using-rna-interference-to-investigate-innate-immune-response-mouse

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

National Jewish Health and University of Colorado School of Medicine

During larval development in C. elegans, some of the cells of the ventral epidermis, the Pn.p cells, fuse with the growing epidermal syncytium hyp7. The pattern of these cell fusions is regulated in a complex, sexually dimorphic manner. It is essential that some Pn.p cells remain unfused in order for some sex-specific mating structures to be generated. The pattern of Pn.p cell fusion is regulated combinatorially by two genes of the C. elegans Hox gene cluster: lin-39 and mab-5. Some of the complexity in the Pn.p cell fusion pattern arises because these two Hox proteins can regulate each other's activities. We describe a zinc-finger transcription factor, REF-2, that is required for the Pn.p cells to be generated and to remain unfused. REF-2 functions with the Hox proteins to prevent Pn.p cell fusion. ref-2 may also be a transcriptional target of the Hox proteins.

The zinc finger protein REF-2 functions with the Hox genes to inhibit cell fusion in the ventral epidermis of C. elegans.

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.

Specificity and complexity of the Caenorhabditis elegans innate immune response.

Exposure 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.

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

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 innate immunity genes and pathways using a comparative genomics approach.

In the nematode Caenorhabditis elegans, 300 of the 959 somatic nuclei present in the adult hermaphrodite are located in syncytia. These syncytia are formed by the fusion of mononucleate cells throughout embryonic and postembryonic development. These cell fusions occur in a well-characterized stereotypical pattern, allowing investigators to study many cell fusion events at the molecular and cellular levels. Using tools that allow visualization of cell membranes, cell junctions, and cell cytoplasm during fusion, genetic screens have identified many C. elegans cell fusion genes, including those that regulate the fusion cell fate decision and two genes that encode components of the cell fusion machinery.

Cell fusion in Caenorhabditis elegans.

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.

Impact of cigarette smoke exposure on innate immunity: a Caenorhabditis elegans model.

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-1beta, IL-6, and TNFalpha, 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.

Identification of novel genes that mediate innate immunity using inbred mice.

The relationship between the mechanisms that control an organism's 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.

The Caenorhabditis elegans germ line regulates distinct signaling pathways to control lifespan and innate immunity.

In humans, there is an interdependent relationship between aging and immune system function, with each process affecting the outcome of the other. Aging can trigger immune system dysfunction, and alterations in the immune response can in turn affect human lifespan. Genetic experiments in model organisms such as C. elegans and Drosophila have led to the identification of numerous genes and signaling pathways that can modulate organismal lifespan and immune system function. Importantly, many of these signaling pathways exhibit conserved function in multiple species, including mammals, suggesting that the research in these simpler models could one day pave the way for the modulation of aging and immunity in humans. Here, we review the recent progress in our understanding of aging, innate immunity and the interaction between these two processes using these simple model systems. Additionally, we discuss what this may tell us about aging and the innate immune system in humans.

Model systems to the rescue: The relationship between aging and innate immunity.

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.

Novel regulators of the systemic response to lipopolysaccharide.

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.

Identification of novel innate immune genes by transcriptional profiling of macrophages stimulated with TLR ligands.

Abbiamo precedentemente identificato Tbc1d23 come regolatore dell&#39;immunit&agrave; innata utilizzando schermi di genomica comparativa RNA interference in Caenorhabditis elegans e mouse macrofagi romanzo candidato. Utilizzando topi knockout Tbc1d23 e macrofagi ingegnerizzati per overexpress Tbc1d23, mostriamo ora che Tbc1d23 &egrave; un inibitore generale dell&#39;immunit&agrave; innata, segnalazione, inibendo fortemente pi&ugrave; TLR e dectin-segnalazione percorsi. Tbc1d23 probabili atti a monte della trascrizione e a valle di adattatori TLR-segnalazione MyD88 e Trif fattore XBP1. Importante, come XBP1, Tbc1d23 colpisce la manutenzione, ma non l&#39;iniziazione, della produzione di citochine infiammatorie indotta da LPS. Tbc1d23 agisce come un RAB-GAP per regolare l&#39;immunit&agrave; innata di segnalazione. Cos&igrave;, Tbc1d23 esercita il suo effetto inibitorio sull&#39;immunit&agrave; innata, segnalando in maniera spatiotemporal. L&#39;identificazione di un romanzo spatiotemporal regolatore di immunit&agrave; innata segnalazione convalida l&#39;approccio di genomica comparativa per la scoperta del gene dell&#39;immunit&agrave; innata.

Spatiotemporal inibizione dell&#39;immunit&agrave; innata segnalazione da Tbc1d23 RAB-GAP.

&#x7DDA;&#x866B;&#x306E;&#x7DDA;&#x866B;&#x3068;&#x30DE;&#x30A6;&#x30B9;&#x306E;&#x30DE;&#x30AF;&#x30ED;&#x30D5;&#x30A1;&#x30FC;&#x30B8;&#x306E;&#x6BD4;&#x8F03;&#x30B2;&#x30CE;&#x30E0; RNA &#x5E72;&#x6E09;&#x753B;&#x9762;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x81EA;&#x7136;&#x514D;&#x75AB;&#x306E;&#x5019;&#x88DC;&#x306F;&#x3001;&#x5C0F;&#x8AAC;&#x306E;&#x8ABF;&#x6574;&#x88C5;&#x7F6E;&#x3068;&#x3057;&#x3066;&#x306E; Tbc1d23 &#x4EE5;&#x524D;&#x8B58;&#x5225;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306F;&#x4ECA; Tbc1d23 &#x30B7;&#x30B0;&#x30CA;&#x30EA;&#x30F3;&#x30B0;&#x3001;&#x5F37;&#x304F;&#x8907;&#x6570; TLR &#x3068;&#x30D2;&#x30C8;&#x30C7;&#x30AF;&#x30C1;&#x30F3; &#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x3092;&#x963B;&#x5BB3;&#x3059;&#x308B;&#x81EA;&#x7136;&#x514D;&#x75AB;&#x306E;&#x4E00;&#x822C;&#x7684;&#x306A;&#x963B;&#x5BB3;&#x5264;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x793A;&#x3059; Tbc1d23 &#x30CE;&#x30C3;&#x30AF;&#x30A2;&#x30A6;&#x30C8; &#x30DE;&#x30A6;&#x30B9; &#x30FB; &#x30DE;&#x30AF;&#x30ED;&#x30D5;&#x30A1;&#x30FC;&#x30B8;&#x306E;&#x904E;&#x5270; Tbc1d23 &#x30EA;&#x30D0;&#x30FC;&#x30B9; &#x30A8;&#x30F3;&#x30B8;&#x30CB;&#x30A2; &#x30EA;&#x30F3;&#x30B0;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x7D4C;&#x8DEF;&#x3002;Tbc1d23 &#x53EF;&#x80FD;&#x6027;&#x304C;&#x9AD8;&#x3044;&#x884C;&#x70BA; TLR &#x30B7;&#x30B0;&#x30CA;&#x30EB; &#x30A2;&#x30C0;&#x30D7;&#x30BF;&#x30FC; MyD88 &#x3068; Trif &#x306E;&#x4E0B;&#x6D41;&#x30FB;&#x4E0A;&#x6D41;&#x8EE2;&#x5199;&#x3059;&#x308B;&#x8EE2;&#x5199;&#x56E0;&#x5B50; XBP1 &#x3092;&#x8981;&#x56E0;&#x3057;&#x307E;&#x3059;&#x3002;&#x91CD;&#x8981;&#x306A;&#x306F;&#x3001;&#x3059;&#x308B;&#x8EE2;&#x5199;&#x56E0;&#x5B50; XBP1 &#x306E;&#x3088;&#x3046;&#x306A; Tbc1d23&#x3001;&#x30E1;&#x30F3;&#x30C6;&#x30CA;&#x30F3;&#x30B9;&#x304C; LPS &#x306B;&#x3088;&#x3063;&#x3066;&#x8A98;&#x5C0E;&#x3055;&#x308C;&#x308B;&#x708E;&#x75C7;&#x6027;&#x30B5;&#x30A4;&#x30C8;&#x30AB;&#x30A4;&#x30F3;&#x751F;&#x7523;&#x306E;&#x958B;&#x59CB;&#x3057;&#x306A;&#x3044;&#x5F71;&#x97FF;&#x3057;&#x307E;&#x3059;&#x3002;Tbc1d23 &#x306F;&#x3001;&#x81EA;&#x7136;&#x514D;&#x75AB;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B; RAB &#x30AE;&#x30E3;&#x30C3;&#x30D7;&#x3068;&#x3057;&#x3066;&#x6A5F;&#x80FD;&#x3057;&#x307E;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;Tbc1d23 &#x306F;&#x305D;&#x306E;&#x6642;&#x7A7A;&#x9593;&#x306E;&#x30D5;&#x30A1;&#x30C3;&#x30B7;&#x30E7;&#x30F3;&#x3067;&#x30B7;&#x30B0;&#x30CA;&#x30EA;&#x30F3;&#x30B0;&#x81EA;&#x7136;&#x514D;&#x75AB;&#x6291;&#x5236;&#x52B9;&#x679C;&#x3092;&#x767A;&#x63EE;&#x3057;&#x307E;&#x3059;&#x3002;&#x81EA;&#x7136;&#x514D;&#x75AB;&#x30B7;&#x30B0;&#x30CA;&#x30EA;&#x30F3;&#x30B0;&#x306E;&#x5C0F;&#x8AAC;&#x306E;&#x6642;&#x7A7A;&#x9593;&#x30EC;&#x30AE;&#x30E5;&#x30EC;&#x30FC;&#x30BF;&#x306E;&#x540C;&#x5B9A;&#x306E;&#x6BD4;&#x8F03;&#x30B2;&#x30CE;&#x30E0; &#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x306E;&#x81EA;&#x7136;&#x514D;&#x75AB;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x767A;&#x898B;&#x3092;&#x691C;&#x8A3C;&#x3057;&#x307E;&#x3059;&#x3002;

&#x306B;&#x3088;&#x308B; Tbc1d23 RAB &#x30AE;&#x30E3;&#x30C3;&#x30D7;&#x306E;&#x81EA;&#x7136;&#x514D;&#x75AB;&#x306E;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x306E;&#x6642;&#x7A7A;&#x9593;&#x6291;&#x5236;&#x3002;

&#x6211;&#x4EEC;&#x5148;&#x524D;&#x53D1;&#x73B0;&#x7684; Tbc1d23 &#x4F5C;&#x4E3A;&#x5019;&#x9009;&#x4EBA;&#x7684;&#x5929;&#x7136;&#x514D;&#x75AB;&#x4F7F;&#x7528;&#x79C0;&#x4E3D;&#x9690;&#x6746;&#x7EBF;&#x866B;&#x548C;&#x5C0F;&#x9F20;&#x5DE8;&#x566C;&#x7EC6;&#x80DE;&#x4E2D;&#x7684;&#x6BD4;&#x8F83;&#x57FA;&#x56E0;&#x7EC4; RNA &#x5E72;&#x6270;&#x5C4F;&#x5E55;&#x7684;&#x65B0;&#x578B;&#x8C03;&#x8282;&#x3002;&#x4F7F;&#x7528; Tbc1d23 &#x57FA;&#x56E0;&#x6572;&#x9664;&#x5C0F;&#x9F20;&#x548C;&#x5DE8;&#x566C;&#x7EC6;&#x80DE;&#x5DE5;&#x7A0B;&#x4E3A; overexpress Tbc1d23&#xFF0C;&#x6211;&#x4EEC;&#x73B0;&#x5728;&#x663E;&#x793A; Tbc1d23 &#x662F;&#x4E00;&#x79CD;&#x4E00;&#x822C;&#x6291;&#x5236;&#x5242;&#x5929;&#x7136;&#x514D;&#x75AB;&#x4FE1;&#x53F7;&#x3001; &#x5F3A;&#x70C8;&#x6291;&#x5236;&#x591A;&#x4E2A;&#x5355;&#x53CD;&#x548C; dectin &#x4FE1;&#x53F7;&#x901A;&#x8DEF;&#x3002;Tbc1d23 &#x53EF;&#x80FD;&#x884C;&#x4E3A;&#x7684;&#x5355;&#x53CD;&#x4FE1;&#x53F7;&#x9002;&#x914D;&#x63A5;&#x5934; MyD88 &#x548C;&#x7279;&#x91CC;&#x592B;&#x987A;&#x6D41;&#x548C;&#x9006;&#x6D41;&#x7684;&#x8F6C;&#x5F55;&#x56E0;&#x5B50; XBP1&#x3002;&#x91CD;&#x8981;&#x7684;&#x662F;&#xFF0C;&#x50CF; XBP1&#xFF0C;Tbc1d23 &#x4F1A;&#x5F71;&#x54CD;&#x7EF4;&#x4FEE;&#xFF0C;&#x4F46;&#x4E0D;&#x662F;&#x542F;&#x52A8;&#x7684; LPS &#x6240;&#x81F4;&#x7684;&#x708E;&#x6027;&#x7EC6;&#x80DE;&#x56E0;&#x5B50;&#x751F;&#x4EA7;&#x3002;Tbc1d23 &#x4F5C;&#x4E3A; RAB &#x5DEE;&#x8DDD;&#x8C03;&#x8282;&#x5929;&#x7136;&#x514D;&#x75AB;&#x4FE1;&#x53F7;&#x3002;&#x56E0;&#x6B64;&#xFF0C;Tbc1d23 &#x65BD;&#x52A0;&#x5176;&#x5BF9;&#x5929;&#x7136;&#x514D;&#x75AB;&#x5728;&#x65F6;&#x7A7A;&#x7684;&#x65F6;&#x5C1A;&#x4FE1;&#x53F7;&#x7684;&#x6291;&#x5236;&#x4F5C;&#x7528;&#x3002;&#x65B0;&#x578B;&#x65F6;&#x7A7A;&#x8C03;&#x8282;&#x5929;&#x7136;&#x514D;&#x75AB;&#x4FE1;&#x53F7;&#x8BC6;&#x522B;&#x9A8C;&#x8BC1;&#x5148;&#x5929;&#x514D;&#x75AB;&#x57FA;&#x56E0;&#x53D1;&#x73B0;&#x7684;&#x6BD4;&#x8F83;&#x57FA;&#x56E0;&#x7EC4;&#x5B66;&#x65B9;&#x6CD5;&#x3002;

&#x65F6;&#x7A7A;&#x7684;&#x5929;&#x7136;&#x514D;&#x75AB;&#x4FE1;&#x53F7;&#x7531; Tbc1d23 &#x7684; RAB &#x5DEE;&#x8DDD;&#x7684;&#x6291;&#x5236;&#x4F5C;&#x7528;&#x3002;

We previously identified Tbc1d23 as a candidate novel regulator of innate immunity using comparative genomics RNA interference screens in Caenorhabditis elegans and mouse macrophages. Using Tbc1d23 knockout mice and macrophages engineered to overexpress Tbc1d23, we now show that Tbc1d23 is a general inhibitor of innate immunity signaling, strongly inhibiting multiple TLR and dectin-signaling pathways. Tbc1d23 likely acts downstream of the TLR-signaling adaptors MyD88 and Trif and upstream of the transcription factor XBP1. Importantly, like XBP1, Tbc1d23 affects the maintenance, but not the initiation, of inflammatory cytokine production induced by LPS. Tbc1d23 acts as a RAB-GAP to regulate innate immunity signaling. Thus, Tbc1d23 exerts its inhibitory effect on innate immunity signaling in a spatiotemporal fashion. The identification of a novel spatiotemporal regulator of innate immunity signaling validates the comparative genomics approach for innate immunity gene discovery.

Spatiotemporal Inhibition of Innate Immunity Signaling by the Tbc1d23 RAB-GAP.

Nous avons identifi&eacute; pr&eacute;c&eacute;demment Tbc1d23 comme un r&eacute;gulateur de nouveaux candidats de l&#39;immunit&eacute; inn&eacute;e &agrave; l&#39;aide d&#39;&eacute;crans de g&eacute;nomique comparative RNA interference dans les macrophages de Caenorhabditis elegans et la souris. &Agrave; l&#39;aide de souris knock-out Tbc1d23 et macrophages machin&eacute;s pour overexpress Tbc1d23, nous montrons maintenant que Tbc1d23 est un inhibiteur g&eacute;n&eacute;ral de l&#39;immunit&eacute; inn&eacute;e de signalisation, inhibant fortement multiples TLR et dectin-signalisation des voies. Actes susceptibles de Tbc1d23 en aval des signalisation TLR adaptateurs MyD88 et FTBC et en amont de la transcription factor XBP1. Ce qui est important, comme XBP1, Tbc1d23 affecte le maintien, mais pas l&#39;ouverture, de la production de cytokines inflammatoires induite par le LPS. Tbc1d23 agit comme un RAB-GAP pour r&eacute;guler l&#39;immunit&eacute; inn&eacute;e de signalisation. Ainsi, Tbc1d23 exerce son effet inhibiteur sur l&#39;immunit&eacute; inn&eacute;e de signalisation de fa&ccedil;on spatio-temporelle. L&#39;identification d&#39;un nouveau r&eacute;gulateur spatio-temporelle de signalisation de l&#39;immunit&eacute; inn&eacute;e valide l&#39;approche g&eacute;nomique comparative pour la d&eacute;couverte de g&egrave;nes de l&#39;immunit&eacute; inn&eacute;e.

Inhibition spatio-temporelle de l&#39;immunit&eacute; inn&eacute;e de signalisation par l&#39;&eacute;cart Tbc1d23 RAB.

Wir haben vorher Tbc1d23 als Kandidat Roman Regulator der angeborenen Immunit&auml;t &uuml;ber vergleichende Genomik-RNA-Interferenz-Bildschirme in Caenorhabditis Elegans und Maus Makrophagen identifiziert. Mit Tbc1d23-Knockout-M&auml;use und Makrophagen konstruiert, HER2 Tbc1d23, wir jetzt zeigen, dass Tbc1d23 eine allgemeine Inhibitor der angeborenen Immunit&auml;t Signalisierung, stark behindern die mehrere TLR und Dectin-Signalisierung Wege. Tbc1d23 wahrscheinlich Handlungen von der TLR-Signalisierung-Adaptern MyD88 und Trif downstream und upstream der Transkription Faktor XBP1. Wichtig ist, betrifft die Tbc1d23 wie XBP1, die Pflege, aber nicht die Einleitung, der entz&uuml;ndliche Cytokine Produktion durch LPS induziert. Tbc1d23 fungiert als eine RAB-L&uuml;cke, Angeborene Immunit&auml;t Signalisierung zu regulieren. Daher &uuml;bt Tbc1d23 seine hemmende Wirkung auf Angeborene Immunit&auml;t signalisieren in eine raumzeitliche Mode. Die Identifizierung eines neuartigen raumzeitliche Regulators Angeborene Immunit&auml;t zu signalisieren &uuml;berpr&uuml;ft den vergleichende Genomik-Ansatz f&uuml;r Angeborene Immunit&auml;t Gen-Entdeckung.

Raumzeitliche Hemmung der angeborenen Immunit&auml;t Signalisierung durch die Tbc1d23-RAB-L&uuml;cke.

&#xC6B0;&#xB9AC;&#xB294; &#xC774;&#xC804;&#xC5D0; Caenorhabditis elegans&#xC640; &#xB9C8;&#xC6B0;&#xC2A4; &#xB300; &#xC2DD; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xBE44;&#xAD50; &#xC720;&#xC804;&#xCCB4;&#xD559; RNA &#xAC04;&#xC12D; &#xC2A4;&#xD06C;&#xB9B0;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xD0C0;&#xACE0; &#xB09C; &#xBA74;&#xC81C;&#xC758; &#xD6C4;&#xBCF4; &#xC18C;&#xC124; &#xC870;&#xC815;&#xAE30;&#xB85C; tbc1d23&#xB97C; &#xBC1C;&#xACAC;. &#xC6B0;&#xB9AC; &#xC9C0;&#xAE08; Tbc1d23 &#xC2E0;&#xD638;, &#xB2E4;&#xC911; TLR &#xBC0F; dectin &#xC2E0;&#xD638;&#xB97C; &#xAC15;&#xB825; &#xD558; &#xAC8C; &#xC5B5;&#xC81C; &#xD558;&#xB294; &#xD0C0;&#xACE0; &#xB09C; &#xBA74;&#xC81C;&#xC758; &#xC77C;&#xBC18;&#xC801;&#xC778; &#xC5B5;&#xC81C;&#xC81C;&#xB294; &#xD45C;&#xC2DC; Tbc1d23 &#xB179;&#xC544;&#xC6C3; &#xB9C8;&#xC6B0;&#xC2A4; &#xBC0F; overexpress Tbc1d23 &#xC124;&#xACC4; &#xD558;&#xB294; &#xB300; &#xC2DD; &#xC138;&#xD3EC;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xACBD;&#xB85C;. Tbc1d23 &#xAC00;&#xB2A5;&#xC131;&#xC774; &#xD589;&#xC704; TLR &#xC2E0;&#xD638; &#xC5B4;&#xB311;&#xD130; MyD88 &#xBC0F; Trif&#xC758; &#xB2E4;&#xC6B4;&#xC2A4;&#xD2B8;&#xB9BC; &#xBC0F; &#xC5C5;&#xC2A4;&#xD2B8;&#xB9BC; &#xC804;&#xC0AC;&#xC758; XBP1 &#xC694;&#xC18C;. &#xC911;&#xC694; &#xD55C; &#xAC83;&#xC740;, XBP1, &#xAC19;&#xC740; tbc1d23&#xC5D0;&#xB294; &#xC720;&#xC9C0; &#xBCF4;&#xC218;, &#xD558;&#xC9C0;&#xB9CC; &#xAC1C;&#xC2DC;, LPS&#xC5D0; &#xC758;&#xD574; &#xC720;&#xB3C4; &#xB41C; &#xC5FC;&#xC99D; &#xC131; cytokine &#xC0DD;&#xC0B0;&#xC774;&#xC5D0; &#xC801;&#xC6A9; &#xB429;&#xB2C8;&#xB2E4;. Tbc1d23 &#xD0C0;&#xACE0; &#xB09C; &#xBA74;&#xC5ED; &#xC2E0;&#xD638; &#xC870;&#xC808; &#xD558; &#xB7A9; &#xAC04;&#xACA9;&#xC73C;&#xB85C; &#xC791;&#xB3D9; &#xD569;&#xB2C8;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, Tbc1d23 &#xD0C0;&#xACE0; &#xB09C; &#xBA74;&#xC81C; spatiotemporal &#xBC29;&#xC2DD;&#xC5D0;&#xC11C; &#xC2E0;&#xD638;&#xC5D0; &#xBBF8;&#xCE58;&#xB294; &#xC5B5;&#xC81C; &#xC601;&#xD5A5;&#xC744; &#xBBF8;&#xCE58;&#xB294;. &#xD0C0;&#xACE0; &#xB09C; &#xBA74;&#xC5ED; &#xC2E0;&#xD638;&#xC758; &#xC18C;&#xC124; spatiotemporal &#xB808; &#xADE4; &#xB808;&#xC774; &#xD130;&#xC758; &#xD0C0;&#xACE0; &#xB09C; &#xBA74;&#xC5ED; &#xC720;&#xC804;&#xC790; &#xBC1C;&#xACAC;&#xC744; &#xC704;&#xD55C; &#xBE44;&#xAD50; &#xC720;&#xC804;&#xCCB4;&#xD559; &#xC811;&#xADFC; &#xBC29;&#xC2DD;&#xC744; &#xD655;&#xC778;&#xD569;&#xB2C8;&#xB2E4;.

Tbc1d23 &#xB7A9; &#xCC28;&#xC774; &#xC758;&#xD574; &#xD0C0;&#xACE0; &#xB09C; &#xBA74;&#xC5ED; &#xC2E0;&#xD638; spatiotemporal &#xC800;&#xD574;.

Previamente se identificaron Tbc1d23 como un regulador nuevo candidato de la inmunidad innata en pantallas de interferencia de RNA de gen&oacute;mica comparativa de Caenorhabditis elegans y rat&oacute;n macr&oacute;fagos. Utilizando ratones knockout de Tbc1d23 y macr&oacute;fagos dirigidos a sobrexpresan Tbc1d23, mostramos ahora que Tbc1d23 es un inhibidor general de inmunidad innata, se&ntilde;alizaci&oacute;n, fuertemente inhibir m&uacute;ltiples TLR y dectin-se&ntilde;alizaci&oacute;n v&iacute;as. Tbc1d23 probables actos posteriores de los adaptadores de TLR-se&ntilde;alizaci&oacute;n MyD88 y Trif y aguas arriba de la transcripci&oacute;n factor XBP1. Lo importante, como XBP1, Tbc1d23 afecta el mantenimiento, pero no la iniciaci&oacute;n, de la producci&oacute;n de citoquinas inflamatorias inducida por LPS. Tbc1d23 act&uacute;a como un RAB-GAP para regular la inmunidad innata de se&ntilde;alizaci&oacute;n. As&iacute;, Tbc1d23 ejerce su efecto inhibitorio sobre la inmunidad innata, se&ntilde;alizaci&oacute;n de manera espacio-temporal. La identificaci&oacute;n de un nuevo regulador espaciotemporal de se&ntilde;alizaci&oacute;n de la inmunidad innata valida el enfoque de la gen&oacute;mica comparativa para el descubrimiento de genes de inmunidad innata.

Inhibici&oacute;n espacio-temporal de la inmunidad innata se&ntilde;alizaci&oacute;n por el RAB Tbc1d23-boquete.

The innate immune response plays a critical role in pathogen clearance. However, dysregulation of innate immunity contributes to acute inflammatory diseases such as sepsis and many chronic inflammatory diseases including asthma, arthritis, and Crohn's disease. Pathogen recognition receptors including the Toll-like family of receptors play a pivotal role in the initiation of inflammation and in the pathogenesis of many diseases with an inflammatory component. Studies over the last 15 years have identified complex innate immune signal transduction pathways involved in inflammation that have provided many new potential therapeutic targets to treat disease. We are investigating several novel genes that exert spatial and in some cases temporal regulation on innate immunity signaling pathways. These novel genes include Tbc1d23, a RAB-GAP that inhibits innate immunity. In this review, we will discuss inflammation, the role of inflammation in disease, innate immune signal transduction pathways, and the use of spatiotemporal regulators of innate immunity as potential targets for discovery and therapeutics.

Identifying novel spatiotemporal regulators of innate immunity.

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.

An evolutionarily conserved innate immunity protein interaction network.

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.

Limiting of the innate immune response by SF3A-dependent control of MyD88 alternative mRNA splicing.

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.

Article	Year
The zinc finger protein REF-2 functions with the Hox genes to inhibit cell fusion in the ventral epidermis of C. elegans. Development (Cambridge, England)\| PubMed ID: 12091304	2002
Specificity and complexity of the Caenorhabditis elegans innate immune response. Molecular and cellular biology\| PubMed ID: 17526726	2007
Toxicogenomic analysis of Caenorhabditis elegans reveals novel genes and pathways involved in the resistance to cadmium toxicity. Genome biology\| PubMed ID: 17592649	2007
Identification of innate immunity genes and pathways using a comparative genomics approach. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 18463287	2008
Cell fusion in Caenorhabditis elegans. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 18979238	2008
Impact of cigarette smoke exposure on innate immunity: a Caenorhabditis elegans model. PloS one\| PubMed ID: 19718433	2009
Identification of novel genes that mediate innate immunity using inbred mice. Genetics\| PubMed ID: 19805818	2009
The Caenorhabditis elegans germ line regulates distinct signaling pathways to control lifespan and innate immunity. The Journal of biological chemistry\| PubMed ID: 19923212	2010
Model systems to the rescue: The relationship between aging and innate immunity. Communicative & integrative biology\| PubMed ID: 21057627	2010
Novel regulators of the systemic response to lipopolysaccharide. American journal of respiratory cell and molecular biology\| PubMed ID: 21131441	2011
Identification of novel innate immune genes by transcriptional profiling of macrophages stimulated with TLR ligands. Molecular immunology\| PubMed ID: 21665277	2011
Spatiotemporal Inhibition of Innate Immunity Signaling by the Tbc1d23 RAB-GAP. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 22312129	2012
Identifying novel spatiotemporal regulators of innate immunity. Immunologic research\| PubMed ID: 22926826	2013
An evolutionarily conserved innate immunity protein interaction network. The Journal of biological chemistry\| PubMed ID: 23209288	2013
Limiting of the innate immune response by SF3A-dependent control of MyD88 alternative mRNA splicing. PLoS genetics\| PubMed ID: 24204290	2013
Comparative Genomics RNAi Screen Identifies Eftud2 as a Novel Regulator of Innate Immunity. Genetics\| PubMed ID: 24361939	2013

Scott Alper

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