BACKGROUND
the hallmark of sepsis is a systemic overwhelming inflammatory response induced by an infectious agent.
despite extensive research, sepsis still remains a leading cause of death in the united states  <cit> .
multiple immunological molecules and pathways with redundant roles contribute to the outcome of sepsis.
microarray based transcription profiling for understanding the pathophysiology of sepsis has predominantly focused on the blood profiles from sepsis patients or animal models  <cit> , with few reports on the primary organs in which the infection originated, such as lung  <cit>  and brain  <cit> .
sepsis caused by intra-abdominal infections arising from a ruptured appendix, penetrating trauma and peritonitis accounts for about 20% of all sepsis cases  <cit> , with a mortality rate of 25-35%  <cit> .
although sepsis is a systemic infection, studies indicate that the inflammatory response is variegated amongst different organs and peripheral blood  <cit> .
this variation can be attributed to the nature of the insult or infection, and the cellular composition and the microenvironment of each organ system  <cit> .
several different studies have provided evidence of differential expression of cytokines and chemokines in different organs or blood in animal models of sepsis  <cit> .
a study in a mouse endotoxemia model showed that the mrna levels of il-1β, tnf-α and mip- <dig> are significantly different in neutrophils derived from lungs as compared to those from peripheral blood  <cit> .
similar differences in il-1β levels in lung and blood have been reported in immunohistochemistry studies  <cit> .
organ specific expression of cxc chemokines  and a cc chemokine  has been shown in a mouse model of peritonitis and endotoxemia  <cit> .
in addition to differences in the cytokine and chemokine expression, there is evidence that the signaling and the receptors leading to the inflammatory response also vary depending upon the anatomical location of the leukocytes involved.
this is exemplified by a study that showed that lps induced nf-κb activation in liver is mediated through tnf-α and il- <dig> receptor dependent pathways while in the lungs it is largely independent of these receptors <cit> .
tolerance to endotoxin in a mouse model has also been shown to be compartmentalized depending upon the cell type in a given organ  <cit> .
therefore, it is important to evaluate the inflammatory responses at the primary focus of the infection which leads to sepsis in order to understand the progression of sepsis from the primary location of infection to a severe systemic condition and then to resolution, as the inducing microbes are cleared.
to date, genome wide transcriptional changes in cells isolated from the peritoneal fluid, the primary site of infection in intra-abdominal sepsis have not been reported.
here, using microarrays, we describe temporal  changes in inflammatory responses in cells from the peritoneal fluid of mice infected with a non-pathogenic strain of escherichia coli.
the mouse model used in this study involves the intraperitoneal administration of a non-pathogenic strain of e. coli.
this is designed to represent the patient population in which sepsis is caused by microbial contamination of the peritoneal cavity due to conditions that compromise the normal mucosal barrier.
the infections often start as polymicrobial but in more than half of the cases, e. coli is typically the only type of bacterium isolated  <cit> .
this model of sepsis causes time-dependent changes that are consistent within treatment groups  <cit> , whereas cecal ligation puncture and the fecal pellet models exhibit considerable variation within groups at early time points and thus are not well suited for assessment of time dependent changes  <cit> .
RESULTS
animal model, bacterial and differential cell counts in peritoneal fluid
the mouse model of intra-abdominal sepsis used here is based on our previous study  <cit> , which indicates that  <dig> h is about the latest time point for sampling, without the risk of animal mortality or morbidity.
levels of pro-inflammatory cytokines  are elevated as early as  <dig> h while most other cytokines peak within  <dig> h of infection  <cit> .
therefore, we chose two early  and one late  time point in this study.
the peritoneal fluid bacterial counts were highest at  <dig> h , consistent with our previous findings  <cit> .
by  <dig> h most of the bacteria were cleared , which should allow the resolution of inflammation.
the survival study also indicates that most of the mice survive at this dose of infection .
at  <dig> h and  <dig> h > 85% of the cells in the peritoneal fluid are macrophages , and at  <dig> h they are predominantly neutrophils  .
the percentage of macrophages that contain  <dig> or more e. coli cells increases at  <dig> h as compared to  <dig> h indicating increased uptake of bacteria.
thus, the changes in gene expression noted in this study between early and late time points undoubtedly reflect both changes in gene expression and changes in the predominant cell type.
however, this change in cell types represents the natural response to sepsis and the beginning of the process of resolution and the results reflect overall gene expression in peritoneal cells during this time period.
therefore, we believe that these results are useful, even though it cannot be determined with certainty which type of cells express the genes detected.
microarray analysis
we used the affymetrix  <dig>  <dig>  mouse microarray platform to evaluate the early  and late  changes in the transcriptional profiles of peritoneal cells from e. coli infected and control mice.
the distribution of the  <dig> differentially expressed  genes shared among the different time points and those unique to each time point are shown as a venn diagram in figure  <dig>  a majority of the de genes were unique to  <dig> h, while most of the de genes at  <dig> h were shared with other time points, although in some cases the direction of change is different.
analysis of gene expression at early and late time points
differences in the transcription profiles between early and late time points, to some extent, will reflect the shift from macrophages to neutrophils.
for example, cd <dig>  a co-stimulatory molecule present on macrophages and dendritic cells, implicated in early phases of the immune response,  <cit>  was significantly up-regulated at  <dig> h .
expression of cd <dig> or nb <dig> a neutrophil specific antigen  <cit>  increased only at  <dig> h .
the gene designated h2-ob is the mouse ortholog of hla-dob, which forms the β chain in the mhc ii molecules expressed on macrophages, dendritic cells and b cells.
expression of this gene was elevated at the two early time points and significantly decreased at  <dig> h .
expression of cr <dig> , prkca , which are expressed by macrophages and are important for complement activation and neutrophil recruitment  <cit> , had a similar pattern of expression .
to confirm that the chosen time points describe molecular events at the early and late stages in sepsis, we looked for expression of markers specific to these stages of sepsis.
adrenomedullin is a member of the calcitonin peptide superfamily, and is a reliable early prognostic marker of sepsis in humans  <cit> , with a prognostic accuracy similar to the apacheii score and procalcitonin expression  <cit> .
besides being a potent vasodilating agent, adrenomedullin is an immune-modulating and antibacterial mediator and a potential therapeutic target for sepsis  <cit> .
in abdominal sepsis, adrenomedullin stabilizes the gut barrier function  <cit> .
in our mouse model of sepsis, adrenomedullin was significantly up-regulated at the early time points  while it was down-regulated at  <dig> h  which corresponds with the recovery from sepsis.
other cell surface markers like cd <dig>  <cit>  and cd <dig>  <cit>  whose expression increases in severe sepsis and decreases with the resolution of sepsis were significantly up- regulated at early time points, and decreased at  <dig> h .
de genes unique to time points
gene expression that is unique to early or late time points is indicative of specific host responses at these stages of infection.
the top  <dig> functions and canonical pathways represented by de genes unique to early and late time points identified by ingenuity pathway analysis  are shown in table  <dig>  the overall effect of the increased/decreased gene expression on the significant molecular functions  at  <dig> h and  <dig> h was assessed by gomodeler  <cit>  workflow.
gomodeler enables gene ontology based hypothesis-driven interrogation of high throughput data.
since the total number of de genes  and thus the de genes associated with each molecular function  at  <dig> h was too limited for meaningful interpretation by gomodeler, they were excluded from analysis.
the net effect of de at  <dig> h is positive for “antigen presentation”, “cellular development”, “cellular function and maintenance”, and “cellular growth and proliferation” .
some of the top canonical pathways at  <dig> h are “dendritic cell maturation”, “trem signaling” and “role of jak family kinase in il- <dig> cytokine signaling” .
taken together these results indicate the initiation of an inflammatory response at  <dig> h in response to the bacteria.
the up-regulation of genes associated with dendritic cell maturation could indicate the presence of maturing dendritic cells in peritoneal fluid, or perhaps more likely, the activation of macrophages , which express many of the same markers as dendritic cells.
a significant number of de genes  uniquely expressed at  <dig> h are associated with cell death.
most of these include apoptotic genes involved in the apoptosis of immune cells.
gomodeler analysis shows a net positive bias for “cell death”.
pro- apoptotic genes like bcl <dig>  bcl2l <dig> and fas are up-regulated at  <dig> h. de genes associated with “cell to cell signaling and interaction” had a net negative bias .
lps mediated mapk signaling  identified at  <dig> h by ipa shows that genes associated with induction of cytokine mediated innate immune response are down-regulated while inhibitors of nf-κb are up-regulated.
the overall pattern of gene expression at  <dig> h indicates an initiation of resolution of the acute inflammatory response observed at the early time points.
analysis of de genes common to all time points
de genes  common to all time points were subjected to hierarchical clustering and short time series expression miner  analysis.
the dendrogram associated with the hierarchical clustering revealed that  <dig> h and  <dig> h groups were similar to each other compared to  <dig> h .
stem based significant clusters show similar trends of expression  at the early time points.
the top functions of the de genes common to all time points are related to immune or inflammatory response.
figure  <dig> represents the temporal trend of the transcriptional changes of the immune response related genes and the corresponding clusters which were statistically significant in stem analysis.
a majority of these genes code for cytokines, transcription regulators, kinases, and transmembrane receptors.
key pro- and anti- inflammatory mediators like il-1α, mip-1β, rantes, cxcl- <dig>  il- <dig> and il- <dig>  implicated to play a role in sepsis were up-regulated at both the early and late time points .
this trend was not surprising, as it is recognized that both pro- and anti- inflammatory responses are regulated simultaneously from early stages of sepsis.
<cit> .
our earlier findings  <cit>  and current results demonstrate that a small number of viable e. coli are present in peritoneal lavage fluid at  <dig> h. therefore, it is not surprising that a number of molecules related to immunity and inflammation are still expressed at  <dig> h.
two profiles  have opposing trends at early and late time points .
some of the immune response related genes with opposing trends of expression at early  and late  time points includes, prkca , cr <dig> , and cd3g involved in t cell co-stimulation.
prkca is important for fcγr mediated phagocytosis by macrophages  <cit> , neutrophil chemotaxis  <cit> , and respiratory burst in neutrophils  <cit> .
it is interesting to note that most of the immune response genes belonged to cluster  <dig>  a significant stem cluster that represents the trend where most of the genes were up-regulated at  <dig> h and their expression began to decrease by  <dig> h. this pattern indicates that by  <dig> h the peritoneal cells exhibit the initiation of resolution of infection.
in addition to transcriptional regulators like rel , whose role in inflammation and sepsis is well established, the list of de immune response related genes common to all time points also has two potentially novel transcription regulators , not previously known to be involved in sepsis .
the plagl <dig> gene product induces the expression of a pro-apoptotic protein nip- <dig> which causes cellular apoptosis  <cit> .
compared to the early time points, the expression of plagl <dig> was higher at  <dig> h. expression of the ebf <dig> gene , which decreases the differentiation of mouse pro b lymphocytes  <cit> , was up-regulated at early time points and down-regulated at  <dig> h. expanding the transcription regulators analysis to all de genes with ≤ − <dig> or ≥ 2-fold expression change, and an immune response related function, we identified additional potentially novel transcription regulators in sepsis.
in all,  <dig> potentially novel transcription regulators involved in sepsis were identified .
de: differential expression.
to increase confidence in the biological interpretation of our microarray data, we determined the concentrations of three cytokines and a chemokine , using a multiplex kit.
the overall trends in cytokine concentrations were consistent with those observed with microarray except for il- <dig>  il- <dig> protein concentration was highest at  <dig> h while the gene expression was maximal at  <dig> h .
however, expression was increased compared to untreated controls at  <dig> and  <dig> h.
discussion
sepsis is the 10th leading cause of death in the united states  <cit> , and the mortality rate has not changed substantially in many years, in spite of the use a wide range of experimental therapeutics.
biological based therapies that work well in rodent models when given before challenge have typically not worked in human sepsis patients.
this could be due to ineffectiveness of administration of these drugs after sepsis is established or the complex biological response with many partially redundant mediators, causing inhibition of a single molecule to be ineffective.
a better understanding of the full range of gene expression changes associated with sepsis will be necessary to understand these issues, and this was the purpose of the present study.
sepsis is a result of a complex systemic dysregulation of inflammation that results when the host is unable to contain an infection that usually starts in a primary organ.
the inflammatory response to sepsis involves two distinct phases which are not always mutually exclusive.
the initial acute phase is the systemic inflammatory response, which is closely followed by the compensatory anti-inflammatory response  <cit> .
the dysregulation of the immune response observed in sepsis is a combination of the local immune response at the primary site of infection and the systemic immune response.
the site of primary infection plays a key role as it produces inflammatory molecules that modulate the systemic immune response.
the cellular environment of each primary organ involved in sepsis including the immunological functional cells  is very specific also the microbial aetiology leading to the infection also differs considerably.
therefore it is expected that the local immune response in sepsis varies depending on the site of primary infection and thus it is important to evaluate the organ specific immune responses.
the abdomen is the second most common primary site of infection in sepsis.
intra-abdominal infections account for about 20% of all sepsis cases.
we used a widely accepted  <cit>  model of intraperitoneal inoculation of a non-pathogenic strain of e. coli to induce sepsis.
two early time points  and one late time point  were used to evaluate the changes in the transcriptional profiles of cells in the peritoneal cavity of the infected animals.
as described in the results, the early time points that are associated with increased bacterial counts are expected to reflect an acute inflammatory response.
in our experience the sepsis model used in this study is associated with very low mortality, only one out of the ten mice in the survival study succumbed to infection .
this is expected since a non-pathogenic strain of e. coli was used for infection and most animals were able to fully clear the bacterial load by about  <dig> h post infection  <cit> .
thus the  <dig> h time point chosen in the study gives a view of the transcriptional changes involved in initiation of the resolution of infection.
the coordinated response of professional phagocytes  is essential for the resolution of infection.
the bacteria were recognized by the resident peritoneal macrophages because of their proximity to the site of infection.
thus, most of the cells in the peritoneal fluid at the early time points were macrophages .
the significantly increased expression of markers associated with macrophages was also reflected in the microarray data at the early time points.
the cytokines released by the macrophages recruit the neutrophils from the blood stream to the site of infection.
so as the infection progresses, the cell population in peritoneal fluid predominantly consists of neutrophils .
this shift was also reflected in the microarray data at  <dig> h. most of the cells at the early time points were macrophages while at  <dig> h neutrophils were the predominant cell type in the peritoneal fluid .
the  <dig> h time point in our mouse model is expected to correspond to the initiation of the resolution of infection.
the significant de genes at this time point were found to be associated with cell death of immune cells.
the apoptosis of neutrophils is critical for the resolution of infection as it ends the sustained neutrophil recruitment and the phagoctytic clearance of the apoptotic neutrophils also re-programs the macrophages to an anti-inflammatory phenotype  <cit> .
we compared our results with a recent meta-analysis of  <dig> studies that evaluated the blood profiles from early and late stages of sepsis from human subjects  <cit> .
in this meta analysis, there was no clear transition between pro- and anti-inflammatory phases at early or late stages.
the up-regulation of tnf-α, il-1α, il-1β, cxcl- <dig>  socs <dig>  il- <dig> at  <dig> and  <dig> h in the present study was similar to that observed in blood profiles of sepsis patients at early stages  <cit> .
expression of these genes peaked at  <dig> h and was still up-regulated at  <dig> h in this study.
decreased expression of cd <dig>  cd <dig>  and mhc ii genes  in peritoneal cell profiles is consistent with the blood profiles of sepsis patients at late stages of sepsis  <cit> .
however, there are some differences in expression of some genes at  <dig> h, as would be expected due to differences in inflammatory responses in different anatomical locations  <cit> .
for example, tnf-α levels in peritoneal cells is elevated at  <dig> h in our study , but meta-analysis identified a decreased expression of tnf-α in blood profiles  <cit> .
the ccr <dig> and s100a <dig> genes are up-regulated in early stages of sepsis in blood  <cit>  but down-regulated in the peritoneal cells in our sepsis model.
despite a number of similarities, gene expression in peritoneal cells differs from the patterns in blood, suggesting that cell type and anatomical location as well as species differences influence the specific characteristics of the local inflammatory response.
nevertheless, there were many similiarities between the early and late responses to sepsis in humans and mice.
an important finding of this study is the identification of  <dig> transcription regulators among the de genes, whose role in sepsis was not previously known.
some of the transcription regulators expressed uniquely at  <dig> and  <dig> h are known to be involved in t cell regulation.
transcription factor  <dig>  was up-regulated and kruppel like factor  <dig>  was down-regulated at  <dig> h. tcf- <dig> is a critical regulator of t cell specification  <cit>  while klf <dig> induces tgf-β <dig> expression.
tgf-β <dig>  an anti-inflammatory cytokine, also regulates t cell activation  <cit> .
zbtb7b, a zinc finger protein crucial for the commitment of mhcii restricted thymocytes to a cd4+ lineage  <cit>  was up-regulated at  <dig> h. strawberry notch homolgue <dig>  was also up-regulated at  <dig> h. sbno <dig> is induced by il- <dig> and contributes to its downstream anti-inflammatory effects  <cit> .
it is not clear if the expression of t cell-related transcription regulators indicates the presence of t cells in the peritoneal cavity or if these transcription regulators are also expressed in macrophages, and have not been recognized as having a role in these cells.
lymphocytes are present in the peritoneal cavity , and we have previously observed expression of genes unique to t cells , so it is possible that t cell activation is an important event in sepsis.
it is also possible that a set of transcription regulators not previously known to be expressed by macrophages are expressed and are potentially important in regulating inflammation.
whether changes in expression of these regulators at  <dig> h is due to altered expression in macrophages or differential patterns of expression in neutrophils vs. macrophages, they are significant compared to naive mice, thus implicating them in the host response to sepsis.
it is not clear if these transcription factors are activated by known or previously unrecognized signaling pathways.
we report here that the best known transcription factor associated with sepsis, nf-κb, exhibits no alteration in transcript quantity.
however, transcriptomics does not reveal information about activation of signaling or transcription related molecules.
our previous study demonstrated that sepsis substantially increases activation of nf-κb using a transgenic reporter mouse model  <cit> .
this serves as a reminder that the changes in the quantity of transcripts for transcription factors may not correspond with activation of those transcription factors.
all other things being equal, increased expression should yield increased activity, but activity needs to be measured for each transcription factor implicated in this study.
this will be done in future studies.
CONCLUSIONS
in conclusion, the results of this study provide an evaluation of the changes in the transcriptional profile of peritoneal cells at early and late stages of intra-abdominal sepsis.
the pattern of expression of key biomarkers for sepsis in humans is similar to that observed in this study, validating the findings of this study, and suggesting the utility of this model for further studies of sepsis.
we identified several novel transcription regulators which might play an important role in immune response to sepsis and could be useful diagnostic markers or therapeutic targets.
future experimental studies are needed to validate and determine the role of these transcription regulators in sepsis.

 the hallmark of sepsis is a systemic overwhelming inflammatory response induced by an infectious agent. despite extensive research, sepsis still remains a leading cause of death in the united states. multiple immunological molecules and pathways with redundant roles contribute to the outcome of sepsis. microarray based transcription profiling for understanding the pathophysiology of sepsis has predominantly focused on the blood profiles from sepsis patients or animal models, with few reports on the primary organs in which the infection originated, such as lung and brain. sepsis caused by intra-abdominal infections arising from a ruptured appendix, penetrating trauma and peritonitis accounts for about 20% of all sepsis cases, with a mortality rate of 25-35%. although sepsis is a systemic infection, studies indicate that the inflammatory response is variegated amongst different organs and peripheral blood. this variation can be attributed to the nature of the insult or infection, and the cellular composition and the microenvironment of each organ system. several different studies have provided evidence of differential expression of cytokines and chemokines in different organs or blood in animal models of sepsis. a study in a mouse endotoxemia model showed that the mrna levels of il-1β, tnf-α and mip- are significantly different in neutrophils derived from lungs as compared to those from peripheral blood. similar differences in il-1β levels in lung and blood have been reported in immunohistochemistry studies. organ specific expression of cxc chemokines and a cc chemokine has been shown in a mouse model of peritonitis and endotoxemia. in addition to differences in the cytokine and chemokine expression, there is evidence that the signaling and the receptors leading to the inflammatory response also vary depending upon the anatomical location of the leukocytes involved. this is exemplified by a study that showed that lps induced nf-κb activation in liver is mediated through tnf-α and il- receptor dependent pathways while in the lungs it is largely independent of these receptors. tolerance to endotoxin in a mouse model has also been shown to be compartmentalized depending upon the cell type in a given organ. therefore, it is important to evaluate the inflammatory responses at the primary focus of the infection which leads to sepsis in order to understand the progression of sepsis from the primary location of infection to a severe systemic condition and then to resolution, as the inducing microbes are cleared. to date, genome wide transcriptional changes in cells isolated from the peritoneal fluid, the primary site of infection in intra-abdominal sepsis have not been reported. here, using microarrays, we describe temporal changes in inflammatory responses in cells from the peritoneal fluid of mice infected with a non-pathogenic strain of escherichia coli. the mouse model used in this study involves the intraperitoneal administration of a non-pathogenic strain of e. coli. this is designed to represent the patient population in which sepsis is caused by microbial contamination of the peritoneal cavity due to conditions that compromise the normal mucosal barrier. the infections often start as polymicrobial but in more than half of the cases, e. coli is typically the only type of bacterium isolated. this model of sepsis causes time-dependent changes that are consistent within treatment groups, whereas cecal ligation puncture and the fecal pellet models exhibit considerable variation within groups at early time points and thus are not well suited for assessment of time dependent changes.  animal model, bacterial and differential cell counts in peritoneal fluid the mouse model of intra-abdominal sepsis used here is based on our previous study, which indicates that h is about the latest time point for sampling, without the risk of animal mortality or morbidity. levels of pro-inflammatory cytokines are elevated as early as h while most other cytokines peak within h of infection. therefore, we chose two early and one late time point in this study. the peritoneal fluid bacterial counts were highest at h, consistent with our previous findings. by h most of the bacteria were cleared, which should allow the resolution of inflammation. the survival study also indicates that most of the mice survive at this dose of infection. at h and h > 85% of the cells in the peritoneal fluid are macrophages, and at h they are predominantly neutrophils. the percentage of macrophages that contain or more e. coli cells increases at h as compared to h indicating increased uptake of bacteria. thus, the changes in gene expression noted in this study between early and late time points undoubtedly reflect both changes in gene expression and changes in the predominant cell type. however, this change in cell types represents the natural response to sepsis and the beginning of the process of resolution and the results reflect overall gene expression in peritoneal cells during this time period. therefore, we believe that these results are useful, even though it cannot be determined with certainty which type of cells express the genes detected. microarray analysis we used the affymetrix mouse microarray platform to evaluate the early and late changes in the transcriptional profiles of peritoneal cells from e. coli infected and control mice. the distribution of the differentially expressed genes shared among the different time points and those unique to each time point are shown as a venn diagram in figure a majority of the de genes were unique to h, while most of the de genes at h were shared with other time points, although in some cases the direction of change is different. analysis of gene expression at early and late time points differences in the transcription profiles between early and late time points, to some extent, will reflect the shift from macrophages to neutrophils. for example, cd a co-stimulatory molecule present on macrophages and dendritic cells, implicated in early phases of the immune response, was significantly up-regulated at h. expression of cd or nb a neutrophil specific antigen increased only at h. the gene designated h2-ob is the mouse ortholog of hla-dob, which forms the β chain in the mhc ii molecules expressed on macrophages, dendritic cells and b cells. expression of this gene was elevated at the two early time points and significantly decreased at h. expression of cr, prkca, which are expressed by macrophages and are important for complement activation and neutrophil recruitment, had a similar pattern of expression. to confirm that the chosen time points describe molecular events at the early and late stages in sepsis, we looked for expression of markers specific to these stages of sepsis. adrenomedullin is a member of the calcitonin peptide superfamily, and is a reliable early prognostic marker of sepsis in humans, with a prognostic accuracy similar to the apacheii score and procalcitonin expression. besides being a potent vasodilating agent, adrenomedullin is an immune-modulating and antibacterial mediator and a potential therapeutic target for sepsis. in abdominal sepsis, adrenomedullin stabilizes the gut barrier function. in our mouse model of sepsis, adrenomedullin was significantly up-regulated at the early time points while it was down-regulated at h which corresponds with the recovery from sepsis. other cell surface markers like cd and cd whose expression increases in severe sepsis and decreases with the resolution of sepsis were significantly up- regulated at early time points, and decreased at h. de genes unique to time points gene expression that is unique to early or late time points is indicative of specific host responses at these stages of infection. the top functions and canonical pathways represented by de genes unique to early and late time points identified by ingenuity pathway analysis are shown in table the overall effect of the increased/decreased gene expression on the significant molecular functions at h and h was assessed by gomodeler workflow. gomodeler enables gene ontology based hypothesis-driven interrogation of high throughput data. since the total number of de genes and thus the de genes associated with each molecular function at h was too limited for meaningful interpretation by gomodeler, they were excluded from analysis. the net effect of de at h is positive for “antigen presentation”, “cellular development”, “cellular function and maintenance”, and “cellular growth and proliferation”. some of the top canonical pathways at h are “dendritic cell maturation”, “trem signaling” and “role of jak family kinase in il- cytokine signaling”. taken together these results indicate the initiation of an inflammatory response at h in response to the bacteria. the up-regulation of genes associated with dendritic cell maturation could indicate the presence of maturing dendritic cells in peritoneal fluid, or perhaps more likely, the activation of macrophages, which express many of the same markers as dendritic cells. a significant number of de genes uniquely expressed at h are associated with cell death. most of these include apoptotic genes involved in the apoptosis of immune cells. gomodeler analysis shows a net positive bias for “cell death”. pro- apoptotic genes like bcl bcl2l and fas are up-regulated at h. de genes associated with “cell to cell signaling and interaction” had a net negative bias. lps mediated mapk signaling identified at h by ipa shows that genes associated with induction of cytokine mediated innate immune response are down-regulated while inhibitors of nf-κb are up-regulated. the overall pattern of gene expression at h indicates an initiation of resolution of the acute inflammatory response observed at the early time points. analysis of de genes common to all time points de genes common to all time points were subjected to hierarchical clustering and short time series expression miner analysis. the dendrogram associated with the hierarchical clustering revealed that h and h groups were similar to each other compared to h. stem based significant clusters show similar trends of expression at the early time points. the top functions of the de genes common to all time points are related to immune or inflammatory response. figure represents the temporal trend of the transcriptional changes of the immune response related genes and the corresponding clusters which were statistically significant in stem analysis. a majority of these genes code for cytokines, transcription regulators, kinases, and transmembrane receptors. key pro- and anti- inflammatory mediators like il-1α, mip-1β, rantes, cxcl- il- and il- implicated to play a role in sepsis were up-regulated at both the early and late time points. this trend was not surprising, as it is recognized that both pro- and anti- inflammatory responses are regulated simultaneously from early stages of sepsis.. our earlier findings and current results demonstrate that a small number of viable e. coli are present in peritoneal lavage fluid at h. therefore, it is not surprising that a number of molecules related to immunity and inflammation are still expressed at h. two profiles have opposing trends at early and late time points. some of the immune response related genes with opposing trends of expression at early and late time points includes, prkca, cr, and cd3g involved in t cell co-stimulation. prkca is important for fcγr mediated phagocytosis by macrophages, neutrophil chemotaxis, and respiratory burst in neutrophils. it is interesting to note that most of the immune response genes belonged to cluster a significant stem cluster that represents the trend where most of the genes were up-regulated at h and their expression began to decrease by h. this pattern indicates that by h the peritoneal cells exhibit the initiation of resolution of infection. in addition to transcriptional regulators like rel, whose role in inflammation and sepsis is well established, the list of de immune response related genes common to all time points also has two potentially novel transcription regulators, not previously known to be involved in sepsis. the plagl gene product induces the expression of a pro-apoptotic protein nip- which causes cellular apoptosis. compared to the early time points, the expression of plagl was higher at h. expression of the ebf gene, which decreases the differentiation of mouse pro b lymphocytes, was up-regulated at early time points and down-regulated at h. expanding the transcription regulators analysis to all de genes with ≤ − or ≥ 2-fold expression change, and an immune response related function, we identified additional potentially novel transcription regulators in sepsis. in all, potentially novel transcription regulators involved in sepsis were identified. de: differential expression. to increase confidence in the biological interpretation of our microarray data, we determined the concentrations of three cytokines and a chemokine, using a multiplex kit. the overall trends in cytokine concentrations were consistent with those observed with microarray except for il- il- protein concentration was highest at h while the gene expression was maximal at h. however, expression was increased compared to untreated controls at and h. discussion sepsis is the 10th leading cause of death in the united states, and the mortality rate has not changed substantially in many years, in spite of the use a wide range of experimental therapeutics. biological based therapies that work well in rodent models when given before challenge have typically not worked in human sepsis patients. this could be due to ineffectiveness of administration of these drugs after sepsis is established or the complex biological response with many partially redundant mediators, causing inhibition of a single molecule to be ineffective. a better understanding of the full range of gene expression changes associated with sepsis will be necessary to understand these issues, and this was the purpose of the present study. sepsis is a result of a complex systemic dysregulation of inflammation that results when the host is unable to contain an infection that usually starts in a primary organ. the inflammatory response to sepsis involves two distinct phases which are not always mutually exclusive. the initial acute phase is the systemic inflammatory response, which is closely followed by the compensatory anti-inflammatory response. the dysregulation of the immune response observed in sepsis is a combination of the local immune response at the primary site of infection and the systemic immune response. the site of primary infection plays a key role as it produces inflammatory molecules that modulate the systemic immune response. the cellular environment of each primary organ involved in sepsis including the immunological functional cells is very specific also the microbial aetiology leading to the infection also differs considerably. therefore it is expected that the local immune response in sepsis varies depending on the site of primary infection and thus it is important to evaluate the organ specific immune responses. the abdomen is the second most common primary site of infection in sepsis. intra-abdominal infections account for about 20% of all sepsis cases. we used a widely accepted model of intraperitoneal inoculation of a non-pathogenic strain of e. coli to induce sepsis. two early time points and one late time point were used to evaluate the changes in the transcriptional profiles of cells in the peritoneal cavity of the infected animals. as described in the results, the early time points that are associated with increased bacterial counts are expected to reflect an acute inflammatory response. in our experience the sepsis model used in this study is associated with very low mortality, only one out of the ten mice in the survival study succumbed to infection. this is expected since a non-pathogenic strain of e. coli was used for infection and most animals were able to fully clear the bacterial load by about h post infection. thus the h time point chosen in the study gives a view of the transcriptional changes involved in initiation of the resolution of infection. the coordinated response of professional phagocytes is essential for the resolution of infection. the bacteria were recognized by the resident peritoneal macrophages because of their proximity to the site of infection. thus, most of the cells in the peritoneal fluid at the early time points were macrophages. the significantly increased expression of markers associated with macrophages was also reflected in the microarray data at the early time points. the cytokines released by the macrophages recruit the neutrophils from the blood stream to the site of infection. so as the infection progresses, the cell population in peritoneal fluid predominantly consists of neutrophils. this shift was also reflected in the microarray data at h. most of the cells at the early time points were macrophages while at h neutrophils were the predominant cell type in the peritoneal fluid. the h time point in our mouse model is expected to correspond to the initiation of the resolution of infection. the significant de genes at this time point were found to be associated with cell death of immune cells. the apoptosis of neutrophils is critical for the resolution of infection as it ends the sustained neutrophil recruitment and the phagoctytic clearance of the apoptotic neutrophils also re-programs the macrophages to an anti-inflammatory phenotype. we compared our results with a recent meta-analysis of studies that evaluated the blood profiles from early and late stages of sepsis from human subjects. in this meta analysis, there was no clear transition between pro- and anti-inflammatory phases at early or late stages. the up-regulation of tnf-α, il-1α, il-1β, cxcl- socs il- at and h in the present study was similar to that observed in blood profiles of sepsis patients at early stages. expression of these genes peaked at h and was still up-regulated at h in this study. decreased expression of cd cd and mhc ii genes in peritoneal cell profiles is consistent with the blood profiles of sepsis patients at late stages of sepsis. however, there are some differences in expression of some genes at h, as would be expected due to differences in inflammatory responses in different anatomical locations. for example, tnf-α levels in peritoneal cells is elevated at h in our study, but meta-analysis identified a decreased expression of tnf-α in blood profiles. the ccr and s100a genes are up-regulated in early stages of sepsis in blood but down-regulated in the peritoneal cells in our sepsis model. despite a number of similarities, gene expression in peritoneal cells differs from the patterns in blood, suggesting that cell type and anatomical location as well as species differences influence the specific characteristics of the local inflammatory response. nevertheless, there were many similiarities between the early and late responses to sepsis in humans and mice. an important finding of this study is the identification of transcription regulators among the de genes, whose role in sepsis was not previously known. some of the transcription regulators expressed uniquely at and h are known to be involved in t cell regulation. transcription factor was up-regulated and kruppel like factor was down-regulated at h. tcf- is a critical regulator of t cell specification while klf induces tgf-β expression. tgf-β an anti-inflammatory cytokine, also regulates t cell activation. zbtb7b, a zinc finger protein crucial for the commitment of mhcii restricted thymocytes to a cd4+ lineage was up-regulated at h. strawberry notch homolgue was also up-regulated at h. sbno is induced by il- and contributes to its downstream anti-inflammatory effects. it is not clear if the expression of t cell-related transcription regulators indicates the presence of t cells in the peritoneal cavity or if these transcription regulators are also expressed in macrophages, and have not been recognized as having a role in these cells. lymphocytes are present in the peritoneal cavity, and we have previously observed expression of genes unique to t cells, so it is possible that t cell activation is an important event in sepsis. it is also possible that a set of transcription regulators not previously known to be expressed by macrophages are expressed and are potentially important in regulating inflammation. whether changes in expression of these regulators at h is due to altered expression in macrophages or differential patterns of expression in neutrophils vs. macrophages, they are significant compared to naive mice, thus implicating them in the host response to sepsis. it is not clear if these transcription factors are activated by known or previously unrecognized signaling pathways. we report here that the best known transcription factor associated with sepsis, nf-κb, exhibits no alteration in transcript quantity. however, transcriptomics does not reveal information about activation of signaling or transcription related molecules. our previous study demonstrated that sepsis substantially increases activation of nf-κb using a transgenic reporter mouse model. this serves as a reminder that the changes in the quantity of transcripts for transcription factors may not correspond with activation of those transcription factors. all other things being equal, increased expression should yield increased activity, but activity needs to be measured for each transcription factor implicated in this study. this will be done in future studies.  in conclusion, the results of this study provide an evaluation of the changes in the transcriptional profile of peritoneal cells at early and late stages of intra-abdominal sepsis. the pattern of expression of key biomarkers for sepsis in humans is similar to that observed in this study, validating the findings of this study, and suggesting the utility of this model for further studies of sepsis. we identified several novel transcription regulators which might play an important role in immune response to sepsis and could be useful diagnostic markers or therapeutic targets. future experimental studies are needed to validate and determine the role of these transcription regulators in sepsis. 
expression pattern for key biomarkers in this study is similar to that reported in human sepsis, indicating the suitability of this model for future studies of sepsis, and the observed differences in gene expression suggest species differences or differences in the response of blood leukocytes and peritoneal leukocytes. gene ontology based hypothesis testing identified a positive bias of differentially expressed genes associated with cellular development and cell death at h and h respectively. intra-abdominal infections are the second most common cause of sepsis. we conducted microarray analysis of the peritoneal cells of mice infected with a non-pathogenic strain of escherichia coli. peritoneal fluid is the primary site of infection in these cases. a multiplexed bead array analysis was used to confirm protein expression for several cytokines which showed differential expression at different time points based on the microarray data. a microarray-based approach was used to study the temporal changes in cells from the peritoneal cavity of septic mice and to identify potential biomarkers and therapeutic targets for this subset of sepsis patients. transcriptional regulators like plagl ebf tcf klf and sbno previously not described in sepsis, are differentially expressed at early and late time points. the events leading to sepsis start with an invasive infection of a primary organ of the body followed by an overwhelming systemic response. differentially expressed genes were identified at two early and one late time point. most differentially expressed genes common to all time points had an immune response related function, consistent with the observation that a few bacteria are still present at h.
 the events leading to sepsis start with an invasive infection of a primary organ of the body followed by an overwhelming systemic response. intra-abdominal infections are the second most common cause of sepsis. peritoneal fluid is the primary site of infection in these cases. a microarray-based approach was used to study the temporal changes in cells from the peritoneal cavity of septic mice and to identify potential biomarkers and therapeutic targets for this subset of sepsis patients.  we conducted microarray analysis of the peritoneal cells of mice infected with a non-pathogenic strain of escherichia coli. differentially expressed genes were identified at two early and one late time point. a multiplexed bead array analysis was used to confirm protein expression for several cytokines which showed differential expression at different time points based on the microarray data. gene ontology based hypothesis testing identified a positive bias of differentially expressed genes associated with cellular development and cell death at h and h respectively. most differentially expressed genes common to all time points had an immune response related function, consistent with the observation that a few bacteria are still present at h.  transcriptional regulators like plagl ebf tcf klf and sbno previously not described in sepsis, are differentially expressed at early and late time points. expression pattern for key biomarkers in this study is similar to that reported in human sepsis, indicating the suitability of this model for future studies of sepsis, and the observed differences in gene expression suggest species differences or differences in the response of blood leukocytes and peritoneal leukocytes. intra-abdominal sepsismicroarrayperitoneal leukocytes 