(500h) Unique Hepatic Responses to Burn, Sepsis and Trauma: The Adaptability of Innate Immunity in the Face of Different Stimuli

The systemic immune
response is considered to be a core response to external insults upon mammalian
systems, and consists of a wide variety of effects, including dilation of the
vasculature, hypermetabolism and lean body mass loss, acute phase protein synthesis,
and macrophage activation., which, if untreated, can eventually lead to
multiple organ failure and death [1]. Because of its central role in the
metabolic aspects of this systemic response, as well as protein synthesis and
innate immune activation, the liver represents a central player in this
response whose transcriptional activity following injury is an indicator of
critical pathways which are affected by the response, as well as a biomarker
for the long term resolution of the host response. Traditionally, the acute
phase of the inflammatory response is considered to be critically dangerous,
however recent evidence suggests that the long term response to injuries can
also result in serious patient complications, including mortality [2]. In order to understand the long term effects of
injuries on pathways involved in the inflammatory response, we utilized sepsis
and/or burn injury in rats, to assess their whole liver transcriptional
response to multiple different injury conditions over the course of 8 days. We used
statistical and clustering algorithms to investigate the
genetic signatures of hepatic response to this double-injury model and
evaluated the progression of the response in a temporal manner, in order to
resolve critical pathways involved in the long term, as well as patterns of
resolution in each injury.

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
analysis.
Co-expressed and injury-responsive
gene groups in the microarray analysis were identified between CLP and SCLP using
a ?consensus clustering? approach [3] , 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 [4]
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.  
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.

References:

1 . Beal, A. L. and F. B. Cerra, Multiple organ
failure syndrome in the 1990s. Systemic
inflammatory response and organ dysfunction. Jama, 1994. 271(3): p.
226-33.

2 . Sasse,
Kent C., et al., Long-term survival after
intensive care unit admission with sepsis. Critical Care
Medicine, 1995. 23(6): p. 1040-1047.

3 . Nguyen,
T. T., R. S. Nowakowski, and I. P. Androulakis, Unsupervised selection of highly coexpressed and noncoexpressed
genes using a consensus clustering approach. Omics, 2009. 13(3): p.
219-37.

4 . Tusher,
Virginia Goss, Robert Tibshirani, and Gilbert Chu, Significance analysis of microarrays applied
to the ionizing radiation response. Proceedings of the
National Academy of Sciences, 2001. 98(9): p. 5116-5121.