OF MICE AND MEN:COMMONALITY OF THE MURINE AND HUMAN RESPONSE TO BURN
Alex G. Cuenca MD1, Lori F. Gentile MD1, M. Cecilia Lopez MS2, Wenzhong Xiao PhD3, Michael N. Mindrinos PhD3 ,
Richard L. Gamelli MD6, Ravi Shankar PhD5, Celeste C. Finnerty PhD6, David N. Herndon MD6, Lyle L.
MoldawerPhD1, Ronald G. Tompkins MD4, Henry V. Baker PhD1,2,8, and the Inflammation and Host Response to
Injury Consortium7
Department of 1Surgery and 2Molecular Genetics and Microbiology, University of Florida College of Medicine,
Gainesville, FL; 3Stanford Genome Technology Center, Palo Alto, CA; 4Department of Surgery, Massachusetts
General Hospital, Boston, MA; 5 Department of Surgery, Loyola Medical College, Chicago, IL, 6Department of
Surgery, University of Texas Medical Branch, Galveston, TX.
Background: Although significant advances in resuscitation and critical care have improved clinical outcomes, burn
injury remains a significant health care issue. To investigate the immunological and inflammatory response to
injury, investigators have commonly employed murine models. The mouse has been ideal because of its small size,
ease of breeding, and relatively low maintenance. More recently, the in-bred mouse has become an essential prerequisite because of its uniform genetic background, and the generation and commercial availability of knockouts
and transgenic strains (1). Genomic sequencing studies suggest that humans share 99% of their genes with the
mouse, making it an ideal mammalian model for studying molecular and genetic mechanisms in human disease (2).
Despite these similarities, some of the most promising therapeutic modalities developed in mouse models of
sepsis and systemic injury have failed to improve survival in human clinical trials (3). Therefore, the question
remains, is the mouse an appropriate model for the study of human disease?
There are a large number of possible explanations for the contrasting responses by humans and mice to
sepsis and injury. Despite the obvious differences in metabolic rate between mice and humans as well as
anatomy, most murine studies have used inbred, juvenile, previously-healthy mice that do not reflect the
heterogeneity of the human patient with regards to their age, pre-existing co-morbidities, immune, and nutritional
status (1, 3). Additionally, mouse models of critical illness do not provide the complex physiological support that
humans receive in the critical care setting, such as aggressive fluid, nutritional, cardiac and ventilator support. With
the increasing sophistication of techniques such as deep sequencing and microarrays, much is known about gene
structure and function in humans and in mice. However, less is known about gene expression and regulation,
particularly after critical illness, such as severe burn injury (4).
Objectives: To examines changes in gene expression from whole blood leukocytes in response to burn injury, and
compares burn responsive genes in the human to those in the mouse.
Methods: For human studies, whole blood leukocyte RNA from juvenile male subjects, aged 5-15 years and
experiencing greater than 20% total body surface are. In addition, 12 healthy male subjects between the ages of 017 years with a similar age distribution made up the control group. There were 102 blood samples collected at
various time intervals from 77 burn patients up to post burn day 28, and 12 samples from 12 healthy volunteers.
GeneChip™ Human Genome U133 Plus 2.0 Arrays (Affymetrix), were processed as described previously (28). For
murine studies, juvenile mice, aged eight weeks, exposed to a 25% total body surface area scald burn, was
collected at several time points post injury (5). The mouse samples were processed as previously described (5).
GeneChip™ Mouse Genome 430 2.0 Arrays were processed at Washington University St. Louis as described
previously. 1-2ug total RNA was used to make antisense cDNA with the NuGEN Technologies (San Carlo, CA) and
processed according to Affymetrix standard protocols.
Results
The human and mouse genomic response to burn injury is fundamentally similar.Though many murine models of
injury exist to study the pathologic and physiologic perturbations associated with trauma, sepsis, or infection,
there is a paucity of evidence demonstrating whether these models actually recapitulate the human condition (1).
In both cohorts, severe burn produced dramatic changes in the whole blood leukocyte transcriptome. Following
Alex G Cuenca, MD
1600 SW Archer RD
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alex.cuenca@surgery.ufl.edu
burn injury, 2,383 murine probe sets and 7,042 human probesets
1.
Overall transcriptomic
changed significantly over time (using a false discovery adjusted Figure
response
to
burn
injury in mice and
probability of Q<0.001). The magnitude of the genomic changes in the
humans.
(A
and
B) Heat map of
burned patients is very similar to that seen in humans after endotoxin
probesets
found
to
be significantly
administration (6) or severe trauma (manuscript in press), and has
increased
or
decreased
via K-means
been termed, a ‘genomic storm’.
clustering.
For
the
human
set
there were
These genes could be organized into two main expression
4107
genes
differentially
expressed
and
clusters based on whether their expression increased or decreased
for
the
mice,
there
were
2383
genes
following burn injury (Figure 1A and B). When comparing the human
transcriptomic response of burn injury to that of the mouse at the differentially expressed (Q <0.001).
level of individual genes, a total of 4107 and 1332 Ingenuity
Function/Pathway AnalysisTM (IPA) eligible genes changed significantly
(p<0.001), representing between 21%-61% of the respective mouse or
human genomes. Although one of the limitations of the analysis is
that there is a dramatic difference in the time course during which
samples were collected in humans compared to mice, the time periods
were obtained when changes in expression was maximal for each
species, suggesting that the majority of the responses to severe burn
injury were still captured in both data sets.
While at the level of the probe set or gene at the stringent
significance threshold used here there do not appear to be many
similarities between humans and mice, analysis of the signaling
pathways that were either up- or down- regulated as a result of burn injury demonstrate a remarkable similarity
between both species. Of the 20 pathways most upregulated in both species, Ingenuity Pathway Analysis™ (IPA)
revealed that the immune related pathways for Integrin signaling, IL-10 signaling, Fc Receptor Mediated
Phagocytosis in Macrophages and Monocytes, and B cell Receptor Signaling were all highly increased in both mice
and humans. Pathway analysis also revealed that Molecular Mechanisms of Cancer and Germ Cell-Sertoli Junction
Signaling were also increased in both mice and humans. Though the significance of these latter two pathways in
response to injury let alone burn injury is less clear, the upregulation of these pathways following burn is more
than likely related to proliferation and repair of infiltrating leukocytes.
There was also significant overlap between the top 20 down-regulated pathways in mice and humans
following burn injury. IPA revealed that iCOS-iCOSL signaling in T cells, CD28 Signaling in T Helper Cells, PKC
Signaling in T lymphocytes, T cell Receptor, Antigen presentation pathway and B cell Receptor Signaling were all
significantly decreased in both mice and humans following burn injury. These data support the hypothesis that at
the level of individual pathways or ontologies, there is indeed a broadly similar transcriptomic response to severe
injury following burn injury between mice and humans. This becomes particularly important with regards to the
focus of many studies which assume that because the deletion or modulation of a single protein, such as TNF-, IL1, HMGB1 or even the more recently studied SPHK1, improves outcome in murine investigations, it should
similarly work well in clinical trials (7-9). Unfortunately, the reality is that therapeutic design using these strategies
rarely succeeds and while the murine model may give important insight into disease mechanism, the mouse may
be less suitable for use in preclinical therapeutic studies, due to the observed differences between species at the
gene level (10, 11).
This is captured in further analyses of specific pathways that are similarly upregulated in both mouse and
human. For example, though the Toll like receptor pathway is upregulated in both mouse and humans, CD14,
MAP2K4, MyD88, TIRAP, and TLR6 are upregulated in the mouse but not the human and conversely, IKKalpha,
LBP, MEKK1, TLR1, TLR4, TLR5, TLR8, and TOLLIP are significantly upregulated in the human but not the mouse.
These data suggest that although there are mechanistic similarities between humans and mice in the overall
response to thermal injury within a given pathway, there are differences in individual gene expression. These
observations hold true when pathways involved in adaptive immune signaling, stress hormone responses, and cell
proliferation are analyzed. In addition, when we compare the IL-1 signaling pathway between mouse and human in
response to thermal injury, we observe that again, though the pathway is similarly altered following burn, the
specific genes that are altered within the pathway are different. These differences may be why in preclinical
studies, blockade of IL-1 appeared to improve survival to sepsis whereas in human trials, had little to no effect.
Conclusions: This study presents a novel comparison of the mouse and human leukocyte transcriptome following
severe burn injury. As the mouse has been and continues to be extensively used to investigate the human burn
injury response, as well as test the efficacy of therapeutics, validation of a common response to a given stimuli is
critical to the use of this animal as a model for the human condition. Though these findings are somewhat
expected, the whole blood leukocyte transcriptomic response has never been explored with such detail and then
subsequently compared between both species. Although the concordance in expression at the individual gene is
not particularly strong, there was remarkable overlap in gene expression when evaluated at the level of individual
functional modules, pathways or ontologies.
Given these data, however, the question arises of why then do preclinical therapeutic trials in mice often
succeed whereas the clinical trials fail? The answers are clearly complex and multifactoral. But partly, the results
appear to rest with the choice of therapeutic and its putative target. In many cases, the intervention targets the
activity or response of an individual gene or protein, and concordance between mice and humans is dependent
upon the functional overlap of the individual gene, and not necessarily the pathway involved. This raises the
intriguing possibility that although there is commonality in the pathways affected following burn injury, the genes
within each pathway may not have identical behavior between species, and therefore molecular targets affecting
single genes may affect the pathologic process differently in one species than in the other. Although the overall
responses to burn injury are remarkably similar, the studies here strongly suggest that individual therapies must
take into consideration the differences in gene expression patterns.
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