The rat model receiving a 20 % total body surface area burn (B) followed 2
days later by cecal ligation and puncture (CLP) to induce sepsis was used.
Control groups were given a sham-burn (S) treatment, which is identical to burn treatment
but the animals were immersed in 37°C water instead of boiling water, or sham cecal ligation
and puncture treatment (SCLP), which utilized an identical procedure to the CLP
treatment, but did not ligate or puncture the cecum (SCLP condition). Animals were sacrificed at different time
points during the first 10 days to collect liver samples for microarray
Co-expressed and injury-responsive
gene groups in the microarray analysis were identified between CLP and SCLP using
a “consensus clustering” approach  , which
selected for temporal variations in the gene expression profiles of both control and treatment groups. Principle component analysis was then used to
identify key differences in temporal progression between CLP and SCLP over the
entire time course. In order to assess the combined effects of the BCLP injury,
a technique known as SAM (Significant Analysis of Microarrays) was utilized 
to determine a set of significantly varying genes following injury. This method
uses multiple gene specific t-tests to verify the significance of the fold
change of the double hit injury relative to sepsis alone, followed by a false
discovery rate test, which aims to exclude genes whose variations are the
result of random chance.
The consensus clustering
method identified a total of 3 major responses in the CLP condition when
compared directly to the SCLP control. These clusters are all strongly related to
the inflammatory response, with two of the major clusters representing
co-expressed sets of pro inflammatory genes, while the third cluster represents
co-expressed anti inflammatory genes. Principle component analysis of the
concatenated data set (Figure 1) reveals that CLP and SCLP have polar opposite
responses along the first, major principle component, while they have almost
identical responses along the second. Analysis of genes contributing to each of
the principle components reveals that principle component 1 is mapping out a
completely unique inflammatory response profile for each injury: the septic
injury (CLP) reveals an early pro inflammatory response followed by severe
immunosuppression, while the trauma injury (SCLP) shows the opposite dynamics,
with early immunosuppression followed by severe pro inflammatory activity.
The SAM analysis identified a total of 957
genes that were either reduced in expression level or increased by a factor of
2 between the CLP condition, and the CLP condition influenced by prior burn
injuries. Critical genes can be seen in Figure 2, which divides the functional
differences into metabolic and immune changes: one can observe an up regulation
in amino acid degradation over the first day of the response, as well as an
early immunosuppression that quickly resolves into increased innate immune
function. The long term response appears to be affected in its method of
recuperation through a shift towards amino acid biosynthesis and wound repair.
| Figure 1: Principle component analysis of the progression of the long term CLP response compared to SCLP control |
| Figure 2: Critical genes altered by the burn injury over the progression of the subsequent CLP response |
In conclusion, the differences in the responses of the innate immune
response to a variety of stimuli necessitate a new paradigm of studying these
conditions: inflammation is not a single condition, but rather a collection of
responses unique to the injury. By utilizing transcriptional analysis, we were
able to observe that the trauma injury has a completely different hepatic
response to the septic injury, while burn priming does not appear to change the
fundamental underlying response. Clinical strategies aiming to mitigate the
disruptive effects of SIRS should therefore be tailored to account for the
adaptability of the innate immune system in the face of various stimuli.
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