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The immune system is capable of mounting robust responses
against a seemingly endless array of pathogens. Most pathogens are
cleared efficiently by the immune system but some pathogens have
also evolved strategies to escape immune destruction and can persist
in the host for extended periods of time. Infection of mice with
the clone 13 variant of lymphocytic choriomeningitis virus (LCMV)
leads suppression of CD8 T cell responses and persistence of the
virus. The objective of this research study is to understand the
mechanism by which LCMV clone13 suppresses the immune response.
This immuno suppression could be mediated by inhibitory cytokines.
Cytokines are proteins that regulate lymphocyte proliferation and
differentiation. Certain cytokines inhibit immune response, while
others stimulate it. Interleukin–10 (IL-10) is an example
of inhibitory, anti inflammatory cytokine which inhibits immune
responses. We hypothesize that LCMVclone13 causes immuno suppression
by inducing the production of the IL-10 family of inhibitory cytokines.
The experiment to test this hypothesis would involve isolating purified
cell populations (T, B, macrophage, neutrophil cells) from naïve
uninfected or persistently infected mice and analyzing their RNA
for IL-10 expression by real-time polymerase chain reaction. The
first step in this process is to design and standardize real-time
PCR assays . In the past two months, I have successfully designed
and developed real time PCR assays for IL-10. The data will be presented.
The immune system is capable of mounting robust responses
against a seemingly endless array of pathogens. Most pathogens are
cleared efficiently by the immune system but some pathogens have
also evolved strategies to escape immune destruction and can persist
in the host for extended periods of time. Infection of mice with
the Armstrong strain of lymphocytic choriomeningitis virus (LCMVarm)
leads to robust immune response and clearance of the virus. In contrast,
infection of mice with the clone 13 variant of lymphocytic choriomeningitis
virus (LCMVclone13) leads to immune suppression and persistence
of the virus. The mechanism by which LCMVclone13 causes immune supression
is unknown. It could be mediated by inhibitory cytokines. Cytokines
are proteins that regulate lymphocyte proliferation and differentiation.
Certain cytokines inhibit immune responses, while others stimulate
it. Interleukin–10 (IL-10) is an inhibitory, anti inflammatory
cytokine that inhibits immune responses. IL-10 regulates the proliferation
and differentiation of special helper T cells (TH1) which appears
to control immune response in vivo (Moore, de Waal Malefyt, Coffman
& O’Garra 2001). Previous studies using human immune cells
stimulated with either LPS from E.coli 0127B8, IL-2, IL-12, or anti-CD3
suggest that IL-10 is produced by T-cells (Wolk, Kunz, Asadullah
& Sabat 2002). In different cell types, the duration of stimulation
affects IL-10 expression differentially. In T cells IL-10 expression
occurs shortly after stimulation and the levels of IL-10 increases
with the duration of stimulation. Natural killer cells on the other
hand requires prolonged stimulation (18 hours) to express IL-10
(Wolk et al 2002 ). We hypothesize that LCMVclone13 causes immuno
suppression by inducing the production of the IL-10 family of inhibitory
cytokines. Here, we have analyzed IL-10 gene expression in various
cell types of LCMV immune mice and chronically infected mice.
Animals
- 6-8 week old Black 6 with genotype C57bL/6J mice were used.
- Naïve mice were housed in a specific pathogen-free vivarium.
- Immune mice were generated by infecting mice with LCMV armstrong
(acute strain).
- Chronic mice were generated by infecting mice with the LCMV
clone 13 strain.
Methodology
- Based on the research by Kerwin et al, we will investigate cytokine
expression in various cell types of naïve, immune or chronically
infected mice. The cell types that will be analyzed are CD8+ and
CD4+ T-cells, B-cells, macrophages, natural killer cells and neutrophils.
- Purified populations of various cell types will be acquired
by cell sorting using FACS.
- Trizol RNA isolation harvest of RNA from the various cell lines
- DNase treatment eliminating the genomic DNA from the RNA samples
to prevent spurious PCR amplification
- Omniscript Reverse Transcription reaction conversion of RNA
into cDNA
- Real time PCR using the ABI 7700 sequence detector method of
quantitative DNA amplification.
In naïve mice, IL-10 is expressed in macrophages
and natural killer cells. In immune mice, IL-10 is detected in B
cells and CD8 T cells, with B cells expressing more than 10 times
more IL-10 than CD8. In chronic mice, IL-10 expression was observed
in all cell types with the largest expression in macrophages and
significant expression in natural killer cells.
Conclusions
- Differential IL-10 expression was observed in different cell
types in naïve, LCMV immune and chronically infected mice
- Interestingly, macrophages produced the most IL-10 in chronic
mice.
- Macrophages are responsible for regulating CD4 T cell-mediated
immune responses
- An increased production of IL-10 by macrophages in chronically
infected mice suggests that blocking IL-10 production could enable
the host to clear LCMV clone 13
Future
- Analyze IL-10 receptor expression on different cell types -Investigate
IL-10 gene expression at different time points (days 2, 8, 15)
during the course of LCMVARM and LCMV clone 13 infection
- Analyze the expression of IL-10 homologs such as IL-20,
IL-22 and IL-24.
Howard Hughes Medical Institute Grant No. 52003071.
Cytokines are proteins that are released that can
either inhibit or excite the immune system. The logic behind cytokines
is that the inhibitory cytokines, IL-10, shuts down the immune system
which is why a virus is never cleared. The virus I'm using is LMCV
clone 13 and the objective of my experiment is to understand the
nature by which LCMV induces a chronic infection(one that is never
cleared). I hypothesized that increased levels of IL-10 induce the
shut down of the immune system, thereby allowing the virus to persist.
In order to do this, I obtained spleen cells from naive, immune
and chronic mice and sorted them into various cell subsets (CD8,
CD4, macrophages, neutrophils, B cells, and NK cells). I then harvested
RNA from each cell type and DNase treated the samples to rid them
of genomic DNA. Next I reverse transcribed the RNA into cDNA such
that I could run Real time PCR afterwards. If the cDNA amplified,
we are assuming that the cells type expressed the IL-10 gene. We
found that chronically infected mice did show an increased level
of IL-10. More importantly, we found that macrophages expressed
the most IL-10 in chronic mice. Because of how macrophages are situated
within the immune system (they "talk" to CD4 cells which
is responsible for cell-mediated and humoral immunity), our findings
suggest that blocking IL-10 productiong in macrophages could enable
the host to clear the virus.
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