Antiphospholipid antibody-mediated effects in an arterial model of thrombosis are dependent on Toll-like receptor 4.

Patients with antiphospholipid syndrome (APS) produce antiphospholipid antibodies (aPL) and develop vascular thrombosis that may occur in large or small vessels in the arterial or venous beds. On the other hand, many individuals produce aPL and yet never develop thrombotic events. Toll-like receptor 4 (TLR4) appears to be necessary for aPL-mediated prothrombotic effects in venous and microvascular models of thrombosis, but its role in arterial thrombosis has not been studied. Here, we propose that aPL alone are insufficient to cause thrombotic events in an arterial model of APS, and that a concomitant trigger of innate immunity (e.g. TLR4 activation) is required. We show specifically that anti-ß2-glycoprotein I (anti-ß2GPI) antibodies, a subset of aPL, accelerated thrombus formation in C57BL/6 wild-type, but not TLR4-deficient, mice in a ferric chloride-induced carotid artery injury model. These aPL bound to arterial and venous endothelial cells, particularly in the presence of ß2GPI, and to human TLR4 by enzyme-linked immunoassay. Arterial endothelium from aPL-treated mice had enhanced leukocyte adhesion, compared to control IgG-treated mice. In addition, aPL treatment of mice enhanced expression of tissue factor (TF) in leukocytes induced by the TLR4 ligand lipopolysaccharide (LPS). aPL also enhanced LPS-induced TF expression in human leukocytes in vitro. Our findings support a mechanism in which aPL enhance TF expression by leukocytes, as well as augment adhesion of leukocytes to the arterial endothelium. The activation of TLR4 in aPL-positive individuals may be required to trigger thrombotic events.

An ENU-induced splicing mutation reveals a role for Unc93b1 in early immune cell activation following influenza A H1N1 infection.

Genetic and immunological analysis of host-pathogen interactions can reveal fundamental mechanisms of susceptibility and resistance to infection. Modeling human infectious diseases among inbred mouse strains is a proven approach but is limited by naturally occurring genetic diversity. Using N-ethyl-N-nitrosourea mutagenesis, we created a recessive loss-of-function point mutation in Unc93b1 (unc-93 homolog B1 (C. elegans)), a chaperone for endosomal Toll-like receptors (TLR)3, TLR7 and TLR9, which we termed Letr for 'loss of endosomal TLR response'. We used Unc93b1(Letr/Letr) mice to study the role of Unc93b1 in the immune response to influenza A/PR/8/34 (H1N1), an important global respiratory pathogen. During the early phase of infection, Unc93b1(Letr/Letr) mice had fewer activated exudate macrophages and decreased expression of CXCL10, interferon (IFN)-γ and type I IFN. Mutation of Unc93b1 also led to reduced expression of the CD69 activation marker and a concomitant increase in the CD62L naive marker on CD4(+) and CD8(+) T cells in infected lungs. Finally, loss of endosomal TLR signaling resulted in delayed viral clearance that coincided with increased tissue pathology during infection. Taken together, these findings establish a role for Unc93b1 and endosomal TLRs in the activation of both myeloid and lymphoid cells during the innate immune response to influenza.

Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4)

Bacterial lipopolysaccharide (LPS) provokes a vigorous, generalized proinflammatory state in the infected host. Genetic regulation of this response has been localized to the Lps locus on mouse chromosome 4, through study of the C3H/HeJ and C57BL/10ScCr inbred strains. Both C3H/HeJ and C57BL/10ScCr mice are homozygous for a mutant Lps allele (Lpsd/d) that confers hyporesponsiveness to LPS challenge, and therefore exhibit natural tolerance to its lethal effects. Genetic and physical mapping of 1,345 backcross progeny segregating this mutant phenotype confined Lps to a 0.9-cM interval spanning 1.7 Mb. Three transcription units were identified within the candidate interval, including Toll-like receptor 4 (Tlr4), part of a protein family with members that have been implicated in LPS-induced cell signaling. C3H/HeJ mice have a point mutation within the coding region of the Tlr4 gene, resulting in a nonconservative substitution of a highly conserved proline by histidine at codon 712, whereas C57BL/ 10ScCr mice exhibit a deletion of Tlr4. Identification of distinct mutations involving the same gene at the Lps locus in two different hyporesponsive inbred mouse strains strongly supports the hypothesis that altered Tlr4 function is responsible for endotoxin tolerance.

Sex differences in the genetic architecture of susceptibility to Cryptococcus neoformans pulmonary infection.

Cryptococcus neoformans is a major cause of fungal pneumonia, meningitis and disseminated disease in the immune compromised host. Here we have used a clinically relevant model to investigate the genetic determinants of susceptibility to progressive cryptococcal pneumonia in C57BL/6J and CBA/J inbred mice. At 5 weeks after infection, the lung fungal burden was over 1000-fold higher in C57BL/6J compared to CBA/J mice. A genome-wide scan performed on 210 male and 203 female (CBA/J x C57BL/6J) F2 progeny using lung colony-forming units as a quantitative trait revealed a sex difference in genetic architecture with three loci (designated Cnes1-Cnes3) associated with susceptibility to cryptococcal pneumonia. Single locus analysis identified significant loci on chromosomes 3 (Cnes1) and 17 (Cnes2) with logarithm of the odds (LOD) scores of 4.09 (P=0.0110) and 7.30 (P<0.0001) that explained 8.9 and 15.9% of the phenotypic variance, respectively, in female CBAB6F2 and one significant locus on chromosome 17 (Cnes3) with a LOD score of 4.04 (P=0.010) that explained 8.6% of the phenotypic variance in male CBAB6F2 mice. Genome-wide pair-wise analysis revealed significant quantitative trait locus interactions in both the female and male CBAB6F2 progeny that collectively explained 43.8 and 19.5% of phenotypic variance in each sex, respectively.

Host resistance to infection: genetic control of lipopolysaccharide responsiveness by TOLL-like receptor genes.

Gram-negative bacterial lipopolysaccharide evokes a protective inflammatory response in the normal host. Through genetic analysis of mutant mice, the gene encoding Toll-like receptor 4 (Tlr4) was recently identified as a critical component of this host defense mechanism. Tlr4 is a member of an ancient gene family that regulates antimicrobial host defense in plants, invertebrates and mammals.

Pasteurella multocida septic shock following liver transplantation treated with drotrecogin alpha (activated).

Severe sepsis and progression to septic shock in solid organ transplant recipients is associated with a high mortality. We describe a fulminant case of septic shock in a liver transplant recipient caused by Pasteurella multocida, a gram-negative coccobacillus most commonly associated with domestic cats and dogs. P. multocida is a rare cause of bacteremia and has not been reported as a cause of septic shock following liver transplantation. In addition to standard therapy, the patient was managed with drotrecogin alpha (activated) recombinant activated protein C (APC), an evidence-based agent that has been shown to significantly improve outcome in severe sepsis in the non-transplant population. The known risk factors, clinical course, and outcomes of severe infection associated with P. multocida are also briefly reviewed. This case illustrates the need for transplant recipients and their healthcare providers to carefully consider the risk of severe infection associated with domestic animal exposure.

Functional analysis and chromosomal mapping of Gata5, a gene encoding a zinc finger DNA-binding protein.

The GATA family of zinc finger proteins are transcriptional regulators with critical functions in lineage differentiation and embryonic development. Based on structural and expression pattern comparisons, the GATA proteins have been subdivided into two groups. The first subgroup consists of GATA-1, -2, and -3, which are all highly expressed in the hematopoietic system, whereas GATA-4, -5, and -6 are present essentially in the heart and gut. We have isolated and functionally characterized the rat GATA-5 cDNA, which encodes a 45-kDa protein with 71%, 73%, and 97% homology to its amphibian, avian, and murine homologs, respectively. Northern blot analysis showed that rat GATA-5 is expressed in a dynamic pattern during embryonic and postnatal development. In the midgestation embryo, GATA-5 transcripts are most abundant in the heart and decrease dramatically in the postnatal heart; in contrast, GATA-5 expression is upregulated in the lung and gut during postnatal development. Functional studies with recombinant GATA-4, -5, and -6 proteins show that GATA-5 has preferential affinity for a subset of GATA elements found on cardiac promoters and differentially activate cardiac gene transcription. Structure-function analysis revealed the presence of an activation domain within the carboxy terminal region of GATA-5 that is essential for transcriptional regulation of target promoters. Linkage analysis localized Gata5 to distal mouse Chromosome (Chr) 2 in a conserved linkage group with genes localized to rat Chr 3q43 and human Chr 20q13.2-q13.3. The results suggest that GATA-5 may have specific downstream targets and that GATA-4, -5, and -6 can only partially substitute for each other in cardiogenesis. Thus, Gata5 probably plays a specialized evolutionary conserved role in cardiac development.

Comparative genomics and host resistance against infectious diseases.

The large size and complexity of the human genome have limited the identification and functional characterization of components of the innate immune system that play a critical role in front-line defense against invading microorganisms. However, advances in genome analysis (including the development of comprehensive sets of informative genetic markers, improved physical mapping methods, and novel techniques for transcript identification) have reduced the obstacles to discovery of novel host resistance genes. Study of the genomic organization and content of widely divergent vertebrate species has shown a remarkable degree of evolutionary conservation and enables meaningful cross-species comparison and analysis of newly discovered genes. Application of comparative genomics to host resistance will rapidly expand our understanding of human immune defense by facilitating the translation of knowledge acquired through the study of model organisms. We review the rationale and resources for comparative genomic analysis and describe three examples of host resistance genes successfully identified by this approach.

The Lps locus: genetic regulation of host responses to bacterial lipopolysaccharide.

Lipopolysaccharide (LPS), an abundant glycolipid of the outer membrane of gram-negative bacteria, is able to provoke a generalized proinflammatory response in the infected host. Genetic regulation of this trait has been localized to the Lps locus on mouse chromosome 4. Several inbred mouse strains, including C3H/HeJ, C57BL/10ScNCr and C57BL/10ScCr, bear mutations at the Lps locus (Lps(d)) that confer hyporesponsiveness to the immunostimulatory properties of LPS and susceptibility to overwhelming gram-negative bacterial infection. The phenotypic expression of Lps(d) is pleiotropic, affecting several cell types crucial to host defense, including the macrophage. By positional cloning, Toll-like receptor 4 (Tlr4), a transmembrane protein with a cytoplasmic domain that bears homology to the Interleukin-1 receptor, has been identified as the gene encoded by Lps. Tlr4 is a member of a novel gene family that participates in host defense against microbial infection in plants, invertebrates and mammals. Discovery of the molecular basis of the Lps mutation represents a significant advance in defining the fundamental mechanisms of cellular activation by LPS.

LPS-hyporesponsiveness of mnd mice is associated with a mutation in Toll-like receptor 4.

Toll-like receptors (Tlrs) are transmembrane proteins that have recently been shown to play a critical role in the innate immune recognition of microbial constituents. Among this family, Tlr4 is a crucial signal transducer for lipopolysaccharide (LPS), the major component of the Gram-negative bacteria outer cell membrane. In this paper, we report that C57BL/6.KB2-mnd mice, a model of neuronal ceroid lipofuscinosis, do not respond to LPS. This defect is associated with a spontaneous mutation in Tlr4 consisting of a large insertion within exon 2 predicting a frameshift mutation and a truncated protein.

A high-resolution map in the chromosomal region surrounding the Lps locus.

The Lps locus on mouse chromosome 4 controls host responsiveness to lipopolysaccharide, a major component of the outer membrane of Gram-negative bacteria. The C3H/HeJ inbred mouse strain is characterized by a mutant Lps allel (Lpsd) that renders it hyporesponsive to LPS and naturally tolerant of its lethal effects. To identify the Lps gene by a positional cloning strategy, we have generated a high-resolution linkage map of the chromosomal region surrounding this locus. We have analyzed a total of 1604 backcross mice from a preexisting interspecific backcross panel of 259 (Mus spretus x C57BL/6J)F1 x C57BL/6J and two novel panels of 597 (DBA/2J x C3H/HeJ)F1 x C3H/HeJ and 748 (C57BL/6J x C3H/HeJ)F1 x C3H/HeJ segregating at Lps. A total of 50 DNA markers have been mapped in a 11.8-cM span overlapping the Lps locus. This positions the Lps locus within a 1.1-cM interval, flanked proximally by a large cluster of markers, including three know genes (Cd301, Hxb, and Ambp), and distally by two microsatellite markers (D4Mit7/D4Mit178). The localization of the Lps locus is several centimorgans proximal to that previously assigned.

Cutting edge: functional characterization of the effect of the C3H/HeJ defect in mice that lack an Lpsn gene: in vivo evidence for a dominant negative mutation.

A point mutation in the Tlr4 gene, which encodes Toll-like receptor 4, has recently been proposed to underlie LPS hyporesponsiveness in C3H/HeJ mice (Lpsd). The data presented herein demonstrate that F1 progeny from crosses between mice that carry a approximately 9-cM deletion of chromosome 4 (including deletion of LpsTlr4) and C3H/HeJ mice (i.e., Lps0 x Lpsd F1 mice) exhibit a pattern of LPS sensitivity, measured by TNF activity, that is indistinguishable from that exhibited by Lpsn x Lpsd F1 progeny and whose average response is "intermediate" to parental responses. Thus, these data provide clear functional support for the hypothesis that the C3H/HeJ defect exerts a dominant negative effect on LPS sensitivity; however, expression of a normal Toll-like receptor 4 molecule is apparently not required.