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1.  Segregation of a Spontaneous Klrd1 (CD94) Mutation in DBA/2 Mouse Substrains 
G3: Genes|Genomes|Genetics  2014;5(2):235-239.
Current model DBA/2J (D2J) mice lack CD94 expression due to a deletion spanning the last coding exon of the Klrd1 gene that occurred in the mid- to late 1980s. In contrast, DBA/2JRj (D2Rj) mice, crosses derived from DBA/2J before 1984, and C57BL/6J (B6) mice lack the deletion and have normal CD94 expression. For example, BXD lines (BXD1–32) generated in the 1970s by crossing B6 and D2J do not segregate for the exonic deletion and have high expression, whereas BXD lines 33 and greater were generated after 1990 are segregating for the deletion and have highly variable Klrd1 expression. We performed quantitative trait locus analysis of Klrd1 expression by using BXD lines with different generation times and found that the expression difference in Klrd1 in the later BXD set is driven by a strong cis-acting expression quantitative trait locus. Although the Klrd1/CD94 locus is essential for mousepox resistance, the genetic variation among D2 substrains and the later set of BXD strains is not associated with susceptibility to the Influenza A virus PR8 strain. Substrains with nearly identical genetic backgrounds that are segregating functional variants such as the Klrd1 deletion are useful genetic tools to investigate biological function.
PMCID: PMC4321031  PMID: 25520036
Klrd1; BXD; DBA/2
2.  Controlling complexity: the clinical relevance of mouse complex genetics 
European Journal of Human Genetics  2013;21(11):1191-1196.
Experimental animal models are essential to obtain basic knowledge of the underlying biological mechanisms in human diseases. Here, we review major contributions to biomedical research and discoveries that were obtained in the mouse model by using forward genetics approaches and that provided key insights into the biology of human diseases and paved the way for the development of novel therapeutic approaches.
PMCID: PMC3798854  PMID: 23632795
Mouse model; forward genetics; review
3.  The Interferon-Induced Gene Ifi27l2a is Active in Lung Macrophages and Lymphocytes After Influenza A Infection but Deletion of Ifi27l2a in Mice Does Not Increase Susceptibility to Infection 
PLoS ONE  2014;9(9):e106392.
Interferons represent one of the first and essential host defense mechanisms after infection, and the activation of the IFN-pathway results in the transcriptional activation of hundreds of interferon-stimulated genes. The alpha-inducible protein 27 like 2A (Ifi27l2a) gene (human synonym: ISG12) is strongly up-regulated in the lung after influenza A infection in mice and has been shown in gene expression studies to be highly correlated to other activated genes. Therefore, we investigated the role of Ifi27l2a for the host defense to influenza A infections in more detail. RT-PCR analyses in non-infected mice demonstrated that Ifi27l2a was expressed in several tissues, including the lung. Detailed analyses of reporter gene expression in lungs from Ifi27l2a-LacZ mice revealed that Ifi27l2a was expressed in macrophages and lymphocytes but not in alveolar cells or bronchiolar epithelium cells. The number of macrophages and lymphocyte strongly increased in the lung after infection, but no significant increase in expression levels of the LacZ reporter gene was found within individual immune cells. Also, no reporter gene expression was found in bronchiolar epithelial cells, alveolar cells or infiltrating neutrophils after infection. Thus, up-regulation of Ifi27l2a in infected lungs is mainly due to the infiltration of macrophages and lymphocytes. Most surprisingly, deletion of Ifi27l2a in mouse knock-out lines did not result in increased susceptibility to infections with H1N1 or H7N7 influenza A virus compared to wild type C57BL/6N mice, suggesting a less important role of the gene for the host response to influenza infections than for bacterial infections.
PMCID: PMC4153650  PMID: 25184786
4.  Cellular Changes in Blood Indicate Severe Respiratory Disease during Influenza Infections in Mice 
PLoS ONE  2014;9(7):e103149.
Influenza A infection is a serious threat to human and animal health. Many of the biological mechanisms of the host-pathogen-interactions are still not well understood and reliable biomarkers indicating the course of the disease are missing. The mouse is a valuable model system enabling us to study the local inflammatory host response and the influence on blood parameters under controlled circumstances. Here, we compared the lung and peripheral changes after PR8 (H1N1) influenza A virus infection in C57BL/6J and DBA/2J mice using virus variants of different pathogenicity resulting in non-lethal and lethal disease. We monitored hematological and immunological parameters revealing that the granulocyte to lymphocyte ratio in the blood represents an early indicator of severe disease progression already two days after influenza A infection in mice. These findings might be relevant to optimize early diagnostic options of severe influenza disease and to monitor successful therapeutic treatment in humans.
PMCID: PMC4110021  PMID: 25058639
5.  Systems Genetics of Liver Fibrosis: Identification of Fibrogenic and Expression Quantitative Trait Loci in the BXD Murine Reference Population 
PLoS ONE  2014;9(2):e89279.
The progression of liver fibrosis in response to chronic injury varies considerably among individual patients. The underlying genetics is highly complex due to large numbers of potential genes, environmental factors and cell types involved. Here, we provide the first toxicogenomic analysis of liver fibrosis induced by carbon tetrachloride in the murine ‘genetic reference panel’ of recombinant inbred BXD lines. Our aim was to define the core of risk genes and gene interaction networks that control fibrosis progression. Liver fibrosis phenotypes and gene expression profiles were determined in 35 BXD lines. Quantitative trait locus (QTL) analysis identified seven genomic loci influencing fibrosis phenotypes (pQTLs) with genome-wide significance on chromosomes 4, 5, 7, 12, and 17. Stepwise refinement was based on expression QTL mapping with stringent selection criteria, reducing the number of 1,351 candidate genes located in the pQTLs to a final list of 11 cis-regulated genes. Our findings demonstrate that the BXD reference population represents a powerful experimental resource for shortlisting the genes within a regulatory network that determine the liver's vulnerability to chronic injury.
PMCID: PMC3938463  PMID: 24586654
6.  Infection- and procedure-dependent effects on pulmonary gene expression in the early phase of influenza A virus infection in mice 
BMC Microbiology  2013;13:293.
Investigating the host response in the early stage of influenza A virus (IAV) infection is of considerable interest. However, it is conceivable that effects due to the anesthesia and/or intranasal infection procedure might introduce artifacts. We therefore aimed to evaluate the effects of anesthesia and/or intranasal infection on transcription of selected pulmonary mRNAs in two inbred mouse strains with differential susceptibility to IAV infection.
DBA/2J and C57BL/6J mice were evaluated in a time course experiment in which lung tissue was sampled after 6, 12, 18, 24, 48 and 120 h. After anesthesia with ketamine and xylazine, a suspension of mouse-adapted IAV strain PR8_Mun in 20 μl sterile buffer, or 20 μl sterile buffer only, was instilled intranasally. The mice receiving anesthesia and PBS only were designated the “mock treatment” group. Pulmonary expression of 10 host mRNAs (Fos, Retnla, Irg1, Il6, Il1b, Cxcl10, Stat1, Ifng, Ifnl2, and Mx1) and viral hemagglutinin (HA) mRNA were determined at the designated time points. As expected, weight loss and viral replication were greater in the DBA/2J strain (which is more susceptible to IAV infection). Four mRNAs (Retnla, Irg1, Il6, and Cxcl10) were procedure-dependently regulated in DBA/2J mice between 6 and 24 h, and two (Retnla and Il6) in C57BL/6J mice, although to a lesser extent. All 10 mRNAs rose after infection, but one (Fos) only in DBA/2J mice. These infection-dependent effects could be separated from procedure-dependent effects beginning around 12 h in DBA/2J and 18 h in C57BL/6J mice. The interferon-related mRNAs Stat1, Ifng, Infl2, and Mx1 were unaffected by mock treatment in either mouse strain. Mx1 and Infl2 correlated best with HA mRNA expression (r = 0.97 and 0.93, respectively, in DBA/2J).
These results demonstrate effects of the anesthesia and/or intranasal infection procedure on pulmonary gene expression, which are detectable between approximately 6 and 24 h post procedure and vary in intensity and temporal evolution depending on the mouse strain used. Mock infection controls should be included in all studies on pulmonary gene expression in the early phase of infection with IAV and, likely, other respiratory pathogens.
PMCID: PMC3880568  PMID: 24341411
Anesthesia; Gene expression; Influenza virus; Interferon; Interferon lambda; Intranasal infection; Mouse model
7.  Tmprss2 Is Essential for Influenza H1N1 Virus Pathogenesis in Mice 
PLoS Pathogens  2013;9(12):e1003774.
Annual influenza epidemics and occasional pandemics pose a severe threat to human health. Host cell factors required for viral spread but not for cellular survival are attractive targets for novel approaches to antiviral intervention. The cleavage activation of the influenza virus hemagglutinin (HA) by host cell proteases is essential for viral infectivity. However, it is unknown which proteases activate influenza viruses in mammals. Several candidates have been identified in cell culture studies, leading to the concept that influenza viruses can employ multiple enzymes to ensure their cleavage activation in the host. Here, we show that deletion of a single HA-activating protease gene, Tmprss2, in mice inhibits spread of mono-basic H1N1 influenza viruses, including the pandemic 2009 swine influenza virus. Lung pathology was strongly reduced and mutant mice were protected from weight loss, death and impairment of lung function. Also, after infection with mono-basic H3N2 influenza A virus body weight loss and survival was less severe in Tmprss2 mutant compared to wild type mice. As expected, Tmprss2-deficient mice were not protected from viral spread and pathology after infection with multi-basic H7N7 influenza A virus. In conclusion, these results identify TMPRSS2 as a host cell factor essential for viral spread and pathogenesis of mono-basic H1N1 and H3N2 influenza A viruses.
Author Summary
Seasonal influenza epidemics and pandemics represent a serious health threat to the human population. Resistance to presently available anti-viral drugs is frequently observed. Therefore, identification of new targets for anti-viral therapy is an urgent need. Host proteases are required for processing of the virus hemagglutinin and may thus represent a suitable target for intervention. Here, we report that the deletion of a single host protease gene, Tmprss2, in mice protects the host against viral spread in infected lungs. Only very mild pathogenesis was observed in Tmprss2 mutant mice after infection with H1N1 virus and less severe pathogenesis was observed after infection with H3N2 virus. Thus, our results suggest that the host protease TMPRSS2 may be a prime target for antiviral intervention.
PMCID: PMC3857797  PMID: 24348248
8.  The bioluminescent Listeria monocytogenes strain Xen32 is defective in flagella expression and highly attenuated in orally infected BALB/cJ mice 
Gut Pathogens  2013;5:19.
In vivo bioluminescence imaging (BLI) is a powerful method for the analysis of host-pathogen interactions in small animal models. The commercially available bioluminescent Listeria monocytogenes strain Xen32 is commonly used to analyse immune functions in knockout mice and pathomechanisms of listeriosis.
To analyse and image listerial dissemination after oral infection we have generated a murinised Xen32 strain (Xen32-mur) which expresses a previously described mouse-adapted internalin A. This strain was used alongside the Xen32 wild type strain and the bioluminescent L. monocytogenes strains EGDe-lux and murinised EGDe-mur-lux to characterise bacterial dissemination in orally inoculated BALB/cJ mice. After four days of infection, Xen32 and Xen32-mur infected mice displayed consistently higher rates of bioluminescence compared to EGDe-lux and EGDe-mur-lux infected animals. However, surprisingly both Xen32 strains showed attenuated virulence in orally infected BALB/c mice that correlated with lower bacterial burden in internal organs at day 5 post infection, smaller losses in body weights and increased survival compared to EGDe-lux or EGDe-mur-lux inoculated animals. The Xen32 strain was made bioluminescent by integration of a lux-kan transposon cassette into the listerial flaA locus. We show here that this integration results in Xen32 in a flaA frameshift mutation which makes this strain flagella deficient.
The bioluminescent L. monocytogenes strain Xen32 is deficient in flagella expression and highly attenuated in orally infected BALB/c mice. As this listerial strain has been used in many BLI studies of murine listeriosis, it is important that the scientific community is aware of its reduced virulence in vivo.
PMCID: PMC3720536  PMID: 23856386
Listeriosis; Flagella; Mouse infection model; Bioluminescent imaging
9.  ATR-FTIR spectroscopy reveals genomic loci regulating the tissue response in high fat diet fed BXD recombinant inbred mouse strains 
BMC Genomics  2013;14:386.
Obesity-associated organ-specific pathological states can be ensued from the dysregulation of the functions of the adipose tissues, liver and muscle. However, the influence of genetic differences underlying gross-compositional differences in these tissues is largely unknown. In the present study, the analytical method of ATR-FTIR spectroscopy has been combined with a genetic approach to identify genetic differences responsible for phenotypic alterations in adipose, liver and muscle tissues.
Mice from 29 BXD recombinant inbred mouse strains were put on high fat diet and gross-compositional changes in adipose, liver and muscle tissues were measured by ATR-FTIR spectroscopy. The analysis of genotype-phenotype correlations revealed significant quantitative trait loci (QTL) on chromosome 12 for the content of fat and collagen, collagen integrity, and the lipid to protein ratio in adipose tissue and on chromosome 17 for lipid to protein ratio in liver. Using gene expression and sequence information, we suggest Rsad2 (viperin) and Colec11 (collectin-11) on chromosome 12 as potential quantitative trait candidate genes. Rsad2 may act as a modulator of lipid droplet contents and lipid biosynthesis; Colec11 might play a role in apoptopic cell clearance and maintenance of adipose tissue. An increased level of Rsad2 transcripts in adipose tissue of DBA/2J compared to C57BL/6J mice suggests a cis-acting genetic variant leading to differential gene activation.
The results demonstrate that the analytical method of ATR-FTIR spectroscopy effectively contributed to decompose the macromolecular composition of tissues that accumulate fat and to link this information with genetic determinants. The candidate genes in the QTL regions may contribute to obesity-related diseases in humans, in particular if the results can be verified in a bigger BXD cohort.
PMCID: PMC3717084  PMID: 23758785
Collagen; Endoplasmic reticulum; Apoptosis; Remodeling; Liver steatosis; Viperin; Collectin-11
10.  Influence of internalin a murinisation on host resistance to orally acquired listeriosis in mice 
BMC Microbiology  2013;13:90.
The bacterial surface protein internalin (InlA) is a major virulence factor of the food-born pathogen Listeria monocytogenes. It plays a critical role in the bacteria crossing the host intestinal barrier by a species-specific interaction with the cell adhesion molecule E-cadherin. In mice, the interaction of InlA with murine E-cadherin is impaired due to sequence-specific binding incompatibilities. We have previously used the approach of ‘murinisation’ to establish an oral listeriosis infection model in mice by exchanging two amino acid residues in InlA. This dramatically increases binding to mouse E-cadherin. In the present study, we have used bioluminescent murinised and non-murinised Listeria strains to examine the spatiotemporal dissemination of Listeria in four diverse mouse genetic backgrounds after oral inoculation.
The murinised Listeria monocytogenes strain showed enhanced invasiveness and induced more severe infections in all four investigated mouse inbred strains compared to the non-murinised Listeria strain. We identified C57BL/6J mice as being most resistant to orally acquired listeriosis whereas C3HeB/FeJ, A/J and BALB/cJ mice were found to be most susceptible to infection. This was reflected in faster kinetics of Listeria dissemination, higher bacterial loads in internal organs, and elevated serum levels of IL-6, IFN-γ, TNF-α and CCL2 in the susceptible strains as compared to the resistant C57BL/6J strain. Importantly, murinisation of InlA did not cause enhanced invasion of Listeria monocytogenes into the brain.
Murinised Listeria are able to efficiently cross the intestinal barrier in mice from diverse genetic backgrounds. However, expression of murinized InlA does not enhance listerial brain invasion suggesting that crossing of the blood brain barrier and crossing of the intestinal epithelium are achieved by Listeria monocytogenes through different molecular mechanisms.
PMCID: PMC3640945  PMID: 23617550
11.  PLAU inferred from a correlation network is critical for suppressor function of regulatory T cells 
Network-based analysis of transcriptome dynamics during activation in two human T-cell subpopulations identifies key regulators, and reveals that PLAU plays a critical role in both human and murine regulatory T-cell function.
We construct a Treg-specific correlation network from a high time-resolution transcriptome of human Tregs versus CD4+ T effector cells measured during their very early activation process.We propose a queen bee-surrounding principle to predict key candidate genes from the simplified undirected correlation network rather than an advanced directed transcription regulatory network. These potential key genes would have not been easily identified by a differential expression analysis.We show that the plasminogen activator urokinase (PLAU) is critical for suppressor function of both human and murine Tregs.We further demonstrate that PLAU is particularly important for memory Tregs and that PLAU mediates Treg suppressor function via STAT5 and ERK signaling pathways.
Human FOXP3+CD25+CD4+ regulatory T cells (Tregs) are essential to the maintenance of immune homeostasis. Several genes are known to be important for murine Tregs, but for human Tregs the genes and underlying molecular networks controlling the suppressor function still largely remain unclear. Here, we describe a strategy to identify the key genes directly from an undirected correlation network which we reconstruct from a very high time-resolution (HTR) transcriptome during the activation of human Tregs/CD4+ T-effector cells. We show that a predicted top-ranked new key gene PLAU (the plasminogen activator urokinase) is important for the suppressor function of both human and murine Tregs. Further analysis unveils that PLAU is particularly important for memory Tregs and that PLAU mediates Treg suppressor function via STAT5 and ERK signaling pathways. Our study demonstrates the potential for identifying novel key genes for complex dynamic biological processes using a network strategy based on HTR data, and reveals a critical role for PLAU in Treg suppressor function.
Network-based analysis of transcriptome dynamics during activation in two human T-cell subpopulations identifies key regulators, and reveals that PLAU plays a critical role in both human and murine regulatory T-cell function.
PMCID: PMC3531908  PMID: 23169000
high time-resolution time series; human CD4 regulatory T cell; infer key genes from undirected gene networks; Plau knockout mice; Treg development and suppressor function
12.  Equivalence of Self- and Staff-Collected Nasal Swabs for the Detection of Viral Respiratory Pathogens 
PLoS ONE  2012;7(11):e48508.
The need for the timely collection of diagnostic biosamples during symptomatic episodes represents a major obstacle to large-scale studies on acute respiratory infection (ARI) epidemiology. This may be circumvented by having the participants collect their own nasal swabs. We compared self- and staff-collected swabs in terms of swabbing quality and detection of viral respiratory pathogens.
Methodology/Principal Findings
We conducted a prospective study among employees of our institution during the ARI season 2010/2011 (December-March). Weekly emails were sent to the participants (n = 84), reminding them to come to the study center in case of new symptoms. The participants self-collected an anterior nasal swab from one nostril, and trained study personnel collected one from the other nostril. The participants self-collected another two swabs (one from each nostril) on a subsequent day. Human β-actin DNA concentration was determined in the swabs as a quality control. Viral respiratory pathogens were detected by multiplex RT-PCR (Seeplex RV15 kit, Seegene, Eschborn, Germany). Of 84 participants, 56 (67%) reported at least one ARI episode, 18 participants two, and one participant three. Self-swabbing was highly accepted by the participants. The amount of β-actin DNA per swab was higher in the self- than in the staff-collected swabs (p = 0.008). β-actin concentration was lower in the self-swabs collected on day 1 than in those collected on a subsequent day (p<0.0001). A respiratory viral pathogen was detected in 31% (23/75) of staff- and in 35% (26/75) of self-collected swabs (p = 0.36). With both approaches, the most frequently identified pathogens were human rhinoviruses A/B/C (12/75 swabs, 16%) and human coronavirus OC43 (4/75 swabs, 5%). There was almost perfect agreement between self- and staff-collected swabs in terms of pathogen detection (agreement = 93%, kappa = 0.85, p<0.0001).
Nasal self-swabbing for identification of viral ARI pathogens proved to be equivalent to staff-swabbing in this population in terms of acceptance and pathogen detection.
PMCID: PMC3498275  PMID: 23155387
13.  Immunization with live virus vaccine protects highly susceptible DBA/2J mice from lethal influenza A H1N1 infection 
Virology Journal  2012;9:212.
The mouse represents an important model system to study the host response to influenza A infections and to evaluate new prevention or treatment strategies. We and others reported that the susceptibility to influenza A virus infections strongly varies among different inbred mouse strains. In particular, DBA/2J mice are highly susceptible to several influenza A subtypes, including human isolates and exhibit severe symptoms after infection with clinical isolates.
Upon intra-muscular immunization with live H1N1 influenza A virus (mouse-adapted PR8M, and 2009 pandemic human HA04), DBA/2J mice mounted virus-specific IgG responses and were protected against a subsequent lethal challenge. The immune response and rescue from death after immunization in DBA/2J was similar to those observed for C57BL/6J mice.
DBA/2J mice represent a suitable mouse model to evaluate virulence and pathogenicity as well as immunization regimes against existing and newly emerging human influenza strains without the need for prior adaptation of the virus to the mouse.
PMCID: PMC3502422  PMID: 22992381
Influenza A virus; Mouse; DBA/2J; Immunization
14.  Distinct gene loci control the host response to influenza H1N1 virus infection in a time-dependent manner 
BMC Genomics  2012;13:411.
There is strong but mostly circumstantial evidence that genetic factors modulate the severity of influenza infection in humans. Using genetically diverse but fully inbred strains of mice it has been shown that host sequence variants have a strong influence on the severity of influenza A disease progression. In particular, C57BL/6J, the most widely used mouse strain in biomedical research, is comparatively resistant. In contrast, DBA/2J is highly susceptible.
To map regions of the genome responsible for differences in influenza susceptibility, we infected a family of 53 BXD-type lines derived from a cross between C57BL/6J and DBA/2J strains with influenza A virus (PR8, H1N1). We monitored body weight, survival, and mean time to death for 13 days after infection. Qivr5 (quantitative trait for influenza virus resistance on chromosome 5) was the largest and most significant QTL for weight loss. The effect of Qivr5 was detectable on day 2 post infection, but was most pronounced on days 5 and 6. Survival rate mapped to Qivr5, but additionally revealed a second significant locus on chromosome 19 (Qivr19). Analysis of mean time to death affirmed both Qivr5 and Qivr19. In addition, we observed several regions of the genome with suggestive linkage. There are potentially complex combinatorial interactions of the parental alleles among loci. Analysis of multiple gene expression data sets and sequence variants in these strains highlights about 30 strong candidate genes across all loci that may control influenza A susceptibility and resistance.
We have mapped influenza susceptibility loci to chromosomes 2, 5, 16, 17, and 19. Body weight and survival loci have a time-dependent profile that presumably reflects the temporal dynamic of the response to infection. We highlight candidate genes in the respective intervals and review their possible biological function during infection.
PMCID: PMC3479429  PMID: 22905720
15.  Global Transcriptome Analysis in Influenza-Infected Mouse Lungs Reveals the Kinetics of Innate and Adaptive Host Immune Responses 
PLoS ONE  2012;7(7):e41169.
An infection represents a highly dynamic process involving complex biological responses of the host at many levels. To describe such processes at a global level, we recorded gene expression changes in mouse lungs after a non-lethal infection with influenza A virus over a period of 60 days. Global analysis of the large data set identified distinct phases of the host response. The increase in interferon genes and up-regulation of a defined NK-specific gene set revealed the initiation of the early innate immune response phase. Subsequently, infiltration and activation of T and B cells could be observed by an augmentation of T and B cell specific signature gene expression. The changes in B cell gene expression and preceding chemokine subsets were associated with the formation of bronchus-associated lymphoid tissue. In addition, we compared the gene expression profiles from wild type mice with Rag2 mutant mice. This analysis readily demonstrated that the deficiency in the T and B cell responses in Rag2 mutants could be detected by changes in the global gene expression patterns of the whole lung. In conclusion, our comprehensive gene expression study describes for the first time the entire host response and its kinetics to an acute influenza A infection at the transcriptome level.
PMCID: PMC3398930  PMID: 22815957
17.  Expression QTL mapping in regulatory and helper T cells from the BXD family of strains reveals novel cell-specific genes, gene-gene interactions and candidate genes for auto-immune disease 
BMC Genomics  2011;12:610.
Regulatory T cells (Tregs) play an essential role in the control of the immune response. Treg cells represent important targets for therapeutic interventions of the immune system. Therefore, it will be very important to understand in more detail which genes are specifically activated in Treg cells versus T helper (Th) cells, and which gene regulatory circuits may be involved in specifying and maintaining Treg cell homeostasis.
We isolated Treg and Th cells from a genetically diverse family of 31 BXD type recombinant inbred strains and the fully inbred parental strains of this family--C57BL/6J and DBA/2J. Subsequently genome-wide gene expression studies were performed from the isolated Treg and Th cells. A comparative analysis of the transcriptomes of these cell populations allowed us to identify many novel differentially expressed genes. Analysis of cis- and trans-expression Quantitative Trait Loci (eQTLs) highlighted common and unique regulatory mechanisms that are active in the two cell types. Trans-eQTL regions were found for the Treg functional genes Nrp1, Stat3 and Ikzf4. Analyses of the respective QTL intervals suggested several candidate genes that may be involved in regulating these genes in Treg cells. Similarly, possible candidate genes were found which may regulate the expression of F2rl1, Ctla4, Klrb1f. In addition, we identified a focused group of candidate genes that may be important for the maintenance of self-tolerance and the prevention of allergy.
Variation of expression across the strains allowed us to find many novel gene-interaction networks in both T cell subsets. In addition, these two data sets enabled us to identify many differentially expressed genes and to nominate candidate genes that may have important functions for the maintenance of self-tolerance and the prevention of allergy.
PMCID: PMC3277499  PMID: 22182475
18.  Dynamic gene network reconstruction from gene expression data in mice after influenza A (H1N1) infection 
The immune response to viral infection is a temporal process, represented by a dynamic and complex network of gene and protein interactions. Here, we present a reverse engineering strategy aimed at capturing the temporal evolution of the underlying Gene Regulatory Networks (GRN). The proposed approach will be an enabling step towards comprehending the dynamic behavior of gene regulation circuitry and mapping the network structure transitions in response to pathogen stimuli.
We applied the Time Varying Dynamic Bayesian Network (TV-DBN) method for reconstructing the gene regulatory interactions based on time series gene expression data for the mouse C57BL/6J inbred strain after infection with influenza A H1N1 (PR8) virus. Initially, 3500 differentially expressed genes were clustered with the use of k-means algorithm. Next, the successive in time GRNs were built over the expression profiles of cluster centroids. Finally, the identified GRNs were examined with several topological metrics and available protein-protein and protein-DNA interaction data, transcription factor and KEGG pathway data.
Our results elucidate the potential of TV-DBN approach in providing valuable insights into the temporal rewiring of the lung transcriptome in response to H1N1 virus.
PMCID: PMC3219564  PMID: 22017961
Gene Regulatory Network; Time Varying Dynamic Bayesian Network; Immune System; Influenza A
19.  Bioinformatics tools and database resources for systems genetics analysis in mice—a short review and an evaluation of future needs 
Briefings in Bioinformatics  2011;13(2):135-142.
During a meeting of the SYSGENET working group ‘Bioinformatics’, currently available software tools and databases for systems genetics in mice were reviewed and the needs for future developments discussed. The group evaluated interoperability and performed initial feasibility studies. To aid future compatibility of software and exchange of already developed software modules, a strong recommendation was made by the group to integrate HAPPY and R/qtl analysis toolboxes, GeneNetwork and XGAP database platforms, and TIQS and xQTL processing platforms. R should be used as the principal computer language for QTL data analysis in all platforms and a ‘cloud’ should be used for software dissemination to the community. Furthermore, the working group recommended that all data models and software source code should be made visible in public repositories to allow a coordinated effort on the use of common data structures and file formats.
PMCID: PMC3294237  PMID: 22396485
QTL mapping; database; mouse; systems genetics
20.  Genome-wide analysis of the mouse lung transcriptome reveals novel molecular gene interaction networks and cell-specific expression signatures 
Respiratory Research  2011;12(1):61.
The lung is critical in surveillance and initial defense against pathogens. In humans, as in mice, individual genetic differences strongly modulate pulmonary responses to infectious agents, severity of lung disease, and potential allergic reactions. In a first step towards understanding genetic predisposition and pulmonary molecular networks that underlie individual differences in disease vulnerability, we performed a global analysis of normative lung gene expression levels in inbred mouse strains and a large family of BXD strains that are widely used for systems genetics. Our goal is to provide a key community resource on the genetics of the normative lung transcriptome that can serve as a foundation for experimental analysis and allow predicting genetic predisposition and response to pathogens, allergens, and xenobiotics.
Steady-state polyA+ mRNA levels were assayed across a diverse and fully genotyped panel of 57 isogenic strains using the Affymetrix M430 2.0 array. Correlations of expression levels between genes were determined. Global expression QTL (eQTL) analysis and network covariance analysis was performed using tools and resources in GeneNetwork
Expression values were highly variable across strains and in many cases exhibited a high heri-tability factor. Several genes which showed a restricted expression to lung tissue were identified. Using correlations between gene expression values across all strains, we defined and extended memberships of several important molecular networks in the lung. Furthermore, we were able to extract signatures of immune cell subpopulations and characterize co-variation and shared genetic modulation. Known QTL regions for respiratory infection susceptibility were investigated and several cis-eQTL genes were identified. Numerous cis- and trans-regulated transcripts and chromosomal intervals with strong regulatory activity were mapped. The Cyp1a1 P450 transcript had a strong trans-acting eQTL (LOD 11.8) on Chr 12 at 36 ± 1 Mb. This interval contains the transcription factor Ahr that has a critical mis-sense allele in the DBA/2J haplotype and evidently modulates transcriptional activation by AhR.
Large-scale gene expression analyses in genetic reference populations revealed lung-specific and immune-cell gene expression profiles and suggested specific gene regulatory interactions.
PMCID: PMC3105947  PMID: 21535883
21.  TMPRSS2 and TMPRSS4 Facilitate Trypsin-Independent Spread of Influenza Virus in Caco-2 Cells▿  
Journal of Virology  2010;84(19):10016-10025.
Proteolysis of influenza virus hemagglutinin by host cell proteases is essential for viral infectivity, but the proteases responsible are not well defined. Recently, we showed that engineered expression of the type II transmembrane serine proteases (TTSPs) TMPRSS2 and TMPRSS4 allows hemagglutinin (HA) cleavage. Here we analyzed whether TMPRSS2 and TMPRSS4 are expressed in influenza virus target cells and support viral spread in the absence of exogenously added protease (trypsin). We found that transient expression of TMPRSS2 and TMPRSS4 resulted in HA cleavage and trypsin-independent viral spread. Endogenous expression of TMPRSS2 and TMPRSS4 in cell lines correlated with the ability to support the spread of influenza virus in the absence of trypsin, indicating that these proteases might activate influenza virus in naturally permissive cells. Indeed, RNA interference (RNAi)-mediated knockdown of both TMPRSS2 and TMPRSS4 in Caco-2 cells, which released fully infectious virus without trypsin treatment, markedly reduced the spread of influenza virus, demonstrating that these proteases were responsible for efficient proteolytic activation of HA in this cell line. Finally, TMPRSS2 was found to be coexpressed with the major receptor determinant of human influenza viruses, 2,6-linked sialic acids, in human alveolar epithelium, indicating that viral target cells in the human respiratory tract express TMPRSS2. Collectively, our results point toward an important role for TMPRSS2 and possibly TMPRSS4 in influenza virus replication and highlight the former protease as a potential therapeutic target.
PMCID: PMC2937781  PMID: 20631123
22.  Inhibition of lung serine proteases in mice: a potentially new approach to control influenza infection 
Virology Journal  2011;8:27.
Host serine proteases are essential for the influenza virus life cycle because the viral haemagglutinin is synthesized as a precursor which requires proteolytic maturation. Therefore, we studied the activity and expression of serine proteases in lungs from mice infected with influenza and evaluated the effect of serine protease inhibitors on virus replication both in cell culture and in infected mice.
Two different inbred mouse strains were investigated: DBA/2J as a highly susceptible and C57Bl/6J as a more resistant strain to influenza virus infection. The serine proteases from lung homogenates of mice exhibited pH optima of 10.00. Using the substrate Bz-Val-Gly-Arg-p-nitroanilide or in zymograms, the intensities of proteolysis increased in homogenates from both mouse strains with time post infection (p.i.) with the mouse-adapted influenza virus A/Puerto Rico/8/34 (H1N1; PR8). In zymograms at day 7 p.i., proteolytic bands were stronger and numerous in lung homogenates from DBA/2J than C57Bl/6J mice. Real-time PCR results confirmed differential expression of several lung proteases before and after infecting mice with the H1N1 virus. The most strongly up-regulated proteases were Gzma, Tmprss4, Elane, Ctrl, Gzmc and Gzmb. Pretreatment of mouse and human lung cell lines with the serine protease inhibitors AEBSF or pAB or a cocktail of both prior to infection with the H1N1 or the A/Seal/Massachusetts/1/80 (H7N7; SC35M) virus resulted in a decrease in virus replication. Pretreatment of C57Bl/6J mice with either AEBSF or a cocktail of AEBSF and pAB prior to infection with the H1N1 virus significantly reduced weight loss and led to a faster recovery of treated versus untreated mice while pAB alone exerted a very poor effect. After infection with the H7N7 virus, the most significant reduction of weight loss was obtained upon pretreatment with either the protease inhibitor cocktail or pAB. Furthermore, pretreatment of C57BL/6J mice with AEBSF prior to infection resulted in a significant reduction in the levels of both the H1N1 and H7N7 nucleoproteins in mice lungs and also a significant reduction in the levels of the HA transcript in the lungs of the H1N1- but not the H7N7-infected mice.
Multiple serine protease activities might be implicated in mediating influenza infection. Blocking influenza A virus infection in cultured lung epithelia and in mice by the used serine protease inhibitors may provide an alternative approach for treatment of influenza infection.
PMCID: PMC3034701  PMID: 21251300
23.  QTLminer: identifying genes regulating quantitative traits 
BMC Bioinformatics  2010;11:516.
Quantitative trait locus (QTL) mapping identifies genomic regions that likely contain genes regulating a quantitative trait. However, QTL regions may encompass tens to hundreds of genes. To find the most promising candidate genes that regulate the trait, the biologist typically collects information from multiple resources about the genes in the QTL interval. This process is very laborious and time consuming.
QTLminer is a bioinformatics tool that automatically performs QTL region analysis. It is available in GeneNetwork and it integrates information such as gene annotation, gene expression and sequence polymorphisms for all the genes within a given genomic interval.
QTLminer substantially speeds up discovery of the most promising candidate genes within a QTL region.
PMCID: PMC2964687  PMID: 20950438
24.  Data-driven assessment of eQTL mapping methods 
BMC Genomics  2010;11:502.
The analysis of expression quantitative trait loci (eQTL) is a potentially powerful way to detect transcriptional regulatory relationships at the genomic scale. However, eQTL data sets often go underexploited because legacy QTL methods are used to map the relationship between the expression trait and genotype. Often these methods are inappropriate for complex traits such as gene expression, particularly in the case of epistasis.
Here we compare legacy QTL mapping methods with several modern multi-locus methods and evaluate their ability to produce eQTL that agree with independent external data in a systematic way. We found that the modern multi-locus methods (Random Forests, sparse partial least squares, lasso, and elastic net) clearly outperformed the legacy QTL methods (Haley-Knott regression and composite interval mapping) in terms of biological relevance of the mapped eQTL. In particular, we found that our new approach, based on Random Forests, showed superior performance among the multi-locus methods.
Benchmarks based on the recapitulation of experimental findings provide valuable insight when selecting the appropriate eQTL mapping method. Our battery of tests suggests that Random Forests map eQTL that are more likely to be validated by independent data, when compared to competing multi-locus and legacy eQTL mapping methods.
PMCID: PMC2996998  PMID: 20849587
25.  Sustained viral load and late death in Rag2-/- mice after influenza A virus infection 
Virology Journal  2010;7:172.
The importance of the adaptive immune response for secondary influenza infections and protection from a lethal challenge after vaccination has been well documented. However, some controversy still exists concerning the specific involvement of B and T cells during a primary infection. Here, we have followed the survival, weight loss, viral load and lung pathology in Rag2-/- knock-out mice after infection with influenza A virus (H1N1). Infected wild type mice initially lost weight early after infection but then cleared the virus and recovered. Rag2-/- mice, however, showed similar weight loss kinetics in the early stages after infection but weight loss continued post infection and culminated in death. In contrast to wild type mice, Rag2-/- mice were not able to clear the virus, despite an increased inflammatory response. Furthermore, they did not recruit virus-specific lymphocytes into the lung in the later stages after infection and exhibited sustained pulmonary lesions.
PMCID: PMC2919473  PMID: 20667098

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