SPACEFLIGHT AFFECTS IMMUNE CELLS

MARIANNE COGOLI-GREUTER, AUGUSTO COGOLI

Zero-g LifeTec GmbH

2007 ELGRA Symposium / XX National Meeting of AIMAS, Florence, September 4 - 7, 2007

STUDIES WITH IMMUNE CELLS IN CULTURE ⇒ ININ--VITRO EXPERIMENTSVITRO EXPERIMENTS- IN SPACE - ON EARTH IN MODELED µG

INVESTIGATIONS ON THE IMMUNE SYSTEM OF HUMANS IN SPACE ⇒ EXEX--VIVO / INVIVO / IN--VIVO STUDIESVIVO STUDIES

SUMMARY OF RESULTS OF SUMMARY OF RESULTS OF

25 YEARS IN25 YEARS IN--VITRO STUDIESVITRO STUDIES

REPORTS IN THE EARLY 70’S REVEALED:

LYMPHOCYTES FROM CREW MEMBERS ON US AND SOVIET SPACE MISSIONS HAD A DECREASED

RESPONSE TO MITOGEN AFTER FLIGHT

• KIMZEY ET AL. - APOLLO AND SKYLAB

• KONSTANTINOVA ET AL. - SOYUZ

EFFECTS OF WEIGHTLESSNESS ON LYMPHOCYTE EFFECTS OF WEIGHTLESSNESS ON LYMPHOCYTE PROLIFERATIONPROLIFERATION

EXPERIMENT PROPOSED BY A. COGOLI FOR SPACELAB-1

EFFECTOR CELL

RESTING CELL

MEMORY CELL

ACTIVATOR

BLAST CELL

ACTIVATION OF T-LYMPHOCYTES

ISOLATION OF LYMPHOCYTESISOLATION OF LYMPHOCYTES

Centrifugation

Blood on Ficoll Lymphocytes

RESULTS FROM SPACELAB-1 1983, Cogoli et al., Science, 225: 228- 30, 1984

ININ--VITRO EXVITRO EX--VIVO / INVIVO / IN--VIVOVIVO

IN-VITRO: ISOLATED CELLS IN CULTURE MEDIUM

EX-VIVO / IN-VIVO: IMMUNE CELLS ARE EXPOSED TO OTHER SYSTEMS, INCLUDING NEUROENDOCRINE SYSTEM

020406080

100

Act

ivat

ion

leve

l in

%

0 x gspace

1 x gspace

1 x gground

MITOGEN ACTIVATED LYMPHOCYTES MITOGEN ACTIVATED LYMPHOCYTES RESULTS FROM SPACELAB D-1, 1985

LYMPHOCYTES IN SPACELAB DLYMPHOCYTES IN SPACELAB D--1, 19851, 19853 DAYS AFTER MITOGENIC ACTIVATION

In-flight 1g control in µg samples reference centrifuge

FAST ROTATING CLINOSTAT

RANDOM POSITIONING MACHINE

APOPTOSIS IN ACTIVATED TAPOPTOSIS IN ACTIVATED T--CELLS IN MODELED CELLS IN MODELED µµGG

0

1

2

3

4

5

6

7

0 2 4 8 12 24 48 72

µg 1g

Ap

op

toti

c b

od

y fo

rmat

ion

*

**

Incubation time (hrs)

Maccarrone et al., J Leukocyte Biology, 2003

IN FLIGHT ACTIVATION OF WHOLE BLOOD IN FLIGHT ACTIVATION OF WHOLE BLOOD LYMPHOCYTES FROM 4 DONORSLYMPHOCYTES FROM 4 DONORS, SLS, SLS--1, 19911, 1991

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

donor 1 donor 2 donor 3 donor 4

0 g0.6 g1 g1.36 g1.75 g

Cogoli et al.,1996

3 SIGNALS ARE REQUIRED FOR FULL T CELL 3 SIGNALS ARE REQUIRED FOR FULL T CELL ACTIVATIONACTIVATION

Antigen/MHCanti-CD3ConA

TCR/CD3

CD28

IL-2R

B7 (Accessory cell / monocytes)

IL-2(Autocrine signal)

SIGNAL TRANSDUCTION IN T CELLS /1SIGNAL TRANSDUCTION IN T CELLS /1

TCR/CD3CD28

B7

G

Cytoskeleton

Motion

PLCPKC

c-fosc-jun AP-1

IL-2

DAG

Pi3

PIP2

IP3

ER

Ca2+

?

SIGNAL TRANSDUCTION IN T CELLS /2SIGNAL TRANSDUCTION IN T CELLS /2

TCR/CD3

IL-2RCD28

B7IL-2

IL-2

PROLIFERATION

JAK

STAT

STATIL-2 IL-2R

DELIVERY OF FIRST SIGNALDELIVERY OF FIRST SIGNAL

• BINDING OF MITOGEN CONCANAVALIN A NOT ALTERED IN µG

• CROSSLINKING OF THE RECEPTORS (PATCHING AND CAPPING) IS DELAYED

DELIVERY OF FIRST SIGNAL NOT DISTURBED BY GRAVITY UNLOADING

(J. Hatton, ESA)

DELIVERY OF SECOND SIGNALDELIVERY OF SECOND SIGNAL

SUSPENDED LYMPHOCYTES ARE HIGHLY MOTILE IN µG AND FORM AGGREGATES

1

55

µm

CELL A

CELL D

µm

55

1

Cell out of plane

CELL G

75 80 85 905

10

15

20

25

86 88 90 92 94 96 98 100 10228

30

32

34

36

38

40

42

µm

55

1

CELL B

125 130 135 140 145 1505

10

15

20

25

30

70 75 80 85 90 9560

65

70

75

80

85

1

55

µm

46 HOURS

78 HOURS

SINGLE CELLS (MAXUS 1)

12 HOURS 46 HOURS

CON A ACTIVATED CELLS (IML-2)

MOTILITY OF MONOCYTES IN MODELED MOTILITY OF MONOCYTES IN MODELED µµGGLOCOMOTION TRACKS OF J-111 ON GOLD-PARTICLE

COATED SLIDES

INCUBATION AT 37°C

1 HOUR

24 HOURS

CONTROL RPM

SINGEL CELL ATTACHED ON COLLOIDAL GOLD SINGEL CELL ATTACHED ON COLLOIDAL GOLD COATED COVER SLIDESCOATED COVER SLIDES

CONTROL RPM

INCUBATION TIME AT 37°C: 24 HOURS

0

20

40

60

80Fr

eque

ncy

%

1 2 3 4 5 6 7 8 9 10 11

Control 24h

FREQUENCY OF MIGRATION FREQUENCY OF MIGRATION

DISTANCE (DISTANCE (µµm) m) DURING DURING

24 HOURS (%)24 HOURS (%)

11 00--44

22 55--99

33 1010--1414

44 1515--1919

55 2020--2424

66 2525--2929

77 3030--3434

88 3535--3939

99 4040--4444

1010 4545--4949

1111 ≥≥500

20

40

60

80

Freq

uenc

y %

1 2 3 4 5 6 7 8 9 10 11

R.P.M. 24h

50

FF--ACTIN IN J111 CELLSACTIN IN J111 CELLS

INCUBATION AT 37° C

1 HOUR

24 HOURS

CONTROL RPM

STRUCTURE OF VIMENTIN IN JURKAT CELLSSTRUCTURE OF VIMENTIN IN JURKAT CELLS

MAXUS 1 (1991)

Ground control 30 sec at µg

MICROTUBULE MORPHOLOGY IN JURKAT CELLSMICROTUBULE MORPHOLOGY IN JURKAT CELLSLewis et al, 1998

Ground control fixed 4h after activation in µg

DELIVERY OF THE THIRD SIGNAL DELIVERY OF THE THIRD SIGNAL

TCR/CD3

IL-2RCD28

B7IL-2

IL-2

PROLIFERATION

JAK

STAT

STATIL-2 IL-2R

SECRETION OF ILSECRETION OF IL--1, IL1, IL--2 AND IL2 AND IL--2R OF CON A 2R OF CON A ACTIVATED LYMPHOCYTES (IMLACTIVATED LYMPHOCYTES (IML--2, 1994)2, 1994)

0

50

100

150

200

Sec

retio

n le

vel i

n %

0 x gspace

1 x gspace

1 x gground

IL-1IL-2IL-2R

IL-2Ralpha

IL-2Rbeta

IL-2Rgamma

IL-2 Receptor

IL-2

T cell membrane

α- and β-subunits: produced during activation process

χ-subunit: constitutively present on membrane

EARLY TRANSCRIPTIONAL RESPONSE OF T-LYMPHOCYTES TO MICROGRAVITY CONDITIONS

MASER 9, 2002

Gene Gene modifiedmodified** Type of Type of modulationmodulationILILββ11 inducedinduced

ILIL--4R4Rαα, IL, IL--13R13Rαα1, IL1, IL--13R13Rαα22 inducedinduced

cytokinecytokine A13A13 inducedinduced

ILIL--88 depresseddepressed

ILIL--2R2Rαα, IL, IL--7R7Rαα, IL, IL--10R10Rββ depresseddepressed

cytokinecytokine A2A2 depresseddepressed

cytokinecytokine A5 (RANTES)A5 (RANTES) depresseddepressed

* compared to inflight 1xg control Cogoli-Greuter et al.

EXPERIMENT LEUKINEXPERIMENT LEUKIN

REMEMBERINGCOLUMBIA STS-107

16TH JAN - 1ST FEB 2003

SEMISEMI--QUANTITATIVE RTPCRQUANTITATIVE RTPCR

T cells activated with Con A / anti CD28 / Experiment in modeled µG

IL-2RαIL-2IFNγ

IL-2RγIL-2Rβ

Blue dots: 217 genes aresignificantly upregulated

T cells activated for 4 h with Con A / anti-CD28

1g 4h

RPM 0h

RPM 4h

GRAVITY-DEPENDENT DISTRIBUTIONOF GENE EXPRESSION IN T CELLS ACTIVATED WITH CON A / ANTI CD28

99 genes upregulated above 2-fold

Drastic drop in gene expression

Little change in gene expression

Key genes and transcription factorsKey genes and transcription factors

IMPORTANT CONTRIBUTIONS TO THE UNDERSTANDING OF THE BEHAVIOUR OF IMMUNE CELLS IN SPACE AND SIMULATED MICROGRAVITY HAVE BEEN MADE BY THE TEAMS OF:

DIDIER SCHMITT

BEN HASHEMI

NEAL PELLIS

CLARENCE SAMS

MARIAN LEWIS

LAURENCE SCHAFFAR

LUDMILA BURAVKOVA

THE IMMUNE SYSTEM OF HUMANS IN SPACE

⇒ EXEX--VIVO / INVIVO / IN--VIVO STUDIESVIVO STUDIES

IMMUNE RESPONSES SHOWN TO BE ALTERED IMMUNE RESPONSES SHOWN TO BE ALTERED DURING OR AFTER SPACEFLIGHTDURING OR AFTER SPACEFLIGHT

• LEUKOCYTE BLASTOGENESIS – SEVERELY INHIBITED

• CYTOKINE PRODUCTION – INTERFERON-α/β INHIBITED

• LEUKOCYTE SUBSET DISTRIBUTION – REDUCTION IN T-CELLS, NK-CELLS, INCREASE IN NEUTROPHILS

• NATURAL KILLER CELL ACTIVITY - REDUCED

• DELAYED-TYPE HYPERSENSITIVE REACTIONS TO COMMON RECALL ANTIGENS - REDUCED

NOT ALTERED: CONTENT OF IMMUNOGLOBULINS

IMMUNOKITIMMUNOKIT flown on MIR(Mission 7, 8 and 9)

DELAYED-TYPE HYPERSENSITIVITY REACTION

Commercially available applicator for “Skin test” (Merieux)

adapted to space

"SKIN TEST" on 5 MIR COSMONAUTS"SKIN TEST" on 5 MIR COSMONAUTS

Score (mm)

05

1015202530

Pre-flight In-flight Post-flight

A

BC

D

E

EFFECT OF SPACEFLIGHT CONDITIONS EFFECT OF SPACEFLIGHT CONDITIONS ON LYMPHOCYTES OF DON LYMPHOCYTES OF D--1 ASTRONAUTS1 ASTRONAUTS

0

20

40

60

80

100

120

140

160

A B C

L-9 dL-2 dL+3 d at 1 gR+0 dR+7 dR+13 d

Crew member

Mitotic indexin cpm

Baseline

MONOCYTE DIFFERENTIATION IN SPACEFLIGHTMONOCYTE DIFFERENTIATION IN SPACEFLIGHT

•• BLOOD SPECIMENS FROM 25 ASTRONAUTS BLOOD SPECIMENS FROM 25 ASTRONAUTS COLLECTEDCOLLECTED POST FLIGHT: POST FLIGHT: MONOCYTES EXHIBITED REDUCTIONS IN ABILITY TO MONOCYTES EXHIBITED REDUCTIONS IN ABILITY TO ENGULF E. COLIENGULF E. COLIELICIT AN OXIDATIVE BURST AND DEGRANULATE ELICIT AN OXIDATIVE BURST AND DEGRANULATE PHAGOCYTIC INDEX WAS SIGNIFICANTLY REDUCEDPHAGOCYTIC INDEX WAS SIGNIFICANTLY REDUCED

•• BLOOD SPECIMENS FROMBLOOD SPECIMENS FROM 27 RETURNING 27 RETURNING ASTRONAUTS: ASTRONAUTS: SIGNIFICANT DECREASE IN PERCENTAGE OF SIGNIFICANT DECREASE IN PERCENTAGE OF CD14+CD16+ MONOCYTE DIFFERENTIATION CD14+CD16+ MONOCYTE DIFFERENTIATION MARKERS IN ALL SUBJECTS TESTEDMARKERS IN ALL SUBJECTS TESTED

Slide provided by M. Hughes-Fulford

CHANGES IN MONOCYTE POPULATION AND CHANGES IN MONOCYTE POPULATION AND PHAGOCYTIC POTENTIAL IN ASTRONAUTSPHAGOCYTIC POTENTIAL IN ASTRONAUTS

16 Day Missions, 12 AstronautsSmall but significantchange in monocyte

5-11 Day Missions, 25 AstronautsLarge and significant change in

Phagocytic potential

Graphed from data in Stowe et al., Aviation, Space and Envir. Med. 2003 Dec;74(19):1281-1284.

Graphed from data in Kaur et al., Brain Behav. Immun. 2005 Nov;19(6):547-54.

Slide provided by M. Hughes-Fulford

INFECTION IN SPACEFLIGHTINFECTION IN SPACEFLIGHT

•• 15 OF THE 29 APOLLO ASTRONAUTS HAD A 15 OF THE 29 APOLLO ASTRONAUTS HAD A BACTERIAL OR VIRAL INFECTION EITHER BACTERIAL OR VIRAL INFECTION EITHER DURING FLIGHT OR WITHIN ONE WEEK AFTER DURING FLIGHT OR WITHIN ONE WEEK AFTER FLIGHTFLIGHT

•• APOLLO 13, ONE ASTRONAUT DEVELOPED AN APOLLO 13, ONE ASTRONAUT DEVELOPED AN URINARY TRACT INFECTION DURING THE FLIGHTURINARY TRACT INFECTION DURING THE FLIGHT

⇒⇒ COUNTERMEASURER: CREW ISOLATION COUNTERMEASURER: CREW ISOLATION BEFORE SPABEFORE SPACEFLIGHTCEFLIGHT

Slide provided by M. Hughes-Fulford

LATENT VIRUS REACTIVATIONLATENT VIRUS REACTIVATION

• VARICELLA ZOSTER VIRUS PRESENT IN 30% OF 200 SALIVA SAMPLES FROM 8 ASTRONAUTS COLLECTED IN AND POSTFLIGHT

• EPSTEIN-BARR VIRUS DNA IN 16% OF INFLIGHT SAMPLES FROM 25 ASTRONAUTS (SIGNIFICANTLY GREATER THAN PRE/POSTFLIGHT)

• 11 OF 28 ASTRONAUTS SHOWED POSTFLIGHT EPSTEIN-BARR VIRUS ACTIVATION

SPACE FLIGHT SPACE FLIGHT ““STRESSORSSTRESSORS”” POSSIBLY ASSOCIATED POSSIBLY ASSOCIATED WITH ALTERED IMMUNE RESPONSEWITH ALTERED IMMUNE RESPONSE

• INTENSE PRE-LAUNCH TRAINING• LAUNCH• MICROGRAVITY• RADIATION• MICROBIAL CONTAMINATION• SLEEP DISRUPTION / CHANGES IN CIRCADIAN

RHYTHM• PSYCHOLOGICAL DYNAMICS OF CONFINED

ENVIRONMENT• PRESSURE OF HIGH PERFORMANCE

EXPLORATION BEYOND LOW EARTH ORBITEXPLORATION BEYOND LOW EARTH ORBIT

POTENTIAL NEW RISKS FOR EXPLORATION POTENTIAL NEW RISKS FOR EXPLORATION MISSIONSMISSIONS

• NEW FORMS OF RADIATION

• CREW ISOLATION WITH NO CHANGE OF RAPID RESCUE OR TREATMENT

• ALTERED IMMUNE SYSTEM WITH POTENTIAL ALTERED VIIRULENCE OF PATHOGENES

• ALTERED NUTRITION

ACKNOWLEDGEMENT

Millie Hughes-Fulford and her team, Univ. California, S. Francisco

Proto Pippia, M.A. Meloni, G. Galleri, G. Pani, A. Saba, L. Sciola, A. Spano, Univ. Sassari

Mauro Macarrone and his team, Univ. Teramo

Otfried Müller, Univ. Bern

Rodolfo Negri and his team, Univ. Rome

Alex Tschopp, Pia Fuchs-Bislin, Birgitt Bechler, Thomas Schopper, ETHZ

Isabelle Walther, Zero-g LifeTec GmbH

The teams of ESA, NASA, IBMP MoscowThe astronauts of SL-1, D-1, SLS-1, IML-1, IML-2The cosmonauts of MIR and Soyuz/ISS

The financial support: ETHZ, ESA/PRODEX, ASI, NASA, SNSF

I THANK YOU FOR YOUR ATTENTION

SPACE AGGREGATE 78 Hours after addition of Con ANIZEMI / Spacelab IML-2, 1994

Cogoli-Greuter et al., 1996

SINGLE CELL MOVING OUT

OF AGGREGATE

MET 3-18:44:01 3-18:48:00 3-18:52:00

3-18:56:00 3-19:00:35

Signaling Pathways Involved in TSignaling Pathways Involved in T--cell Activationcell Activation Fold Change

0.1 1 5.0

Chemokine Receptors

IL2CD28

IL2Rα

CD45

PLCPLC

DAGDAG

PKCPKC

JNKJNK

VAVRAC

ELKELKNFKB1

JUN

RAFRAF

MEKMEK

ERK 1/2ERK 1/2

RASRAS

[Ca++]

IPIP33

NFATNFAT

P38P38MAPKMAPK

con A α-CD28 Ab

CD4

JAK1/3JAK1/3

STAT5STAT5

MYCMYCPI3K

AKT

E2F6

Responsive Genes:BCL2CCL20IER3

A1

JAK

STAT1

IFNγ

IFNγR

TCR/CD3

CCL20CSF2CXCL10WARS

AHRCCL4CYP51EGR1EGR2HK2IL2MAFFMIFNOLC1TNFSF2

BCL2CDK4E2FEZH2IL2RAMYB SHMT2STK12

6

A1BIRC3BCL10CCL3CCL4CCL20CSF2CXCL10EGR1HIST1H3DIER3IL1F9IL2IFNγIRF4MIFMMP1OAS3SELLTNFSF1TNFSF2TNFSF14TNFRSF6TNFRSF9TRAF1XCL1XCL2

We performed a series of experiments using:

Forskolin as PKA activatorH89 as PKA inhibitorGF109203X as PKC inhibitorU0126 as MEK1 inhibitorWestern blot with anti-p-CREBEMSA assay for activated transcription factor AP1

AP-1

Gq

cAMPcAMP

CREB1CREB1

Gαs

PKAPKA

Gαi

TRAF1

BIRC3

1214

69

TNFRSF

TNFSF

Signaling Pathways Involved in TSignaling Pathways Involved in T--cell Activationcell Activation Fold Change

0.1 1 5.0

Chemokine Receptors

IL2CD28

IL2Rα

CD45

PLCPLC

DAGDAG

PKCPKC

JNKJNK

VAVRAC

ELKELKNFKB1

JUN

RAFRAF

MEKMEK

ERK 1/2ERK 1/2

RASRAS

[Ca++]

IPIP33

NFATNFAT

P38P38MAPKMAPK

con A α-CD28 Ab

CD4

JAK1/3JAK1/3

STAT5STAT5

MYCMYCPI3K

AKT

E2F6

Responsive Genes:BCL2CCL20IER3

A1

JAK

STAT1

IFNγ

IFNγR

TCR/CD3

CCL20CSF2CXCL10WARS

AHRCCL4CYP51EGR1EGR2HK2IL2MAFFMIFNOLC1TNFSF2

BCL2CDK4E2FEZH2IL2RAMYB SHMT2STK12

6

A1BIRC3BCL10CCL3CCL4CCL20CSF2CXCL10EGR1HIST1H3DIER3IL1F9IL2IFNγIRF4MIFMMP1OAS3SELLTNFSF1TNFSF2TNFSF14TNFRSF6TNFRSF9TRAF1XCL1XCL2

Conclusions:

1. PKA in addition to PKC and MAPK pathways plays a role in Early T cell activationand induction of IL-2, IL-2Rα and IFNγ Gene expression

2. ERK2 is phosphorylated within minutes of activationwhile P38 and JNK remain unchanged

3. Probable cross-talk between the MEK and PKA pathways and between the PKC and PKA pathways

AP-1

Gq

cAMPcAMP

CREB1CREB1

Gαs

PKAPKA

Gαi

TRAF1

BIRC3

1214

69

TNFRSF

TNFSF

Signaling Pathways Involved in TSignaling Pathways Involved in T--cell Activationcell Activation Fold Change

0.1 1 5.0

Chemokine Receptors

IL2CD28

IL2Rα

CD45

PLCPLC

DAGDAG

PKCPKC

JNKJNK

VAVRAC

ELKELKNFKB1

JUN

RAFRAF

MEKMEK

ERK 1/2ERK 1/2

RASRAS

[Ca++]

IPIP33

NFATNFAT

P38P38MAPKMAPK

con A α-CD28 Ab

CD4

JAK1/3JAK1/3

STAT5STAT5

MYCMYCPI3K

AKT

E2F6

Responsive Genes:BCL2CCL20IER3

A1

JAK

STAT1

IFNγ

IFNγR

TCR/CD3

Gq

cAMPcAMP

CREB1CREB1

Gαs

PKAPKA

Gαi

CCL20CSF2CXCL10WARS

AHRCCL4CYP51EGR1EGR2HK2IL2MAFFMIFNOLC1TNFSF2

BCL2CDK4E2FEZH2IL2RAMYB SHMT2STK12

6

A1BIRC3BCL10CCL3CCL4CCL20CSF2CXCL10EGR1HIST1H3DIER3IL1F9IL2IFNγIRF4MIFMMP1OAS3SELLTNFSF1TNFSF2TNFSF14TNFRSF6TNFRSF9TRAF1XCL1XCL2

AP-1

TRAF1

BIRC3

1214

69

TNFRSF

TNFSF