NASA is concerned with the health risks to astronauts, particularly those risks related to radiation exposure. Both radiation and increased iron stores can independently increase oxidative damage, resulting in protein, lipid and DNA oxidation. Oxidative stress increases the risk of many health problems including cancer, cataracts, and heart disease. This study, a subset of a larger interdisciplinary investigation of the combined effect of iron overload on sensitivity to radiation injury, monitored immune parameters in the peripheral blood of rats subjected to gamma radiation, high dietary iron or both. Specific immune measures consisted of (A) peripheral leukocyte distribution; (B) plasma cytokine levels; (C) cytokine production profiles following whole blood stimulation of either T cells or monocytes

Crucian, Brian E.

Morgan, Jennifer L. L.

Quiriarte, Heather A.

Sams, Clarence F.

Smith, Scott M.

Zwart, Sara R.

Both radiation and increased iron stores can independently increase oxidative damage, resulting in protein, lipid and DNA oxidation. Oxidative stress increases the risk of many health problems including cancer, cataracts, and heart disease. This study, a subset of a larger interdisciplinary investigation of the combined effect of iron overload on sensitivity to radiation injury, monitored immune parameters in the peripheral blood of rats subjected to gamma radiation, high dietary iron or both. Specific immune measures consisted of: (1) peripheral leukocyte distribution, (2) plasma cytokine levels and (3) cytokine production profiles following whole blood mitogenic stimulatio

NASA Technical Reports Server

Combined effects of Gamma Radiation and High Dietary Iron on 
Peripheral Leukocyte Distribution and Function 
Brian E. Crucian, 
Jennifer L.L. Morgan 
Heather A. Quiriarte 
Clarence F. Sams 
Scott M. Smith 
Sara R. Zwart 
https://ntrs.nasa.gov/search.jsp?R=20120007589 2019-08-30T19:58:29+00:00Z
•This study, a subset of a larger interdisciplinary investigation of the combined 
effect of iron overload on sensitivity to radiation injury, monitored 
immune parameters in the peripheral blood of rats subjected to gamma 
radiation, high dietary iron or both.   
 
•Specific immune measures consisted of:  
-peripheral leukocyte distribution 
-plasma cytokine levels 
-cytokine production profiles following whole blood mitogenic stimulation 
•Both radiation and increased iron stores 
can independently increase oxidative 
damage, resulting in protein, lipid and 
DNA oxidation.   
 
•Oxidative stress increases the risk of 
many health problems including cancer, 
cataracts, and heart disease.   
Concept 
THE IMMUNE SYSTEM 
050
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L-180 L-45 Early Mid Late R+0 R+30
Immune dysregulation during long-duration spaceflight 
(SMO-015 mid-study data; n=10)
Cytotoxic CD8+ T cell function CD4+ T cell function CD8+
IL-10 (CD3/CD28) IL-6 (PMA-I) TNFa (PMA-I)
Immune Parameters
Integrated Immune mid-study long duration data (n=10) 
VZV Reactivation 
Immune dysregulation during deep space missions has the capacity to 
synergize with other variables such as oxidative damage or radiation 
exposure.   This would further enhance clinical risk to crewmembers. 
Current Study Design 
Irradiation - Cs-137 source, .375 Gy  Sacrifice
Starting after week 2, irradiation every other day (8 doses);  3 Gy total)
Normal iron diet
Week 1 Week 2 Week 3 Week 4
High iron diet
Normal iron diet
High iron diet
Specific Immunology Assays 
•WBC 
 
•Neutrophil, Lymphocyte, Monocyte 
 
•T cell subsets:  CD4+/CD8+ (Flow cytometry) 
 
•Cytokine Profiles (cytometrix bead array)  
 Adaptive immunity:  IFNg, IL-10, IL-4, IL-2 
 Innate/inflammatory:  TNFa, IL-1b, IL-6) 
Leukocyte Distribution 
0
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12
0 1 2 3 4 5
WBC
Control FE++ IRR FE++/IRR
*
0
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30
40
50
60
0 1 2 3 4 5
Grans
Control FE++ IRR FE++/IRR
*
*
0
20
40
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100
120
0 1 2 3 4 5
Lymph
Control FE++ IRR FE++/IRR
**
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0 1 2 3 4 5
Mono
Control FE++ IRR FE++/IRR
0
10
20
30
40
50
60
70
80
0 1 2 3 4 5
CD4+
Control FE++ IRR FE++/IRR
**
0
10
20
30
40
50
60
0 1 2 3 4 5
CD8+
Control FE++ IRR FE++/IRR
**
00.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 1 2 3 4 5
IFNg
Control FE++ IRR FE++/IRR
0
2
4
6
8
10
12
14
16
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20
0 1 2 3 4 5
IL-10
Control FE++ IRR FE++/IRR
0
20
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60
80
100
120
0 1 2 3 4 5
IL-1b
Control FE++ IRR FE++/IRR
0
5
10
15
20
25
30
35
0 1 2 3 4 5
IL-2
Control FE++ IRR FE++/IRR
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 1 2 3 4 5
IL-4
Control FE++ IRR FE++/IRR
0
20
40
60
80
100
120
0 1 2 3 4 5
IL-6
Control FE++ IRR FE++/IRR
0
5
10
15
20
25
30
0 1 2 3 4 5
TNFa
Control FE++ IRR FE++/IRR
Constitutive Plasma Cytokine 
Levels 
020
40
60
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180
0 1 2 3 4 5
IFNg
Control FE++ IRR FE++/IRR
*
0
50
100
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300
0 1 2 3 4 5
IL-10
Control FE++ IRR FE++/IRR
*
0
10
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40
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70
0 1 2 3 4 5
IL-1b
Control FE++ IRR FE++/IRR
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0 1 2 3 4 5
IL-2
Control FE++ IRR FE++/IRR
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16
0 1 2 3 4 5
IL-4
Control FE++ IRR FE++/IRR
*
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6000
0 1 2 3 4 5
IL-6
Control FE++ IRR FE++/IRR
0
20
40
60
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180
0 1 2 3 4 5
TNFa
Control FE++ IRR FE++/IRR
*
Cytokine Production Profiles (anti-
CD3/28, 48hr) 
010
20
30
40
50
60
0 1 2 3 4 5
IFNg
Control FE++ IRR FE++/IRR
0
10
20
30
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60
70
0 1 2 3 4 5
IL-10
Control FE++ IRR FE++/IRR
***
0
10
20
30
40
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60
70
80
90
0 1 2 3 4 5
IL-1b
Control FE++ IRR FE++/IRR
0
5
10
15
20
25
30
35
40
45
0 1 2 3 4 5
IL-2
Control FE++ IRR FE++/IRR
0
1
2
3
4
5
6
0 1 2 3 4 5
IL-4
Control FE++ IRR FE++/IRR
*
0
100
200
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900
0 1 2 3 4 5
IL-6
Control FE++ IRR FE++/IRR
*
0
10
20
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40
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100
0 1 2 3 4 5
TNFa
Control FE++ IRR FE++/IRR
Cytokine Production Profiles (anti 
LPS, 48hr) 
050
100
150
200
250
0 1 2 3 4 5
IFNg
Control FE++ IRR FE++/IRR
*
*
0
20
40
60
80
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120
0 1 2 3 4 5
IL-10
Control FE++ IRR FE++/IRR
0
10
20
30
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0 1 2 3 4 5
IL-1b
Control FE++ IRR FE++/IRR
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0 1 2 3 4 5
IL-2
Control FE++ IRR FE++/IRR
*
*
0
1
2
3
4
5
6
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8
0 1 2 3 4 5
IL-4
Control FE++ IRR FE++/IRR
**
0
200
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600
800
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1200
0 1 2 3 4 5
IL-6
Control FE++ IRR FE++/IRR
0
20
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80
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120
140
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0 1 2 3 4 5
TNFa
Control FE++ IRR FE++/IRR
Cytokine Production Profiles (anti 
PMA-I, 48hr) 
Conclusions 
(and places we can go…) 
Lutetia (Rosetta) Gaspar (Galileo) Ida (Galileo) 
•Gamma-radiation treatment:  Demonstrable alterations in peripheral 
leukocyte distribution and leukocyte cytokine production following 
mitogenic stimulation.    
 
•High iron diet:  Resulted in an elevated WBC but with a normal subset 
distribution; minimal direct immune effects; abrogated some of the 
radiation-induced functional alterations but not the phenotypic alterations 
 
•Summary:  Radiation induced demonstrable changes in peripheral 
immunity.   Generally, the high iron diet did not, yet did abrogate many of 
the radiation effects. 
ALTERED INNATE AND LYMPHOCYTIC IMMUNITY IN MURINE 
SPLENOCYTES FOLLOWING SHORT-DURATION SPACEFLIGHT 
Brian E. Crucian 
Shen-An Hwang 
Jeffrey K. Actor 
Heather Quiriarte  
Clarence F. Sams 
STS-135 Study Design 
STS-135 (13 day mission) 
Ground controls 
½ spleen; n=6 
Splenocytes harvested 3-4 
hours post-landing 
Phenotype 
T cells:  CD4:CD8 
Dendritic cells: 
CD11c, CD86, MHC-I 
Cytokine Profiles 
CD3/CD28 
PMA/I 
LPS 
(7 cytokine array) 
Activation marker expression and 
cytokine profiles 
24 or 48hr cell culture 
T-cell Function: 
Anti-CD3/28 
SEA/SEB 
PMA/Ionomycin 
CD69 and/or CD25 
DC Function: 
Zymosan (TLR-2) 
LPS (TLR-4) 
Flagellin (TLR-5) 
Expression of MHC-I, CD86 
Altered expression of lymphocytic markers post-flight 
*** 
0.071 
*** 
*** = p<0.001 
% positive cells 
Intensityof marker 
expression 
Post-flight splenocytes have decreased expression of 
antigen presentation and co-stimulatory molecules 
** 
*** 
*** 
** = p<0.01 
*** = p<0.001 
% positive cells 
Intensity of marker 
expression 
Post-flight DCs show decreased expression of presentation 
and co-stimulatory molecules after TLR stimulation 
** = p<0.01 
*** = p<0.001 
MHC I 
CD86 
*** *** *** 
** ** 
No differences in 
MHC II observed 
Post-flight splenocytes demonstrate increased CD25+ in 
CD4+/CD8+CD28+ cells when stimulation bypasses the 2o signal 
*** 
*** = p<0.001 
CD4 
* 
* = p<0.05 
CD8 
Data – T Cell Function, 24h culture, CD69/25 dual positive 
0
5
10
15
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40
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50
GROUND FLIGHT
CD4+
*
0
5
10
15
20
25
30
35
40
45
GROUND FLIGHT
CD8+
*
50
55
60
65
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90
GROUND FLIGHT
CD4+
50
55
60
65
70
75
80
85
90
95
100
GROUND FLIGHT
CD8+
SEA+SEB 
αCD3+CD28 
Data – Cytokine Production Profiles 
(anti CD3/28, 48hr) 
0
10
20
30
40
50
60
GROUND FLIGHT
IL-10
*
0
10
20
30
40
50
60
70
80
GROUND FLIGHT
IL-17a
0
50
100
150
200
250
300
GROUND FLIGHT
TNFa
0
50
100
150
200
250
GROUND FLIGHT
IFNg 1058
0
20
40
60
80
100
120
140
160
GROUND FLIGHT
IL-6
0
1
2
3
4
5
6
GROUND FLIGHT
IL-4
0
50
100
150
200
250
300
350
GROUND FLIGHT
IL-2
Data – Cytokine Production Profiles 
(anti PMA-I, 48hr) 
0
10
20
30
40
50
60
70
GROUND FLIGHT
IL-10
0
20
40
60
80
100
120
140
160
GROUND FLIGHT
IL-17a
0
200
400
600
800
1000
1200
1400
GROUND FLIGHT
TNFa
*
0
500
1000
1500
2000
2500
3000
3500
4000
GROUND FLIGHT
IFNg
0
20
40
60
80
100
120
140
160
180
GROUND FLIGHT
IL-6
*
0
1
2
3
4
5
6
GROUND FLIGHT
IL-4
*
0
500
1000
1500
2000
2500
3000
3500
GROUND FLIGHT
IL-2
Data – Cytokine Production Profiles 
(anti LPS, 48hr) 
0
50
100
150
200
250
300
350
GROUND FLIGHT
TNFa
0
2
4
6
8
10
12
14
16
GROUND FLIGHT
IFNg
234
0
50
100
150
200
250
300
350
400
GROUND FLIGHT
IL-6
*
Conclusions 
•These data indicate that alterations in splenocytes phenotype, function 
and cytokine production patterns are evident following spaceflight.   
 
•The pattern suggests that some innate immune functions are possibly 
enhanced, whereas some adaptive immune parameters may be inhibited.   
 
•Follow up human and in-flight studies will determine if a clinical risk 
related to immune dysregulation exists for astronauts. 
Acknowledgement: 


Combined Effects of Gamma Radiation and High Dietary Iron on Peripheral Leukocyte Distribution and Function

NASA HRP IWG – February 2012, Houston, Texas 
Evaluation of the combined effects of gamma radiation and high dietary iron on 
peripheral leukocyte distribution and function  
 
Brian E. Crucian, Jennifer L.L. Morgan, Heather A. Quiriarte, Clarence F. Sams,  
Scott M. Smith and Sara R. Zwart 
 
Background:  NASA is concerned with the health risks to astronauts, particularly those risks 
related to radiation exposure.  Both radiation and increased iron stores can independently 
increase oxidative damage, resulting in protein, lipid and DNA oxidation.  Oxidative stress 
increases the risk of many health problems including cancer, cataracts, and heart disease.  This 
study, a subset of a larger interdisciplinary investigation of the combined effect of iron overload 
on sensitivity to radiation injury, monitored immune parameters in the peripheral blood of rats 
subjected to gamma radiation, high dietary iron or both.  Specific immune measures consisted 
of (A) peripheral leukocyte distribution; (B) plasma cytokine levels; (C) cytokine production 
profiles following whole blood stimulation of either T cells or monocytes.   
Results:  Both radiation exposure and the high iron diet resulted in an altered leukocyte 
distribution.  Iron alone resulted in a significantly elevated WBC, which appeared to be via a 
lymphocytosis.  While radiation did not significantly alter the total WBC, it induced a significant 
increase in the granulocyte percentage and a reduced CD4:CD8 T cell ratio.   Iron treatment 
alone did not have an effect these cell subsets.  Radiation treatment had a significant effect on 
cytokine production following T cell stimulation.  Production of IFNg, IL-10, IL-4 and TNFa were 
significantly elevated following radiation treatment, however for all cytokines measured the 
high iron diet inhibited the radiation effect.  Following monocyte stimulation, radiation 
treatment resulted in elevated IL-4 and decreased IL-6 production, with both effects abrogated 
by the high iron diet.  Interestingly, the high iron diet, radiation treatment, or both resulted in a 
significant decrease in monocyte IL-10 production.  No significant differences were observed in 
plasma cytokine levels for either treatment.    
Conclusion:  Gamma-radiation treatment resulted in demonstrable alterations in peripheral 
leukocyte distribution and leukocyte cytokine production following mitogenic stimulation.   The 
high iron diet abrogated the functional alterations but not the phenotypic alterations.  The high 
iron diet alone resulted in an elevated WBC but with a normal subset distribution.   In most 
cases, the high iron diet reduced mean cytokine production levels but these changes were not 
significant.   Thus, a high iron diet appears to inhibit both constitutive immune functional 
responses and the elevated functional responses observed following radiation treatment. 
 
 
 
https://ntrs.nasa.gov/search.jsp?R=20110023179 2019-08-30T18:04:26+00:00Z


Evaluation of the Combined Effects of Gamma Radiation and High Dietary Iron on Peripheral Leukocyte Distribution and Function

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