Scott Alper

Specificity and Complexity of the Caenorhabditis elegans Innate Immune Response

ABSTRACT In response to infection, Caenorhabditis elegans produces an array of antimicrobial proteins. To understand the C. elegans immune response, we have investigated the regulation of a large, representative sample of candidate antimicrobial genes. We found that all these putative antimicrobial genes are expressed in tissues exposed to the environment, a position from which they can ward off infection. Using RNA interference to inhibit the function of immune signaling pathways in C. elegans, we found that different immune response pathways regulate expression of distinct but overlapping sets of antimicrobial genes. We also show that different bacterial pathogens regulate distinct but overlapping sets of antimicrobial genes. The patterns of genes induced by pathogens do not coincide with any single immune signaling pathway. Thus, even in this simple model system for innate immunity, striking specificity and complexity exist in the immune response. The unique patterns of antimicrobial gene expression observed when C. elegans is exposed to different pathogens or when different immune signaling pathways are perturbed suggest that a large set of yet to be identified pathogen recognition receptors (PRRs) exist in the nematode. These PRRs must interact in a complicated fashion to induce a unique set of antimicrobial genes. We also propose the existence of an “antimicrobial fingerprint,” which will aid in assigning newly identified C. elegans innate immunity genes to known immune signaling pathways.

Toxicogenomic analysis of Caenorhabditis elegans reveals novel genes and pathways involved in the resistance to cadmium toxicity

BackgroundExposure to cadmium is associated with a variety of human diseases. At low concentrations, cadmium activates the transcription of stress-responsive genes, which can prevent or repair the adverse effects caused by this metal.ResultsUsing Caenorhabditis elegans, 290 genes were identified that are differentially expressed (>1.5-fold) following a 4 or 24 hour exposure to cadmium. Several of these genes are known to be involved in metal detoxification, including mtl-1, mtl-2, cdr-1 and ttm-1, confirming the efficacy of the study. The majority, however, were not previously associated with metal-responsiveness and are novel. Gene Ontology analysis mapped these genes to cellular/ion trafficking, metabolic enzymes and proteolysis categories. RNA interference-mediated inhibition of 50 cadmium-responsive genes resulted in an increased sensitivity to cadmium toxicity, demonstrating that these genes are involved in the resistance to cadmium toxicity. Several functional protein interacting networks were identified by interactome analysis. Within one network, the signaling protein KEL-8 was identified. Kel-8 protects C. elegans from cadmium toxicity in a mek-1 (MAPKK)-dependent manner.ConclusionBecause many C. elegans genes and signal transduction pathways are evolutionarily conserved, these results may contribute to the understanding of the functional roles of various genes in cadmium toxicity in higher organisms.

Identification of innate immunity genes and pathways using a comparative genomics approach

To reveal regulators of innate immunity, we used RNAi assays to monitor the immune response when genes are inhibited in Caenorhabditis elegans and mouse macrophages. Genes that altered innate immune responsiveness in C. elegans were validated in murine macrophages, resulting in the discovery of 11 genes that regulate the innate immune response in both systems and the subsequent identification of a protein interaction network with a conserved role in innate immunity regulation. We confirmed the role of four of these 11 genes in antimicrobial gene regulation using available mutants in C. elegans. Several of these genes (acy-1, tub-2, and tbc-1) also regulate susceptibility to the pathogen Pseudomonas aeruginosa. These genes may prove critical to understanding host defense and represent potential therapeutic targets for infectious and immunological diseases.

Identification of Novel Genes That Mediate Innate Immunity Using Inbred Mice

Innate immunity is the first line of defense against microbial infections. Although polymorphisms in toll-like receptors (TLRs) and downstream signaling molecules (CD14, TLR2, TLR4, TLR5, and IRAK4) affect the innate immune response, these variants account for only a portion of the ability of the host to respond to bacteria, fungi, and viruses. To identify other genes involved in the innate immune response, we challenged 16 inbred murine strains with lipopolysaccharide (LPS) systemically and measured serum concentrations of pro-inflammatory cytokines IL-1β, IL-6, and TNFα, and the chemokine KC 6 hr post-treatment. Loci that segregate with strain phenotypes were identified by whole genome association (WGA) mapping of cytokine concentrations. Published gene expression profiles and quantitative trait loci (QTL) were then utilized to prioritize loci and genes that potentially regulate the host response to LPS. Sixteen loci were selected for further investigation by combining WGA analysis with previously published QTL for murine response to LPS or gram negative bacteria. Thirty-eight genes within these loci were then selected for further investigation on the basis of the significance of the identified locus, transcriptional response to LPS, and biological plausibility. RNA interference-mediated inhibition of 4 of 38 candidate genes was shown to block the production of IL-6 in J774A.1 macrophages. In summary, our analysis identified 4 genes that have not previously been implicated in innate immunity, namely, 1110058L19Rik, 4933415F23Rik, Fbxo9, and Ipo7. These genes could represent potential sepsis biomarkers or therapeutic targets that should be further investigated in human populations.

The Caenorhabditis elegans Germ Line Regulates Distinct Signaling Pathways to Control Lifespan and Innate Immunity

The relationship between the mechanisms that control an organisms lifespan and its ability to respond to environmental challenges are poorly understood. In Caenorhabditis elegans, an insulin-like signaling pathway modulates lifespan and the innate immune response to bacterial pathogens via a common mechanism involving transcriptional regulation by the DAF-16/FOXO transcription factor. The C. elegans germ line also modulates lifespan in a daf-16-dependent manner. Here, we show that the germ line controls the innate immune response of C. elegans somatic cells to two different Gram-negative bacteria. In contrast to the insulin-like signaling pathway, the germ line acts via distinct signaling pathways to control lifespan and innate immunity. Under standard nematode culture conditions, the germ line regulates innate immunity in parallel to a known p38 MAPK signaling pathway, via a daf-16-independent pathway. Our findings indicate that a complex regulatory network integrates inputs from insulin-like signaling, p38 MAPK signaling, and germ line stem cells to control innate immunity in C. elegans. We also confirm that innate immunity and lifespan in C. elegans are distinct processes, as nonoverlapping regulatory networks control survival in the presence of pathogenic and nonpathogenic bacteria. Finally, we demonstrate that the p38 MAPK pathway in C. elegans is activated to a similar extent by both pathogenic and nonpathogenic bacteria, suggesting that both can induce the nematode innate immune response.

Novel Regulators of the Systemic Response to Lipopolysaccharide

Our understanding of the role that host genetic factors play in the initiation and severity of infections caused by gram-negative bacteria is incomplete. To identify novel regulators of the host response to lipopolysaccharide (LPS), 11 inbred murine strains were challenged with LPS systemically. In addition to two strains lacking functional TLR4 (C3H/HeJ and C57BL/6J(TLR4-/-)), three murine strains with functional TLR4 (C57BL/6J, 129/SvImJ, and NZW/LacJ) were found to be relatively resistant to systemic LPS challenge; the other six strains were classified as sensitive. RNA from lung, liver, and spleen tissue was profiled on oligonucleotide microarrays to determine if unique transcripts differentiate susceptible and resistant strains. Gene expression analysis identified the Hedgehog signaling pathway and a number of transcription factors (TFs) involved in the response to LPS. RNA interference-mediated inhibition of six TFs (C/EBP, Cdx-2, E2F1, Hoxa4, Nhlh1, and Tead2) was found to diminish IL-6 and TNF-α production by murine macrophages. Mouse lines with targeted mutations were used to verify the involvement of two novel genes in innate immunity. Compared with wild-type control mice, mice deficient in the E2F1 transcription factor were found to have a reduced inflammatory response to systemic LPS, and mice heterozygote for Ptch, a gene involved in Hedgehog signaling, were found to be more responsive to systemic LPS. Our analysis of gene expression data identified novel pathways and transcription factors that regulate the host response to systemic LPS. Our results provide potential sepsis biomarkers and therapeutic targets that should be further investigated in human populations.

Limiting of the Innate Immune Response by SF3A-Dependent Control of MyD88 Alternative mRNA Splicing

Controlling infectious disease without inducing unwanted inflammatory disease requires proper regulation of the innate immune response. Thus, innate immunity needs to be activated when needed during an infection, but must be limited to prevent damage. To accomplish this, negative regulators of innate immunity limit the response. Here we investigate one such negative regulator encoded by an alternative splice form of MyD88. MyD88 mRNA exists in two alternative splice forms: MyD88L, a long form that encodes a protein that activates innate immunity by transducing Toll-like receptor (TLR) signals; and a short form that encodes a different protein, MyD88S, that inhibits the response. We find that MyD88S levels regulate the extent of inflammatory cytokine production in murine macrophages. MyD88S mRNA levels are regulated by the SF3A and SF3B mRNA splicing complexes, and these mRNA splicing complexes function with TLR signaling to regulate MyD88S production. Thus, the SF3A mRNA splicing complex controls production of a negative regulator of TLR signaling that limits the extent of innate immune activation.

Identification of Novel Innate Immune Genes by Transcriptional Profiling of Macrophages Stimulated with TLR Ligands

Toll-like receptors (TLRs) are key receptors in innate immunity and trigger responses following interaction with pathogen-associated molecular patterns (PAMPs). TLR3, TLR4 and TLR9 recognize double stranded RNA, lipopolysaccharide (LPS) and CpG DNA, respectively. These receptors differ importantly in downstream adaptor molecules. TLR4 signals through MyD88 and TRIF; in contrast, the TLR3 pathway involves only TRIF while TLR9 signals solely through MyD88. To determine how differences in downstream signaling could influence gene expression in innate immunity, gene expression patterns were determined for the RAW264.7 macrophage cell line stimulated with LPS, poly (I:C), or CpG DNA. Gene expression profiles 6 and 24h post-stimulation were analyzed to determine genes, pathways and transcriptional networks induced. As these experiments showed, the number and extent of genes expressed varied with stimulus. LPS and poly (I:C) induced an abundant array of genes in RAW264.7 cells at 6h and 24h following treatment while CpG DNA induced many fewer. By analyzing data for networks and pathways, we prioritized differentially expressed genes with respect to those common to the three TLR ligands as well as those shared by LPS and poly (I:C) but not CpG DNA. The importance of changes in gene expression was demonstrated by experiments indicating that RNA interference-mediated inhibition of two genes identified in this analysis, PLEC1 and TPST1, reduced IL-6 production by J774A.1 and RAW264.7 macrophages stimulated with LPS. Together, these findings delineate macrophage gene response patterns induced by different PAMPs and identify new genes that have not previously been implicated in innate immunity.

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.

An Evolutionarily Conserved Innate Immunity Protein Interaction Network

Background: Innate immunity affects infectious and inflammatory diseases. Results: Using RNAi and proteomic data, we identified a novel evolutionarily conserved protein network that modulates innate immunity. Conclusion: Studies using mutant C. elegans and mice demonstrate the utility of this network for disease investigation. Significance: This innate immunity network provides a novel set of targets for future innate immunity disease studies. The innate immune response plays a critical role in fighting infection; however, innate immunity also can affect the pathogenesis of a variety of diseases, including sepsis, asthma, cancer, and atherosclerosis. To identify novel regulators of innate immunity, we performed comparative genomics RNA interference screens in the nematode Caenorhabditis elegans and mouse macrophages. These screens have uncovered many candidate regulators of the response to lipopolysaccharide (LPS), several of which interact physically in multiple species to form an innate immunity protein interaction network. This protein interaction network contains several proteins in the canonical LPS-responsive TLR4 pathway as well as many novel interacting proteins. Using RNAi and overexpression studies, we show that almost every gene in this network can modulate the innate immune response in mouse cell lines. We validate the importance of this network in innate immunity regulation in vivo using available mutants in C. elegans and mice.