Type I IFN triggers RIG-I/TLR3/NLRP3-dependent inflammasome activation in influenza A virus infected cells.

Influenza A virus (IAV) triggers a contagious and potentially lethal respiratory disease. A protective IL-1ß response is mediated by innate receptors in macrophages and lung epithelial cells. NLRP3 is crucial in macrophages; however, which sensors elicit IL-1ß secretion in lung epithelial cells remains undetermined. Here, we describe for the first time the relative roles of the host innate receptors RIG-I (DDX58), TLR3, and NLRP3 in the IL-1ß response to IAV in primary lung epithelial cells. To activate IL-1ß secretion, these cells employ partially redundant recognition mechanisms that differ from those described in macrophages. RIG-I had the strongest effect through a MAVS/TRIM25/Riplet-dependent type I IFN signaling pathway upstream of TLR3 and NLRP3. Notably, RIG-I also activated the inflammasome through interaction with caspase 1 and ASC in primary lung epithelial cells. Thus, NS1, an influenza virulence factor that inhibits the RIG-I/type I IFN pathway, strongly modulated the IL-1ß response in lung epithelial cells and in ferrets. The NS1 protein derived from a highly pathogenic strain resulted in increased interaction with RIG-I and inhibited type I IFN and IL-1ß responses compared to the least pathogenic virus strains. These findings demonstrate that in IAV-infected lung epithelial cells RIG-I activates the inflammasome both directly and through a type I IFN positive feedback loop.

Mapping of clinical and expression quantitative trait loci in a sex-dependent effect of host susceptibility to mouse-adapted influenza H3N2/HK/1/68.

Seasonal influenza outbreaks and recurrent influenza pandemics present major challenges to public health. By studying immunological responses to influenza in different host species, it may be possible to discover common mechanisms of susceptibility in response to various influenza strains. This could lead to novel therapeutic targets with wide clinical application. Using a mouse-adapted strain of influenza (A/HK/1/68-MA20 [H3N2]), we produced a mouse model of severe influenza that reproduces the hallmark high viral load and overexpression of cytokines associated with susceptibility to severe influenza in humans. We mapped genetic determinants of the host response using a panel of 29 closely related mouse strains (AcB/BcA panel of recombinant congenic strains) created from influenza-susceptible A/J and influenza-resistant C57BL/6J (B6) mice. Combined clinical quantitative trait loci (QTL) and lung expression QTL mapping identified candidate genes for two sex-specific QTL on chromosomes 2 and 17. The former includes the previously described Hc gene, a deficit of which is associated with the susceptibility phenotype in females. The latter includes the phospholipase gene Pla2g7 and Tnfrsf21, a member of the TNFR superfamily. Confirmation of the gene underlying the chromosome 17 QTL may reveal new strategies for influenza treatment.

Genetic control of high density lipoprotein-cholesterol in AcB/BcA recombinant congenic strains of mice.

Epidemiological studies show that high HDL-cholesterol (HDLc) decreases the risk of cardiovascular disease. To map genes controlling lipid metabolism, particularly HDLc levels, we screened the plasma lipids of 36 AcB/BcA RC mouse strains subjected to either a normal or a high-fat/cholesterol diet. Strains BcA68 and AcB65 showed deviant HDLc plasma levels compared with the parental A/J and C57BL/6J strains; they were thus selected to generate informative F2 crosses. Linkage analyses in the AcB65 strain identified a locus on chromosome 4 (Hdlq78) responsible for high post-high fat diet HDLc levels. This locus has been previously associated at genome-wide significance to two regions in the human genome. A second linkage analysis in strain BcA68 identified linkage in the vicinity of a gene cluster known to control HDLc levels. Sequence analysis of these candidates identified a de novo, loss-of-function mutation in the ApoA1 gene of BcA68 that prematurely truncates the ApoA1 protein. The possibility of dissecting the specific effects of this new ApoA1 deficiency in the context of isogenic controls makes the BcA68 mouse a valuable new tool.

Genetic determinants of emotionality and stress response in AcB/BcA recombinant congenic mice and in silico evidence of convergence with cardiovascular candidate genes.

Genomic loci bearing stress-related phenotypes were dissected in recombinant congenic strains (RCS) of mice with C57BL/6J (B6) and A/J progenitors. Adult male mice from 14 A/J and 22 B6 background lines were evaluated for emotional reactivity in open-field (OF) and elevated plus-maze tests. Core temperature was monitored by radio telemetry during immobilization and on standard as well as salt-enriched diets. In addition, urinary electrolytes were measured. Genome-wide linkage analysis of the parameters revealed over 20 significant quantitative trait loci (QTL). The highest logarithm of odds (LOD) scores were within the previously-reported OF emotionality locus on Chr 1 (LOD = 4.6), in the dopa decarboxylase region on Chr 11 for the plus-maze (LOD = 4.7), and within a novel region of calmodulin 1 on Chr 12 for Ca++ excretion after a 24-h salt load (LOD = 4.6). RCS stress QTL overlapped with several candidate loci for cardiovascular (CV) disease. In silico evidence of functional polymorphisms by comparative sequence analysis of progenitor strains assisted to ascertain this convergence. The anxious BcA70 strain showed down regulation of Atp1a2 gene expression in the heart (P < 0.001) and brain (P < 0.05) compared with its parental B6 strain, compatible with the enhanced emotionality described in knock out animals for this gene, also involved in the salt-sensitive component of hypertension. Functional polymorphisms in regulatory elements of candidate genes of the CV/inflammatory/immune systems support the hypothesis of genetically-altered environmental susceptibility in CV disease development.

Identification of novel chromosomal regions associated with airway hyperresponsiveness in recombinant congenic strains of mice.

Airway responsiveness is the ability of the airways to respond to bronchoconstricting stimuli by reducing their diameter. Airway hyperresponsiveness has been associated with asthma susceptibility in both humans and murine models, and it has been shown to be a complex and heritable trait. In particular, the A/J mouse strain is known to have hyperresponsive airways, while the C57BL/6 strain is known to be relatively refractory to bronchoconstricting stimuli. We analyzed recombinant congenic strains (RCS) of mice generated from these hyper- and hyporesponsive parental strains to identify genetic loci underlying the trait of airway responsiveness in response to methacholine as assessed by whole-body plethysmography. Our screen identified 16 chromosomal regions significantly associated with airway hyperresponsiveness (genome-wide P 73 cM), chromosome 7 (>63 cM), chromosome 8 (52-67 cM), chromosome 10 (3-7 cM and >68 cM), and chromosome 12 (25-38 cM and >52 cM). Our data identify several likely candidate genes from the 16 regions, including Ddr2, Hc, Fbn1, Flt3, Utrn, Enpp2, and Tsc.

The AcB/BcA recombinant congenic strains of mice: strategies for phenotype dissection, mapping and cloning of quantitative trait genes.

The AcB/BcA gene discovery platform consists of a series of 36 recombinant congenic strains (RCS) produced from the second backcross generation of the progenitor mouse strains A/J and C57BL/6J. Each individual inbred RCS carries 12.5% of the donor genome in 87.5% of the background genome. As the two parental strains are known to vary in the expression of resistance and susceptibility to a considerable number of mouse models of human diseases, the AcB/BcA RCS platform represents a valuable and versatile genetic tool to study many different phenotypes. RCS can be used to follow the segregation of single gene effects in individual strains, or to look at association/dissociation of mechanistic aspects of complex phenotypes. In addition, one can select strains with fixed alleles at known loci to look for novel gene effects, or use strains with overlapping congenic segments to delineate minimal QTL, intervals. The AcB/BcA RCS platform was used by our group and others to study a series of complex phenotypes including nociception, malaria susceptibility and lipid metabolism. Linkage mapping in secondary crosses and gene expression analysis in targeted organs allowed the identification of chromosomal regions, genes, and biological pathways which might unravel novel targets for preventive and therapeutic interventions.

Mapping cis-acting regulatory variation in recombinant congenic strains.

We present an integrated approach for the enriched detection of genes subject to cis-acting variation in the mouse genome. Gene expression profiling was performed with lung tissue from a panel of recombinant congenic strains (RCS) derived from A/J and C57BL/6J inbred mouse strains. A multiple-regression model measuring the association between gene expression level, donor strain of origin (DSO), and predominant strain background identified over 1,500 genes (P < 0.05) whose expression profiles differed according to the DSO. This model also identified over 1,200 genes whose expression showed dependence on background (P < 0.05), indicating the influence of background genetic context on transcription levels. Sequences obtained from 1-kb segments of 3'-untranslated regions identified single nucleotide polymorphisms in 64% of genes whose expression levels correlated with DSO status, compared with 29% of genes that displayed no association (P < 0.01, Fisher exact test). Allelic imbalance was identified in 50% of genes positive for expression-DSO association, compared with 22% of negative genes (P < 0.05, Fisher exact test). Together, these results demonstrate the utility of RCS mice for identifying the roles of proximal genetic determinants and background genetic context in determining gene expression levels. We propose the use of this integrated experimental approach in multiple tissues from this and other RCS panels as a means for genome-wide cataloging of genetic regulatory mechanisms in laboratory strains of mice.

Anti-tumour necrosis factor agents and tuberculosis risk: mechanisms of action and clinical management.

Cases of active tuberculosis have been reported worldwide with the use of therapeutic agents that inhibit tumour necrosis factor (TNF) alpha. TNFalpha has a central role in mycobacterial infection and disease. Accordingly, progression of recently acquired tuberculosis infection or reactivation of remotely acquired infection should be expected with the use of anti-TNF agents. The available in-vitro and epidemiological evidence for the two currently approved agents, infliximab and etanercept, shows that the risk of development of active tuberculosis is greater with infliximab. Tuberculin skin testing (TST) should be undertaken before any significant immunosuppressive therapy including these agents, though the possibility of false-negative reactions in immunocompromised populations must be borne in mind. A positive TST should be followed by medical assessment and chest radiography, as well as by other tests judged appropriate by the physician to identify active disease. Active tuberculosis must be treated appropriately before initiation of treatment with an anti-TNF agent. Treatment of latent tuberculosis can be considered on an individual basis for TST-negative patients receiving anti-TNF agents when significant risk factors for infection are present.

Provisional mapping of quantitative trait loci modulating the acoustic startle response and prepulse inhibition of acoustic startle.

Prepulse inhibition (PPI) of the acoustic startle response (ASR) is a form of sensorimotor gating, defined as an inhibition of the startle response when a low intensity stimulus, the prepulse, precedes the startling stimulus. Deficits in PPI have been reported in schizophrenia and other psychiatric/neurological disorders, and correlate with symptom severity in schizophrenia, suggesting that deficient PPI per se or abnormalities in neural circuits regulating PPI may cause some symptoms of schizophrenia. If so, then genes conferring reduced PPI may contribute toward genetic vulnerability to schizophrenia. Studies with selectively bred rodent strains indicate that PPI is under genetic control; however, the identity of the relevant genes is unknown. The current study used recombinant congenic mouse strains derived from C57BL/6J and A/J parents to assess genetic variability in PPI and in ASR and to identify provisional quantitative trait loci (QTLs) modulating these phenotypes. Significant between-strain differences in ASR and in PPI at each of several prepulse intensities (75, 80, 85, 90, 95 dB) were found. Correlations between PPI at the various prepulse intensities were highly significant, suggesting appreciable overlap in genetic regulation of PPI across prepulse intensities. Five QTLs (chromosomes 3, 5, 7, 16) associated with PPI across all prepulse intensities, but not with ASR, were identified. Two additional QTLs (chromosomes 2, 11) associated with both PPI and ASR were found. Fifteen QTLs were associated with ASR alone. Data on genotypes of informative congenic strains were used to support probable involvement of loci modulating PPI and to narrow the probable chromosomal location of QTLs. If confirmed, these QTLs may suggest candidate genes directing novel mechanisms for regulation of PPI

Genetic analysis of influences on survival following Toxoplasma gondii infection.

Survival of mice during the acute stage of Toxoplasma gondii infection was not influenced by the MHC Class I gene, L(d), but was influenced by the MHC Class II genes, Ia and Ie. As unexplained variability was noted in our initial studies of influence of the L(d) gene on survival, influence of the L(d) gene region on survival in the presence of a number of variables was studied. Although route of administration and dose of parasites, and age and gender of the mice markedly influenced outcome of T. gondii infection, the Class I L(d) gene did not modify survival in any of these circumstances. In separate studies, using mice with a differing genetic background, i.e. H-2(b), C57BL/10 mice, presence of Ia or Ie alone diminished survival even though presence of Ia reduced parasite burden. When neither or both the Ia and Ie genes were present together, survival was greater. In separate analyses of our studies of AxB BxA recombinant inbred mice, similar influences of MHC genes on survival and parasite burden following peroral infection were confirmed. Previously undescribed associations of novel genetic loci and survival and parasite burden also were identified. Genetic loci associated with enhanced survival included D8Mit42, D1Mit3, Iapls1-16, D8Mit14, Hoxb, Mpmv29, Pmv45, and Emv-2; genetic loci associated with reduced parasite burden included H-2, D17Mit62, D17Mit83, D17Mit21, D17Mit34, D17Mit47, D18Mit4, and Gln3-5. These studies demonstrate the importance of MHC region genes (but not L(d)) for survival, and the influence of other novel genes, and endogenous and exogenous variables on survival and parasite burden specified by host genes following T. gondii infection.

Linkage of leprosy susceptibility to Parkinson's disease genes.

In early 2003, an international team of scientists conducted a genome scan in Vietnamese multiplex leprosy families and found that susceptibility to leprosy was significantly linked to region q25 on the long arm of chromosome 6. Further confirmation of the chromosome 6 locus was provided by high-resolution linkage mapping in simplex leprosy families. Now, in a continuation of these findings, the team has pinpointed the chromosome 6 susceptibility locus to the 5' regulatory promoter region shared by both the Parkinson's disease gene PARK2 and its co-regulated gene PACRG. The surprising discovery has important implications for the understanding of leprosy pathogenesis and for the strategy of genetic analysis of infectious diseases.

Leprosy susceptibility revealed.

In order for these findings to have practical significance in terms of leprosy control and prevention, it will be necessary to extend the linkage of chromosome 6q25 to another region endemic for leprosy. Replicative findings would likely mean that the chromosome 6q25 susceptibility gene is a variant of a common gene that promotes susceptibility to infection per se. Identification of the gene variant will hopefully reveal insight about transmission and disease incidence--the longstanding enigmas of leprosy. Whether a more effective, universal MDT treatment or another type of prevention (either vaccine or environmental) could be based on this knowledge, is an exciting prospect to contemplate.

Recombinant congenic strains of mice: a new tool for the genetic dissection of efficacy and toxicity of adjuvants.

The efficacy of immunization of a vaccine depends on an antigen, an adjuvant, and on the expression of multiple genes in the host. The responsiveness of various strains of mice to adjuvants is therefore dependent on their genetic background present as a complex, multigenic trait similarly as in man. We have recently developed a gene-discovery platform, termed recombinant congenic strains (RCS), that greatly facilitates the dissection, localization and characterization of genes that mediate complex traits such as responsiveness to adjuvants. These recombinant congenic mice, which were constructed from two progenitor strains (A/J and C57BL/6) that are phenotypically different for several spontaneous or infectious diseases, were generated such that they carry 13.5% of the one genome in 85% of the other genome. The use of these RCS mice therefore enables a more efficient identification of genes that mediate the responsiveness of the adjuvant.