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2.  Aged Mice are Unable to Mount an Effective Myeloid Response to Sepsis 
Journal of immunology (Baltimore, Md. : 1950)  2013;192(2):10.4049/jimmunol.1302109.
The elderly have increased morbidity and mortality following sepsis; however, the cause(s) remain unclear. We hypothesized that these poor outcomes are due in part to defects in innate immunity, rather than to an exaggerated early inflammatory response. Juvenile (6–12 weeks) or aged (20–24 months) mice underwent polymicrobial sepsis and subsequently, the aged mice had increased mortality and defective peritoneal bacterial clearance compared to young mice. No differences were found in the magnitude of the plasma cytokine responses. Although septic aged mice displayed equivalent or increased numbers of circulating, splenic and bone marrow myeloid cells, some of these cells exhibited decreased phagocytosis, reactive oxygen species production and chemotaxis. Blood leukocyte gene expression was less altered in aged versus young mice one day after sepsis. Aged mice had a relative inability to upregulate gene expression of pathways related to ‘PMN-mediated protective immunity’, ‘chemokine/chemokine receptor binding’ and ‘responses to exogenous molecules’. Expression of most MHC genes remained more down-regulated in aged mice at day three. Despite their increased myeloid response to sepsis, the increased susceptibility of aged mice to sepsis appears not to be due to an exaggerated inflammatory response, but rather, a failure to mount an effective innate immune response.
PMCID: PMC3884685  PMID: 24337739
Critical care medicine  2014;42(6):1406-1413.
Genomic analyses from blood leukocytes have concluded that mouse injury poorly reflects human trauma at the leukocyte transcriptome. Concerns have focused on the modest severity of murine injury models, differences in murine compared to human age, dissimilar circulating leukocyte populations between species, and whether similar signaling pathways are involved. We sought to examine whether the transcriptomic response to severe trauma in mice could be explained by these extrinsic factors, by utilizing an increasing severity of murine trauma and shock in young and aged mice over time, and examining the response in isolated neutrophil populations.
Pre-clinical controlled in vivo laboratory study and retrospective cohort study
Laboratory of Inflammation Biology and Surgical Science and multi-institution level 1 trauma centers
6–10 week old and 20–24 month old C57BL/6 (B6) mice and two cohorts of 167 and 244 severely traumatized (ISS >15) adult (>18 yo) patients.
Mice underwent one of two severity polytrauma models of injury. Total blood leukocyte and neutrophil samples were collected.
Measurements and Main Results
Fold expression changes in leukocyte and neutrophil genome-wide expression analyses between healthy and injured mice (p<0.001) were compared to human total and enriched blood leukocyte expression analyses of severe trauma patients at 0.5, 1, 4, 7, 14, and 28 days after injury (Glue Grant TRDB). We found that increasing the severity of the murine trauma model only modestly improved the correlation in the transcriptomic response with humans, whereas the age of the mice did not. In addition, the genome-wide response to blood neutrophils (rather than total WBC) was also not well correlated between humans and mice. However, the expression of many individual gene families was much more strongly correlated after injury in mice and humans.
Although overall transcriptomic association remained weak even after adjusting for the severity of injury, age of the animals, timing, and individual leukocyte populations, there were individual signaling pathways and ontogenies that were strongly correlated between mice and humans. These genes are involved in early inflammation and innate/adaptive immunity.
PMCID: PMC4283139  PMID: 24413577
microarray; blunt trauma; shock; mouse model
4.  Differences in outcome between obese and nonobese patients following severe blunt trauma are not consistent with an early inflammatory genomic response 
Critical care medicine  2010;38(1):51-58.
Obesity has been demonstrated to alter a number of acute and chronic medical conditions. The effect of obesity on severely injured patients, however, remains incompletely defined. We sought to unravel potential physiologic and genomic alterations induced by obesity in severely injured blunt trauma patients.
A retrospective review of clinical and genomic information contained in the Inflammation and the Host Response to Injury multicenter trauma-related database examining the relationship between body mass index and the early genomic response from peripheral blood leukocytes to patient outcome following severe blunt trauma was performed.
Multicenter collaboration between university-based academic trauma centers.
Severely injured blunt trauma patients enrolled in the database.
Measurements and Main Results
Univariate analysis of 455 severely injured trauma patients using the National Institutes of Health/World Health Organization body mass index classification system revealed significant increases in morbidity, including longer intensive care unit stays and a greater number of ventilator days, cardiac arrests, episodes of acute renal failure, and patients developing multiple organ failure. Regression modeling identified body mass index class as being independently associated with adverse outcomes and increased morbidity but an inverse relationship with mortality in patients who suffered severe blunt traumatic injury. Initial leukocyte genomic expression patterns between 163 patients in the four different body mass index groupings did not differ; however, analysis of gene differences between body mass index classes occurring over time demonstrated significant changes in 513 probe sets with significant pathway differences being related to cellular metabolism.
Increasing body mass index is associated with increased morbidity following severe blunt trauma. The initial blood leukocyte inflammatory response to blunt trauma does not appear to differ significantly between patients despite increasing body mass index. Resolution of the inflammatory response may differ between patients on the basis of body mass index; however, additional work is needed to clarify the potential causality of this finding.
PMCID: PMC4028684  PMID: 19661803
obesity; trauma; inflammation; genomics; leukocytes
5.  Hemorrhage and Subsequent Allogenic Red Blood Cell Transfusion are Associated With Characteristic Monocyte Messenger RNA Expression Patterns in Patients After Multiple Injury—A Genome Wide View 
The Journal of trauma  2009;67(4):792-801.
As outcome to severe trauma is frequently affected by massive blood loss and consecutive hemorrhagic shock, replacement of red blood cell (RBC) units remains indispensable. Administration of RBC units is an independent risk factor for adverse outcome in patients with trauma. The impact of massive blood transfusion or uncrossmatched blood transfusion on the patients’ immune response in the early posttraumatic period remains unclear.
Thirteen patients presenting with blunt multiple injuries (Injury Severity Score >16) were studied. Monocytes were obtained on admission and at 6, 12, 24, 48, and 72 hours after trauma. Biotinylated complementary RNA targets were hybridized to Affymetrix HG U 133A microarrays. The data were analyzed by a supervised analysis based on whether the patients received massive blood transfusions, and then subsequently, by hierarchical clustering, and by Ingenuity pathway analysis.
Supervised analysis identified 224 probe sets to be differentially expressed (p < 0.001) in patients who received massive blood transfusion, when compared with those who did not. In addition, 331 probe sets were found differentially expressed (p < 0.001) in patients who received uncrossmatched RBC units in comparison with those who exclusively gained crossmatched ones. Functional pathway analysis of the respectively identified gene expression profiles suggests a contributory role by the AKT/PI3Kinase pathway, the mitogen-activated protein-kinase pathway, the Ubiquitin pathway, and the diverse inflammatory networks.
We exhibited for the first time a serial, sequential screening analysis of monocyte messenger RNA expression patterns in patients with multiple trauma indicating a strongly significant association between the patients’ genomic response in blood monocytes and massive or uncross-matched RBC substitution.
PMCID: PMC4026089  PMID: 19820587
Microarray; MAP kinase; Ubiquitin; Allogenic red blood cell transfusion; Hemorrhagic shock
6.  Microfluidic Leukocyte Isolation for Gene Expression Analysis in Critically Ill Hospitalized Patients 
Clinical chemistry  2008;54(5):891-900.
Microarray technology is becoming a powerful tool for diagnostic, therapeutic, and prognostic applications. There is at present no consensus regarding the optimal technique to isolate nucleic acids from blood leukocyte populations for subsequent expression analyses. Current collection and processing techniques pose significant challenges in the clinical setting. Here, we report the clinical validation of a novel microfluidic leukocyte nucleic acid isolation technique for gene expression analysis from critically ill, hospitalized patients that can be readily used on small volumes of blood.
We processed whole blood from hospitalized patients after burn injury and severe blunt trauma according to the microfluidic and standard macroscale leukocyte isolation protocol. Side-by-side comparison of RNA quantity, quality, and genome-wide expression patterns was used to clinically validate the microfluidic technique.
When the microfluidic protocol was used for processing, sufficient amounts of total RNA were obtained for genome-wide expression analysis from 0.5 mL whole blood. We found that the leukocyte expression patterns from samples processed using the 2 protocols were concordant, and there was less variability introduced as a result of harvesting method than there existed between individuals.
The novel microfluidic approach achieves leukocyte isolation in <25 min, and the quality of nucleic acids and genome expression analysis is equivalent to or surpasses that obtained from macroscale approaches. Microfluidics can significantly improve the isolation of blood leukocytes for genomic analyses in the clinical setting.
PMCID: PMC4011019  PMID: 18375483
7.  Development of a genomic metric that can be rapidly used to predict clinical outcome in severely injured trauma patients 
Critical care medicine  2013;41(5):10.1097/CCM.0b013e318277131c.
Many patients following severe trauma have complicated recoveries due to the development of organ injury. Physiological and anatomical prognosticators have had limited success in predicting clinical trajectories. We report on the development and retrospective validation of a simple genomic composite score that can be rapidly used to predict clinical outcomes.
Retrospective cohort study
Multi-institution level 1 trauma centers
Data was collected from 167 severely traumatized (ISS >15) adult (18–55 yo) patients
Microarray-derived genomic data obtained from 167 severely traumatized patients over 28 days were assessed for differences in mRNA abundance between individuals with different clinical trajectories. Once a set of genes was identified based on differences in expression over the entire study period, mRNA abundance from these subjects obtained in the first 24 hours was analyzed in a blinded fashion using a rapid multiplex platform, and genomic data reduced to a single metric.
From the existing genomic data set, we identified 63 genes whose leukocyte expression differed between an uncomplicated and complicated clinical outcome over 28 days. Using a multiplex approach that can quantitate mRNA abundance in less than 12 hours (nanoString™), we reassessed total mRNA abundance from the first 24 hours after trauma, and reduced the genomic data to a single composite score using the difference from reference (DFR). This composite score showed good discriminatory capacity to distinguish patients with a complicated outcome (area under a receiver-operator curve, 0.811, p < 0.001). This was significantly better than the predictive power of either APACHE II or NISS scoring systems.
A rapid genomic composite score obtained in the first 24 hours after trauma can retrospectively identify trauma patients who are likely to develop a complicated clinical trajectories. A novel platform is described in which this genomic score can be obtained within 12 hours of blood collection, making it available for clinical decision making. (300 words; limit 300)
PMCID: PMC3652285  PMID: 23388514
nanostring; microarray; blunt trauma
8.  Host Responses to Sepsis Vary in Different Low-Lethality Murine Models 
PLoS ONE  2014;9(5):e94404.
Animal models for the study of sepsis are being increasingly scrutinized, despite their essential role for early translational research. In particular, recent studies have suggested that at the level of the leukocyte transcriptome, murine models of burns, trauma and endotoxemia markedly differ from their human equivalents, and are only weakly similar amongst themselves. We compared the plasma cytokine and leukocyte transcriptome responses between two different low-lethality murine models of polymicrobial intra-abdominal sepsis.
Six to ten week male C57BL/6j mice underwent either the ‘gold standard’ cecal ligation and puncture (CLP) model of intra-abdominal sepsis or administration of a cecal slurry (CS), where cecal contents are injected intraperitoneally. Surviving mice were euthanized at two hours, one or three days after sepsis.
The murine leukocyte transcriptomic response to the CLP and CS models of sepsis was surprisingly dissimilar at two hours, one, and three days after sepsis. The Pearson correlation coefficient for the maximum change in expression for the entire leukocyte transcriptome that changed significantly over time (n = 19,071) was R = 0.54 (R2 = 0.297). The CS model resulted in greater magnitude of early inflammatory gene expression changes in response to sepsis with associated increased production of inflammatory chemokines and cytokines. Two hours after sepsis, CLP had more significant expression of genes associated with IL-10 signaling pathways, whereas CS had greater expression of genes related to CD28, apoptosis, IL-1 and T-cell receptor signaling. By three days, the changes in gene expression in both sepsis models were returning to baseline in surviving animals.
These analyses reveal that the murine blood leukocyte response to sepsis is highly dependent on which model of intra-abdominal sepsis is employed, despite their similar lethality. It may be difficult to extrapolate findings from one murine model to another, let alone to human sepsis.
PMCID: PMC4006924  PMID: 24788351
Critical care medicine  2013;41(4):1075-1085.
To develop a novel polytrauma model that better recapitulates the immunological response of the severely injured patient by combining long-bone fracture, muscle tissue damage and cecectomy with hemorrhagic shock, resulting in an equivalent Injury Severity Score of greater than 15. We compared this new polytrauma/shock model to historically-used murine trauma-hemorrhage models.
Pre-clinical controlled in vivo laboratory study.
Laboratory of Inflammation Biology and Surgical Science.
6–10 wk old C57BL/6 (B6) mice
Mice underwent 90 minutes of shock (MAP 30 mmHg) and resuscitation via femoral artery cannulation followed by either laparotomy (TH), laparotomy with femur fracture (H+FFx), or laparotomy with cecetomy and femur fracture with muscle tissue damage (PT). Mice were euthanized at two hours, one day and three days post injury.
Measurements and Main Results
The spleen, bone marrow, blood, and serum were collected from mice for analysis at the above time points. None of the models were lethal. Mice undergoing PT exhibited a more robust inflammatory response with significant elevations in cytokine/chemokine concentrations when compared to traditional models. PT was the only model to induce neutrophilia (Ly6G+CD11b+ cells) on days 1 and 3 (p<0.05). PT, as compared to TH and H+FFx, induced a loss of circulating CD4+ T cell with simultaneous increased cell activation (CD69+ and CD25+), similar to human trauma. There was a prolonged loss of MHCII expression on monocytes in the PT model (p<0.05). Results were confirmed by genome-wide expression analysis which revealed a greater magnitude and duration of blood leukocyte gene expression changes in the PT model than the TH and sham models.
This novel polytrauma model better replicates the human leukocyte, cytokine, and overall inflammatory response following injury and hemorrhagic shock.
PMCID: PMC3666582  PMID: 23399937
hemorrhage; gene expression; femur fracture; inflammation; immune suppression; trauma
10.  Microfluidics-Based Capture of Human Neutrophils for Expression Analysis in Blood and Bronchoalveolar Lavage 
Gene expression analysis can be a powerful tool in predicting patient outcomes and identifying patients who may benefit from targeted therapies. However, isolating human blood neutrophils (PMNs) for genomic analysis has been challenging. We employed a novel microfluidic technique that isolates PMNs by capturing CD66b+ cells and compared it to dextran-Ficoll gradient isolation. We also employed microfluidic isolation techniques to blood and bronchoalveolar lavage (BAL) samples of patients with ARDS to evaluate PMN genomic alterations secondary to pulmonary sequestration. PMNs obtained from ex vivo lipopolysaccharide (LPS)-stimulated or unstimulated whole blood from five healthy volunteers were isolated by either dextran-Ficoll gradient, microfluidics capture, or a combination of the two techniques. Blood and BAL fluid PMNs were also isolated using microfluidics from seven hospitalized patients with ARDS. Gene expression was inferred from extracted RNA using Affymetrix U133 Plus 2.0 GeneChips™. All methods of PMN isolation produced similar quantities of high-quality RNA, when adjusted for recovered cell number. Unsupervised analysis and hierarchal clustering indicated that LPS stimulation was the primary factor affecting gene expression patterns among all ex vivo samples. Patterns of gene expression from blood and BAL PMNs differed significantly from each other in the patients with ARDS. Isolation of PMNs by microfluidics can be applied to both blood and BAL specimens from critically ill, hospitalized patients. Unique genomic expression patterns are obtained from the blood and BAL fluid of critically ill patients with ARDS, and these differ significantly from genomic patterns seen after ex vivo LPS stimulation.
PMCID: PMC3957199  PMID: 21931299
Neutrophil isolation; microfluidics; genomics; dextran; ficoll
11.  A Genomic Analysis of Clostridium difficile Infections in Blunt Trauma Patients 
The journal of trauma and acute care surgery  2013;74(1):10.1097/TA.0b013e3182789426.
Evidence demonstrates that susceptibility to Clostridium difficile infection (C. diff. ) is related as much to host risk factors as bacterial potency. Using blood leukocyte genome-wide expression patterns of severe blunt trauma patients obtained by the NIGMS sponsored Glue Grant “Inflammation and the Host Response to Injury” we examined C. diff. patients’ leukocyte genomic profiles to determine pre- and post-infection gene expression changes.
The genomic responses of 21 severe trauma patients were analyzed (5 C. diff. 16 controls matched for age and severity of injury). After elimination of probe sets whose expression was below baseline or were unchanged, remaining probe sets underwent hierarchical clustering and principal component analysis. Molecular pathways were generated through Ingenuity Pathways Analysis ®.
Supervised analysis demonstrated 118 genes whose expression in C. diff. patients varied before and after their infection. Supervised analysis comparing C. diff. to matched non-C. diff. patients prior to infection suggested that the expression of 501 genes were different in the two groups with up to 87% class prediction (p<0.05). Many of these genes are related to cell-mediated immune responses, signaling and interaction.
Genomic analysis of severe blunt trauma patients reveals a distinct leukocyte expression profile of C. diff. both prior to and after infection. We conclude that an association may exist between a severe trauma patient’s leukocyte genomic expression profile and subsequent susceptibility to C. diff. Further prospective expression analysis of this C. diff. population may reveal potential therapeutic interventions and allow early identification of C. diff. susceptible patients.
Level of Evidence
Level III prognostic/diagnostic study.
PMCID: PMC3864116  PMID: 23271108
12.  Murine Lupus Susceptibility Locus Sle1c2 Mediates CD4+ T cell Activation and Maps to Estrogen-Related Receptor Gamma Esrrg 
Sle1c is a sublocus of the NZM2410-derived Sle1 major lupus susceptibility locus. We have previously shown that Sle1c contributes to lupus pathogenesis by conferring increased CD4+ T cell activation and increased susceptibility to chronic graft-versus-host disease (cGVHD), which mapped to the centromeric portion of the locus. In this study, we have refined the centromeric sublocus to a 675Kb interval, termed Sle1c2. Mice from recombinant congenic strains expressing Sle1c2 exhibited increased CD4+ T cell intrinsic activation and cGVHD susceptibility, similar to mice with the parental Sle1c. In addition, B6.Sle1c2 mice displayed a robust expansion of IFNγ expressing T cells. NZB complementation studies showed that Sle1c2 expression exacerbated B cell activation, autoAb production, and renal pathology, verifying that Sle1c2 contributes to lupus pathogenesis. The Sle1c2 interval contains two genes, only one of which, Esrrg, is expressed in T cells. B6.Sle1c2 CD4+ T cells expressed less Esrrg than B6 CD4+ T cells, and Esrrg expression was negatively correlated to CD4+ T cell activation. Esrrg encodes for an orphan nuclear receptor that regulates oxidative metabolism and mitochondrial functions. In accordance with a reduced Esrrg expression, B6.Sle1c2 CD4+ T cells present reduced mitochondrial mass and altered mitochondrial functions, as well as altered metabolic pathway utilization when compared to B6. Taken together, we propose Esrrg as a novel lupus susceptibility gene regulating CD4+ T cell function through their mitochondrial metabolism.
PMCID: PMC3392454  PMID: 22711888
13.  Transcriptomic Analysis of PNN- and ESRP1-Regulated Alternative Pre-mRNA Splicing in Human Corneal Epithelial Cells 
We investigated the impact of PININ (PNN) and epithelial splicing regulatory protein 1 (ESRP1) on alternative pre-mRNA splicing in the corneal epithelial context.
Isoform-specific RT-PCR assays were performed on wild-type and Pnn knockout mouse cornea. Protein interactions were examined by deconvolution microscopy and co-immunoprecipitation. For genome-wide alternative splicing study, immortalized human corneal epithelial cells (HCET) harboring doxycycline-inducible shRNA against PNN or ESRP1 were created. Total RNA was isolated from four biological replicates of control and knockdown HCET cells, and subjected to hGlue3_0 transcriptome array analysis.
Pnn depletion in developing mouse corneal epithelium led to disrupted alternative splicing of multiple ESRP-regulated epithelial-type exons. In HCET cells, ESRP1 and PNN displayed close localization in and around nuclear speckles, and their physical association in protein complexes was identified. Whole transcriptome array analysis on ESRP1 or PNN knockdown HCET cells revealed clear alterations in transcript profiles and splicing patterns of specific subsets of genes. Separate RT-PCR validation assays confirmed successfully specific changes in exon usage of several representative splice variants, including PAX6(5a), FOXJ3, ARHGEF11, and SLC37A2. Gene ontologic analyses on ESRP1- or PNN-regulated alternative exons suggested their roles in epithelial phenotypes, such as cell morphology and movement.
Our data suggested that ESRP1 and PNN modulate alternative splicing of a specific subset of target genes, but not general splicing events, in HCET cells to maintain or enhance epithelial characteristics.
Genome-wide PNN- and ESRP1-regulated alternative pre-mRNA splicing events were identified in human corneal epithelial cells through whole transcriptome array analysis. The study suggests specific roles of PNN and ESRP1 in the regulation of epithelial-specific alternative splicing.
PMCID: PMC3559072  PMID: 23299472
14.  Polymicrobial periodontal pathogens transcriptomes in calvarial bone and soft tissue 
Molecular oral microbiology  2011;26(5):303-320.
Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia are consistently associated with adult periodontitis. This study sought to document the host transcriptome to a P. gingivalis, T. denticola, and T. forsythia challenge as a polymicrobial infection using a murine calvarial model of acute inflammation and bone resorption. Mice were infected with P. gingivalis, T. denticola, and T. forsythia over the calvaria, after which the soft tissues and calvarial bones were excised. A Murine GeneChip® array analysis of transcript profiles showed that 6997 genes were differentially expressed in calvarial bones (P < 0.05) and 1544 genes were differentially transcribed in the inflamed tissues after the polymicrobial infection. Of these genes, 4476 and 1035 genes in the infected bone and tissues were differentially expressed by upregulation. Biological pathways significantly impacted by the polymicrobial infection in calvarial bone included leukocyte transendothelial migration (LTM), cell adhesion molecules, adherens junction, major histocompatibility complex antigen, extracellular matrix-receptor interaction (ECM), and antigen processing and presentation resulting in inflammatory/cytokine/chemokine transcripts stimulation in bone and soft tissue. Intense inflammation and increased activated osteoclasts was observed in calvarias compared to sham-infected controls. Quantitative real-time RT-PCR analysis confirmed mRNA level of selected genes corresponded with the microarray expression. The polymicrobial infection regulated several LTM and extracellular membrane (ECM) pathway genes in a manner distinct from monoinfection with P. gingivalis, T. denticola, or T. forsythia. To our knowledge, this is the first definition of the polymicrobial induced transcriptome in calvarial bone and soft tissue in response to periodontal pathogens.
PMCID: PMC3170131  PMID: 21896157
P. gingivalis; T. denticola; T. forsythia; polymicrobial infection; gene expression; calvarial bone; tissue; microarray
15.  Severe Injury Is Associated With Insulin Resistance, Endoplasmic Reticulum Stress Response, and Unfolded Protein Response 
Annals of Surgery  2012;255(2):370-378.
We determined whether postburn hyperglycemia and insulin resistance are associated with endoplasmic reticulum (ER) stress/unfolded protein response (UPR) activation leading to impaired insulin receptor signaling.
Inflammation and cellular stress, hallmarks of severely burned and critically ill patients, have been causally linked to insulin resistance in type 2 diabetes via induction of ER stress and the UPR.
Twenty severely burned pediatric patients were compared with 36 nonburned children. Clinical markers, protein, and GeneChip analysis were used to identify transcriptional changes in ER stress and UPR and insulin resistance–related signaling cascades in peripheral blood leukocytes, fat, and muscle at admission and up to 466 days postburn.
Burn-induced inflammatory and stress responses are accompanied by profound insulin resistance and hyperglycemia. Genomic and protein analysis revealed that burn injury was associated with alterations in the signaling pathways that affect insulin resistance, ER/sarcoplasmic reticulum stress, inflammation, and cell growth/apoptosis up to 466 days postburn.
Burn-induced insulin resistance is associated with persistent ER/sarcoplasmic reticulum stress/UPR and subsequent suppressed insulin receptor signaling over a prolonged period of time.
PMCID: PMC3395076  PMID: 22241293
16.  Bacillus anthracis’ lethal toxin induces broad transcriptional responses in human peripheral monocytes 
BMC Immunology  2012;13:33.
Anthrax lethal toxin (LT), produced by the Gram-positive bacterium Bacillus anthracis, is a highly effective zinc dependent metalloprotease that cleaves the N-terminus of mitogen-activated protein kinase kinases (MAPKK or MEKs) and is known to play a role in impairing the host immune system during an inhalation anthrax infection. Here, we present the transcriptional responses of LT treated human monocytes in order to further elucidate the mechanisms of LT inhibition on the host immune system.
Western Blot analysis demonstrated cleavage of endogenous MEK1 and MEK3 when human monocytes were treated with 500 ng/mL LT for four hours, proving their susceptibility to anthrax lethal toxin. Furthermore, staining with annexin V and propidium iodide revealed that LT treatment did not induce human peripheral monocyte apoptosis or necrosis. Using Affymetrix Human Genome U133 Plus 2.0 Arrays, we identified over 820 probe sets differentially regulated after LT treatment at the p <0.001 significance level, interrupting the normal transduction of over 60 known pathways. As expected, the MAPKK signaling pathway was most drastically affected by LT, but numerous genes outside the well-recognized pathways were also influenced by LT including the IL-18 signaling pathway, Toll-like receptor pathway and the IFN alpha signaling pathway. Multiple genes involved in actin regulation, signal transduction, transcriptional regulation and cytokine signaling were identified after treatment with anthrax LT.
We conclude LT directly targets human peripheral monocytes and causes multiple aberrant gene responses that would be expected to be associated with defects in human monocyte’s normal signaling transduction pathways and function. This study provides further insights into the mechanisms associated with the host immune system collapse during an anthrax infection, and suggests that anthrax LT may have additional downstream targets outside the well-known MAPK pathway.
PMCID: PMC3475123  PMID: 22747600
17.  A genomic storm in critically injured humans 
The Journal of Experimental Medicine  2011;208(13):2581-2590.
Critical injury in humans induces a genomic storm with simultaneous changes in expression of innate and adaptive immunity genes.
Human survival from injury requires an appropriate inflammatory and immune response. We describe the circulating leukocyte transcriptome after severe trauma and burn injury, as well as in healthy subjects receiving low-dose bacterial endotoxin, and show that these severe stresses produce a global reprioritization affecting >80% of the cellular functions and pathways, a truly unexpected “genomic storm.” In severe blunt trauma, the early leukocyte genomic response is consistent with simultaneously increased expression of genes involved in the systemic inflammatory, innate immune, and compensatory antiinflammatory responses, as well as in the suppression of genes involved in adaptive immunity. Furthermore, complications like nosocomial infections and organ failure are not associated with any genomic evidence of a second hit and differ only in the magnitude and duration of this genomic reprioritization. The similarities in gene expression patterns between different injuries reveal an apparently fundamental human response to severe inflammatory stress, with genomic signatures that are surprisingly far more common than different. Based on these transcriptional data, we propose a new paradigm for the human immunological response to severe injury.
PMCID: PMC3244029  PMID: 22110166
18.  Incidence, clinical predictors, genomics, and outcome of acute kidney injury among trauma patients 
Annals of Surgery  2010;252(1):158-165.
To determine clinical and genomic characteristics and in-hospital mortality risk associated with acute kidney injury (AKI) in the multicenter prospective cohort of patients with blunt trauma.
Summary Background Data
Less severe stages of AKI characterized by small changes in serum creatinine (sCr) are inadequately studied among trauma patients.
We performed a secondary analysis of the “Inflammation and the Host Response to Injury” (GlueGrant) database to include adult blunt trauma patients without history of kidney disease. AKI was defined by the RIFLE (Risk, Injury, Failure, Loss, and End-stage Kidney) classification, which requires a 50% increase in sCr and stratifies patients into three severity stages: risk, injury, and failure. Association between all stages of AKI and in-hospital mortality was analyzed using a multivariable logistic regression analysis. Genome-wide expression analysis was performed on whole blood leukocytes obtained within 12 hours of trauma.
AKI occurred in 26% of 982 patients. The adjusted risk for hospital death was three times higher for patients with AKI compared to patients without AKI (odds ratio [OR] 3.05 (95% confidence interval [CI], (1.73, TO 5.40). This risk was evident in a dose-response manner and even patients with mild AKI had OR for dying of 2.57 (95% CI, 1.19 to 5.50) compared to patients without AKI. Genome-wide expression analysis failed to show a significant number of genes whose expression could discriminate among patients with and without AKI.
In a multi-center prospective cohort of blunt trauma patients, AKI characterized by small changes in sCr was associated with an independent risk of hospital death.
PMCID: PMC3357629  PMID: 20562612
trauma; inflammation; genomics; leukocytes
19.  Partitioning Transcript Variation in Drosophila: Abundance, Isoforms, and Alleles 
G3: Genes|Genomes|Genetics  2011;1(6):427-436.
Multilevel analysis of transcription is facilitated by a new array design that includes modules for assessment of differential expression, isoform usage, and allelic imbalance in Drosophila. The ∼2.5 million feature chip incorporates a large number of controls, and it contains 18,769 3′ expression probe sets and 61,919 exon probe sets with probe sequences from Drosophila melanogaster and 60,118 SNP probe sets focused on Drosophila simulans. An experiment in D. simulans identified genes differentially expressed between males and females (34% in the 3′ expression module; 32% in the exon module). These proportions are consistent with previous reports, and there was good agreement (κ = 0.63) between the modules. Alternative isoform usage between the sexes was identified for 164 genes. The SNP module was verified with resequencing data. Concordance between resequencing and the chip design was greater than 99%. The design also proved apt in separating alleles based upon hybridization intensity. Concordance between the highest hybridization signals and the expected alleles in the genotype was greater than 96%. Intriguingly, allelic imbalance was detected for 37% of 6579 probe sets examined that contained heterozygous SNP loci. The large number of probes and multiple probe sets per gene in the 3′ expression and exon modules allows the array to be used in D. melanogaster and in closely related species. The SNP module can be used for allele specific expression and genotyping of D. simulans.
PMCID: PMC3276160  PMID: 22384353
SNP chip
20.  Roles of Vaccinia Virus Genes E3L and K3L and Host Genes PKR and RNase L during Intratracheal Infection of C57BL/6 Mice ▿  
Journal of Virology  2010;85(1):550-567.
The importance of the 2′-5′ oligoadenylate synthetase (OAS)/RNase L and double-stranded RNA (dsRNA)-dependent protein kinase (PKR) pathways in host interferon induction resulting from virus infection in response to dsRNA has been well documented. In poxvirus infections, the interactions between the vaccinia virus (VV) genes E3L and K3L, which target RNase L and PKR, respectively, serve to prevent the induction of the dsRNA-dependent induced interferon response in cell culture. To determine the importance of these host genes in controlling VV infections, mouse single-gene knockouts of RNase L and PKR and double-knockout mice were studied following intratracheal infection with VV, VVΔK3L, or VVΔE3L. VV caused lethal disease in all mouse strains. The single-knockout animals were more susceptible than wild-type animals, while the RNase L−/− PKR−/− mice were the most susceptible. VVΔE3L infections of wild-type mice were asymptomatic, demonstrating that E3L plays a critical role in controlling the host immune response. RNase L−/− mice showed no disease, whereas 20% of the PKR−/− mice succumbed at a dose of 108 PFU. Lethal disease was routinely observed in RNase L−/− PKR−/− mice inoculated with 108 PFU of VVΔE3L, with a distinct pathology. VVΔK3L infections exhibited no differences in virulence among any of the mouse constructs, suggesting that PKR is not the exclusive target of K3L. Surprisingly, VVΔK3L did not disseminate to other tissues from the lung. Hence, the cause of death in this model is respiratory disease. These results also suggest that an unanticipated role of the K3L gene is to facilitate virus dissemination.
PMCID: PMC3014211  PMID: 20943971
21.  Porphyromonas gingivalis infection-induced tissue and bone transcriptional profiles 
Molecular oral microbiology  2010;25(1):61-74.
Porphyromonas gingivalis has been associated with subgingival biofilms in adult periodontitis. However, the molecular mechanisms of its contribution to chronic gingival inflammation and loss of periodontal structural integrity remain unclear. The objectives of this investigation were to examine changes in the host transcriptional profiles during a P. gingivalis infection using a murine calvarial model of inflammation and bone resorption.
P. gingivalis FDC 381 was injected into the subcutaneous soft tissue over the calvaria of BALB/c mice for 3 days, after which the soft tissues and calvarial bones were excised. RNA was isolated from infected soft tissues and calvarial bones and analyzed for transcript profiles using Murine GeneChip® arrays to provide a molecular profile of the events that occur following infection of these tissues.
After P. gingivalis infection, 5517 and 1900 probe sets in the infected soft tissues and calvarial bone, respectively, were differentially expressed (P ≤ 0.05) and up-regulated. Biological pathways significantly impacted by P. gingivalis infection in tissues and calvarial bone included cell adhesion (immune system) molecules, Toll-like receptors, B cell receptor signaling, TGF-β cytokine family receptor signaling, and MHC class II antigen processing pathways resulting in proinflammatory, chemotactic effects, T cell stimulation, and down regulation of antiviral and T cell chemotactic effects. P. gingivalis-induced inflammation activated osteoclasts, leading to local bone resorption.
This is the first in vivo evidence that localized P. gingivalis infection differentially induces transcription of a broad array of host genes that differed between inflamed soft tissues and calvarial bone.
PMCID: PMC3001241  PMID: 20331794
P. gingivalis; gene expression; calvarial tissue; bone; microarray
22.  A limited innate immune response is induced by a replication-defective herpes simplex virus vector following delivery to the murine central nervous system 
Journal of neurovirology  2009;15(5-6):411-424.
Herpes simplex virus type 1 (HSV-1)–based vectors readily transduce neurons and have a large payload capacity, making them particularly amenable to gene therapy applications within the central nervous system (CNS). Because aspects of the host responses to HSV-1 vectors in the CNS are largely unknown, we compared the host response of a nonreplicating HSV-1 vector to that of a replication-competent HSV-1 virus using microarray analysis. In parallel, HSV-1 gene expression was tracked using HSV-specific oligonucleotide-based arrays in order to correlate viral gene expression with observed changes in host response. Microarray analysis was performed following stereotactic injection into the right hippocampal formation of mice with either a replication-competent HSV-1 or a nonreplicating recombinant of HSV-1, lacking the ICP4 gene (ICP4−). Genes that demonstrated a significant change (P < .001) in expression in response to the replicating HSV-1 outnumbered those that changed in response to mock or nonreplicating vector by approximately 3-fold. Pathway analysis revealed that both the replicating and nonreplicating vectors induced robust antigen presentation but only mild interferon, chemokine, and cytokine signaling responses. The ICP4− vector was restricted in several of the Toll-like receptor-signaling pathways, indicating reduced stimulation of the innate immune response. These array analyses suggest that although the nonreplicating vector induces detectable activation of immune response pathways, the number and magnitude of the induced response is dramatically restricted compared to the replicating vector, and with the exception of antigen presentation, host gene expression induced by the non-replicating vector largely resembles mock infection.
PMCID: PMC2975456  PMID: 20095947
gene therapy; host response; HSV; vector
23.  Role of the ATM-Checkpoint Kinase 2 Pathway in CDT-Mediated Apoptosis of Gingival Epithelial Cells 
PLoS ONE  2010;5(7):e11714.
The cytolethal distending toxin (CDT) of the oral pathogen Aggregatibacter actinomycetemcomitans induces cell cycle arrest and apoptosis in various cell types. Western analysis, pharmacological inhibition and siRNA silencing were performed in human immortalized gingival keratinocytes (HIGK) to dissect the functional role of the ataxia telangiectasia mutated (ATM) pathway in the signal transduction steps triggered by the CDT. Infection of HIGK was associated with a time-dependent induction of cytoplasmic histone-associated DNA fragmentation. However, in the absence of CDT, infected HIGK underwent reversible DNA strand breaks but not apoptosis, while caspase 3 activity, p21 levels, and HIGK viability were unaffected. Caspase 9 activity was attenuated in the CDT mutant-infected HIGK compared to wild-type infected cells. Pharmacological inhibition and siRNA-silencing of the ATM downstream effector, the protein kinase checkpoint kinase 2 (Chk2), significantly impacted CDT-mediated apoptosis. Together, these findings provide insight on the specificity of the ATM-Chk2 pathway in response to the CDT of A. actinomycetemcomitans in oral epithelial cells, which ultimately leads to apoptosis. We further propose the existence of an unidentified factor that is distinct from the CDT, and involved with a reversible DNA fragmentation that does not trigger terminal apoptosis in oral epithelial cells. This model potentially explains conflicting reports on the biological activity of the A. actinomycetemcomitans CDT.
PMCID: PMC2909199  PMID: 20668524
24.  The degree of microbiome complexity influences the epithelial response to infection 
BMC Genomics  2009;10:380.
The human microflora is known to be extremely complex, yet most pathogenesis research is conducted in mono-species models of infection. Consequently, it remains unclear whether the level of complexity of a host's indigenous flora can affect the virulence potential of pathogenic species. Furthermore, it remains unclear whether the colonization by commensal species affects a host cell's response to pathogenic species beyond the direct physical saturation of surface receptors, the sequestration of nutrients, the modulation of the physico-chemical environment in the oral cavity, or the production of bacteriocins. Using oral epithelial cells as a model, we hypothesized that the virulence of pathogenic species may vary depending on the complexity of the flora that interacts with host cells.
This is the first report that determines the global epithelial transcriptional response to co-culture with defined complex microbiota. In our model, human immortalized gingival keratinocytes (HIGK) were infected with mono- and mixed cultures of commensal and pathogenic species. The global transcriptional response of infected cells was validated and confirmed phenotypically. In our model, commensal species were able to modulate the expression of host genes with a broad diversity of physiological functions and antagonize the effect of pathogenic species at the cellular level. Unexpectedly, the inhibitory effect of commensal species was not correlated with its ability to inhibit adhesion or invasion by pathogenic species.
Studying the global transcriptome of epithelial cells to single and complex microbial challenges offers clues towards a better understanding of how bacteria-bacteria interactions and bacteria-host interactions impact the overall host response. This work provides evidence that the degree of complexity of a mixed microbiota does influence the transcriptional response to infection of host epithelial cells, and challenges the current dogma regarding the potential versus the actual pathogenicity of bacterial species. These findings support the concept that members of the commensal oral flora have evolved cellular mechanisms that directly modulate the host cell's response to pathogenic species and dampen their relative pathogenicity.
PMCID: PMC2736203  PMID: 19689803
25.  The Addition of Tumor Necrosis Factor plus Beta Interferon Induces a Novel Synergistic Antiviral State against Poxviruses in Primary Human Fibroblasts▿ †  
Journal of Virology  2008;83(2):498-511.
Tumor necrosis factor (TNF) and members of the interferon (IFN) family have been shown to independently inhibit the replication of a variety of viruses. In addition, previous reports have shown that treatment with various combinations of these antiviral cytokines induces a synergistic antiviral state that can be significantly more potent than addition of any of these cytokines alone. The mechanism of this cytokine synergy and its effects on global gene expression, however, are not well characterized. Here, we use DNA microarray analysis to demonstrate that treatment of uninfected primary human fibroblasts with TNF plus IFN-β induces a distinct synergistic state characterized by significant perturbations of several hundred genes which are coinduced by the individual cytokines alone, as well as the induction of more than 850 novel host cell genes. This synergy is mediated directly by the two ligands, not by intermediate secreted factors, and is necessary and sufficient to completely block the productive replication and spread of myxoma virus in human fibroblasts. In contrast, the replication of two other poxviruses, vaccinia virus and tanapox virus, are only partially inhibited in these cells by the synergistic antiviral state, whereas the spread of both of these viruses to neighboring cells was efficiently blocked. Taken together, our data indicate that the combination of TNF and IFN-β induces a novel synergistic antiviral state that is highly distinct from that induced by either cytokine alone.
PMCID: PMC2612348  PMID: 18971273

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