No abstract

With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled "superpedigrees" are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening.N-ethyl-N-nitrosourea | genetic mapping | forward genetics | mutagenesis | massively parallel sequencing P henotypic variation in mice can be induced with N-ethyl-Nnitrosourea (ENU), which creates single base pair substitutions in germ line DNA. However, the positional cloning of ENU-induced mutations causative for phenotypes of interest has historically been a time-consuming process, beginning with generation of an outcrossed recombinant mapping population of phenotypically mutant and WT mice, genotyping individual mice at genetic markers across the genome to create a linkage map, and finally targeted sequencing to identify the causative mutation within the critical region. The advent of massively parallel sequencing techniques has given rise to more rapid "mapping-bysequencing" methods in which genome-wide marker genotyping and DNA sequencing are combined into a single step applied to either individual or pooled groups of organisms (1). For ENUmutagenized mice, early experiments used massively parallel sequencing for mutation identification within a critical region defined by traditional or bulk segregation mapping using recombinant mapping populations produced by outcrossing the mutant to another inbred laboratory strain and backcrossing or intercrossing a second time (2-4). Later reports demonstrated mapping with the identified sequence variants themselves as markers, which eliminated...

Sepsis is the leading cause of death amongst critically ill patients in intensive care units, and treatment options are limited. Therapies developed against the pro-inflammatory stage have failed clinically; therefore new approaches that target the host immune response in sepsis are necessary. Increasing evidence suggests that a major pathophysiological event in sepsis is immune suppression, often resulting in secondary fungal, bacterial, or viral infections. Recent studies from animal sepsis models and patient samples suggest that cytokines such as IL-7, IL-15, GM-CSF as well as co-inhibitory molecule blockade, such as anti-PD-1 and anti-BTLA, may have utility in alleviating the clinical morbidity associated with sustained sepsis. This review discusses some of these novel immunomodulatory agents and evaluates their potential use as therapeutics.

PD-1 and PD-L1 have been reported to provide peripheral tolerance by inhibiting TCR-mediated activation. We have reported that PD-L1-/- animals are protected from sepsis-induced mortality and immune suppression. Whereas studies indicate that LSECs normally express PD-L1, which is also thought to maintain local immune liver tolerance by ligating the receptor PD-1 on T lymphocytes, the role of PD-L1 in the septic liver remains unknown. Thus, we hypothesized initially that PD-L1 expression on LSECs protects them from sepsis-induced injury. We noted that the increased vascular permeability and pSTAT3 protein expression in whole liver from septic animals were attenuated in the absence of PD-L1. Isolated LSECs taken from septic animals, which exhibited increased cell death, declining cell numbers, reduced cellular proliferation, and VEGFR2 expression (an angiogenesis marker), also showed improved cell numbers, proliferation, and percent VEGFR2(+) levels in the absence of PD-L1. We also observed that sepsis induced an increase of liver F4/80(+)PD-1(+)-expressing KCs and increased PD-L1 expression on LSECs. Interestingly, PD-L1 expression levels on LSECs decreased when PD-1(+)-expressing KCs were depleted with clodronate liposomes. Contrary to our original hypothesis, we document here that increased interactions between PD-1(+) KCs and PD-L1(+) LSECs appear to lead to the decline of normal endothelial function-essential to sustain vascular integrity and prevent ALF. Importantly, we uncover an underappreciated pathological aspect of PD-1:PD-L1 ligation during inflammation that is independent of its normal, immune-suppressive activity.