Nitric Oxide Engages an Anti-inflammatory Feedback Loop Mediated by Peroxiredoxin 5 in Phagocytes.

Phagocyte microbiocidal mechanisms and inflammatory cytokine production are temporally coordinated, although their respective interdependencies remain incompletely understood. Here, we identify a nitric-oxide-mediated antioxidant response as a negative feedback regulator of inflammatory cytokine production in phagocytes. In this context, Keap1 functions as a cellular redox sensor that responds to elevated reactive nitrogen intermediates by eliciting an adaptive transcriptional program controlled by Nrf2 and comprised of antioxidant genes, including Prdx5. We demonstrate that engaging the antioxidant response is sufficient to suppress Toll-like receptor (TLR)-induced cytokine production in dendritic cells and that Prdx5 is required for attenuation of inflammatory cytokine production. Collectively, these findings delineate the reciprocal regulation of inflammation and cellular redox systems in myeloid cells.

TMEM258 Is a Component of the Oligosaccharyltransferase Complex Controlling ER Stress and Intestinal Inflammation.

Significant insights into disease pathogenesis have been gleaned from population-level genetic studies; however, many loci associated with complex genetic disease contain numerous genes, and phenotypic associations cannot be assigned unequivocally. In particular, a gene-dense locus on chromosome 11 (61.5-61.65 Mb) has been associated with inflammatory bowel disease, rheumatoid arthritis, and coronary artery disease. Here, we identify TMEM258 within this locus as a central regulator of intestinal inflammation. Strikingly, Tmem258 haploinsufficient mice exhibit severe intestinal inflammation in a model of colitis. At the mechanistic level, we demonstrate that TMEM258 is a required component of the oligosaccharyltransferase complex and is essential for N-linked protein glycosylation. Consequently, homozygous deficiency of Tmem258 in colonic organoids results in unresolved endoplasmic reticulum (ER) stress culminating in apoptosis. Collectively, our results demonstrate that TMEM258 is a central mediator of ER quality control and intestinal homeostasis.

Functional genomics identifies negative regulatory nodes controlling phagocyte oxidative burst.

The phagocyte oxidative burst, mediated by Nox2 NADPH oxidase-derived reactive oxygen species, confers host defense against a broad spectrum of bacterial and fungal pathogens. Loss-of-function mutations that impair function of the Nox2 complex result in a life-threatening immunodeficiency, and genetic variants of Nox2 subunits have been implicated in pathogenesis of inflammatory bowel disease (IBD). Thus, alterations in the oxidative burst can profoundly impact host defense, yet little is known about regulatory mechanisms that fine-tune this response. Here we report the discovery of regulatory nodes controlling oxidative burst by functional screening of genes within loci linked to human inflammatory disease. Implementing a multi-omics approach, we define transcriptional, metabolic and ubiquitin-cycling nodes controlled by Rbpj, Pfkl and Rnf145, respectively. Furthermore, we implicate Rnf145 in proteostasis of the Nox2 complex by endoplasmic reticulum-associated degradation. Consequently, ablation of Rnf145 in murine macrophages enhances bacterial clearance, and rescues the oxidative burst defects associated with Ncf4 haploinsufficiency.

Human leucine-rich repeat proteins: a genome-wide bioinformatic categorization and functional analysis in innate immunity.

In innate immune sensing, the detection of pathogen-associated molecular patterns by recognition receptors typically involve leucine-rich repeats (LRRs). We provide a categorization of 375 human LRR-containing proteins, almost half of which lack other identifiable functional domains. We clustered human LRR proteins by first assigning LRRs to LRR classes and then grouping the proteins based on these class assignments, revealing several of the resulting protein groups containing a large number of proteins with certain non-LRR functional domains. In particular, a statistically significant number of LRR proteins in the typical (T) and bacterial + typical (S+T) categories have transmembrane domains, whereas most of the LRR proteins in the cysteine-containing (CC) category contain an F-box domain (which mediates interactions with the E3 ubiquitin ligase complex). Furthermore, by examining the evolutionary profiles of the LRR proteins, we identified a subset of LRR proteins exhibiting strong conservation in fungi and an enrichment for "nucleic acid-binding" function. Expression analysis of LRR genes identifies a subset of pathogen-responsive genes in human primary macrophages infected with pathogenic bacteria. Using functional RNAi, we show that MFHAS1 regulates Toll-like receptor (TLR)-dependent signaling. By using protein interaction network analysis followed by functional RNAi, we identified LRSAM1 as a component of the antibacterial autophagic response.

Ubiquitin accumulation in autophagy-deficient mice is dependent on the Nrf2-mediated stress response pathway: a potential role for protein aggregation in autophagic substrate selection.

Genetic ablation of autophagy in mice leads to liver and brain degeneration accompanied by the appearance of ubiquitin (Ub) inclusions, which has been considered to support the hypothesis that ubiquitination serves as a cis-acting signal for selective autophagy. We show that tissue-specific disruption of the essential autophagy genes Atg5 and Atg7 leads to the accumulation of all detectable Ub-Ub topologies, arguing against the hypothesis that any particular Ub linkage serves as a specific autophagy signal. The increase in Ub conjugates in Atg7(-/-) liver and brain is completely suppressed by simultaneous knockout of either p62 or Nrf2. We exploit a novel assay for selective autophagy in cell culture, which shows that inactivation of Atg5 leads to the selective accumulation of aggregation-prone proteins, and this does not correlate with an increase in substrate ubiquitination. We propose that protein oligomerization drives autophagic substrate selection and that the accumulation of poly-Ub chains in autophagy-deficient circumstances is an indirect consequence of activation of Nrf2-dependent stress response pathways.

Integrative systems biology and networks in autophagy.

The growing recognition that autophagy has important roles in many biological pathways, physiological systems, and infection and disease states necessitates a multidimensional perspective and systems-wide understanding of how autophagy is triggered or modulated by diverse stimuli. To delineate the nonlinearity and combinatorial complexity of biological networks and signaling pathways impinging on autophagy requires an integrative framework that brings together diverse information from genome-scale data (genomics, transcriptomics, proteomics, interactomics, functional RNAi screens) to dynamic time-series analyses and biochemical assays across a variety of biological and clinical contexts. We outline recent applications of genome-wide approaches to studying autophagy and highlight how some of these could be integrated to derive sub-networks that are more functionally focused. Viewed from a network perspective, the extensive interconnectivity between pathway systems converging on autophagy provides the essential foundation from which to systematically elucidate the regulatory nuances and crosstalks that orchestrate autophagic processes in different pathophysiological contexts.

Virus-plus-susceptibility gene interaction determines Crohn's disease gene Atg16L1 phenotypes in intestine.

It is unclear why disease occurs in only a small proportion of persons carrying common risk alleles of disease susceptibility genes. Here we demonstrate that an interaction between a specific virus infection and a mutation in the Crohn's disease susceptibility gene Atg16L1 induces intestinal pathologies in mice. This virus-plus-susceptibility gene interaction generated abnormalities in granule packaging and unique patterns of gene expression in Paneth cells. Further, the response to injury induced by the toxic substance dextran sodium sulfate was fundamentally altered to include pathologies resembling aspects of Crohn's disease. These pathologies triggered by virus-plus-susceptibility gene interaction were dependent on TNFalpha and IFNgamma and were prevented by treatment with broad spectrum antibiotics. Thus, we provide a specific example of how a virus-plus-susceptibility gene interaction can, in combination with additional environmental factors and commensal bacteria, determine the phenotype of hosts carrying common risk alleles for inflammatory disease.

Identifying relationships among genomic disease regions: predicting genes at pathogenic SNP associations and rare deletions.

Translating a set of disease regions into insight about pathogenic mechanisms requires not only the ability to identify the key disease genes within them, but also the biological relationships among those key genes. Here we describe a statistical method, Gene Relationships Among Implicated Loci (GRAIL), that takes a list of disease regions and automatically assesses the degree of relatedness of implicated genes using 250,000 PubMed abstracts. We first evaluated GRAIL by assessing its ability to identify subsets of highly related genes in common pathways from validated lipid and height SNP associations from recent genome-wide studies. We then tested GRAIL, by assessing its ability to separate true disease regions from many false positive disease regions in two separate practical applications in human genetics. First, we took 74 nominally associated Crohn's disease SNPs and applied GRAIL to identify a subset of 13 SNPs with highly related genes. Of these, ten convincingly validated in follow-up genotyping; genotyping results for the remaining three were inconclusive. Next, we applied GRAIL to 165 rare deletion events seen in schizophrenia cases (less than one-third of which are contributing to disease risk). We demonstrate that GRAIL is able to identify a subset of 16 deletions containing highly related genes; many of these genes are expressed in the central nervous system and play a role in neuronal synapses. GRAIL offers a statistically robust approach to identifying functionally related genes from across multiple disease regions--that likely represent key disease pathways. An online version of this method is available for public use (http://www.broad.mit.edu/mpg/grail/).