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.     

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.     

Genome-wide investigation reveals pathogen-specific and shared signatures in the response of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>to infection

Background  

There are striking similarities between the innate immune systems of invertebrates and vertebrates. Caenorhabditis elegans is increasingly used as a model for the study of innate immunity. Evidence is accumulating that C. elegans mounts distinct responses to different pathogens, but the true extent of this specificity is unclear. Here, we employ direct comparative genomic analyses to explore the nature of the host 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.     

The Alkaloid Compound Harmane Increases the Lifespan of Caenorhabditis elegans during Bacterial Infection,by Modulating the Nematode’s Innate Immune Response

The nematode Caenorhabditis elegans has in recent years been proven to be a powerful in vivo model for testing antimicrobial compounds. We report here that the alkaloid compound Harmane (2-methyl-β-carboline) increases the lifespan of nematodes infected with a human pathogen, the Shiga toxin-producing Escherichia coli O157:H7 strain EDL933 and several other bacterial pathogens. This was shown to be unrelated to the weak antibiotic effect of Harmane. Using GFP-expressing E. coli EDL933, we showed that Harmane does not lower the colonization burden in the nematodes. We also found that the expression of the putative immune effector gene F35E12.5 was up-regulated in response to Harmane treatment. This indicates that Harmane stimulates the innate immune response of the nematode; thereby increasing its lifespan during bacterial infection. Expression of F35E12.5 is predominantly regulated through the p38 MAPK pathway; however, intriguingly the lifespan extension resulting from Harmane was higher in p38 MAPK-deficient nematodes. This indicates that Harmane has a complex effect on the innate immune system of C. elegans. Harmane could therefore be a useful tool in the further research into C. elegans immunity. Since the innate immunity of C. elegans has a high degree of evolutionary conservation, drugs such as Harmane could also be possible alternatives to classic antibiotics. The C. elegans model could prove to be useful for selection and development of such drugs.     

Cranberry Extract Standardized for Proanthocyanidins Promotes the Immune Response of Caenorhabditis elegans to Vibrio cholerae through the p38 MAPK Pathway and HSF-1

Botanicals are rich in bioactive compounds, and some offer numerous beneficial effects to animal and human health when consumed. It is well known that phytochemicals in cranberries have anti-oxidative and antimicrobial activities. Recently, an increasing body of evidence has demonstrated that cranberry phytochemicals may have potential benefits that promote healthy aging. Here, we use Caenorhabditis elegans as a model to show that water-soluble cranberry extract standardized to 4.0% proanthocyanidins (WCESP), a major component of cranberries, can enhance host innate immunity to resist against Vibrio cholerae (V. cholerae; wild type C6706 (O1 El Tor biotype)) infection. Supplementation of WCESP did not significantly alter the intestinal colonization of V. cholerae, but upregulated the expression of C. elegans innate immune genes, such as clec-46, clec-71, fmo-2, pqn-5 and C23G10.1. Additionally, WCESP treatment did not affect the growth of V. cholerae and expression of the major bacterial virulence genes, and only slightly reduced bacterial colonization within C. elegans intestine. These findings indicate that the major components of WCESP, including proanthocyanidins (PACs), may play an important role in enhancing the host innate immunity. Moreover, we engaged C. elegans mutants and identified that the p38 MAPK signaling, insulin/IGF-1 signaling (IIS), and HSF-1 play pivotal roles in the WCESP-mediated host immune response. Considering the level of conservation between the innate immune pathways of C. elegans and humans, the results of this study suggest that WCESP may also play an immunity-promoting role in higher order organisms.     

Impact of Cigarette Smoke Exposure on Innate Immunity: A Caenorhabditis elegans Model

Background

Cigarette smoking is the major cause of chronic obstructive pulmonary disease (COPD) and lung cancer. Respiratory bacterial infections have been shown to be involved in the development of COPD along with impaired airway innate immunity.

Methodology/Principal Findings

To address the in vivo impact of cigarette smoke (CS) exclusively on host innate defense mechanisms, we took advantage of Caenorhabditis elegans (C. elegans), which has an innate immune system but lacks adaptive immune function. Pseudomonas aeruginosa (PA) clearance from intestines of C. elegans was dampened by CS. Microarray analysis identified 6 candidate genes with a 2-fold or greater reduction after CS exposure, that have a human orthologue, and that may participate in innate immunity. To confirm a role of CS-down-regulated genes in the innate immune response to PA, RNA interference (RNAi) by feeding was carried out in C. elegans to inhibit the gene of interest, followed by PA infection to determine if the gene affected innate immunity. Inhibition of lbp-7, which encodes a lipid binding protein, resulted in increased levels of intestinal PA. Primary human bronchial epithelial cells were shown to express mRNA of human Fatty Acid Binding Protein 5 (FABP-5), the human orthologue of lpb-7. Interestingly, FABP-5 mRNA levels from human smokers with COPD were significantly lower (p = 0.036) than those from smokers without COPD. Furthermore, FABP-5 mRNA levels were up-regulated (7-fold) after bacterial (i.e., Mycoplasma pneumoniae) infection in primary human bronchial epithelial cell culture (air-liquid interface culture).

Conclusions

Our results suggest that the C. elegans model offers a novel in vivo approach to specifically study innate immune deficiencies resulting from exposure to cigarette smoke, and that results from the nematode may provide insight into human airway epithelial cell biology and cigarette smoke exposure.     

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.     

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.     

Selenite Enhances Immune Response against Pseudomonas aeruginosa PA14 via SKN-1 in Caenorhabditis elegans

Background

Selenium (Se) is an important nutrient that carries out many biological processes including maintaining optimal immune function. Here, inorganic selenite (Se(IV)) was evaluated for its pathogen resistance and potential-associated factors in Caenorhabditis elegans. The immune effects of Se(IV) were investigated by examining the responses of C. elegans to Pseudomonas aerugonisa PA14 strain.

Principal Findings

Se(IV)-treated C. elegans showed increased survival under PA14 infection compared with untreated controls. The significant pathogen resistance of Se(IV) on C. elegans might not be attributed to the effects of Se(IV) on PA14 as Se(IV) showed no effect on bacterial quorum-sensing and virulence factors of PA14. This study showed that Se(IV) enhanced the expression of a gene pivotal for the innate immunity in C. elegans. The study found that the pathogen-resistant phenotypes contributed by Se(IV) was absent from the skn-1 mutant worms. Moreover, Se(IV) influenced the subcellular distribution of SKN-1/Nrf in C. elegans upon PA14 infection. Furthermore, Se(IV) increased mRNA levels of SKN-1 target genes (gst-4 and gcs-1).

Conclusions

This study found evidence of Se(IV) protecting C. elegans against P. aeruginosa PA14 infection by exerting effects on the innate immunity of C. elegans that is likely mediated via regulation of a SKN-1-dependent signaling pathway.     

Components of the Cultivated Red Seaweed Chondrus crispus Enhance the Immune Response of Caenorhabditis elegans to Pseudomonas aeruginosa through the pmk-1, daf-2/daf-16, and skn-1 Pathways

Marine macroalgae are rich in bioactive compounds that can, when consumed, impart beneficial effects on animal and human health. The red seaweed Chondrus crispus has been reported to have a wide range of health-promoting activities, such as antitumor and antiviral activities. Using a Caenorhabditis elegans infection model, we show that C. crispus water extract (CCWE) enhances host immunity and suppresses the expression of quorum sensing (QS) and the virulence factors of Pseudomonas aeruginosa (strain PA14). Supplementation of nematode growth medium with CCWE induced the expression of C. elegans innate immune genes, such as irg-1, irg-2, F49F1.6, hsf-1, K05D8.5, F56D6.2, C29F3.7, F28D1.3, F38A1.5 ZK6.7, lys-1, spp-1, and abf-1, by more than 2-fold, while T20G5.7 was not affected. Additionally, CCWE suppressed the expression of PA14 QS genes and virulence factors, although it did not affect the growth of the bacteria. These effects correlated with a 28% reduction in the PA14-inflicted killing of C. elegans. Kappa-carrageenan (K-CGN), a major component of CCWE, was shown to play an important role in the enhancement of host immunity. Using C. elegans mutants, we identified that pmk-1, daf-2/daf-16, and skn-1 are essential in the K-CGN-induced host immune response. In view of the conservation of innate immune pathways between C. elegans and humans, the results of this study suggest that water-soluble components of C. crispus may also play a health-promoting role in higher animals and humans.     

Thirty years of resistance: Zig-zag through the plant immune system

Understanding the plant immune system is crucial for using genetics to protect crops from diseases. Plants resist pathogens via a two-tiered innate immune detection-and-response system. The first plant Resistance (R) gene was cloned in 1992 . Since then, many cell-surface pattern recognition receptors (PRRs) have been identified, and R genes that encode intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) have been cloned. Here, we provide a list of characterized PRRs and NLRs. In addition to immune receptors, many components of immune signaling networks were discovered over the last 30 years. We review the signaling pathways, physiological responses, and molecular regulation of both PRR- and NLR-mediated immunity. Recent studies have reinforced the importance of interactions between the two immune systems. We provide an overview of interactions between PRR- and NLR-mediated immunity, highlighting challenges and perspectives for future research.

A review of major research advances in plant immunity during the last three decades and individual characterized immune receptors, their immune signaling pathways, and interactions between immune systems     

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.     

mir-233 Modulates the Unfolded Protein Response in C. elegans during Pseudomonas aeruginosa Infection

The unfolded protein response (UPR), which is activated by perturbations of the endoplasmic reticulum homeostasis, has been shown to play an important role in innate immunity and inflammation. However, little is known about the molecular mechanisms underlying activation of the UPR during immune responses. Using small RNA deep sequencing and reverse genetic analysis, we show that the microRNA mir-233 is required for activation of the UPR in Caenorhabditis elegans exposed to Pseudomonas aeruginosa PA14. P. aeruginosa infection up-regulates the expression of mir-233 in a p38 MAPK-dependent manner. Quantitative proteomic analysis identifies SCA-1, a C. elegans homologue of the sarco/endoplasmic reticulum Ca²⁺-ATPase, as a target of mir-233. During P. aeruginosa PA14 infection, mir-233 represses the protein levels of SCA-1, which in turn leads to activation of the UPR. Whereas mir-233 mutants are more sensitive to P. aeruginosa infection, knockdown of sca-1 leads to enhanced resistance to the killing by P. aeruginosa. Our study indicates that microRNA-dependent pathways may have an impact on innate immunity by activating the UPR.