microgravity alters host immune responses in vitro · launch date: july 8, 2011 landing date: july...
TRANSCRIPT
Nabarun Chakraborty
Integrative System Biology Program US Army Center for Environmental Health Research
Fort Detrick, MD, 21702
Microgravity Alters Host Immune Responses in vitro:
Pan-omics Approaches
Disclaimers: The views, opinions, and/or findings contained in this report are those of the authors and should not be construed as official Department of the Army position, policy, or decision, unless so designated by other official documentation. Citations of commercial organizations or trade names in this report do not constitute an official Department of the Army endorsement or approval of the products or services of these organizations.
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Study Aims
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• A comprehensive understanding of the effect of microgravity on host immunity wound healing tissue regeneration
• Identification of microgravity specific molecular markers/ putative therapeutic targets enabling
enhancement of host immunity improvement of healing and regeneration process
• Understand the efficacy of some of the available bone growth factors in extraterrestrial environment
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Long Term Benefits
• Ensure the betterment of astronauts’ health
• Ensure improved performance and lifespan of space missions
• Ensure a safe environment in microgravity for extended duration
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Comprehensive Project Outline
In vitro Study
In vivo Study
PAN-OMICS
BIOMARKERS
Flight Requirements: Cell culture supporting platform
Assay types- target molecule: Genomics – Genes/RNA Proteomics- proteins Metabolomics—metabolites/chemicals Epigenomics—environmental impact on DNA Microbiomics—microbes that live in or on the body
Flight Requirements: Mouse habitat
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Launch date: July 8, 2011 Landing date: July 21, 2011
The presence of bacterial colonies could threaten the performance of astronauts
Astronauts are more vulnerable to pathogenic infection! – Microgravity inhibits host defense, and promotes virulence of pathogens – Aggressive infection may cause sepsis….and death!
Host-pathogen relationship in spaceflight might adopt networks different from terrestrial paradigms
Motivation to Study Wound Model
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Bacterial colonization (biofilm) on the fire detector in Mir
Biological Model
Wound model in vitro:
Human blood microvascular endothelial cells infected by lipopolysaccharide (LPS), a common outer membrane protein of gram-negative bacteria
Cell Culture Module (CCM): capable of supporting cell growth in spaceflight
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Novelty of this model: Minimum terrestrial bias to host-pathogen
relationship encountered in spaceflight
Spaceflight Study
Human cells exposed to LPS
Ground Study
Human cells exposed to LPS
Microgravity specific markers
Experimental Strategy
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Molecular markers of
terrestrial LPS insult
Molecular markers of
extraterrestrial LPS assault
Experimental Design
0-4 hr
Launch
Cells nourished until Day 10
4-8 hr
Rest of the flight
LPS bioreactors 1 & 2
LPS bioreactors 3 & 4
Fix bioreactors 1-6
Collect Samples
Same protocol was followed concurrently on the ground
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Pan-omics Study
Start of 11th day
Transcriptomics and
microRNA Genomics
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Genomics/ Transcriptomics Workflow
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RNA extraction
Transcriptomic assay (high throughput microarray)
microRNA genomics (qPCR assay)
Cell Culture Module (CCM) Bioreactor
Detect, identify and measure gene expression
Cells
Principal Component Analysis: Significantly Altered Genes of Interest
PC1 82%
PC
2 1
7%
G = Ground
C = Untreated control LPS = LPS treatment 4/8 = Treatment duration (h)
Hierarchical Clustering
4h 4h8h 8hGround Space
B. Cytokines/ Chemokines
4h 4h8h 8hGround Space
C. Apoptosis
4h 4h8h 8hGround Space
D. Growth factorsA. Space-Control
Cyt
oki
ne
s/ C
he
mo
kin
es
Ap
op
tosi
sG
row
th f
acto
rs
2.5
-2.54h 4h8h 8hGround Space
B. Cytokines/ Chemokines
4h 4h8h 8hGround Space
C. Apoptosis
4h 4h8h 8hGround Space
D. Growth factorsA. Space-Control
Cyt
oki
ne
s/ C
he
mo
kin
es
Ap
op
tosi
sG
row
th f
acto
rs
2.5
-2.54h 4h8h 8hGround Space
B. Cytokines/ Chemokines
4h 4h8h 8hGround Space
C. Apoptosis
4h 4h8h 8hGround Space
D. Growth factorsA. Space-Control
Cyt
oki
ne
s/ C
he
mo
kin
es
Ap
op
tosi
sG
row
th f
acto
rs
2.5
-2.5
4h 4h8h 8hGround Space
B. Cytokines/ Chemokines
4h 4h8h 8hGround Space
C. Apoptosis
4h 4h8h 8hGround Space
D. Growth factorsA. Space-Control
Cyt
oki
ne
s/ C
he
mo
kin
es
Ap
op
tosi
sG
row
th f
acto
rs
2.5
-2.5
Enrichment Profile of the Pathways
> 2x
< -2x
< 2x & > -2x
Toll-like Receptor (TLR) Pathway: LPS Signaling in the Cell
2.5-2.5
S-C G-4h S-4h
2.5-2.5
S-C G-4h S-4h
G-LPS S-LPS
mir-146b-5p
mir-382
mir-611, 199a-3p, 191 and 431
mir-200a
mir-212
mir-125b
mir-148a, 133b, 635, 875-3p, 554 and23b
2.7
-2.7
miRNA mRNA targetsmRNA targets miRNA
microRNA Signatures at 4h LPS Exposures
Gro
un
d
Spacefligh
t
Oxidative stress, a consequence of energy depletion
Alcoholic stress
Endotoxic, e.g. LPS assault
Cytokine signaling; Diminished LPS recognition ability
Summary from Genomics Results
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Impact of microgravity on host-pathogen relationship is significant
Micro-gravitational stress mediates immune compromise
Microgravity impairs the early pathogen recognition process, potentially facilitating opportunistic pathogenic invasion
Targeting the molecules responsible for early pathogen detection but affected by microgravity could be viable therapeutic strategy for healing wounds in spaceflight
We also identified potential microRNA targets for therapeutic intervention specific for spaceflight
Metabolomics
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Metabolomics Workflow
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UPLC/QTOFMS
Cell Culture Module (CCM)
Sample Collection bag
Superior resolution - UPLC High mass accuracy - QTOFMS
Separate and identify metabolites
Principal Component Analysis: Significantly Altered Metabolites
-15 -10 -5 0 5 10 15
-4-2
02
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PC1
PC
2
GC
SC
SC
GLPS
GLPS
SLPS
SLPS
PC1 74%
PC
2 1
4%
G = Ground
C = Untreated control LPS = LPS treatment 4 = Treatment duration (h)
Gene Metabolite Relationship: Networks of Metabolism
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Gene Metabolite Relationship: Networks of Cell Survival and Cell Death
Summary from Metabolomics Results
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Microgravity negatively influenced the cellular metabolism pathways
Consequent energy depletion may result in comprehensive deterioration of cellular health, including immune blunting and impaired pathogen recognition
Identified metabolomic markers associated with cellular health and cell death that could be putative therapeutic markers
Additional confirmatory assays are underway
Future Projects
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Organ Specific Immunological Markers: in vivo Approach
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Brain
Lungs
Heart
Liver Stomach
Spleen
Small intestine
Kidney
Pan-omics study
SpX-6: December 5, 2014 A sample sharing program with the CASIS PI
Wound Tissue Model
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in vitro 10 Bioreactors
Bioculture system SpX-7: April 2, 2015
in vivo ~40 mice
ISS Rodent Habitat SpX-10: Feb. 19, 2016
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Acknowledgements
USACEHR family Rasha Hammamieh, PhD Marti Jett, PhD Aarti Gautam, PhD Seid Muhie, PhD Duncan Donohue, PhD Stacy Ann-Miller Allison Hoke Edward Waddy William Santos
Georgetown University Amrita Cheema, PhD
Indiana University, School of Medicine Melissa Kacena, PhD
Tissue Genesis Inc. Tom Canon Cris Kosnik and team
NASA staff at Kennedy Space Center, FL
DoD Space Test Program
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Space Tissue Loss Payload Team
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Additional Slides
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Flowpath of Cell Culture Module (CCM)
Culture of Human Blood Microvascular Endothelial Cells, Dermal-Derived (HMVEC-dBl).
LPS assault was carried out for 4 hours and 8 hours. Control underwent no LPS treatment.
RLT buffer (Qiagen, Inc.) was used to lyse the cells onboard and to maintain the integrity of the nucleic acids at near room temperature until the shuttle returned.
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Bioreactor Pump
Sump Media LPS Fixing
Solution