microgravity alters host immune responses in vitro · launch date: july 8, 2011 landing date: july...

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


C. Apoptosis


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



C. Apoptosis



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



C. Apoptosis



Cyt

oki

ne

s/ C

he

mo

kin

es

Ap

op

tosi

sG

row

th f

acto

rs

2.5

-2.5




C. Apoptosis



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

46

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

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

microgravity alters host immune responses in vitro · launch date: july 8, 2011 landing date: july...

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