Functional assessment of Nramp-like metal transporters and manganese in Caenorhabditis elegans

Nramp1 (natural resistance-associated macrophage protein-1) is a functionally conserved iron-manganese transporter in macrophages. Manganese (Mn), a superoxide scavenger, is required in trace amounts and functions as a cofactor for most antioxidants. Three Nramp homologs, smf-1, smf-2, and smf-3, have been identified thus far in the nematode Caenorhabditis elegans. A GFP promoter assay revealed largely intestinal expression of the smf genes from early embryonic through adult stages. In addition, smf deletion mutants showed increased sensitivity to excess Mn and mild sensitivity to EDTA. Interestingly, these smf deletion mutants demonstrated hypersensitivity to the pathogen Staphylococcus aureus, an effect that was rescued by Mn feeding or knockdown of the Golgi calcium/manganese ATPase, pmr-1, indicating that Mn uptake is essential for the innate immune system. This reversal of pathogen sensitivity by Mn feeding suggests a protective and therapeutic role of Mn in pathogen evasion systems. We propose that the C. elegans intestinal lumen may mimic the mammalian macrophage phagosome and thus could be a simple model for studying Mn-mediated innate immunity.     

The Evolutionarily Conserved Mediator Subunit MDT-15/MED15 Links Protective Innate Immune Responses and Xenobiotic Detoxification

Metazoans protect themselves from environmental toxins and virulent pathogens through detoxification and immune responses. We previously identified a small molecule xenobiotic toxin that extends survival of Caenorhabditis elegans infected with human bacterial pathogens by activating the conserved p38 MAP kinase PMK-1 host defense pathway. Here we investigate the cellular mechanisms that couple activation of a detoxification response to innate immunity. From an RNAi screen of 1,420 genes expressed in the C. elegans intestine, we identified the conserved Mediator subunit MDT-15/MED15 and 28 other gene inactivations that abrogate the induction of PMK-1-dependent immune effectors by this small molecule. We demonstrate that MDT-15/MED15 is required for the xenobiotic-induced expression of p38 MAP kinase PMK-1-dependent immune genes and protection from Pseudomonas aeruginosa infection. We also show that MDT-15 controls the induction of detoxification genes and functions to protect the host from bacteria-derived phenazine toxins. These data define a central role for MDT-15/MED15 in the coordination of xenobiotic detoxification and innate immune responses.     

Comparative Genomics RNAi Screen Identifies Eftud2 as a Novel Regulator of Innate Immunity

The extent of the innate immune response is regulated by many positively and negatively acting signaling proteins. This allows for proper activation of innate immunity to fight infection while ensuring that the response is limited to prevent unwanted complications. Thus mutations in innate immune regulators can lead to immune dysfunction or to inflammatory diseases such as arthritis or atherosclerosis. To identify novel innate immune regulators that could affect infectious or inflammatory disease, we have taken a comparative genomics RNAi screening approach in which we inhibit orthologous genes in the nematode Caenorhabditis elegans and murine macrophages, expecting that genes with evolutionarily conserved function also will regulate innate immunity in humans. Here we report the results of an RNAi screen of approximately half of the C. elegans genome, which led to the identification of many candidate genes that regulate innate immunity in C. elegans and mouse macrophages. One of these novel conserved regulators of innate immunity is the mRNA splicing regulator Eftud2, which we show controls the alternate splicing of the MyD88 innate immunity signaling adaptor to modulate the extent of the innate immune response.     

Characterization of peripheral blood mononuclear cells gene expression profiles of pediatric Staphylococcus aureus persistent and non-carriers using a targeted assay

Defects in innate immunity affect many different physiologic systems and several studies of patients with primary immunodeficiency disorders demonstrated the importance of innate immune system components in disease prevention or colonization of bacterial pathogens. To assess the role of the innate immune system on nasal colonization with Staphylococcus aureus, innate immune responses in pediatric S. aureus nasal persistent carriers (n = 14) and non-carriers (n = 15) were profiled by analyzing co-clustered gene sets (modules). We stimulated previously frozen peripheral blood mononuclear cells (PBMCs) from these subjects with i) a panel of TLR ligands, ii) live S. aureus (either a mixture of strains or stimulation with respective carriage isolates), or iii) heat-killed S. aureus. We found no difference in responses between carriers and non-carriers when PBMCs were stimulated with a panel of TLR ligands. However, PBMC gene expression profiles differed between persistent and non-S. aureus carriers following stimulation with either live or dead S. aureus. These observations suggest that individuals susceptible to persistent carriage with S. aureus may possess differences in their live/dead bacteria recognition pathway and that innate pathway signaling is different between persistent and non-carriers of S. aureus.     

Enhanced Monocyte Response and Decreased Central Memory T Cells in Children with Invasive Staphylococcus aureus Infections

Staphylococcus aureus has emerged as a significant pathogen causing severe invasive disease in otherwise healthy people. Despite considerable advances in understanding the epidemiology, resistance mechanisms, and virulence factors produced by the bacteria, there is limited knowledge of the in vivo host immune response to acute, invasive S. aureus infections. Herein, we report that peripheral blood mononuclear cells from patients with severe S. aureus infections demonstrate a distinctive and robust gene expression profile which is validated in a distinct group of patients and on a different microarray platform. Application of a systems-wide modular analysis framework reveals significant over-expression of innate immunity genes and under-expression of genes related to adaptive immunity. Simultaneous flow cytometry analyses demonstrated marked alterations in immune cell numbers, with decreased central memory CD4 and CD8 T cells and increased numbers of monocytes. CD14+ monocyte numbers significantly correlated with the gene expression levels of genes related to the innate immune response. These results demonstrate the value of applying a systems biology approach that reveals the significant alterations in the components of circulating blood lymphocytes and monocytes in invasive S. aureus infections.     

Insights into the functions of the death associated protein kinases from C. elegans and other invertebrates

The death associated protein kinases (DAPK) are a phylogenetically widespread family of calcium-regulated serine/threonine kinases, initially identified from their roles in apoptosis. Subsequent studies, principally in vertebrate cells or models, have elucidated the functions of the DAPK family in autophagy and tumor suppression. Invertebrate genetic model organisms such as Drosophila and C. elegans have revealed additional functions for DAPK and related kinases. In the nematode C. elegans, the sole DAPK family member DAPK-1 positively regulates starvation-induced autophagy. Genetic analysis in C. elegans has revealed that DAPK-1 also acts as a negative regulator of epithelial innate immune responses in the epidermis. This negative regulatory role for DAPK in innate immunity may be analogous to the roles of mammalian DAPK in inflammatory responses.     

Activation of Imd pathway in hemocyte confers infection resistance through humoral response in Drosophila

Upon microbial invasion the innate immune system of Drosophila melanogaster mounts a response that comes in two distinct but complimentary forms, humoral and cellular. A screen to find genes capable of conferring resistance to the Gram-positive Staphylococcus aureus upon ectopic expression in immune response tissues uncovered imd gene. This resistance was not dependent on cellular defenses but rather likely a result of upregulation of the humoral response through increased expression of antimicrobial peptides, including a Toll pathway reporter gene drosomycin. Taken together it appears that Imd pathway is capable of playing a role in resistance to the Gram-positive S. aureus, counter to notions of traditional roles of the Imd pathway thought largely to responsible for resistance to Gram-negative bacteria.     

A trade-off switch of two immunological memories in Caenorhabditis elegans reinfected by bacterial pathogens

Recent studies have suggested that innate immune responses exhibit characteristics associated with memory linked to modulations in both vertebrates and invertebrates. However, the diverse evolutionary paths taken, particularly within the invertebrate taxa, should lead to similarly diverse innate immunity memory processes. Our understanding of innate immune memory in invertebrates primarily comes from studies of the fruit fly Drosophila melanogaster, the generality of which is unclear. Caenorhabditis elegans typically inhabits soil harboring a variety of fatal microbial pathogens; for this invertebrate, the innate immune system and aversive behavior are the major defensive strategies against microbial infection. However, their characteristics of immunological memory remains infantile. Here we discovered an immunological memory that promoted avoidance and suppressed innate immunity during reinfection with bacteria, which we revealed to be specific to the previously exposed pathogens. During this trade-off switch of avoidance and innate immunity, the chemosensory neurons AWB and ADF modulated production of serotonin and dopamine, which in turn decreased expression of the innate immunity-associated genes and led to enhanced avoidance via the downstream insulin-like pathway. Therefore, our current study profiles the immune memories during C. elegans reinfected by pathogenic bacteria and further reveals that the chemosensory neurons, the neurotransmitter(s), and their associated molecular signaling pathways are responsible for a trade-off switch between the two immunological memories.     

O-GlcNAc cycling: Emerging roles in development and epigenetics

The nutrient-sensing hexosamine signaling pathway modulates the levels of O-linked N-acetylglucosamine (O-GlcNAc) on key targets impacting cellular signaling, protein turnover and gene expression. O-GlcNAc cycling may be deregulated in neurodegenerative disease, cancer, and diabetes. Studies in model organisms demonstrate that the O-GlcNAc transferase (OGT/Sxc) is essential for Polycomb group (PcG) repression of the homeotic genes, clusters of genes responsible for the adult body plan. Surprisingly, from flies to man, the O-GlcNAcase (OGA, MGEA5) gene is embedded within the NK cluster, the most evolutionarily ancient of three homeobox gene clusters regulated by PcG repression. PcG repression also plays a key role in maintaining stem cell identity, recruiting the DNA methyltransferase machinery for imprinting, and in X-chromosome inactivation. Intriguingly, the Ogt gene resides near the Xist locus in vertebrates and is subject to regulation by PcG-dependent X-inactivation. OGT is also an enzymatic component of the human dosage compensation complex. These ‘evo-devo’ relationships linking O-GlcNAc cycling to higher order chromatin structure provide insights into how nutrient availability may influence the epigenetic regulation of gene expression. O-GlcNAc cycling at promoters and PcG repression represent concrete mechanisms by which nutritional information may be transmitted across generations in the intra-uterine environment. Thus, the nutrient-sensing hexosamine signaling pathway may be a key contributor to the metabolic deregulation resulting from prenatal exposure to famine, or the ‘vicious cycle’ observed in children of mothers with type-2 diabetes and metabolic disease.     

Non-Classical ProIL-1beta Activation during Mammary Gland Infection Is Pathogen-Dependent but Caspase-1 Independent

Infection of the mammary gland with live bacteria elicits a pathogen-specific host inflammatory response. To study these host-pathogen interactions wild type mice, NF-kappaB reporter mice as well as caspase-1 and IL-1beta knockout mice were intramammarily challenged with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The murine mastitis model allowed to compare the kinetics of the induced cytokine protein profiles and their underlying pathways. In vivo and ex vivo imaging showed that E. coli rapidly induced NF-kappaB inflammatory signaling concomitant with high mammary levels of TNF-alpha, IL-1 alpha and MCP-1 as determined by multiplex analysis. In contrast, an equal number of S. aureus bacteria induced a low NF-kappaB activity concomitant with high mammary levels of the classical IL-1beta fragment. These quantitative and qualitative differences in local inflammatory mediators resulted in an earlier neutrophil influx and in a more extensive alveolar damage post-infection with E. coli compared to S. aureus. Western blot analysis revealed that the inactive proIL-1beta precursor was processed into pathogen-specific IL-1beta fragmentation patterns as confirmed with IL-1beta knockout animals. Additionally, caspase-1 knockout animals allowed to investigate whether IL-1beta maturation depended on the conventional inflammasome pathway. The lack of caspase-1 did not prevent extensive proIL-1beta fragmentation by either of S. aureus or E. coli. These non-classical IL-1beta patterns were likely caused by different proteases and suggest a sentinel function of IL-1beta during mammary gland infection. Thus, a key signaling nodule can be defined in the differential host innate immune defense upon E. coli versus S. aureus mammary gland infection, which is independent of caspase-1.     

EGL-9 Controls C. elegans Host Defense Specificity through Prolyl Hydroxylation-Dependent and -Independent HIF-1 Pathways

Understanding host defense against microbes is key to developing new and more effective therapies for infection and inflammatory disease. However, how animals integrate multiple environmental signals and discriminate between different pathogens to mount specific and tailored responses remains poorly understood. Using the genetically tractable model host Caenorhabditis elegans and pathogenic bacterium Staphylococcus aureus, we describe an important role for hypoxia-inducible factor (HIF) in defining the specificity of the host response in the intestine. We demonstrate that loss of egl-9, a negative regulator of HIF, confers HIF-dependent enhanced susceptibility to S. aureus while increasing resistance to Pseudomonas aeruginosa. In our attempt to understand how HIF could have these apparently dichotomous roles in host defense, we find that distinct pathways separately regulate two opposing functions of HIF: the canonical pathway is important for blocking expression of a set of HIF-induced defense genes, whereas a less well understood noncanonical pathway appears to be important for allowing the expression of another distinct set of HIF-repressed defense genes. Thus, HIF can function either as a gene-specific inducer or repressor of host defense, providing a molecular mechanism by which HIF can have apparently opposing roles in defense and inflammation. Together, our observations show that HIF can set the balance between alternative pathogen-specific host responses, potentially acting as an evolutionarily conserved specificity switch in the host innate immune response.     

Endoplasmic Reticulum Stress Pathway Required for Immune Homeostasis Is Neurally Controlled by Arrestin-1

In response to pathogen infection, the host innate immune system activates microbial killing pathways and cellular stress pathways that need to be balanced because insufficient or excessive immune responses have deleterious consequences. Recent studies demonstrate that two G protein-coupled receptors (GPCRs) in the nervous system of Caenorhabditis elegans control immune homeostasis. To investigate further how GPCR signaling controls immune homeostasis at the organismal level, we studied arrestin-1 (ARR-1), which is the only GPCR adaptor protein in C. elegans. The results indicate that ARR-1 is required for GPCR signaling in ASH, ASI, AQR, PQR, and URX neurons, which control the unfolded protein response and a p38 mitogen-activated protein kinase signaling pathway required for innate immunity. ARR-1 activity also controlled immunity through ADF chemosensory and AFD thermosensory neurons that regulate longevity. Furthermore, we found that although ARR-1 played a key role in the control of immunity by AFD thermosensory neurons, it did not control longevity through these cells. However, ARR-1 partially controlled longevity through ADF neurons.     

An RIG-I-Like RNA Helicase Mediates Antiviral RNAi Downstream of Viral siRNA Biogenesis in Caenorhabditis elegans

Dicer ribonucleases of plants and invertebrate animals including Caenorhabditis elegans recognize and process a viral RNA trigger into virus-derived small interfering RNAs (siRNAs) to guide specific viral immunity by Argonaute-dependent RNA interference (RNAi). C. elegans also encodes three Dicer-related helicase (drh) genes closely related to the RIG-I-like RNA helicase receptors which initiate broad-spectrum innate immunity against RNA viruses in mammals. Here we developed a transgenic C. elegans strain that expressed intense green fluorescence from a chromosomally integrated flock house virus replicon only after knockdown or knockout of a gene required for antiviral RNAi. Use of the reporter nematode strain in a feeding RNAi screen identified drh-1 as an essential component of the antiviral RNAi pathway. However, RNAi induced by either exogenous dsRNA or the viral replicon was enhanced in drh-2 mutant nematodes, whereas exogenous RNAi was essentially unaltered in drh-1 mutant nematodes, indicating that exogenous and antiviral RNAi pathways are genetically distinct. Genetic epistatic analysis shows that drh-1 acts downstream of virus sensing and viral siRNA biogenesis to mediate specific antiviral RNAi. Notably, we found that two members of the substantially expanded subfamily of Argonautes specific to C. elegans control parallel antiviral RNAi pathways. These findings demonstrate both conserved and unique strategies of C. elegans in antiviral defense.