The elderly are particularly susceptible to trauma, and their outcomes are frequently dismal. Such patients often have complicated clinical courses and ultimately die from infection and sepsis. Recent research has revealed that although elderly subjects have increased baseline inflammation as compared to their younger counterparts, the elderly do not respond to severe infection/injury with an exaggerated inflammatory response. Initial retrospective analysis of clinical data from the Glue Grant trauma database demonstrated that despite a similar frequency, elderly trauma patients have worse outcomes to pneumonia than younger subjects. Subsequent analysis with a murine trauma model also demonstrated that elderly mice had increased mortality after post-trauma Pseudomonas pneumonia. Blood, bone marrow, and bronchoalveolar lavage sample analyses from juvenile and 20–24 month old mice showed that increased mortality to trauma combined with secondary infection in the aged are not due to an exaggerated inflammatory response. Rather, they are due to a failure of bone marrow progenitors, blood neutrophils, and bronchoalveolar lavage cells to initiate and complete an ‘emergency myelopoietic’ response, engendering myeloid cells that fail to clear secondary infection. In addition, the elderly appeared unable to effectively resolve their inflammatory response to severe injury.
Neonates manifest a unique host response to sepsis even among other children. Preterm neonates may experience sepsis soon after birth or during often-protracted birth hospitalizations as they attain physiologic maturity. We examined the transcriptome using genome-wide expression profiling on prospectively collected peripheral blood samples from infants evaluated for sepsis within 24 h after clinical presentation. Simultaneous plasma samples were examined for alterations in inflammatory mediators. Group designation (sepsis or uninfected) was determined retrospectively on the basis of clinical exam and laboratory results over the next 72 h from the time of evaluation. Unsupervised analysis showed the major node of separation between groups was timing of sepsis episode relative to birth (early, <3 d, or late, ≥3 d). Principal component analyses revealed significant differences between patients with early or late sepsis despite the presence of similar key immunologic pathway aberrations in both groups. Unique to neonates, the uninfected state and host response to sepsis is significantly affected by timing relative to birth. Future therapeutic approaches may need to be tailored to the timing of the infectious event based on postnatal age.
Populations encompassing extremes of age, including neonates and elderly, have greater mortality from sepsis. We propose that the increased mortality observed in the neonatal and elderly populations after sepsis is due to fundamental differences in host protective immunity, and are manifested at the level of the leukocyte transcriptome. Neonatal (5–7 days), young adult (6–12 weeks), or elderly (20–24 months) mice underwent a cecal slurry model of intra-abdominal sepsis. Both neonatal and elderly mice exhibited significantly greater mortality to sepsis (p<0.05). Neonates in particular exhibited significant attenuation of their inflammatory response (p<0.05), as well as reductions in cell recruitment and reactive oxygen species production (both p<0.05), all of which could be confirmed at the level of the leukocyte transcriptome. In contrast elderly mice were also more susceptible to abdominal peritonitis, but this was associated with no significant differences in the magnitude of the inflammatory response, reduced bacterial killing (p<0.05), reduced early myeloid cell activation (p<0.05) and a persistent inflammatory response that failed to resolve. Interestingly, elderly mice expressed a persistent inflammatory and immunosuppressive response at the level of the leukocyte transcriptome, with failure to return to baseline by three days. This study reveals that neonatal and elderly mice have profoundly different responses to sepsis that are manifested at the level of their circulating leukocyte transcriptome, although the net result of increased mortality, is similar. Considering these differences are fundamental aspects of the genomic response to sepsis, interventional therapies will require individualization based on the age of the population.
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.
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.
microarray; blunt trauma; shock; mouse model
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.
obesity; trauma; inflammation; genomics; leukocytes
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)
nanostring; microarray; blunt trauma
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.
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.
Neutrophil isolation; microfluidics; genomics; dextran; ficoll
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.
P. gingivalis; T. denticola; T. forsythia; polymicrobial infection; gene expression; calvarial bone; tissue; microarray
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.
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.
trauma; inflammation; genomics; leukocytes
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.
P. gingivalis; gene expression; calvarial tissue; bone; microarray
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.
We have previously described the transcriptional changes that occur in the hippocampal CA1 field of aged rats following a Morris Water Maze (MWM) training paradigm. In this report we proceed with the analysis of the dentate region from the same animals. Animals were first identified as age learning-impaired or age-superior learners when compared to young rats based on their performance in the MWM. Messenger RNA was isolated from the dentate gyrus of each animal to interrogate Affymetrix RAE 230A rat genome microarrays. Microarray profiling identified 1129 genes that were differentially expressed between aged and young rats as a result of aging, and independent of their behavioral training (p<0.005). We applied Ingenuity Pathway Analysis (IPA) algorithms to identify the significant biological processes underlying age-related changes in the dentate gyrus. The most significant functions, as calculated by IPA, included cell movement, cell growth and proliferation, nervous system development and function, cellular assembly and organization, cell morphology and cell death. These significant processes are consistent with age-related changes in neurogenesis, and the neurogenic markers were generally found to be downregulated in senescent animals. In addition, statistical analysis of the different experimental groups of aged animals recognized 85 genes (p<0.005) that were different in the dentate gyrus of aged rats that had learned the MWM when compared to learning impaired and a number of controls for stress, exercise and non-spatial learning. The list of learning-related genes expressed in the dentate adds to the set of genes we previously described in the CA1 region. This long list of genes constitutes a starting tool to elucidating the molecular pathways involved in learning and memory formation.
aging; hippocampus; learning and memory; Morris water maze; nervous system; gene expression; learning impaired; superior learner; dentate gyrus; microarray; class prediction; pathway analysis; neurogenesis
Aldehyde dehydrogenase isozymes ALDH1A1 and ALDH3A1 are highly expressed in non small cell lung cancer. Neither the mechanisms nor the biologic significance for such over expression have been studied.
We have employed oligonucleotide microarrays to analyze changes in gene profiles in A549 lung cancer cell line in which ALDH activity was reduced by up to 95% using lentiviral mediated expression of siRNA against both isozymes (Lenti 1+3). Stringent analysis methods were used to identify gene expression patterns that are specific to the knock down of ALDH activity and significantly different in comparison to wild type A549 cells (WT) or cells similarly transduced with green fluorescent protein (GFP) siRNA.
We confirmed significant and specific down regulation of ALDH1A1 and ALDH3A1 in Lenti 1+3 cells and in comparison to 12 other ALDH genes detected. The results of the microarray analysis were validated by real time RT-PCR on RNA obtained from Lenti 1+3 or WT cells treated with ALDH activity inhibitors. Detailed functional analysis was performed on 101 genes that were significantly different (P < 0.001) and their expression changed by ≥ 2 folds in the Lenti 1+3 group versus the control groups. There were 75 down regulated and 26 up regulated genes. Protein binding, organ development, signal transduction, transcription, lipid metabolism, and cell migration and adhesion were among the most affected pathways.
These molecular effects of the ALDH knock-down are associated with in vitro functional changes in the proliferation and motility of these cells and demonstrate the significance of ALDH enzymes in cell homeostasis with a potentially significant impact on the treatment of lung cancer.
MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression by binding to 3′-untranslated regions (3′UTRs) of target mRNAs. Kaposi's sarcoma-associated herpesvirus (KSHV), a virus linked to malignancies including primary effusion lymphoma (PEL), encodes 12 miRNA genes, but only a few regulatory targets are known. We found that KSHV-miR-K12-11 shares 100% seed sequence homology with hsa-miR-155, an miRNA frequently found to be up-regulated in lymphomas and critically important for B-cell development. Based on this seed sequence homology, we hypothesized that both miRNAs regulate a common set of target genes and, as a result, could have similar biological activities. Examination of five PEL lines showed that PELs do not express miR-155 but do express high levels of miR-K12-11. Bioinformatic tools predicted the transcriptional repressor BACH-1 to be targeted by both miRNAs, and ectopic expression of either miR-155 or miR-K12-11 inhibited a BACH-1 3′UTR-containing reporter. Furthermore, BACH-1 protein levels are low in cells expressing either miRNA. Gene expression profiling of miRNA-expressing stable cell lines revealed 66 genes that were commonly down-regulated. For select genes, miRNA targeting was confirmed by reporter assays. Thus, based on our in silico predictions, reporter assays, and expression profiling data, miR-K12-11 and miR-155 regulate a common set of cellular targets. Given the role of miR-155 during B-cell maturation, we speculate that miR-K12-11 may contribute to the distinct developmental phenotype of PEL cells, which are blocked in a late stage of B-cell development. Together, these findings indicate that KSHV miR-K12-11 is an ortholog of miR-155.
Transcriptional profiling and ontology tools were utilized to define the biological pathways of gingival epithelial cells modulated by coculture with the oral commensal Streptococcus gordonii and the opportunistic commensal Fusobacterium nucleatum. Overall, F. nucleatum and S. gordonii perturbed the gingival epithelial cell transcriptome much less significantly than the oral pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans perturbed the transcriptome, indicating that there was a greater degree of host adaptation by the commensal species (M. Handfield, J. J. Mans, G. Zheng, M. C. Lopez, S. Mao, A. Progulske-Fox, G. Narasimhan, H. V. Baker, and R. J. Lamont, Cell. Microbiol. 7:811-823, 2005). The biological pathways significantly impacted by F. nucleatum and S. gordonii included the mitogen-activated protein kinase (MAPK) and Toll-like receptor signaling pathways. Differential regulation of GADD45 and DUSP4, key components of the MAPK pathway, was confirmed at the protein level by Western blotting. Modulation of the MAPK pathway is likely to affect host cell proliferation and differentiation. In addition, both the MAPK and Toll-like receptor pathways ultimately converge on cytokine gene expression. An enzyme-linked immunosorbent assay of secreted interleukin-6 (IL-6) and IL-8 demonstrated that F. nucleatum induced production of these cytokines, whereas S. gordonii inhibited secretion from the epithelial cells. Stimulation of secretion of proinflammatory cytokines from epithelial cells may reflect the invasive phenotype of F. nucleatum and contribute to the greater pathogenic potential of F. nucleatum than of S. gordonii.
MicroRNAs (miRNAs) are 19 to 23 nucleotide–long RNAs that post-transcriptionally regulate gene expression. Human cells express several hundred miRNAs which regulate important biological pathways such as development, proliferation, and apoptosis. Recently, 12 miRNA genes have been identified within the genome of Kaposi sarcoma–associated herpesvirus; however, their functions are still unknown. To identify host cellular genes that may be targeted by these novel viral regulators, we performed gene expression profiling in cells stably expressing KSHV-encoded miRNAs. Data analysis revealed a set of 81 genes whose expression was significantly changed in the presence of miRNAs. While the majority of changes were below 2-fold, eight genes were down-regulated between 4- and 20-fold. We confirmed miRNA-dependent regulation for three of these genes and found that protein levels of thrombospondin 1 (THBS1) were decreased >10-fold. THBS1 has previously been reported to be down-regulated in Kaposi sarcoma lesions and has known activity as a strong tumor suppressor and anti-angiogenic factor, exerting its anti-angiogenic effect in part by activating the latent form of TGF-β. We show that reduced THBS1 expression in the presence of viral miRNAs translates into decreased TGF-β activity. These data suggest that KSHV-encoded miRNAs may contribute directly to pathogenesis by down-regulation of THBS1, a major regulator of cell adhesion, migration, and angiogenesis.
Kaposi sarcoma–associated herpesvirus (KSHV) is a gamma-herpesvirus associated with Kaposi sarcoma, primary effusion lymphoma, and a subset of muticentric Castleman disease. Recently, it was found that KSHV encodes 12 microRNAs (miRNAs) within its latency-associated region. miRNAs are small ∼22 nucleotide-long single-stranded RNA molecules that act to inhibit gene expression by binding to target messenger RNAs (mRNAs). Because miRNAs bind to these targets with limited base pairing, it has been difficult to find targets. The goal of our study was to identify cellular mRNAs targeted by KSHV-encoded miRNAs. Microarray analysis of cells expressing the KSHV miRNAs revealed a set of 81 genes that were changed. Several genes are regulators of important functions such as blood vessel growth, cell proliferation, and cell death. One target, thrombospondin 1, is a potent inhibitor of blood vessel growth and is known to be down-regulated in Kaposi sarcoma tumors. Thrombospondin 1, which is targeted by multiple miRNAs, also showed reduced protein levels in our study. To our knowledge, our data describe the first targets for tumorvirus-encoded miRNAs and suggest that these novel regulators may have roles in pathogenesis.