Download Assessing the Impact of Microgravity on the Innate Immune System

1. Mentor: Dr. Jamie Foster
2. Name of organization
Department of Microbiology and Cell Science, University of Florida
3. Title of the project
Assessing the impact of microgravity on the innate immune system using the squidvibrio model system
4. Abstract
The overall goal of this project is to examine the impact of microgravity on the cellular
interactions between animals and beneficial microbes. Space flight causes numerous
changes in the growth, physiology and virulence of animal-associated microbes.
However, most studies have focused on pathogenic organisms, which comprise less
than 5% of all microbes known to associate with animal tissues. The effects of
microgravity on mutualistic microbes are virtually unknown. Here, in this summer project
the student will help to examine the impact of space flight on gene expression in Vibrio
fischeri, a beneficial microbe known to be essential for the normal development of the
squid Euprymna scolopes. Specifically, the student will have following objective:
Examine the mechanism(s) associated with the delay of bacteria-induced immune
cell trafficking that occurs in simulated microgravity.
Background: Hemocytes are macrophage-like cells that circulate in the blood stream of
many animals and are key mediators of the innate immune response. Hemocytes are
also through to play a key role in normal development of the squid light organ and
facilitate the regression of the ciliated epithelial appendages, however, the mechanisms
are unknown. Preliminary evidence has shown that there is a delay in the infiltration of
hemocytes in symbiotic animals under microgravity conditions. In simulated microgravity
conditions there is a 10 h delay in significant hemocyte infiltration and levels of
hemocytes in the host light organ never reach that of gravity controls. These results
suggest that the activation of the host innate immune response is delayed and lower in
microgravity conditions.
Approach: Examine changes in NF-B pathway using quantitative real time PCR. The
nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B) pathway is found
in almost all animal cell types and is a key regulator of the host innate immune
response. The NF-B pathway, which has been identified in E. scolopes, is often
activated by MAMPs including LPS and peptidoglycan. Simulated microgravity has been
shown to trigger the NK-B pathway in eukaryotes, although the mechanism(s)
associated with the activation is not well resolved. The intern will examine the
expression of key components of this pathway using quantitative PCR.
Aposymbiotic and symbiotic animals exposed to simulated microgravity conditions in the
HARV chambers will be examined every 2 h for the first 24 h post inoculation.
Additionally, aposymbiotic animals exposed to peptidoglycan (50 g per ml of FSSW)
will also be examined. Gravity controls will be conducted for all treatments. Total RNA
will be extracted from host light organs and converted to cDNA using commercially
available kits. Primers will be designed using the Primer Express software and qPCR
will be performed using a BioRad RTPCR machine. The expression of genes
associated with receptors (e.g. peptidoglycan recognition protein (PGRP1-4); Toll-like
receptor (TLR)), signal transducers (e.g. interleukin-1 receptor associated kinase
(IRAK4)) and targets of the NF-B pathway (e.g. serine kinases or IB kinases (IKKs))
will be initially examined. This approach will enable us to determine if microgravity is
impacting the expression of genes associated with a key pathway of the host innate
immune system in both apo- and symbiotic squid.
5. Expected contribution that the student will do on this project
The intern would initially start by shadowing the mentor to learn the molecular biology
procedures required to complete this project. Depending on the student’s experience
level the student would be personally shown how to complete the task and then
gradually left to continue the task independently. It will be expected that will the
mentor’s guidance and training by the end of the internship the student will be trained in
the described methods and computer programs.
6. Expected student working hours (8 hours a day, 5 days a week)
It will be expected that the student will work for a full 40-h workweek. However,
depending on the specific procedures some days may be longer and others shorter. For
example RNA isolation and clean up may require a 10 h day (with lunch), however, the
student would be allowed to leave earlier on another day.