-Fc or control Ig. Total cells in the spleen and thymus were counted. Fas-Fc treatment prevented HU-induced splenocyte losses, but had no effect on thymocyte reductions. *p<0.001 vs. control.

Fig. 4. HU inhibition of T cell prolifera-tion depends on the Fas-FasL interaction. Mice were subjected to HU for 2 d. with or without administration of Fas-Fc. Splenocytes were isolated and cultured in 96-well plates with or without stimulation with soluble anti-CD3. After 5 d, proliferation was measured by [3H]Tdr incorporation using traditional methods. Stress significantly reduced the proliferative capacity of lymphocytes; neutralization of FasL with Fas fusion protein during HU abrogated this effect.

Fig. 5. HU treatment inhibits ovalbumin-specific lymphocyte proliferation. Mice were immunized with ovalbumin (OVA, 100 g) and subjected to HU for 2 d. After 5 more days, splenocytes were harvested and cultured in 96 well plates with or with-out OVA stimulation (1 g/ml), and proliferation measured after 5 days. OVA-specific proliferation was drastically reduced by HU stress.

1. HU in mice, which mimics some of the conditions of space flight, causes a severe loss of lymphocytes in the spleen and thymus. This effect is mediated by apoptosis, and depends on opioid receptors and corticosteroids receptors in the spleen, but only corticosteroid receptors in the thymus.

2. Lymphocyte apoptosis induced by HU, as well as chronic restraint, is mediated by the Fas-FasL interaction in the spleen, but not the thymus. Furthermore, CD4+CD25+ Treg are required.

3. Many parameters of immune responsiveness in vitro and in vivo are inhibited by HU, indicating a potent generalized immunosuppressive effect of this model.

4. Radiation exposure synergizes with HU to induce high levels of lymphocyte apoptosis.

5. Osteopontin, a pleiotropic cytokine, plays a critical role HU-induced lymphocyte reduction.

6. The haptoglobin and transferrin proteins are upregulated during HU and may thus potentially serve as practical serum biomarkers for “stress” levels in astronauts.

7. Promising candidates for dietary supplement countermeasures: Vitamin C protects thymic lymphocytes from HU-mediated depletion; kelp extract protects from radiation-induced lymphocyte loss.

Fig. 1. Hindlimb unloading (HU) causes a severe loss of splenocytes and thymocytes. HU simulates some of the deleterious conditions of space flight, including lack of load-bearing, fluid shift to the head, and psychological stress. In this model, mice are suspended by tail traction so that only the forelegs contact the cage floor. Male BALB/c mice (8 wk old) were subjected to HU for the indicated time, after which lymphoid organs were harvested, single-cell suspensions prepared, cell subpopulations analyzed by immunofluorescence staining and flow cytometry, and absolute numbers of each phenotype determined (mean + SD x 106 ).

RESULTSRESULTS

Fig. 8. Radiation synergizes with HU stress to induce apoptosis. Mice were irradiated with 100 rads gamma rays then subjected to HU. Splenocytes and thymocytes were isolated and apoptosis detected by staining for total DNA content with propidium iodide in a permeabilizing buffer. The hypodiploid peak reveals apoptotic cells that have lost DNA due to cleavage.

Fig. 12. High-dose vitamin C protects the thymus from cell depletion. Mice were fed water supplemented with vitamin C (ascorbic acid, 2 mg/ml) for 2 weeks before HU. The loss of thymocytes was significantly abrogated by vitamin C. Other antioxidants, including mela-tonin and N-acetylcysteine were less effective. Vitamin C did not prevent splenocyte reduction.

CONCLUSIONSCONCLUSIONS

Spleen

0 2 4 6 8 100

20

40

60

CD4+ T cells CD8+ T cells

B cells

To

tal

cell

nu

mb

er (

x10

6)

Thymus

0 2 4 6 8 100

20

40

60

80

100

CD4+CD8+

CD4 SP

CD8 SP

Days HU treatment

control saline naltrexone RU4860

20

40

60

80

100

120 splenocytesthymocytes

HU treated

**

*

To

tal

cell

no

. (X

106

)

Splenocytes

control saline Fas-Fc n.s IgG0

20

40

60

80

100

120

HU treated

* *

Tot

al c

ell n

o. (

x 10

6)

Thymocytes

control saline Fas-Fc n.s IgG0

20

40

60

80

100

HU treated

* **

Normal: 0.43% Immun: 0.78% Immun+HU: 0.13%

CD8-FITC

O

VA

-spe

c te

tra

1.2% 1.7%

19.6%53.3%

Control

Radiation+ Uuloading

Unloading

Radiation

4.2% 4.9%

22.3%35.6%

DNA Content

Control

Radiation+Uuloading

Unloading

Radiation

Spleen Thymus

1.2% 1.7%

19.6%53.3%

Control

Radiation+ Uuloading

Unloading

Radiation

1.2% 1.7%

19.6%53.3%

1.2% 1.7%

19.6%53.3%

Control

Radiation+ Uuloading

Unloading

Radiation

4.2% 4.9%

22.3%35.6%

DNA Content

Control

Radiation+Uuloading

Unloading

Radiation

Spleen Thymus

1.2% 1.7%

19.6%53.3%

Control

Radiation+ Uuloading

Unloading

Radiation

1.2% 1.7%

19.6%53.3%

1.2% 1.7%

19.6%53.3%

Control

Radiation+ Uuloading

Unloading

Radiation

4.2% 4.9%

22.3%35.6%

DNA Content

Control

Radiation+Uuloading

Unloading

Radiation

Spleen Thymus

1.2% 1.7%

19.6%53.3%

1.2% 1.7%

19.6%53.3%

Control

Radiation+ Uuloading

Unloading

Radiation

1.2% 1.7%

19.6%53.3%

1.2% 1.7%

19.6%53.3%

Control

Radiation+ Uuloading

Unloading

Radiation

4.2% 4.9%

22.3%35.6%

DNA Content

Control

Radiation+Uuloading

Unloading

Radiation

Spleen Thymus

1.2% 1.7%

19.6%53.3%

1.2% 1.7%

19.6%53.3%

Control

Radiation+ Uuloading

Unloading

Radiation

4.2% 4.9%

22.3%45.6%

DNA Content

Control

Radiation+Uuloading

Unloading

Radiation

Spleen Thymus

1.2% 1.7%

19.6%53.3%

Control

Radiation+ Uuloading

Unloading

Radiation

1.2% 1.7%

19.6%53.3%

1.2% 1.7%

19.6%53.3%

Control

Radiation+ Uuloading

Unloading

Radiation

4.2% 4.9%

22.3%35.6%

DNA Content

Control

Radiation+Uuloading

Unloading

Radiation

Spleen Thymus

1.2% 1.7%

19.6%53.3%

1.2% 1.7%

19.6%53.3%

Control

Radiation+ Uuloading

Unloading

Radiation

1.2% 1.7%

19.6%53.3%

1.2% 1.7%

19.6%53.3%

Control

Radiation+ Uuloading

Unloading

Radiation

4.2% 4.9%

22.3%35.6%

DNA Content

Control

Radiation+Uuloading

Unloading

Radiation

Spleen Thymus

1.2% 1.7%

19.6%53.3%

1.2% 1.7%

19.6%53.3%

Control

Radiation+ Uuloading

Unloading

Radiation

4.2% 4.9%

22.3%35.6%

DNA Content

Control

Radiation+Uuloading

Unloading

Radiation

Spleen Thymus

1.2% 1.7%

19.6%53.3%

1.2% 1.7%

19.6%53.3%

Control

Radiation+ Uuloading

Unloading

Radiation

1.2% 1.7%

19.6%53.3%

1.2% 1.7%

19.6%53.3%

Control

Radiation+ Uuloading

Unloading

Radiation

4.2% 4.9%

22.3%35.6%

DNA Content

Control

Radiation+Uuloading

Unloading

Radiation

Spleen Thymus

1.2% 1.7%

19.6%53.3%

1.2% 1.7%

19.6%53.3%

Control

Radiation+ Uuloading

Unloading

Radiation

1.2% 1.7%

19.6%53.3%

1.2% 1.7%

19.6%53.3%

Control

Radiation+ Uuloading

Unloading

Radiation

4.2% 4.9%

22.3%35.6%

DNA Content

Control

Radiation+Uuloading

Unloading

Radiation

Spleen Thymus

1.2% 1.7%

19.6%53.3%

1.2% 1.7%

19.6%53.3%

Control

Radiation+ Uuloading

Unloading

Radiation

4.2% 4.9%

22.3%45.6%

DNA Content

Control

Radiation+Uuloading

Unloading

Radiation

Spleen Thymus

DNA Content

Control

Radiation+Uuloading

Unloading

Radiation

Spleen Thymus Spleen ThymusSplenocytes Thymocytes

Rad+HU+HU

Rad+HURadRad

HUHU

Thymus Cellularity

control Vitamin C0

50

100

150

200

250 controlHU

To

tal

cell

no

. (x

106

)

Fig. 11. Haptoglobin (Hpg) and transferrin increase during HU. Sera from mice subjected to HU were analyzed by 2-D PAGE for changes in proteins (shown). Microsequencing of excised spots revealed high levels of Hpg and transferrin after HU. By ELISA, serum levels of Hpg increased 10-20 fold. Serum samples from astronauts pre- and post-flight are currently being analyzed. Preliminary results indicate that Hpg is increased immediately after space flight.

*

Fig. 6. Activated T cells are more sensitive to HU. Mice were immunized with OVA with or without HU treatment, as in Fig.5, and splenocytes stained by 2-color immunofluorescence to reveal CD8+ T cells with OVA-specific T cell receptor using fluorescent MHC class I tetramer. Flow cytometry revealed CD8+ T cells positive for tetramer (circled red); their numbers as a fraction of total CD8+ T cells in the spleen is given. HU treatment nearly eliminated OVA-specific T cells, indicating suppression of antigen-driven T cell expansion.

HU

Con

Fig. 10. Osteopontin-deficiency prevents HU-induced lymphocyte loss. Osteopontin-KO mice were subjected to HU for 3 days. OPN-KO mice had fewer splenic lymphocytes, but these were less sensitivity HU depletion. Results were similar with thymocytes. In comparison, OPN-deficiency did not protect lymphocyte from depletion in response to restraint stress, confinement in a 50 ml tube for alternating 12 h periods.

Thymocytes

control HU0

20

40

60

80

100

120Splenocyte

control HU0

20

40

60

80

100

120

WTKO

cell

nu

mb

er x

10

6

splenocytes

control 100 rads0

2

4

6

8

controlkelp

tota

l ce

lls

(x 1

06)

thymocytes

control 100 rads0

2

4

6

control

kelp

Fig. 9. CD4+CD25+ Regulatory T cells (Treg) are critical in HU-induced splenocyte depletion. Treg were eliminated in vivo by injecting anti-CD25 Ab one day before subjecting mice to HU. Total splenocyte counts revealed strong protection by depletion of Treg. The effect was similar to that found by Ab-neutralization of FasL.

control HU

10

30

50

70

90

110

130

150

170

Anti-CD25

Saline

Anti-FasL

Sp

len

ocy

te n

um

ber

(x1

06)

Fig. 13. Kelp extract protects lymphocytes from radiation-induced depletion. Mice were fed with a polysaccharide extract of the brown seaweed, Laminaria japonica (part of the Eastern diet for centuries), for 3 weeks before gamma irradiation. Lymphocyte losses in spleen and thymus were significantly improved by this dietary supplement.