Silvia M. Vidal

Natural resistance to infection with intracellular parasites: Isolation of a candidate for Bcg

Natural resistance to infection with intracellular parasites is controlled by a dominant gene on mouse chromosome 1, called Bcg, Lsh, or Ity. Bcg affects the capacity of macrophages to destroy ingested intracellular parasites early during infection. We have assembled a 400 kb bacteriophage and cosmid contig within the genomic interval containing Bcg. A search for transcription units by exon amplification identified six novel genes in this contig. RNA expression studies showed that one of them, designated Nramp, was expressed exclusively in macrophage populations from reticuloendothelial organs and in the macrophage line J774A. Nramp encodes an integral membrane protein that has structural homology with known prokaryotic and eukaryotic transport systems, suggesting a macrophage-specific membrane transport function. Susceptibility to infection (Bcgs) in 13 Bcgr and Bcgs strains tested is associated with a nonconservative Gly-105 to Asp-105 substitution within predicted transmembrane domain 2 of Nramp.

Susceptibility to mouse cytomegalovirus is associated with deletion of an activating natural killer cell receptor of the C-type lectin superfamily

Cytomegalovirus is the leading cause of congenital viral disease and the most important opportunistic infection in immunocompromised patients. We have used a mouse experimental infection model (MCMV) to study the genetic parameters of host/virus interaction. Susceptibility to infection with MCMV is controlled by Cmv1, a chromosome 6 locus that regulates natural killer (NK) cell activity against virally infected targets. Here, we use a positional cloning strategy to isolate the gene mutated at the Cmv1 locus. Cmv1 maps within a 0.35-cM interval defined by markers D6Ott8 and D6Ott115, which corresponds to a physical distance of 1.6 Mb (refs. 6–8). A transcript map of the region identified 19 genes, including members of the killer cell lectin-like receptor family a (Klra, formerly Ly49; refs. 9–12), which encode inhibitory or activating NK cell receptors that interact with MHC class I molecules. Klra genes have different copy numbers and genomic organization, and are highly polymorphic among inbred strains, making it difficult to distinguish between normal allelic variants and distinct Klra genes, or possible mutations associated with Cmv1. The recombinant inbred strain BXD-8/Ty (BXD-8; ref. 18), derived from Cmv1r C57BL/6 (B6, resistant) and Cmv1s DBA/2 (susceptible), is of particular interest because it is highly susceptible to MCMV infection despite having a B6 haplotype at Cmv1. We determined that MCMV susceptibility in BXD-8 is associated with the deletion of Klra8 (formerly Ly49h).

Birc1e is the gene within the Lgn1 locus associated with resistance to Legionella pneumophila

In inbred mouse strains, permissiveness to intracellular replication of Legionella pneumophila is controlled by a single locus (Lgn1), which maps to a region within distal Chromosome 13 that contains multiple copies of the gene baculoviral IAP repeat–containing 1 (Birc1, also called Naip; refs. 1–3). Genomic BAC clones from the critical interval were transferred into transgenic mice to functionally complement the Lgn1-associated susceptibility of A/J mice to L. pneumophila. Here we report that two independent BAC clones that rescue susceptibility have an overlapping region of 56 kb in which the entire Lgn1 transcript must lie. The only known full-length transcript coded in this region is Birc1e (also called Naip5).

Natural resistance to infection with intracellular parasites: molecular genetics identifies Nramp1 as the Bcg/Ity/Lsh locus

Natural resistance to infection with intracellular parasites is controlled in the mouse by the expression of a locus or group of loci on chromosome 1 alternatively named Bcg, Lsh, and Ity. Bcg affects the capacity of mature tissue macrophages to restrict the intracellular proliferation of ingested parasites in the reticuloendothelial organs of the host during the early phase of infection. This review summarizes our molecular genetic approach to the isolation and characterization of the Bcg locus. We have used a positional cloning strategy based on genetic and physical mapping, YAC cloning, and exon trapping to isolate a candidate gene for Bcg, named Nramp1, which codes for a macrophage‐specific polytopic protein with 12 predicted transmembrane domains and a consensus transport motif. Sequence analysis of Nramp1 cDNA clones from 27 Bcgr and Bcgr mouse strains reveals that susceptibility to infection (Bcgr) is associated with a single nonconservative Gly to Asp substitution at position 169 within predicted transmembrane domain 4 of the Nramp protein. Cloning experiments and homology search in available databases demonstrated that the Nramp1 gene belongs to a small gene family with several members in vertebrates and in such distantly related species as yeast and plants. Nramp proteins share a remarkable degree of similarity, with strong amino acid sequence conservation in the transmembrane domains, suggesting a common transport function for the Nramp family. Finally, we generated Nramp1−/‐ gene knockout mice, and analysis of their phenotypic characteristics established that (1) Nramp1 plays a key role in natural defense against infection with intracellular parasites and therefore demonstrated allelism between Nramp1 and Bcg/Ity/Lsh, (2) Nramp1 functions by a novel cytocidal/cytostatic mechanism distinct from those expressed by the activated macrophage, and (3) the Nramp1Asp169 allele of Bcgr inbred strains is a null allele, pointing to a critical role of this residue in Nramp1 function.

Epistasis between mouse Klra and major histocompatibility complex class I loci is associated with a new mechanism of natural killer cell-mediated innate resistance to cytomegalovirus infection

Experimental infection with mouse cytomegalovirus (MCMV) has been used to elucidate the intricate host-pathogen mechanisms that determine innate resistance to infection. Linkage analyses in F2 progeny from MCMV-resistant MA/My (H2 k) and MCMV-susceptible BALB/c (H2 d) and BALB.K (H2 k) mouse strains indicated that only the combination of alleles encoded by a gene in the Klra (also called Ly49) cluster on chromosome 6, and one in the major histocompatibility complex (H2) on chromosome 17, is associated with virus resistance. We found that natural killer cell–activating receptor Ly49P specifically recognized MCMV-infected cells, dependent on the presence of the H2 k haplotype. This binding was blocked using antibodies to H-2Dk but not antibodies to H-2Kk. These results are suggestive of a new natural killer cell mechanism implicated in MCMV resistance, which depends on the functional interaction of the Ly49P receptor and the major histocompatibility complex class I molecule H-2Dk on MCMV-infected cells.

Activating receptors promote NK cell expansion for maintenance, IL-10 production, and CD8 T cell regulation during viral infection

Natural killer (NK) cells have the potential to deliver both direct antimicrobial effects and regulate adaptive immune responses, but NK cell yields have been reported to vary greatly during different viral infections. Activating receptors, including the Ly49H molecule recognizing mouse cytomegalovirus (MCMV), can stimulate NK cell expansion. To define Ly49Hs role in supporting NK cell proliferation and maintenance under conditions of uncontrolled viral infection, experiments were performed in Ly49h−/−, perforin 1 (Prf1)−/−, and wild-type (wt) B6 mice. NK cell numbers were similar in uninfected mice, but relative to responses in MCMV-infected wt mice, NK cell yields declined in the absence of Ly49h and increased in the absence of Prf1, with high rates of proliferation and Ly49H expression on nearly all cells. The expansion was abolished in mice deficient for both Ly49h and Prf1 (Ly49h−/−Prf1−/−), and negative consequences for survival were revealed. The Ly49H-dependent protection mechanism delivered in the absence of Prf1 was a result of interleukin 10 production, by the sustained NK cells, to regulate the magnitude of CD8 T cell responses. Thus, the studies demonstrate a previously unappreciated critical role for activating receptors in keeping NK cells present during viral infection to regulate adaptive immune responses.

A stuttering model for paramyxovirus P mRNA editing.

Paramyxovirus P genes are transcribed into two mRNAs which differ from each other by either one (measles and Sendai virus) or two (SV5 and mumps virus) G insertions, and which code for either the P or V proteins. The G insertions always occur within a short run of Gs, and a stuttering mechanism for the insertions has been suggested in which the viral polymerase reiteratively copies a template C residue during mRNA synthesis. Support for this mechanism was obtained by varying the reaction conditions during Sendai virus mRNA synthesis in vitro. A stuttering model is proposed which accounts for how the ratio of inserted to uninserted mRNAs is controlled, and why some paramyxoviruses insert one G and others two Gs when insertions occur.

Transgenic Expression of the Activating Natural Killer Receptor Ly49H Confers Resistance to Cytomegalovirus in Genetically Susceptible Mice

Natural resistance to infection with mouse cytomegalovirus (MCMV) is controlled by a dominant locus, Cmv1. Cmv1 is linked to the Ly49 family of natural killer receptors on distal chromosome 6. While some studies localized Cmv1 as distal to the Ly49 gene cluster, genetic and functional analysis identified Ly49h as a pivotal factor in resistance to MCMV. The role of these two independent genomic domains in MCMV resistance was evaluated by functional complementation using transgenesis of bacterial artificial chromosomes (BAC) in genetically susceptible mice. Phenotypic and genetic characterization of the transgenic animals traced the resistance gene to a single region spanning the Ly49h gene. The appearance of the Ly49H protein in NK cells of transgenic mice coincided with the emergence of MCMV resistance, and there was a threshold Ly49H protein level associated with full recovery. Finally, transgenic expression of Ly49H in the context of either of the two independent susceptibility alleles, Cmv1 sBALB or Cmv1 sFVB, conferred resistance to MCMV infection. These results demonstrate that Ly49h is necessary and sufficient to confer MCMV resistance, and formally demonstrate allelism between Cmv1 and Ly49h. This panel of transgenic animals provides a unique resource to study possible pleiotropic effect of Cmv1.

Cloning and characterization of a second human NRAMP gene on chromosome 12q13

The mouse Nramp1 gene was initially identified by positional cloning as a candidate for the host resistance locus Bcg on mouse Chromosome (Chr) 1. Subsequent studies have shown that Nramp is a small gene family of at least three members in the mouse genome. We report the isolation and characterization of a cDNA clone corresponding to the second member of the human NRAMP family, NRAMP2. Predicted amino acid sequence analysis of the NRAMP2 polypeptide identifies a polytopic membrane protein highly homologous to NRAMP1, with 66% identical residues (80% overall homology), resulting in identical predicted secondary structures for the two proteins. Sequence conservation is particularly high in the predicted transmembrane domains (90%), suggesting that these regions play a key role in the structural and functional aspects common to both proteins. As opposed to its NRAMP1 counterpart, whose expression is restricted to phagocytic cells, Northern blotting analyses indicate that NRAMP2 mRNA transcripts are expressed at low levels in all tissues analyzed. Fluorescence in situ hybridization has identified 12q13 as the chromosomal location for human NRAMP2.

Type I interferon restricts type 2 immunopathology through the regulation of group 2 innate lymphoid cells

Viral respiratory tract infections are the main causative agents of the onset of infection-induced asthma and asthma exacerbations that remain mechanistically unexplained. Here we found that deficiency in signaling via type I interferon receptor led to deregulated activation of group 2 innate lymphoid cells (ILC2 cells) and infection-associated type 2 immunopathology. Type I interferons directly and negatively regulated mouse and human ILC2 cells in a manner dependent on the transcriptional activator ISGF3 that led to altered cytokine production, cell proliferation and increased cell death. In addition, interferon-γ (IFN-γ) and interleukin 27 (IL-27) altered ILC2 function dependent on the transcription factor STAT1. These results demonstrate that type I and type II interferons, together with IL-27, regulate ILC2 cells to restrict type 2 immunopathology.